JP2003521932A - Water-soluble ligand binding proteins and analogs of ligand-gated ion channels, crystals thereof, and their use for screening ligands for ligand-gated ion channels - Google Patents

Abstract

  (57) [Summary] Provided are mollusk-derived water-soluble ligand binding proteins and analogs of ligand-gated ion channels, their crystals, and their use for screening ligands for ligand-gated ion channels. In particular, a water-soluble ligand-binding protein capable of forming a multimer and crystallizing is provided. A crystal structure of one of these proteins, acetylcholine binding protein (AChBP), is provided, which is used to create a 3D model of the extracellular ligand binding domain of a ligand-gated ion channel, and thus It can be used to screen for drugs that act on ion channels. Further, a chimeric protein capable of binding a ligand of a ligand-dependent receptor, comprising at least an AChBP amino acid determining the solubility of AChBP at the same position as AChBP, and further including an amino acid determining the binding to the ligand. Provided.

Description

Detailed Description of the Invention

[0001] having substantially ligand binding profile similar to the ligand binding profile of the summary ligand-gated ion channels of the present invention, a novel water-soluble ligand binding protein was identified and isolated. DNA molecules encoding such proteins have been cloned and characterized. The biological and structural properties of these proteins are disclosed, as are the amino acid and nucleotide sequences. Recombinant DNA molecules and portions thereof are useful for isolating homologues of DNA molecules, identifying and isolating genomic equivalents of DNA molecules, and identifying, detecting, or isolating mutant forms of DNA molecules. Recombinant expression systems have been used to functionally produce functional DNA molecules encoding water-soluble ligand binding proteins as well as chimeras. In addition, water-soluble ligand-binding proteins can be crystallized, which reveals a three-dimensional (3D) structure and 3D of the ligand-binding domain of ligand-gated ion channels.
Structural modeling has become possible. The present invention is further in the field of the development of new drugs that can selectively intervene in neuronal signaling pathways. The present invention is
More particularly, it relates to providing new analogs of channel-coupled receptors, their crystal structures, and their use in screening ligands for these receptors.

[0002] Some documents are cited by name throughout the text or are referred to by numbers in parentheses. The complete bibliographic information can be found at the end of the specification, just before the claims. Each document cited herein, including any manufacturer's specifications, instructions, etc., is hereby incorporated by reference. However, none of the cited documents is admitted to be prior art to the present invention.

[0003] transmission in the background the central nervous system (CNS) of the invention is caused by a complex interaction of the electrical signals and chemical signals. Molecules that carry chemical information are called neurotransmitters. Chemical information is translated into electrical currents in the postsynaptic membrane that specializes in the recognition and binding of neurotransmitters by protein receptors. Specific binding of a ligand to one such type of receptor, the ionotropic receptor, induces rapid opening of the ion channel coupled to the receptor. An important group of ion channel receptors are:
7-aminobutyric acid (GABA _A ) receptors, glycine receptors, serotonin-3 (5-HT3) receptors, and both neuronal and muscular nicotinic acetylcholine receptors (nA)
ChR), including a superfamily of channel-coupled receptors, also called ligand-gated receptors. These receptors have certain structural features, such as (
1) a 15-residue cysteine loop between amino acids 128 and 142 corresponding to the Torpedo AChRα unit, (2) four transmembrane domains, (3) a similar subunit arrangement, and (4) amino acids Shares sequence homology. Activation of these receptors causes changes in current and hyperpolarization of cell membranes, resulting in inhibition of cell electrical activity. GABA _A and glycine receptors are coupled to chloride-selective channels, so inhibition of electrical activity leads to inhibition of cellular responses. On the other hand, 5-HT3 receptors and nAChR are cation-selective channels (Na +,
Since they are linked to K +, Ca2 +), their activation triggers an excitatory response in cells. AChRs are the best studied of the ligand-dependent receptors; for a review, see Arias, Brain Research Reviews, 25 (1997) 133-191 and Arias, Neurochem. Int. 36 (2000), 595-645). Mutations in these ligand-gated ion channels (LGICs) lead to congenital myasthenia gravis, epilepsy, startle syndrome, and alcohol sensitivity (Vafa and Schofield, Int. Rev. Neurobiol.
42, 285-332; 1998). NAChR mediates nicotine addiction in long-term smokers.
Because nicotine binding to these receptors also has a positive effect on Alzheimer's, Parkinson's, and schizophrenia, these receptors represent important drug targets (Paterson and Nordbreg ( Nordberg A., "Neuronal nicotinic receptors in the human brain
the human brain) "Prog. Neurobiol. 61, 75-111; 2000).

The development of new pharmacologically active compounds that can selectively bind, or in some cases non-selectively bind to channel-coupled receptors, has led to an understanding of the processes that occur in the nervous system and It is of paramount importance in the treatment of disorders of the nervous state. The development of such pharmacologically active compounds requires the availability of reliable model systems of the corresponding receptors. To date, the primary structural features (amino acid sequences) of various receptors have been well elucidated. It has been discovered that certain subunits of AChR are determinants of the pharmacological specificity or affinity of this receptor for its ligands (Corringer et al., J. Neuroscience 18 (1
998), 648-657). However, studies of the ligand binding properties of receptor proteins have been hampered by the still unknown unknown protein spatial structure critical for ligand binding. This is in part because crystallization of receptor proteins has been unsuccessful to date.

The technical problems defined above are solved by the present invention by providing the embodiments characterized in the claims.

Accordingly, in one aspect, the present invention relates to a mollusc-derived water-soluble protein capable of binding a ligand of a ligand-dependent receptor.

It has been discovered by the present invention that certain mollusc acetylcholine binding proteins (AChBPs) exhibit striking structural similarities to channel-coupled receptors on the one hand and interesting physical properties such as water solubility on the other hand. Was done. Mollusc AChBPs form multimers, especially pentamers, and are capable of binding specific toxins such as α-bungarotoxin. These multimers may be of the same kind (the same unit) or may be of different kinds (the different units). These properties make AChBP well suited as a model system to study the binding of candidate ligands to channel-coupled receptors. It was possible to produce these mollusc AChBPs in a recombinant system. Therefore, simple and large-scale production of AChBP is possible. In addition, mollusk ACh
It is possible to construct hybrid proteins that share the physical properties of BP and the pharmacological properties of the (human) channel-coupled receptors, and thus for this purpose to screen ligands for these receptors. New dedicated tools are provided.

AChBP is a natural analogue of the extracellular domain of the neuronal nicotinic acetylcholine receptor (nAChR) α subunit. It lacks the domain that forms the transmembrane ion channel, in contrast to nAChR, but assembles into a homopentamer similar to nAChR (FIG. 6). Moreover, AChBP has ligand binding properties typical of nicotinic receptors. The three-dimensional structure of AChBP was elucidated by X-ray crystallography at 2.7Å resolution (current R _factor = 27.9%, R _free = 30.0%). Unwin, S
truct. Struct. As measured in EM studies by Biol 121 (1998), 181-190, AChBP forms stable homopentamers with dimensions comparable to those of the nAChR ligand binding domain in crystals as in solution. To do. The high-resolution crystal structure of AChBP, along with biochemical and pharmacological data, makes it an excellent mimic of the ligand-binding domain of ligand-gated ion channels including nAChR, 5-HT3R, GABA _A , _C R, and GlyR. It supports the extrapolation of AChBP.

[0009] Four types of AChBPs according to the present invention are exemplified herein, Lymnaea stagnalis (L-AChBP T1 and L-AChBP T2) and Bulinus truncatus (B-AChBP T1 and B-AChBP It was isolated and cloned from the CNS of T2). L-AChBP T1 and 2 are 229 amino acid proteins with a signal sequence of 19 amino acids (B-AChBP 21 and 224 amino acids for T1 and 2, respectively; see also Figure 1), having sequence homology with the extracellular domain of the subunit of the ligand-gated ion channel (Figure 3),
In particular, it has sequence homology with the extracellular domain of the subunit of nAChR (Fig. 4 and
Five). The mass of purified AChBP from Limnaea has been determined by mass spectrometry. The glycosylated form has a mass of about 24720 Da and the deglycosylated form has a mass of about 23832 Da. Glycosylated AChBP migrates between 14 and 26kDA marker proteins on SDS-PAGE. A hydrophobicity plot of AChBP is shown in FIG. This reveals regions of the ligand-binding protein that are significantly hydrophobic and therefore can be replaced at least partially or with their essential amino acids in the ligand-binding domain of the ligand-gated ion channel. Sequence conservation is the so-called loop region (Arias, Ne containing the residues involved in ligand binding.
urochem. Int. 36 (2000), 595-645). A cysteine residue characteristic of the Cys loop family of ligand-dependent receptors is conserved in AChBP. The double cysteine commonly found in the α subunit of nAChR (dou
ble cysteine) also exists. The AChBP protein sequence ends at the beginning of the first predicted transmembrane domain in nAChR. The ligand binding properties of AChPBs are described in Example 4 and summarized in Table 2.

The terms “channel-coupled receptor”, “ligand-gated receptor”, “ligand-gated ion channel” are used interchangeably herein. However, for natural, especially human, molecules, preferably "ligand-gated ion channels"
Is used. The water-soluble ligand-binding proteins of the invention also have at least 10%, more preferably at least 12%, even more preferably at least 15%, and most preferably at least 20% vertebrate ligand-gated ion channels.
Can be characterized as a ligand-binding protein having the amino acid sequence identity of, but lacking any transmembrane domain. The ligand-gated receptors of the present invention have substantially the same ligand binding properties of vertebrate, preferably mammalian, most preferably human ligand-gated ion channels, but with at least one change in the original amino acid sequence. Characterized in that this change determines or contributes to the water solubility of a water-soluble ligand protein found in molluscs, especially snails as described in more detail below. Brings the presence of amino acids.

The terms “ligand-binding protein”, “ligand-binding domain”, and “ligand-binding receptor” refer to water-soluble ligand-binding proteins or corresponding modified ligand-gated ion channels that are required for ligand binding. Means that at least a portion is included. The ligand binding domain consists, at a minimum, of peptides containing this domain. However, the use of this term is meant to include a ligand binding domain or protein that is composed of a larger portion of the ligand-gated ion channel, such as, for example, the completely reconstituted nicotinic acetylcholine receptor.

As shown in FIG. 3, the nicotinic acetylcholine receptor (nAChR) belongs to a well understood member of the ligand-gated ion channel superfamily. Members of this signaling protein group, including 5-HT3, glycine, GABA _A , and GABA _C receptors, share a common secondary structure based on a high degree of sequence similarity, 3
It is thought to share the secondary and quaternary structures. Therefore, the novel findings for the exemplified AchBP are expected to apply equally to other members of the mentioned ligand-gated ion channel superfamily. Therefore, water-soluble proteins capable of binding ligands for any of these ligand-gated ion channels may be found in mollusks, and their 5-HT3, GABA _A , and glycine receptors are associated with their binding. It may be modified to substantially retain the affinity.

Therefore, the ligand of the water-soluble ligand binding protein is preferably acetylcholine, γ-aminobutyric acid (GABA), glycine, nicotine, or serotonin. Isolation of such a water soluble ligand binding protein can be performed as described in Example 1 for AChBPs of the invention. Instead of α-bungarotoxin, other well known ligands can be used for affinity purification. Most preferably, the water soluble ligand binding protein of the present invention is acetylcholine binding protein (AChBP). Preferably, the ligand binding protein substantially exhibits the binding properties shown in Table 2.

The acetylcholine binding proteins used in accordance with the present invention may be used in aquatic mollusc species, in particular oysters (Gastropoda), in particular pulmonary oysters (lus).
First obtained from a species derived from nged snail) (Pulmonata). Pulmonaries are classified into the basic eye (Basommatophora) (mostly aquatic mussels), systemomatophora (Systellommatophora), and stigma (Stylommatophora) (mostly land mussels). The group of the eye-eyes is the genus Acroloxidae.
) (For example, the genus Acroloxus), the family Lynnaeidae (Lymnaeidae)
) (Eg Galba, Stagnicola, Radix (R
adix), Limnaea (genus Lymnaea), Phalaenopsis family (Physidae) (for example,
Physa and Aplexa), and the mussel family (P
lanorbidae) (eg, paranorbis (Planorbis), anisus (Anisus), ancillus (Ancylus), giraura (Gyraulus), biophalaria (Biomphalari)
a), and the genus Bulinus). Examples of suitable species are Limnaea stagnalis (pond snail) and Brinnus truncatas. Isolation of AChBPs from these snails, cDNAs encoding these AChBPs
Cloning and their characterization, including the complete amino acid sequence, is described in the examples. The cDNA and amino acid sequences of AChBP of Limnaea stagnaris are shown in SEQ ID NOs: 1 and 2 (L-AChBP T1) and SEQ ID NOs: 3 and 4 (L-AChBP T2
). The cDNA and amino acid sequences of A. chrysalis of B. truncata are SEQ ID NOs: 5 and 6 (B-AChBP T1) and SEQ ID NOs: 7 and 8 (B-AChBP T2). The characteristics of these proteins are further described in the examples and accompanying figures.

Although water-soluble ligand binding proteins derived from pulmonary species, preferably primordial species, are preferred, the present invention generally comprises a ligand comprising an amino acid sequence selected from the group consisting of: It will be understood that it relates to any water soluble protein capable of binding a ligand for a dependent receptor: (a) the amino acid set forth in any one of SEQ ID NOs: 2, 4, 6 or 8. A sequence or its functional equivalent, or a fragment of at least 5 consecutive amino acids thereof;
(B) an amino acid sequence having at least 30% amino acid identity with the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, or 8; and (c) preferably with a binding affinity of at least 10 ^-7 M. An amino acid sequence that provides a protein detectable by a monoclonal antibody or a polyclonal antibody that recognizes the protein containing the amino acid sequence of (a) or (b).

As is well known in the art, identity or similarity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences determined by sequence comparison. In the art, identity also refers to the degree of sequence relatedness between polypeptide sequences or between polynucleotide sequences, as the case may be, as determined by the match between the strings of polypeptide or polynucleotide sequences. means. Both identity and similarity can be easily calculated ("Computational Molecular Biology", edited by Lesk, Oxford Un.
iversity Press, New York, 1988; “Biocomputing: Informatics and Genome Pro
”, Smith, Ed., Academic Press, New York, 1993;“ Computer Analysis of Sequence Data, Part I
)), Griffin and Griffin, Humana Press, New Jersey
, 1994; "Sequence Analysis in Molecular Bio
logy ”, von Heinje, Academic Press, 1987; and“ Sequence Analysis Primer ”, Gribsk.
ov) and Devereux, M Stockton Press, New York, 1991)
. There are numerous methods for determining identity and similarity between two polynucleotide sequences or between two polypeptide sequences, but both terms are well known to those of skill in the art (Von Heinge, supra; Gribskov and Develeux, supra; and Carillo and Lipman, SIAM J. Applied Math. 48.
(1988), 1073). Commonly used methods for determining identity or similarity between sequences include, but are not limited to, those disclosed in Carilo and Lippmann (see above). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs. A preferred computer program method for determining the identity and similarity between two sequences is the GCG program package (Debereux et al., Nucleic Acids Rese
arch 12 (1984), 387), BLASTP, BLASTN, psiBLAST, and FASTA (Atschul et al., J. Molec. Biol. 215 (1990), 403).

In another aspect, the invention relates to a water-soluble protein capable of binding a ligand of a ligand-dependent receptor, including: (a) a position the same as or corresponding to the position of said water-soluble protein. In, at least the amino acid of said water-soluble protein that determines the solubility of the water-soluble protein; and (b) at least 4 amino acids that determine the binding to the ligand.

Protein expression studies show that mollusc Limnaea stagnaris wild-type AChBP
It has been found that can be produced in the yeast Pichia pastoris. Yeast cells express AChBP in the homopentamer form and secrete the protein complex into the medium. Large amount of AChBP per volume of medium produced (2m / l medium)
and up to a large amount of culturable yeast allow large-scale production of AChBP. Wild type
In addition to AChBP, various AChBP variants have been produced in Pichia pastoris. As these variants, the following 1-point mutation (the number means the amino acid position in the Limnair stagnaris AChBP sequence shown in SEQ ID NO: 2 counted from the first amino acid of the signal peptide. The character before the number was originally Of the amino acids, and the letter after the number indicates the mutant amino acid) N85D, H164Y, D194N, Y204P, Y211P, and D2
Examples include mutants containing 13N.

Accordingly, the present invention relates to a mollusc-derived water-soluble protein, preferably acetylcholine binding protein (AChBP), capable of forming multimers and binding a ligand of a ligand-dependent receptor. These proteins, on the one hand, contain at least the AChBP amino acids that determine the solubility of AChBP in the same positions as AChBP and, on the other hand, the amino acids that determine the binding of ligand-dependent receptors to the ligand. The degree of identity with a mollusc AChBP sequence is, for example, at least 15%, preferably 20%, more preferably 30%, and more preferably as determined using the BLAST algorithm well known in the art. More preferably 40%, preferably at least 50%, even at least 60%, preferably more than 70%, more preferably more than 80%, most preferably at least 90% identity or more It can be defined by amino acid identity. The amino acids that determine binding to the ligand correspond to the receptor sequence and preferably differ from the corresponding AChBP amino acids by at least 4 amino acids, preferably at least 6 amino acids, or even at least 8 amino acids (one (Including at least 3 or 4 amino acids following). Preferred embodiments of these proteins are defined further below. Typically, the water-soluble ligand binding protein, or domain as part of a chimeric ligand-gated ion channel, for example, comprises 200-240 amino acids.

The ligand is preferably acetylcholine, nicotine, lophotoxin (lophot).
oxin), d-tubocurarine, carbamylcholine, galantamine, or epibatidine. The ligand-dependent receptor may be derived from arthropods (preferably insects), plants (preferably higher plants, most preferably seed plants), or vertebrates (preferably mammals, most preferably humans) Well, preferably, the ligand-dependent receptor is the nicotinic acetylcholine receptor.

Generally, the amino acids that determine solubility in the water soluble ligand binding proteins of the invention are at the same position as AChBP having the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6 or 8. . The region that determines solubility is based on solvent accessibility in the structure. For example, each amino acid residue can be selected according to Figure 10 or 11 where solvent accessible regions are shown. Preferably, the water soluble ligand binding protein of the invention is SEQ ID NO: 2.
, 4, 6, or 8 comprising an amino acid sequence having at least 40% amino acid identity with the amino acid sequence of mature AChBP, wherein the ligand-binding amino acid corresponds to the ligand-dependent receptor. Is replaced with an amino acid that

In one embodiment of the protein of the present invention, the amino acid (a) that determines solubility is 20-44, 73-81, 86-92, 112-120, 135- of SEQ ID NO: 2. 152nd, 166
Includes hydrophilic amino acids (Asp, Glu, Arg, Lys) from the sequence at positions -189, 196-20, 209-213, and / or 219-227.

L-AChBP The amino acid sequences of T1 (SEQ ID NO: 2) and T2 (SEQ ID NO: 4) are almost the same. Always L-AChBP for easy understanding Reference is made to T1 (SEQ ID NO: 2). However, Arg (167) becomes Gly (167), and Thr (203)
With the notable exception of Ile (203), all references to amino acid residues apply to both T1 and T2. Furthermore, L-AChBP With respect to amino acid residues (domains) from T1 and the corresponding residues from B-AChBP, the list below lists L-AChBP and
B-AChBP shows different amino acid positions. All amino acid residue numbers below are
It corresponds to the position (counting from methionine (1)) in the amino acid sequence of the immature protein. It is also possible to start the numbering from the beginning of the amino acid sequence of the mature sequence (L (1) DRAD for L-AChBP, Q (1) IRW for B-AChBP). When this second method is used (the 1st amino acid of the mature sequence = 1st position), 19 from the position number of L-AChBP is changed to B-AChBP.
Simply subtract 21 from the position number of (eg, Asp (36) becomes Asp (17) in L-AChBP and Asp (15) in B-AChBP). For further embodiments, the position of L-AChBP T1 (SEQ ID NO: 2) is shown, followed by L-AChBP T1. T2 (SEQ ID NO: 4) and B-
AChBP T1 (SEQ ID NO: 6) and B-AChBP The corresponding amino acid position in the amino acid sequence of T2 (SEQ ID NO: 8) is (L-AChBP T1 and T2: B-AChBP Shown in the form of T1 and T2).

In a preferred embodiment, the amino acid (a) that determines solubility is the amino acids Asp (36), Asp (68), Glu (115), Arg (137), Asp (143), Asp (SEQ ID NO: 2). 148
), Glu (150), Arg (167), Arg (189), Glu (215), where Glu is A
It may be replaced with sp (and vice versa) or Arg with Lys (and vice versa). (L-AchBP T1 and T2: B-AchBP T1 and T2; Asp (36): Asp (36
); Asp (68): Asp (68); Glu (115): Glu (116); Arg (137): Arg (138)
Asp (143): Asp (144); Asp (148): Asp (149); Glu (150): Glu (151)
Arg (167): Gly (167) (L-AChBPT2): Lys (168); Arg (189): Lys (190
); Glu (215): Glu (216)).

In an even more preferred embodiment, the water soluble ligand binding protein is amino acids Cys (142), Thr (149), Ala (153), Thr (154), Cys (155) of SEQ ID NO: 2.
), Arg (156), Ile (157) and / or Lys (158) (L-AChBP T1 and T2: B-AChBP T1 and T2; Cys (142): Cys (143); Thr (149): Thr (150)
Ala (153): Ala (154); Thr (154): Thr (155); Cys (155): Cys (156)
Arg (156): Arg (157); Ile (157): Ile (158); Lys (158): Lys (159)
). In a further embodiment, the water soluble ligand binding protein is additionally or alternatively amino acid (b) Pro (39), Trp (77), Trp (101), Pro of SEQ ID NO: 2.
(103), Asp (194), and / or Ser (161) (L-AChBP T1 and T2
: B-AChBP T1 and T2; Pro (39): Pro (39); Trp (77): Trp (77); Trp (1
01): Trp (102); Pro (103): Pro (104); Ser (161): Ser (162); Asp (1
94): Ser (195)).

In a still further aspect, the water soluble ligand binding protein is in addition to, or as an alternative to, the above aspects, at positions 165-169 of SEQ ID NO: 2 and / or
The amino acid sequence at positions 200-203 is exchanged with the corresponding sequence of the ligand-dependent receptor (L-AChBP T1 and T2: B-AChBP T1 and T2; His (165) to Iso (169): As
p (166) ~ Phe (170) (B-AChBP T1): Asp (166) ~ Leu (170) (B-AChBP T2)
Asn (200) ~ Thr (203); Iso (203) (L-AChBP T2): Asn (201) ~ Lys (204
).

Amino acids that determine the binding of nicotinic acetylcholine receptors to ligands include three stretches in the nAChRα subunit. These stretches are conserved throughout the various nAChRα subunits and contain amino acids essential for ligand binding. These stretches (corresponding to the torpedo α subunit) are (numbering of nAChRα7 shown in SEQ ID NO: 9): Trp (108) and Tyr
Trp (108) to Tyr (115), which require (115); amino acids Trp (171) and Tyr (
173) essential Trp (171) to Tyr (173); amino acids Tyr (210), Cys (212)
, Cys (213), and Tyr (217) are essential Tyr (210) to Tyr (217).
In the chimeric proteins according to the invention, at least one essential amino acid of these stretches is replaced instead of the corresponding amino acid.
Preferably the entire stretch is replaced.

In a particularly preferred embodiment of the invention, the water-soluble ligand binding protein is capable of binding a ligand for the acetylcholine receptor, wherein the protein has Trp (101) -Tyr (T108), Trp (162) of SEQ ID NO: 2. ~ His (164), and Tyr (204) ~
At least one of the amino acid sequences of Tyr (211) is exchanged with the corresponding sequence of acetylcholine receptor (L-AChBP T1 and T2: B-AChBP T1 and T2; Trp (101)
~ Tyr (108): Trp (102) ~ Tyr (109); Trp (162) ~ His (164): Trp (163)
~ His (165), (B ~ AChBP T1): Trp (163) ~ Phe (165) (B-AChBP T2); Tyr
(204) to Tyr (211): Tyr (205) to Tyr (212).

On the basis of homology with AChBP, it is possible to change an amino acid residue in the original amino acid sequence of the ligand-gated ion channel, which amino acid residue
Although not critical for receptor ligand binding or essential for tertiary and quaternary structure, amino acid residues that contribute to water solubility can be substituted according to AChBP (particularly its crystal structure). As a result, the ligand-gated ion channel, or its ligand binding domain, or the respective monomer and pentamer can be more easily expressed, for example in recombinant expression systems, and more importantly, crystalline. It is expected that this will allow the construction of a three-dimensional model of the ligand binding domain.

Accordingly, in another aspect, the invention provides a method of making a water-soluble ligand-dependent receptor or a corresponding ligand-binding domain, or the water-solubility of such receptor or domain comprising The present invention relates to a method for improving the easiness of crystallization. This method determines the water solubility of the above water soluble ligand binding proteins of the invention, or at least 1 at the position corresponding to the amino acid that contributes to the water solubility.
Changing the amino acid sequence of the extracellular domain of the ligand-dependent receptor by the substitution, addition, deletion or modification of one amino acid. The methods of the present invention can be performed using conventional techniques well known in the art, such as one or more amino acid deletions, insertions, substitutions, additions and / or recombinations, and / or in the art. Any other known modification can be used alone or in combination. Methods of introducing such modifications into the DNA sequence underlying the amino acid sequence of the ligand binding domain of the ligand-gated ion channel are well known to those of skill in the art; see, eg, Sambrook, "Molecular Cloning Laboratory Manual (Molecular). Cloning A Laboratory Manual), Cold Spring Harbor La
boratory (1989) N. Y. checking ... The resulting ligand-gated receptor or ligand-binding domain retains ligand-binding activity comparable to that of the ligand-gated ion channel in vitro, preferably in vivo, and more importantly, X Allows complete crystallization of the protein so that it can be characterized by line crystallography. X-ray crystallographic data can be used, for example, in the identification and construction of potential therapeutic compounds in the treatment of various disease states.

As discussed earlier herein, nACh, 5-HT3, glycine, GABA _A and GA
BA _C receptors, and ligand-gated ion channel superfamily including invertebrates glutamate ion channels and MOD-1 serotonin channel, AChB
It contains an extracellular ligand binding domain homologous to P. Many of these receptors are promising drug targets. Thus, the modified ligand-dependent receptor is preferably one of the mentioned superfamily of receptors, most preferably nAChR.

Information on the nucleotide and amino acid sequences of nAch, 5-HT3, glycine, GABA _A and GABA _c receptors, structural elements, functional assays can be found in the prior art. For example, Collinger et al., Annu. Rev. Pharmacol. Toxicol. 40 (2000
, 431-458, nicotinic receptors are described at the amino acid level.
Means for searching nucleotide and amino acid sequences and performing sequence alignments to identify the most likely important amino acid residues are described below and in the Examples; for more general information, see Ferder ( Felder) et al., Phar
mSci. 1 (2) (1999), "A review of the ancient protein module, periplasmic binding protein (PBP), present in multidrug receptors (the review on p.
eriplasmic binding protein (PBP), an ancient protein module present in
multiple drug receotors) ”.

In a preferred embodiment of the method of the present invention at least one amino acid is changed to a corresponding amino acid or an equivalent amino acid of the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6 or 8. Here, preferably, the solubility-determining amino acid comprises a solvent-accessible region in the crystal structure according to FIG. 10 or 11. Preferred amino acid sequence positions and amino acid substitutions are described above for AChBP and can be applied generally in the methods of the invention.

Insertion of the Cys123-Cys136 loop of mature AChBP SEQ ID NO: 2 into the equivalent region of the mature nicotinic α7 homopentamer ligand binding domain (Cys127-Cys141) results in an easily expressed form of this protein. Is expected to be brought. Similarly, this loop or equivalent loops from other water-soluble ligand proteins of the invention may be
Ligand-gated ion channels, for example, can be inserted into the equivalent regions of the glycine receptor and other homopentameric ligand binding domains of the 5-HT3 receptor, resulting in easily expressed forms of these proteins. it can.

Accordingly, in one embodiment, the invention provides the method according to any one of the above methods, wherein the loop of Cys123 to Cys136 of SEQ ID NO: 2 is inserted into the corresponding region of the ligand binding domain of the ligand dependent receptor. Regarding one.

The water-soluble ligand-dependent receptor or the corresponding ligand-binding domain is
Usually created by directed mutagenesis of the underlying coding polynucleotide. Once the corresponding polynucleotide is made, it can be used to express the altered ligand-dependent receptor or the corresponding ligand binding domain. Thus, the method of the invention generally comprises the following steps: (a) a host cell which is transfected with a polynucleotide comprising a nucleotide sequence encoding an altered amino acid sequence and is capable of expressing this polynucleotide. And (b) recovering the water-soluble ligand-dependent receptor or the corresponding ligand-binding domain from the culture.

Methods of expressing and purifying the water-soluble ligand-dependent receptors of the present invention or the corresponding ligand binding domains are described further below. Preferably, the expression system described in Examples 4 and 5 or the corresponding expression system is used.

The invention also relates to the water-soluble ligand-dependent receptor and ligand binding domain obtainable by said method of the invention. Preferably, the water-soluble ligand-dependent receptor is 10-fold, more preferably 100-fold, even more preferably 1000-fold, most preferably 1-fold that of the corresponding wild-type, preferably human ligand-dependent receptor.
Shows 0000 times higher water solubility. However, an improvement in water solubility of about 2-5 fold is also already advantageous. The average hydrophobicity may be in the range -100 to -400. Therefore, the present invention provides
Provided are methods for predicting and making variants and chimeras of the ligand binding domain of homopentameric acetylcholine receptor subtypes and other homopentameric ion channels with increased solubility.

In one embodiment, the water-soluble ligand binding protein of the present invention further comprises a spacer sequence that allows binding to the carrier body. The spacer sequence may be an amino acid sequence that can be encoded by a polynucleotide or may be another molecule such as the polymethylene anchor group commonly used in chip technology. The chimeric protein of the present invention may further comprise a spacer sequence which allows the protein to be attached to the unibody. Such a spacer sequence may be, for example, an oligohistidine stretch attached to the C-terminus of the protein. Such oligohistidine stretches can be attached to Talon (copyright) metal affinity beads or similar carriers. Such binding stretches have no detectable effect on the pharmacological properties of the protein. The chimeric proteins according to the invention can be used for screening for specific binding of potential drugs, especially for screening modulators of ion channel opening. Conventional in vitro screening techniques such as phage display technology can be used for this purpose. High throughput assays may also be used, possibly in combination with combinatorial chemistry. Specific binding of a test compound to the (immobilized) chimeric protein of the present invention can be performed by, for example, a competitive binding assay method using α-bungarotoxin as a competitor. The invention also relates to a test kit comprising said protein together with further means for carrying out a screening test (eg carriers, labels, diluents, other chemicals etc.).

Furthermore, the present invention relates to a fusion protein comprising the water-soluble ligand binding protein of the present invention or a binding fragment thereof and a fragment of a ligand-dependent receptor. The term "fusion protein" as used herein means a protein construct that is the result of combining domains or linker regions to obtain the combined function of multiple protein domains or linker regions. This can be accomplished by molecularly cloning the nucleotide sequence encoding the domain and the like to create a novel polynucleotide sequence encoding the desired fusion protein. Or
Creation of a fusion protein can be accomplished by chemically linking the two proteins. Preferably, the fusion protein of the present invention comprises at least a ligand binding domain of AChBP or a ligand-gated ion channel, which has been modified according to the above method.

The nicotinic acetylcholine receptor consists of five subunits selected from a related family of subunit proteins. These neuronal subunits are classified into two major types depending on the presence or absence of a pair of vicinal cysteines close to the acetylcholine binding site. Thus, all α subunits contain a pair of cysteine residues that are thought to play a role in binding nicotinic agents (
Aplin and Wonnacott, 48 (1994), 473-477)
, Β subunit is not included. There are 10 known α subunits α1 to α10, and at least four β subunits β1 to β4. The receptor contains at least one α subunit, and in some cell types the α subunit is the β subunit,
In some cases it is associated with the γ, δ and ε subunits. For example, AChR at the neuromuscular junction is believed to have (α1) 2β1γδ stoichiometry. There is significant diversity within the group of α subunits, such that a complete functional nAChR is formed. The majority of α subunits form functional receptors only when bound as a heteropentamer with the β subunit in the CNS (McGehee and Role, Annual Review of Physiology 57 (1995)
), 521-546). However, the α7, α8, and α9 nAChR subunits and related 5-HT3A subunits can form functional homopentameric receptors. In this respect, a phylogenetic relationship between nAChR subunits suggests that α7, α8, α9, and related 5-HT3A subunits are more closely related to each other than subunits that form only heteropentameric receptors. That is interesting. Sequence homology is α
It has been shown that the 7, α8, and α9 subunits form distinct subgroups of α subunits.

As is apparent from the above, said water-soluble ligand binding protein or receptor or its ligand binding domain comprises a dimer, pentamer, consisting of at least one monomer of the mentioned protein of the invention. It can be used to form homo- or hetero-multimeric complexes such as dimers or decamers. Preferably, these multimers constitute a functional ligand-dependent receptor. Preferably the ligand dependent receptor is associated with nAchR.

The present invention also relates to the generation of synthetic heteropentamers resembling heteropentamer-gated ion channels with AChBP mutations, using knowledge of the crystal structure for primary and secondary contact regions; checking ... Preferably, the synthetic heteropentamer resembles a heteropentameric nicotinic acetylcholine receptor. Accordingly, the present invention more generally relates to ligand-dependent inclusion of any one of the aforementioned water soluble ligand binding proteins or receptors of the present invention as a monomer, homo or hetero dimer or pentamer. Regarding ion channels. Thus, the method provides for nicotinic acetylcholine receptors that are insensitive or more sensitive to the binding of toxins, such as bungarotoxin, rhotoxin, conotoxin, and other toxins that inhibit ligand-gated ion channels. And allows the prediction and generation of variants and chimeras of other ligand-gated ion channels. Preferably, the ligand-gated ion channel is less sensitive or more sensitive to binding of toxins such as bungarotoxin, rhotoxin, or conotoxin as compared to the wild-type ligand-gated ion channel. .

Further information and examples of how to make chimeric ligand binding proteins according to the present invention are provided in Example 10.

Nucleotide and amino acid sequences of acetylcholine, 5-HT3, glycine, GABA _A and GABA _c receptors can be found in public databases, eg http: ///www.ncbi.nlm.
It can be easily retrieved from the Internet using .nih.gov / Entrez. The citations also include references to the corresponding publications that also report the functional expression of each receptor.

The use of recombinant acetylcholine-gated ion channels and functional assays in the discovery of putative novel ligands is described by Cosford, Pharm. Acta Helv
． (2000), 74 (2-3), 125-130. Furthermore, cell-free expression and functional reconstitution of homo-oligomeric α7 nicotinic acetylcholine receptors into planar lipid bilayers has been demonstrated by Lyford and Rosenberg, J.
． Biol. Chem. (1999), 274 (36), 25675-25681. The use of cloned and native muscarinic acetylcholine receptor functional assays to determine toxin-selectivity profiles is described by Olianas (Oliana).
s) et al. (J. Pharmacol. Exp. Ther. 288 (1999), 164-170). Systems for assessing pharmacological differences and similarities between cells stably transfected with the 5-HT3 receptor have been described, for example, by Bruss et al., Naunyn-Schmiedebe.
rgs Archives of Pharmacology 360 (1999), 225-33. 5-H
The primary structure and functional expression of the T3 receptor is described by Maricq et al., Science 254 (1
991), 432-437. Similarly, stable expression of human glycine α1 and α2 receptor monomers in mouse L (tk-) cells, and use of the monomers to study the physiological and pharmacological properties of functional glycine receptors. Is a wick (
Wick) et al., J. Neurosci. Methods 87 (1999), 97-103. An example for measuring the pharmacological properties of recombinant GABA _A receptor subtypes is described by Simpson et al. Neurosci. Methods 99 (2000), 91-100. Further examples of assay systems are shown below.

For example: (1) for expressing and characterizing the water-soluble ligand-binding proteins and ligand-gated ion channels of the invention; and (2) for expressing novel ligands, stable expression of said ligand-gated ion channels. The methods described above as well as other methods well known to those of skill in the art can be used to use cells transfected into.

The present invention also relates to polynucleotides encoding the water-soluble ligand binding proteins and ligand-gated ion channels of the invention and multimers thereof, preferably dimers or pentamers. Such a polynucleotide may be DNA, such as cDNA, RNA, such as mRNA, or any other form of nucleic acid, including synthetic or modified derivatives, interrupted by one continuous or intervening sequence. Multiple sequences may encode a polypeptide. A polynucleotide, in any form present, is an isolated polynucleotide in that it has been removed from its natural state. This aspect of the invention is based on the cloning of the cDNA of the ligand binding protein. In a preferred embodiment, the polynucleotide is SEQ ID NO: 1,3.
, 5, or 7, which nucleotide sequence optionally comprises one or more mutations or one or more mutations that do not substantially affect the activity of the polypeptide encoded by the polynucleotide. Contains deletions. Such mutations include mutations resulting from the degeneracy of the genetic code as well as mutations resulting in any of the amino acid mutations or deletions described above. The polynucleotide of the present invention preferably comprises: (a) a nucleotide sequence having at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 1, 3, 5, or 7; Or a degenerate nucleotide sequence thereof; or (b) a nucleotide sequence capable of hybridizing to the nucleotide sequence of (a) under stringent hybridization conditions.

Generally, at least about 55% sequence identity over the stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% sequence identity. Hybridization occurs in the presence of any of the following: Kanehisa, Nucleic Acids Res. 12 (1984), 203-213. Nucleic acid hybridization, as will be readily appreciated by those skilled in the art,
It is affected by conditions such as salt concentration, temperature, solvent, base composition of the hybridizing species, length of complementary regions, and number of nucleotide base mismatches between the hybridizing nucleic acids.

In the context of nucleic acid hybridization experiments, “stringent hybridization conditions” and “stringent wash conditions” depend on a number of different physical parameters. The most important parameters include hybridization temperature, nucleic acid base composition, salt concentration, and nucleic acid length. One of ordinary skill in the art knows how to modify these parameters to achieve a particular stringency of hybridization. Generally, "stringent hybridization" is performed at a temperature about 25 ° C lower than the melting temperature (Tm) of a specific DNA hybrid under specific conditions.

The “stringent washing” is performed at a temperature about 5 ° C. lower than the Tm to which a specific DNA hybridizes under specific conditions. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe; see Sambrook et al., Page 9.51, incorporated herein by reference. The Tm of a particular DNA-DNA hybrid can be estimated by the following formula: Tm = 81.5 ° C. + 16.6 (log10 [Na +]) + 0.41 (fraction G + C) -0.63 (% formamide)-(600 / l), where l is the length of the hybrid (base pairs). The Tm of a particular RNA-RNA hybrid can be estimated by the formula:
Tm = 79.8 ° C + 18.5 (log10 [Na +]) + 0.58 (fraction G + C) +11.8 (fraction G + C) 2-0.
35 (% formamide)-(820 / l). The Tm of a particular RNA-DNA hybrid can be estimated by the formula:
Tm = 79.8 ° C + 18.5 (log10 [Na +]) + 0.58 (fraction G + C) +11.8 (fraction G + C) 2-0.
50 (% formamide)-(820 / l).

In general, the Tm is reduced by 1-1.5 ° C for every 1% mismatch between two nucleic acid sequences.
Therefore, one of skill in the art can alter the hybridization and / or wash conditions to obtain sequences with higher or lower sequence identity to the target nucleic acid. For example, to obtain a hybridizing nucleic acid containing up to 10% mismatch from the target nucleic acid sequence, subtract 10-15 ° C from the calculated Tm for a perfectly matching hybrid, and then adjust the hybridization and wash temperatures accordingly. Adjust. The probe sequences can also specifically hybridize to double-stranded DNA under certain conditions to form triplex or other higher order DNA complexes. The production of such probes and suitable hybridization conditions are well known in the art. An example of stringent hybridization conditions for hybridization of complementary nucleic acid sequences with more than 100 complementary residues on filters in Southern or Northern blots or for screening libraries is 50% formamide / 6x SSC at 42 ° C for at least 10 hours. Another example of stringent hybridization conditions is 6 × SSC at 68 ° C. for at least 10 hours. An example of low stringency hybridization conditions for hybridization of complementary nucleic acid sequences having more than 100 complementary residues on filters in Southern or Northern blots, or for screening libraries is 6 × SSC,
At least 10 hours at 42 ° C. Hybridization conditions for identifying similar, but not identical, nucleic acid sequences are by experimentally varying the hybridization temperature from 68 ° C to 42 ° C while keeping the salt concentration constant (6xSSC), or Constant hybridization temperature and salt concentration (eg 42 ° C and 6xSSC
) And changing the formamide concentration from 50% to 0%. Hybridization buffers may also include blocking agents to reduce background. These agents are well known in the art; Sambrook et al., P. 8.46, and incorporated herein by reference.
See pages 9.46-9.58. Washing conditions may also be altered to alter stringency conditions. An example of stringent wash conditions is 0.2 x SSC wash solution at 65 ° C.
15 minutes (see Sunbrook et al. For SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove excess probe. An exemplary medium stringency wash for double-stranded DNA greater than 100 base pairs is 1 × SSC, 45 ° C., 15 minutes. An exemplary low stringency wash for such a duplex is 4 × SSC, 40 ° C. for 15 minutes. Generally, a signal-to-noise ratio that is more than 2-fold higher than that observed for unrelated probes in a particular hybridization assay indicates detection of specific hybridization.

Nucleotide sequences of SEQ ID NOs: 1, 3, 5 and 7 and SEQ ID NOs: 2, 4, 6
, And an identical or similar nucleic acid molecule encoding a water-soluble ligand-binding protein from another species or organism (particularly from a mammal, an orthologous water-soluble ligand-binding protein). It is possible to isolate). As used herein, the term "orthologous" refers to homologous sequences in different species that result from a common ancestral gene during speciation. Orthologous genes may have similar functions,
You may not be responsible; for example, "The latest bioinformatics guide (Tren
ds Guide to Bioinformatics) See Trends Supplement 1998, Elsevier Science Glossary.

In a further aspect, the present invention provides a recombinant polynucleotide containing a vector containing the polynucleotide of the present invention. Many suitable vectors are well known to those of skill in molecular biology, and the choice of vector depends on the desired function, including plasmids, cosmids, viruses, and bacteriophages, as well as other vectors conventionally used in genetic engineering. including. Methods well known to those of skill in the art can be used to construct various plasmids and vectors; see, eg, Sambrook, "Molecular Cloning A Labora.
tory Manual) ”, Cold Spring Harbor Laboratory (1989) N. Y. And Ausubel, "Current Protocols i
n Molecular Biology) '' Green Publishing Associates and Wiley Interscienc
e, N. Y. See the techniques described in (1989), (1994). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells. Cloning vectors were used to isolate individual sequences of DNA, as discussed in further detail below. The relevant sequences can be transferred into an expression vector where expression of the particular polypeptide is required. Common cloning vectors include pBscptsk, pGEM, pUC9, pBR322, and pGBT9. Common expression vectors include pTRE, pCAL-n-EK, pESP-1, pOP13CAT
, PET, pGEX, pMALC, pPIC9, pBac.

Therefore, in a preferred embodiment of the invention said polynucleotide alone,
Alternatively, it is present in a vector and is linked to control sequences that allow expression of the polynucleotide in prokaryotic and / or eukaryotic cells.

The term “control sequences” means the regulatory DNA sequences necessary to bring about the expression of linked coding sequences. The nature of such control sequences will vary depending on the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site, and terminator. In eukaryotes, control sequences generally include promoters, terminators and, in some cases, enhancers, trans-acting factors or transcription factors. The term "regulatory sequence" is intended to include at least all components whose presence is necessary for expression, and may include additional advantageous components.

The term “operably linked” refers to the juxtaposition in which the components so described are in a relationship capable of functioning as intended. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. It will be apparent to the person skilled in the art that preferably double-stranded nucleic acid is used when the control sequence is a promoter.

Therefore, the vector of the present invention is preferably an expression vector. An "expression vector" refers to a construct that can be used to transform a selected host cell, resulting in expression of the coding sequence in the selected host cell. The expression vector may be, for example, a cloning vector, a binary vector, or an integrating vector. Expression preferably involves transcription of the nucleic acid molecule into a translatable mRNA. Regulatory elements ensuring expression in prokaryotic and / or eukaryotic cells are well known to those of skill in the art. In eukaryotic cells, these usually contain a promoter to ensure transcription initiation, and optionally a poly A signal to ensure transcription termination and transcript stabilization. Possible regulatory elements allowing expression in prokaryotic host cells include, for example, the E. coli PL, lac, trp, T7 or tac promoters, examples of regulatory elements allowing expression in eukaryotic host cells are: The yeast AOX1 or GAL1 promoter, or the CMV, SV40, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells. In this regard, the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDN
A3 (In-vitrogene), pSPORT1 (Gibco BRL
Suitable expression vectors such as IBCO BRL)) are well known in the art. An alternative expression system that can be used to express the protein is the insect system. In one such system, Autographa calif
ornica) nuclear polyhedrosis virus (AcNPV), Spodoptera frugiperda (Spodopt
era frugiperda) cells or Trichoplusia larvae as a vector for expressing foreign genes. The coding sequence of the nucleic acid molecule of the present invention may be cloned into a nonessential region of the virus (eg polyhedrin gene) and placed under the control of the polyhedrin promoter. Successful insertion of the coding sequence will inactivate the polyhedrin gene resulting in a recombinant virus lacking the coat protein coat. The recombinant virus is then used to transform S. Infected Frugiperda cells or Trichoprussia larvae, where the proteins of the invention are expressed (Smith, J. Virol. 46 (1983), 584; Engelhard).
), Proc. Nat. Acad. Sci. USA 91 (1994), 3224-3227).

Commonly used promoters in plants are the polyubiquitin promoter and the actin promoter for universal expression. The commonly used termination signals are derived from the nopaline synthase promoter or the CAMV 35S promoter. A commonly used plant translation enhancer is the TMVω sequence, and the inclusion of introns (eg, intron-1 from the maize Shrunken gene) has been shown to increase expression levels up to 100-fold (Mait, Transge
nic Research 6 (1997), 143-156; Ni (Ni), Plant Journal 7 (1995), 661.
~ 676). Additional regulatory elements may include transcription enhancers as well as translation enhancers. Advantageously, said vector of the invention comprises a selectable marker and / or a scoreable marker.
Selectable marker genes useful in the selection of transformed cells, such as plant tissues and plants, are well known to those of skill in the art and include, for example, dhfr (which confer methotrexate resistance).
Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149)
Npt (Herrera-Estrella, EMBO J. 2 (1983), 9) that confers resistance to aminoglycoside neomycin, kanamycin, and paromycin
87-995) as well as hygro (Marsh (Marsh
), Gene32 (1984), 481-485), including antimetabolite resistance as the basis for selection.

Useful scoreable markers are also well known to those of skill in the art and are commercially available. Advantageously, this marker is luciferase (Giacomin,
Pl. Sci. 116 (1996), 59-72; Scikantha, J. et al. Bact. 178 (1996
), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996)
), 44-47), or β-glucuronidase (Jefferson, EM
BO J. 6 (1987), 3901-3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing the vectors of the invention.

Ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, size exclusion chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Proteins can be recovered and purified from recombinant cell culture by well-known methods including chromatography. Most preferably, high performance liquid chromatography ("HPLC
)) Or FPLC is used for purification.

Furthermore, the present invention introduces into a host cell a nucleic acid molecule which, when present in a cell, mediates the expression of a gene encoding a water soluble ligand binding protein, or a polynucleotide or vector as described above or a polynucleotide of the invention. Relating to a host cell, wherein the polynucleotide and / or nucleic acid molecule is exogenous to the host cell. "Foreign" means that the polynucleotide or nucleic acid molecule is heterologous with respect to the host cell (which means derived from a cell or organism with a different genomic background), or homologous with respect to the host cell, It is meant to be located in a genomic environment that is different from the natural corresponding genomic environment of the nucleic acid molecule. This means that when the nucleic acid molecule is homologous with respect to the host cell, it is not located in its natural position in the genome of the host cell, in particular:
It means being surrounded by different genes. In this case, the polynucleotide may be under the control of its own promoter or may be under the control of a heterologous promoter. The vector or nucleic acid molecule of the invention present in a host cell may be integrated into the host cell genome or may be maintained extrachromosomally in some form. In this respect, it should also be understood that the nucleic acid molecules of the invention can be used to restore or create mutant genes by homologous recombination.

The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant or animal cell.

The term “prokaryote” is meant to include all bacteria that can be transformed or transfected with DNA or RNA molecules to express the proteins of the invention. E. coli and S. typhimurium as prokaryotic hosts
, Serratia marcescens, and Bacillus
s subtilis) as well as Gram-negative bacteria as well as Gram-positive bacteria. The term "eukaryote" is meant to include yeast, higher plants, insects, and preferably mammalian cells. Depending on the host used in a recombinant production procedure, the protein encoded by the polynucleotide of the invention may be glycosylated or non-glycosylated. The water soluble ligand binding protein of the present invention may or may not include an initiating methionine amino acid residue. The polynucleotides of the invention can be used to transform or transfect a host using any technique commonly known to those of skill in the art. Further, methods of making fused and operably linked genes and expressing them in, for example, mammalian cells and bacteria are well known in the art (Sambrook, "Molecular Cloning: A Laboratory"). Manual) "
, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

Accordingly, the present invention provides cells capable of expressing the polypeptides discussed herein. Cells usually include recombinant host cells which have incorporated the polynucleotide. Preferably, the host cell has incorporated the polynucleotide as a recombinant polynucleotide. Again, any suitable host cell can be selected depending on the intended purpose. Suitable host cells include XLI-BLUE, B21 (DE3) pLysS, H
B101, SOLR, and SP-Q01 (Saccharomyces pombe)
) Is included.

Using a suitable combination of host cells, vectors and polynucleotides,
Expression systems can be constructed to provide polypeptides useful in the invention. It may include a fusion polypeptide encoded by a recombinant polynucleotide and partly by a vector. Vectors typically have an inducible promoter region, which is linked to the promoter
Connects to the 5'code region. By appropriate manipulation, the polynucleotide encoding the polypeptide can be attached in frame to the 5'coding region. Expression of nucleotide sequences downstream of the promoter region thus results in a fusion polypeptide that includes a polypeptide of the invention.

The present invention also consists of at least 5 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6 or 8 and / or preferably of at least 10 ^-7 M It relates to an antigen containing an epitope that can be detected by a monoclonal or polyclonal antibody that recognizes the above-mentioned protein of the present invention with binding affinity. In the present invention, “epitope” means a fragment of the AChBP of the present invention which has antigenic or immunogenic activity in animals. A preferred embodiment of the invention relates to a polynucleotide encoding an antigen containing an epitope as well as fragments thereof. The region of the protein molecule to which an antibody can bind is defined as the "antigen epitope." In contrast, an "immunogenic epitope" is defined as the part of a protein that elicits an antibody response; see, for example, Geysen, Proc. Na
tl. Acad. Sci. USA 81 (1983); 3998-4002. Fragments that function as epitopes can be generated by any conventional means; see, eg, Houghten, Proc., Further described in US Pat. No. 4,631,211. N
atl. Acad. Sci. See USA 82 (1985), 5131-5135. In the present invention,
The antigenic epitope preferably comprises at least 5,6,7, more preferably at least 9, most preferably about 15 to about 30 amino acids. Antigenic epitopes are useful for producing antibodies, including monoclonal antibodies, that specifically bind the epitope; eg, Wilson, Cell 37 (1984), 767-778; Sutcliffe, Science 219. (1983), 660-666). Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art; eg, Satcliffe, supra; Wilson,
Said; Chow, Proc. Natl. Acad. Sci. USA 82 (1985), 910-914;
And Bittle, J. Gen. Virol. 66 (1985); 2347-2354. Preferred immunogenic epitopes include soluble proteins. The immunogenic epitope can be presented to an animal system (eg, rabbit or mouse) with a carrier protein (eg, albumin) or, if sufficiently long (at least about 25 amino acids), no carrier. However, an immunogenic epitope containing as few as 8-10 amino acids is sufficient to produce at least an antibody (eg, in Western blotting) capable of binding a linear epitope in a denatured polypeptide. It is shown.

The invention also relates to antibodies which specifically recognize the water-soluble ligand binding proteins and ligand-gated ion channels of the invention, in particular the antigens or epitopes mentioned above. The term “antibody” (Ab) or “monoclonal antibody” (Mab) as used herein refers to intact molecules and antibody fragments (eg, Fab and F) that are capable of specifically binding to a protein. (Ab ') ₂ fragment). Fab and F (ab ') ₂ fragments lack the Fc fragment of a whole antibody, may disappear more rapidly from circulation, and may have less nonspecific tissue binding than whole antibodies; for example, Wahl, J. Nucl. Med. 24 (1983), 316-325. Therefore, these fragments, as well as the products of FAB or other immunoglobulin expression libraries, are preferred. In addition, the antibodies of the present invention include chimeric antibodies, single chain antibodies, and humanized antibodies; see also below. An antibody is a monoclonal antibody, a polyclonal antibody, which specifically binds a peptide or polypeptide,
It may be a single chain antibody, a human antibody or a humanized antibody, a primatized antibody, a chimerized antibody, or a fragment thereof, a bispecific antibody, a synthetic antibody, an antibody fragment (for example, Fab, Fv,
Or scFv fragments), or chemically modified derivatives of any of these. General methods for antibody production are well known and are described, for example, in Kohler and Milstein, Nature 256 (1975), 494, J. Am. G. R. Hurrel, "Monoclonal Hybridoma Antibodies: Techniques and Applications (Mo
noclonal Hybridoma Antibodies: Techniques and Applications), CRC Pr
ess Inc., Boco Raron, FL (1982), and L. T. Mimms
) Et al., Virology 176 (1990), 604-619. Furthermore, antibodies against said peptides or fragments thereof are eg described in Harlow and lane (
Lane) "Antibodies, A Laboratory Manual", CSH Pre
ss, Cold Spring Harbor, 1988. For the production of antibodies in experimental animals, various hosts such as goats, rabbits, rats, mice are immunized by injection with a polypeptide of the invention or any immunogenic fragment or oligopeptide or derivative thereof. be able to. Techniques for producing and processing polyclonal antibodies are well known in the art, among others, Mayer and Walker, eds., Immunochemical Methods in Cell and Molec.
regular Biology) ”, Academic Press, London (1987). Polyclonal antibodies can also be obtained from animals (preferably mammals) previously infected with the virus of the invention. Methods of purifying antibodies are well known in the art and include, for example, immunoaffinity chromatography. Various adjuvants or immunological carriers may be used to enhance the immune response, depending on the host species. Such adjuvants include Freund's complete or incomplete adjuvants, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and dinitrophenol. Not limited to. For example,
An example of a carrier that can bind the peptides of the present invention is keyhole limpet hemocyanin (KLH). Surface plasmon resonance used in the BIAcore system can be used to enhance the efficiency of phage antibodies to bind to epitopes of the peptides or polypeptides of the invention if the derivative of the antibody was obtained by a phage display technique (Shell). (Schier), Human Antibodies Hybridoma
s 7 (1996), 97-105; Malmborg, J. Am. Immunol. Methods 183
(1995), 7-13). In many cases, the phenomenon of antibody binding to antigen is comparable to other ligand / antiligand binding.

In another embodiment, the invention relates to an oligonucleotide probe having at least 15 contiguous nucleotides of a polynucleotide of the invention and / or comprising a nucleotide sequence encoding the above antigen. Such an oligonucleotide usually hybridizes specifically with the polynucleotide encoding the water-soluble ligand-binding protein of the present invention. Specific hybridization preferably occurs under stringent conditions and means no or little cross-hybridization with nucleotide sequences that do not encode proteins or nucleotide sequences that encode substantially different proteins. . Such nucleic acid molecules can be used as probes and / or as controls for gene expression. Nucleic acid probe technology is well known to those of skill in the art and it will be readily appreciated by those skilled in the art that such probes may vary in length. 17-35
Nucleotide length nucleic acid probes are preferred. Of course, it may be suitable to use nucleic acids of up to 100 nucleotides in length and more than 100 nucleotides in length. The nucleic acid probe of the present invention is useful in various applications. On the one hand, the nucleic acid probes of the invention can be used as PCR primers for amplifying the polynucleotides according to the invention. Another use is as a hybridization probe to identify polynucleotides that hybridize to the polynucleotides of the invention by homology screening of genomic DNA libraries.
The nucleic acid molecule of this preferred embodiment of the invention which is complementary to a polynucleotide as described above may also be used to suppress the expression of a gene comprising such a polynucleotide, eg by an antisense or triple helix effect, or of the invention Suitable ribozymes that specifically cleave the (pre) mRNA of the gene containing the polynucleotide (eg, EP 0 291 533 (EP-B1 0 291 533), EP 0 321 201 (EP-
A1 0 321 201), European Patent 0 360 257 (see EP-A2 0 360 257)). Selection of appropriate target sites and corresponding ribozymes is described, for example, in Steinecke, "Ribozymes, Methods in Cell Biology" 50, Galbraith (Ga.
Lbraith) et al., Academic Press, Inc. (1995), 449-460. Standard methods related to antisense technology have also been described (Melani Cancer Res. 51 (1991), 2897-2901). The nucleic acid molecule may be chemically synthesized or may be transcribed by a suitable vector containing a chimeric gene that allows transcription of the nucleic acid molecule in cells. Such a nucleic acid molecule may further include the ribozyme sequence described above.

In this regard, the polynucleotides of the present invention are “gene-targeted” for restoring a mutant gene by homologous recombination or for creating a mutant gene.
It should also be understood that it can be used for and / or "gene replacement";
For example, Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990),
4712-4716; Joyner, “Gene targeting practice approach (
Gene Targeting, A Practical Approach) ", Oxford University Press.

Furthermore, such a nucleic acid probe may be provided with a marker suitable for a particular application, such as detecting the presence of a polynucleotide of the invention in a sample obtained from an organism, especially a mammal, preferably a human. It is also well known to those skilled in the art that labeling is possible. Many companies such as Pharmacia Biotech (Piscataway NJ), Promega (Madison WI), and US Biochemical Corp (Cleveland OH) have commercial kits for these procedures. And protocol. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent agents, chemiluminescent or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles and the like. As patents disclosing the use of such a label, U.S. Patent No. 3,817,837 (US-A-3,817,837), U.S. Patent No. 3,850,752 (US-A-3,850,752), U.S. Patent No. 3,939,350 (US-A-3,939,350).
, U.S. Patent No. 3,996,345 (US-A-3,996,345), U.S. Patent No. 4,227,437 (US-A-4
, 227,437), US Pat. No. 4,275,149 (US-A-4,275,149), and US Pat.
66,241 (US-A-4,366,241) is included. Similarly, recombinant immunoglobulins can be produced, as set forth in US Pat. No. 4,816,567 (US-A-4,816,567), incorporated herein by reference.

In addition, so-called "peptide nucleic acid" (PNA) techniques can be used to detect or inhibit expression of the polynucleotides of the invention. For example, binding of PNA to complementary and various single-stranded RNA and DNA nucleic acid molecules can be performed by heat denaturation and BI
It can be systematically investigated using the Acore surface interaction technique (Jen
Sen), Biochemistry 36 (1997), 5072-5077).

The present invention also includes a transgenic non-human animal, preferably trans, which comprises introducing the polynucleotide or vector of the present invention into a germ cell, an embryonic cell, a stem cell, or an egg, or a cell derived therefrom. A method for making a transgenic mouse. Non-human animals can be used according to the inventive screening methods described herein. Generation of transgenic embryos and their screening are described, for example, in A. et al. L. Joiner, "Gene Targeting, A Practical Approach" (1993), Oxford Unive
It can be performed as described in rsity Press. For example, Southern blots with appropriate probes can be used to analyze embryonic membrane DNA of embryos; see above. The present invention also includes a transgenic mouse, rat, hamster, dog, monkey, rabbit, pig, nematode (C. elegans), and a torpedo ray containing the polynucleotide or vector of the present invention or obtained by the above method. (To
rpedo fish) such as transgenic non-human animals such as fish. Preferably here the polynucleotide or vector is stably integrated into the genome of the non-human animal, preferably the presence of the polynucleotide or vector allows the water-soluble protein of the invention to be expressed.

Furthermore, the present invention provides the aforementioned water-soluble ligand-binding protein of the present invention, a multimer such as a dimer or pentamer thereof, a ligand-gated ion channel, a polynucleotide, a vector, a host cell, an antigen, an antibody. , Or any one of the oligonucleotide probes; and, optionally, suitable means for detection or for conducting a ligand-receptor binding assay. In this regard, the invention also relates to a method of identifying an agonist / activator or antagonist / inhibitor of a ligand-gated receptor, comprising the steps of: (a) a water-soluble ligand of the invention Detection of a binding protein, a multimer such as a dimer or pentamer thereof, or a ligand-gated ion channel of the present invention, or a cell expressing the protein and a compound to be screened in response to ligand binding Contacting under conditions that allow binding of the compound to the ligand binding protein in the presence of a component capable of producing a possible signal; and (b) the presence of a signal resulting from the binding activity of the ligand binding protein or It is a step of detecting the absence, and the presence / absence and absence / decrease of the signal are the agonists of the ligand-dependent receptor, respectively. / Activator and antagonist / inhibitor stage.

Ligand-dependent receptors are expressed by natural polyamines such as spermine, as well as by polyamine amides such as philanthotoxin-34.
3)) and polymethylene tetraamine (eg methoctramine) (Usherwood, Farmaco. 55 (2000), 20)
2-205) and the like, which are allosterically regulated by polyamine derivatives, such compounds containing or based on such entities can be used as starting materials for screening. An antagonist or agonist that "modulates" the activity of a polypeptide and causes a change in signal, such as a response in a cell, causes a protein to react differently in the presence of a compound than in the absence of the compound. By a compound is meant that alters the activity of the protein as indicated. In general, the effects of antagonists are observed to block agonist-induced receptor activation. Antagonists include competitive as well as non-competitive antagonists. Competitive antagonists (or competitive blockers) interact with or near the specific site to which the agent binds. Non-competitive antagonists or blockers inactivate the function of the receptor by interacting with sites other than the agent interaction site. As will be appreciated by one of skill in the art, bioassay methods for identifying compounds that modulate the activity of receptors such as the proteins of the invention generally require comparison to a control. One type of "control" is a cell or treatment treated substantially the same as a test cell or test culture exposed to a compound, except that the "control" cell or culture is not exposed to the compound. It is a culture. For example, in methods that use voltage clamp electrophysiological procedures, the same cells can be treated in the presence or absence of the compound by simply exchanging the external fluid containing the cells. Thus, the response of the transfected cells and "control" cells or cultures to the same compound under the same reaction conditions. However, "control data" from the literature can also be used.

As described in Example 6, the three-dimensional structure of AChBP could be elucidated by X-ray crystallography at 2.7Å resolution (current R _factor = 27.9%, R _free = 30.0%). . Unwin and collaborators; see above, as measured in the EM study,
AChBPs form stable homopentamers in crystals as in solution, with dimensions comparable to those of the ligand-binding domain of the ligand-gated ion channel, especially comparable to nAChR. Structural analysis revealed that the monomer had an immunoglobulin-like topology in the AChBP homopentamer. The ligand binding site is located at each interface of the 5 subunits and all residues are consistent with biochemical data. At this site, buffer molecules (HERPES) pile up on tryptophan due to cation-π interactions, like acetylcholine binding. The AChBP structure has been implicated, for example, in drug development for Alzheimer's disease and nicotine addiction. The high-resolution crystal structure of AChBP, along with biochemical and pharmacological data,
Supporting the present disclosure that the water-soluble ligand-binding proteins of the invention, such as BP, are excellent mimetics of the ligand-binding domain of ligand-gated ion channels.

The invention therefore relates to crystals of the water-soluble ligand-binding protein of the invention, preferably in the form of multimers such as dimers, pentamers or decamers. In one embodiment, the crystal comprises a protein-ligand complex. Methods of how to use and analyze such crystals are well known to those of skill in the art; for example, US Pat. See 5,872,011 (US-A-5,872,011). From the crystal structure of the ligand-dependent receptor-ligand binding region in a complex with a ligand, preferably an antagonist or agonist, the determinants of receptor-antagonist / agonist interaction, and the specificity and affinity of ligand binding It will become clear how sex changes with the redox state of distant residues and conserved disulfide bonds. This structure may also indicate a mechanism for allosteric effector action and ligand-induced channel gating. How information about the crystal structure of the ligand-binding domain in the complex with the ligand can be used to develop agonists and antagonists, see Structure of the glutamate receptor ligand-binding core in the complex with kainate. (Arms Strong (Ar
mstrong) et al., Nature 395 (1998), 913-917).

Crystals of the invention may be a complex of a protein with a ligand containing an N-alkylated hydroxyalkyl and / or a quaternary ammonium ion, especially when including a nAChR-related protein. However, other ligands can also be used. Preferred ligands are 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), B-bipinatin (B-bippinatin), rhotoxin, d-tubocurarine, nicotine, acetylcholine, conotoxin, carbamylcholine, galantamine, It contains epibatidine or α-bungarotoxin, or a derivative thereof.

Different aspects of X-ray crystallography, such as data collection, structure elucidation, molecular structure determination from X-ray diffraction, refinement, have been described in the prior art, eg Powell, An.
nu. Rep. Prog. Chem., Sect. C: Phys. Chem. 96 (2000), 139-175, and "Methods in Enzymology", 276-277, Carter (C
See Arter) and Sweet (ed.), Academic Press, 1997. X
State-of-the-art methods and optimization algorithms for refinement of wire crystal structures are described in Van Der Maelen Uria, Crystallogr. Rev. 7 (1999), 12
5 to 180.

The crystals of the invention efficiently diffract X-rays for the determination of the atomic coordinates of proteins or protein-ligand complexes up to resolutions above 5.0 Å, preferably above 4.0 Å. In a preferred embodiment, the crystals efficiently diffract X-rays for the determination of atomic coordinates of protein-ligand complexes, up to a resolution of greater than 3.0 Å. In a more preferred embodiment, the crystal efficiently diffracts X-rays for the determination of atomic coordinates of protein-ligand complexes, up to a resolution of greater than 2.0Å. In one embodiment, the crystal efficiently diffracts X-rays for the determination of atomic coordinates of protein-ligand complexes, up to a resolution of about 2.7Å.

Preferably, the crystals of the present invention differ from the amino acids 20-223 of SEQ ID NO: 2 only by having an amino acid sequence of amino acids 20-223 of SEQ ID NO: 2, or conservative substitutions. It is formed by a protein having an amino acid sequence that does not. As described in the Examples, crystals of AChBP contain decameric forms of proteins. To facilitate the use of the AChBP protein for analysis and crystallography, mutations were made to residues Asp2 and Asp5 of mature AChBP SEQ ID NO: 2 or 4 to remove the calcium binding site and It may interfere with the production. This deletion can be carried out, for example, by oligonucleotide-directed mutagenesis. Alternatively, crystals can be grown in low calcium concentrations or in the absence of calcium.

The crystal of the present invention preferably comprises (1) a space group of P2 ₁ 2 ₁ 2 ₁ and a = 120.6Å,
Unit cell with dimensions b = 137.0Å and c = 161.5Å; (2) Space group P4 ₂ 2 ₁ 2 and unit cell with dimensions a = b = 141.6Å and c = 120.8Å; or (3) P2 ₁ space group, and has a a = 121.1Å, b = 162.1Å, c = 139.4Å, unit cell dimensions of β = 90.1 °.

Crystals of the invention are preferably derived from proteins having secondary structural elements that include α-helices and antiparallel β-sheets, as shown and described in FIGS. 7, 10, 11 and / or 12. Is a crystal of. Most preferably, the crystals of the present invention have a three dimensional structure as defined by the atomic coordinates shown in Table 1. Those skilled in the art understand that the set of structural coordinates determined by X-ray crystallography is within standard error. For the purposes of the present invention, AChBP having a protein backbone atom (N, Cα, C, and O) root mean square deviation of less than 0.75Å when superposed with backbone atoms in the structural coordinates listed in Table 1 or Any set of structural coordinates of AChBP variants shall be considered to be identical.

In the most preferred embodiment of the present invention, the crystal has a binding cavity as shown in FIGS. 6, 8, 9, and / or 13.

In accordance with the findings of the present invention, a detailed blueprint of the receptor binding site of the ligand-gated ion channel superfamily (most preferably nAChR), including nACh, 5-HT3, glycine, GABA _A , and GABA _C. It is proposed to use a mollusc water soluble ligand binding protein as. The availability of X-ray structures and cloned sequences provides a unique opportunity to understand these receptors at the molecular level, possibly elucidating the dynamic changes that occur during ligand binding and allowing other protein modules to The tertiary and quaternary structure of the receptor can be predicted with a degree of confidence that is higher than is possible. This should facilitate the design of ligands selective for any multiple subtypes in any of these receptor families. AChBP-like structures can be used for computational docking into homology models leading to a priori discovery of novel ligands before laboratory experiments begin optimizing drug candidates.

Accordingly, the present invention also relates to methods of using the crystals of the invention in a drug screening assay, including, for example, the steps of: (a) a structure with a three-dimensional structure determined for the crystal. Drug design by (structur
e assisted drug design) to select potential ligands, the selection being performed with computer modeling; (b) potential ligand and ligand dependence in in vitro or in vivo assays. Contacting with a ligand binding domain of a receptor; and (c) detecting binding of a potential ligand to the ligand binding domain.

Polymer crystallography as a tool for investigating drug-receptor interactions, particularly the use of structure-based drug design, is described by Oakley and Wilce, C.
lin. Exp. Pharmacol. Physiol. 27 (2000), 145-151. The desired drug can be an inhibitor or agent that mimics an endogenous transmitter or ligand. Once the 3D structure of the relevant target is known, computer processes can be used to search compound databases to identify compounds that may interact strongly with the target. Of the complex of the lead compound and its biological target
3D structures can be used to improve lead compounds. The activity of selected compounds can then be tested in a functional assay, such as one of the methods described herein.

Preferably, the potential drug is selected based on its affinity for the ligand-binding domain of its ligand-dependent receptor is greater than the affinity of the standard ligand for the ligand-binding domain of the ligand-dependent receptor. It However, the affinity of the selected compound may be less than that of the standard ligand. Such compounds are useful, for example, as leads for developing additional analogs that may have enhanced binding affinity or beneficial therapeutic properties. On the other hand, the selected compounds may bind to sites of ligand-dependent receptors other than known ligands. In a preferred embodiment, the ligand dependent receptor is the nicotinic acetylcholine receptor.

In a further embodiment, the method of the present invention further comprises the following steps: (d) protein-ligand conjugation by co-crystallizing or soaking crystals of the water soluble ligand binding protein and the potential drug. Body supplemental crystals (supplemental cr
ystal) formation, in which the crystals are efficiently X-rayed for the determination of atomic coordinates of the protein-ligand complex up to a resolution of more than 5.0 Å, preferably more than 4.0 Å, more preferably more than 3 Å (E) a step of determining the three-dimensional structure of the auxiliary crystal; (f) a step of selecting a candidate drug by designing a drug with a structure using the three-dimensional structure determined for the auxiliary crystal Wherein the selection is performed with computer modeling; optionally, (g) contacting the candidate drug with cells expressing the ligand-dependent receptor; and (h) detecting a cellular response. The stage at which a candidate drug is identified as a drug when the cellular response is altered compared to cells not contacted with the candidate compound.

The method may further comprise an initial step performed prior to step (a),
The initial step consists of determining the three-dimensional structure of a crystal containing a protein-ligand complex formed by a water-soluble ligand binding protein and a ligand of the ligand-dependent receptor, the crystal being greater than 5.0 Å, preferably Efficiently diffracts X-rays for resolution of atomic coordinates of protein-ligand complexes up to resolutions above 4.0Å. Preferably, the resolution of crystal diffraction in said method is at least 3.0Å, most preferably at least about 2.7Å.

In a still further aspect, the present invention relates to a method of growing a crystal of a protein-ligand complex comprising the steps of: (a) said water soluble ligand binding protein and a ligand of a ligand dependent receptor; Of water-soluble ligand-binding protein to form a protein-ligand complex with the ligand; and (b) growing a crystal of the protein-ligand complex, wherein the crystal is 5.0 Å
Efficiently diffracting X-rays for the determination of the atomic coordinates of the protein-ligand complex to a resolution of more than 4.0, preferably more than 4.0, more preferably at least 3.0, and most preferably at least about 2.7. .

The crystals of the present invention may also be described, for example, in Nienaber et al., Nature Biotechno.
l. 18 (2000), 1105-1108, can be used in the screening technique by X-ray crystallography that combines the steps of lead identification, structure evaluation, and optimization. This crystallographic screening method (called CrystaLEAD) has been used to detect ligands by sampling a large compound library and monitoring changes in the electron density map of the crystal compared to the unbound form. . This electron density map yields high-resolution images of the ligand-protein complex that provide important information for the structure-directed drug discovery process. Bound ligands are visualized directly in the electron density map. The ligand bound away from the target site can be eliminated.

The method described above can be combined with state-of-the-art experimental data collection equipment, including CCD detectors and data collection robotics.

Further embodiments which can be used in accordance with the ligand binding proteins and receptors of the present invention have been described in the prior art, eg ligand screening and design by X-ray crystallography is described in WO 99/45379 and International Publication No. 99
/ 45389. WO 00/14105 discloses a computer algorithm for identifying receptor agonists and antagonists, obtaining crystal coordinates and creating a model that can be applied to iterative processing of various molecular structures. Assaying candidate compounds for their ability to interact with modified receptor tyrosine kinases, including application to. All these methods are equally applicable to the proteins and crystals of the invention.

In one preferred embodiment, the invention relates to a drug screening assay, comprising the steps of: immersing the crystals of the invention in a solution of the compound to be screened, and the compound to the ligand binding protein. Detecting binding of. A possible procedure is also described in Example 9. In addition to the ligand binding detection methods described above in the cited documents and examples, the detection may also be based on measuring the release of ligand in preformed crystals of the protein-ligand complex. As explained herein above, the ligand preferably comprises an alkylated nitrogen and / or a quaternary ammonium ion or may be one of the ligands mentioned above.

The structural information of the crystals of the invention can also be used to increase or decrease the affinity of the drug for ligand-dependent receptors. Such a method may include the following steps: a structural drug design using the determined three-dimensional structure for the crystal, the drug design being carried out with computer modeling; and Modifying the drug to alter or eliminate some of the drug suspected of interacting with the binding site or non-specific binding site of the protein's binding cavity. This method may, of course, be combined with one or more steps of any of the aforementioned screening methods or other screening methods well known in the art. Methods of clinical compound discovery include, for example, ultra high throughput screening for lead identification (Sundberg,
Curr. Opin. Biotechnol. 11 (2000), 47-53) and structure-based drug design for lead optimization (Verlinde and Hol, Structu.
re 2 (1994), 577-587 and combinatorial chemistry (Sale
mme) et al., Structure 15 (1997), 319-324). Further information available to date for the localization of agonist and competitive antagonist binding sites at the nicotinic acetylcholine receptor, which can be taken into account for drug selection and drug design, was recently reviewed (Arias, Neurochem. Int. 36 (2000), 595-6450; Collinger et al., 1999). Once a drug has been selected, the method is used to perform rational drug design with the modified drug and to assess whether the modified drug exhibits better affinity, for example by interaction / energy analysis. You may have the further step of repeating.

A related method of the invention for drug design uses the structural coordinates of a water-soluble ligand-binding protein crystal, including the coordinates in Table 1, to identify the ligand-binding site or non-specific binding site of the ligand-binding protein. Computational evaluation of chemical entities for binding. This approach, realized and made possible by the present invention, is based on AChBP
A small molecule database is computationally screened for chemical entities or compounds capable of binding to all or part of. In this screen, the quality of fit of such an entity or compound at the binding site can be judged by shape complementarity or putative interaction energies. Meng et al., J. Coma. Chem. 13 (1992), 505-524. Furthermore, according to the present invention, AChBP mutants or chimeras can be crystallized in co-complex with known ligand-gated ion channel inhibitors. The crystal structure of a series of such complexes was then solved by molecular replacement (for review see, for example, Brunger et al., Prog. Biophys. Mol. Biol. 72 (1999), 135-155 and therein. See cited references), and can be compared with the crystal structure of wild-type AChBP. Thus, potential sites for modification within the various binding sites of the ligand binding domain can be identified. This information provides an additional tool to determine the most efficient binding interactions (eg, increased hydrophobic interactions) between AChPB and chemical entities or compounds.

Designing compounds that bind or inhibit the ligand-gated ion channels of the present invention generally involves considering two factors.

First, the compound must be able to physically and structurally bind the ligand binding domain. Non-covalent molecular interactions important for the binding of a ligand binding domain to its ligand include hydrogen bonding, van der Waals, and hydrophobic interactions.

Second, the compound must be able to assume a conformation that allows it to bind to the ligand binding domain. Certain parts of the compound are not directly involved in this binding, but may still affect the overall conformation of the molecule.
This, in turn, can have a significant impact on efficacy. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound at all or part of the binding site, or between the functional groups of the compound containing several chemical entities that interact directly with AChBP. The intervals are.

When the theoretical structure of a particular compound suggests poor interaction and association between that compound and AChBP, compound synthesis and testing becomes unnecessary. However, if computer modeling shows strong interactions, synthesize the molecule,
The ability to bind to AChPB or ligand-gated ion channels can be tested and functionally tested according to the methods described above. In this way, the synthesis of non-working compounds can be avoided. Once the appropriate chemical entity or fragment is selected,
They can be assembled into a single compound or inhibitor. The assembly may be performed after visually inspecting the relationship of the fragments to each other on the three-dimensional image displayed on the computer screen with respect to the structural coordinates of AChBP. This is followed by manual model building using software such as Quanta or Sybyl. CAVEAT (Bartlett et al., “CAVEAT: a program that facilitates the structural design of biologically active molecules (CAVEAT: A Program to Facilitate the Structure-Derived
Design of Biologically Active Molecules "," Molecular Recognition in Chemical and Biological P
roblems) ", Special Pub., Royal Chem. Soc. 78 (1989), 182-196; MACCS-
3D (Martin, J. Med. Chem. 35 (1992), 2145-2154) and HO
OK (Molecular Simulations), Burlington
, Mass.) Like 3D database systems. Instead of step-by-step (one fragment or chemical entity at a time as described above) entering into the construction of the AChBP ligand, the AChBP-binding compound is used with an empty active site or selectively with some of the known ligands. Can be designed as a whole or "newly". These methods include LUDI (Bohm, J. ComR. Aid. Molec. Desig
n 6 (1992), 61-78); LEGEND (Nishibata and Itai)
, Tetrahedron 47 (1991), 8985); and LeapFrog (Tripos Associates, St. Louis, Mo.). Other molecular modeling techniques can also be used in accordance with the present invention; eg, Cohen, J.
． Med. Chem. 33 (1990), 883-894, and Navia and Murco (
Murcko), Current Opinions in Structural Biology 2 (1992), 202-210.

Such computer modeling is preferably performed by the docking program (
Dunbrack et al., Protein Sci. 6 (1997), 1661 to 1681 and Fold
ing Des. 2 (1997), R27 to R42).

Methods for identifying drugs or corresponding lead compounds in computer prescreens using X-ray crystal structures have been described in the prior art (Berlinde and Hor, Structure 2 (1994), 577-587; Kuntz. ), Science 257 (1
992), 1078-1082; Shuker et al., Science 274 (1996), 1531-1534.
Fejzo et al., Chem. Biol. 6 (1999), 755-769; International Publication No. 98.
/ 58961). Structural information can be investigated to efficiently optimize leads. Using the elucidated X-ray crystal structure, very small molecules or functional groups (GRID:
Goodford, J.M. Med. Chem. 28 (1985), 849-857; MCSS: Caflish et al. Med. Chem. 36 (1993), 2142 to 2167) and potential lead skeleton (DOCK: Kuntz et al., Accounts Chem. Res. 27 (1994), 1).
Computer programs have been written to identify compounds ranging from 17-123). Another method is to computer prescreen the compound library,
Individual "hits" are tested experimentally by X-ray crystallography to reduce the size of the screening library (Berlinde et al., J. Comput. Aided Mol. De).
s. 6 (1992), 131-147). In addition, an experimental approach has been developed to discover organic solvents that bind to the active site that can recombine to become lead macromolecules (Allen et al., J. Phys. Chem. 100 (1996), 2605. ~ 2611).

Once a compound has been designed or selected by the methods described above, the efficiency with which the compound can bind to AChBP or the corresponding ligand binding domain can be tested and optimized by computer evaluation. For example, a compound designed or selected to function as an inhibitor should preferably exhibit a relatively small energy difference between the bound and free states (ie, a small binding deformation energy).
Therefore, preferably the most efficient inhibitor should be designed with a binding deformation energy of about 10 kcal / mole or less, preferably 7 kcal / mole or less. Inhibitors may interact with the ligand binding domain in more than one conformation with similar total binding energies. In such cases, the binding deformation energy is considered to be the difference between the energy of the free compound and the average energy of the conformation observed when the inhibitor binds to AChBP.

Compounds designed or selected to bind to AChBP have the following properties:
It can be further computationally optimized to preferably lack repulsive electrostatic interactions with the target ligand binding domain. Such non-complementary (eg, electrostatic) interactions include repulsive charge-charge interactions, dipole-dipole interactions, and charge-dipole interactions. In particular, the sum of all electrostatic interactions between the ligand and AChBP when the ligand binds to AChBP preferably makes a neutral or favorable contribution to the enthalpy of binding. Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. As an example of a program designed for such an application, Ga
ussian 92, Revision C (Frisch, Gaussian, Inc.), Pitt
sburgh, Pa.); AMBER, version 4.0 (Kollman, University of California at San Francisco)
QUANTA / CHARMM (Molecular Simulations, Burlington, Mass.);
And Insight II / Discover (Biosysm Technol
ogies Inc.), San Diego, Calif.). These programs are available, for example, on Silicon Graphics workstations, IRIS 4
It can run on a D / 35, IBM RISC / 6000 Workstation Model 550, or better a Unix workstation (SGI, Alpha, Sun, etc.), or any Linux PC. Other hardware systems and software packages will be known to those skilled in the art.

Once the AChBP binding compound has been optimally selected or designed as described above, substitutions can be made at some of the atoms or side groups to improve or modify its binding properties. Generally, the initial substitution is a conservative substitution, that is, the substituent has about the same size, shape, hydrophobicity, and charge as the original group. Of course, it should be understood that changing the conformation must avoid ingredients known in the art. Such substituted compounds can then be analyzed for efficiency of matching to AChBP by the same computational methods detailed above. As mentioned above, the method of the invention described above can also be used as an initial drug screening assay prior to performing the classical drug screening assay using biochemical assays well known in the art.

Methods of preparing compounds, chemical derivatives, and analogs are well known to those of skill in the art,
For example, Beilstein, Handbook of Organic Chemistry (Handb)
ook of Organic Chemistry), Springer, New York Inc., 17
5 Fifth Avenue, New York, N. Y. It is described in.

In one embodiment of the methods of the invention, the identified drug interferes with or promotes the correct assembly of ligand-gated ion channels. Thus, the selected drug may, for example, interfere with the monomer contact area of the ligand-gated ion channel or serve as a scaffold for assembly. In the latter case, the drug may, for example, be based on antibodies that bind to the contact regions of two or more monomers when assembled, thus facilitating the assembly process. The preferred contact areas for AChBP and related nicotinic acetylcholine receptors are shown below. In a still further aspect of the method, the drug can be selected to bind to a non-specific binding site of a ligand-gated ion channel. Non-specific binding sites may include, for example, contact regions that are highly conserved between monomers of ligand-gated ion channels.

Once the drug has been selected according to any one of the above methods of the invention, a therapeutically effective amount of the drug or prodrug thereof can be synthesized. The term "therapeutically effective amount" as used herein has a meaningful patient benefit, ie, the treatment, cure, prevention, or amelioration of symptoms associated with ligand-gated ion channels, or the therapeutic rate of such symptoms. , The total amount of drug or prodrug sufficient to show an increase in cure rate, prevention rate, or improvement rate. Additionally, or alternatively, and particularly with respect to preclinical testing of a drug, the term "therapeutically effective amount" is sufficient in a non-human animal test to elicit a physiological response when the drug binds to a target ligand-gated ion channel. Includes total drug or prodrug.

The present invention also relates to a drug produced by any one of the above methods of the invention or a prodrug thereof. Preferably, the drug or prodrug thereof is present alone or in a composition in a therapeutically effective amount.

The drug obtained by the method of the present invention can be characterized by its interaction with the binding site in the binding cavity defined by the coordinates of the crystal structure of the protein-ligand complex. See, for example, US Pat. No. 5,798,247 (US-A-5,798,247) for examples of such characterization. Preferably, the drug, eg a potential inhibitor, forms a non-covalent bond with one or more amino acids at the active site based on the crystal structure. On the other hand, the drug may bind to the contact regions of individual monomers of the pentameric ligand-dependent receptor. For example, Limnair
The multimer contact region in Stagnaris AChBP (SEQ ID NO: 2) has been identified.
Contiguous regions have at least every other residue involved in contact with other monomers. Contact was defined as two atoms within a distance of 4.2Å in the 2.7Å structure. The primary contact region (residue from A that contacts B) in mature AChBP is 15-21.
Position, 44-47th, 85-87th, 91-94th, 122-124th, 143-146th, 149th, 185-18th
Position 7, the complementary contact region (residue from B that contacts A (identical to the residue in A that contacts E)) is positions 3-4, 7-8, 11 and 37-39. , 53rd, 75-77th, 96-104th
The positions are 114-118 and 163-170. See also Figure 14.

Accordingly, in one preferred embodiment, the drug of the present invention interacts with a ligand-dependent receptor comprising a pentamer having monomers A to E, wherein the drug is SEQ ID NO:
One of the 2 to 15-position, 44-47 position, 85-87 position, 91-94 position, 122-124 position, 143-146 position, 149 position, 185-187 position amino acid residue or Further, the primary contact region of the monomer, and / or SEQ ID NO: 2, 3-4, 7-8, 11-position, 37-39.
Position 53, position 75-77, position 96-104, position 114-118, and position 163-170, the complementary contact region of one or more other monomers ( A residue from B that contacts A (identical to the residue in A that contacts E)); or one of the contact regions shown in FIG. 14; or the corresponding contact region of the monomer of the ligand-gated ion channel. Bind to. Preferably, the ligand-gated ion channel is the nicotinic acetylcholine receptor and the order of the monomers is αγαδβ.

Crystal data and / or data disclosed herein or obtained from independent analysis of crystalline AChBP proteins or other water-soluble ligand binding proteins of the invention
Alternatively, any available method can be used to construct such a model from amino acid sequence data. Such models include HKL, MOSFILM, XDS, C
CP4, SHARP, PHASES, HEAVY, XPLOR, TNT, NMRCOMPASS, NMRPIPE, DIANA, NMRDR
AW, FELIX, VNMR, MADIGRAS, QUANTA, BUSTER, SOLVE, O, FRODO, RASMOL, CNS
, REFMAC, ARP / WARP, XTALVIEW, and CHAIN can be used to build from the available analytical data points. Models constructed from these data are then used, for example, in Silicon Graphics, Evans and Sutherland.
Can be visualized using available systems including Sun, SUN, Hewlett Packard, Apple Macintosh, Apple Macintosh, Deck, DEC, IBM, and Compaq . The invention also provides a device, such as a computer system, that includes the model of the invention and the hardware used to build, process, and / or visualize the model of the invention. A further aspect provides a computer system including the computer hardware and model of the present invention. Studies on the interaction between candidate species and models are conducted by QUANTA, RASMOL, O, CHAIN, FRODO, INSIGHT, DOCK, MCSS.
/ HOOK, CHARMM, LEAPFROG, CAVEAT (UC Berkley), CAVEAT (MSI), MODELLER,
It can be done using available software platforms including CATALYST, XTALVIEW, and ISIS. Crystalline AC disclosed herein or according to the invention
Floppy disks, CD ROMs, tapes, and any other storage means containing crystallographic and / or nucleotide / amino acid sequence data obtained from independent analysis of hBP proteins or other water soluble ligand binding proteins or Computer-readable media, such as processing means, are also the subject of the present invention. Advantageously, any one of the described means and devices can be used to model antagonists / inhibitors or agonists / activators of ligand-dependent receptors.

The invention further relates to the construction of a theoretical three-dimensional (3D) model of the ligand-binding domain of the ligand-gated ion channel by computational molecular modeling of the water-soluble ligand-binding proteins of the invention using X-ray coordinates. These 3D models may correspond to the entire ligand binding domain (~ 220 to 240 extracellular amino acids) or may be restricted to the ligand binding site.

The concept of using the 3D structure of mollusc ligand binding proteins for molecular modeling, and tools for structural prediction of ligand-gated ion channels in, for example, mammals, and particularly humans, include vertebrate glutamate receptor channels and The ligand-binding domain of bacterial periplasmic substrate-binding proteins (PBPs) displays a similar 3D structure despite the very low sequence similarity (12%) between the ionotropic glutamate receptor subunit and PBPs used as templates. It is supported by the observation of sharing; for a review, Paas et al., TiPS 21 (2000).
, 87-92 and the references cited therein.

Thus, selectively, for example, based on computational molecular modeling complemented by functional studies of site-directed variants, crystal structures of the ligand-binding domains of ligand-gated ion channels and theoretical 3D models of these domains. Can be predicted. These models can then be used for structural drug design. Ligand binding sites that affect, for example, (1) agonist-induced channel activation and desensitization, (2) inhibition of channel activity by various competitive receptor antagonists, or (3) binding of various ligands. The predicted model can be further refined by monitoring the effects of mutations at amino acid residues arguably located in Experimental settings for analyzing such effects are well known to those of skill in the art, see also the literature cited for functional assay systems for ligand-gated ion channels.

Thus, aspects of the invention allow for various possibilities of identifying and modeling new ligands for ligand-gated ion channels, as well as modifying the ion channels themselves. Therefore, the present invention provides the above-mentioned polynucleotides, proteins, dimers and pentamers, ligand-gated ion channels, vectors, host cells,
It relates to the use of antigens, antibodies, oligonucleotide probes, crystals, their structural coordinates, as well as methods of screening or profiling putative ligands for ligand-dependent receptors.

Lead generation methods in protein-based drug discovery, as well as mass spectrometry (Cheng et al. J. Am. Chem. Soc. 117 (1995), 8859-8860), and some nuclear magnetic resonance (NMR). ) Method (Fejzo et al., Chem. Biol. 6 (1999), 755.
~ 769; Lin et al., J. Am. Org. Chem. 62 (1997), 8930-8931).

The newly identified drugs, antagonists / obtained by the method of the present invention
The inhibitors or agents / activators can be used in the preparation of a pharmaceutical composition for treating disorders mediated by ligand-gated ion channels, or disorders associated with ligand-gated ion channels. Such disorders are well known to those of skill in the art. For example, possible applications of agonists and antagonists to nAChR have been shown to involve certain neuropsychiatric disorders (Alzheimer's disease and Parkinson's disease, Too Rett syndrome,
Schizophrenia, depression, etc.) is involved in the etiology of. For most of these disorders, the use of nAChR antagonists can be prophylactic (especially Alzheimer's and Parkinson's) or symptomatic; for a review, see, for example, Mihailescu and Drucker-Colin. )
, Arch. Med. Res. 31 (2000), 131-144.

The medicinal chemistry and molecular biology of GABA-activating ligand-gated ion channels, which are also related to the structural profiles of agonists and antagonists, have been described by Chebib et al.
J. Med. Chem. 43 (2000), 1447-1447.

Disorders of glycine receptors and glycinergic neurotransmission have been reported by Rajendra et al., Pharmacol. Ther. 73 (1997), 121-146 and Barry et al., Clin. Exp. Pharmacol. Physiol. 26 (1999), 935-936.

The major role of 5-HT3 receptors in CNS disorders and antagonists of 5-HT3 receptors have been reported by Bloom and Morales, Neurochemical Research 23 (199).
8), 653-659 and Higgins and Kilpatrick.
), Expert Opin. Invest. Drugs 8 (1999), 2183-2188.

In one embodiment, the antagonist / inhibitor is or is derived from a protein, antigen, antibody of a ligand-gated ion channel or is derived from a toxin. Similarly, the agent / activator can be derived from a ligand-gated ion channel protein, antigen, antibody, or toxin. Possible starting points are eg peptide toxins such as conotoxin (IMI) and α-bungarotoxin, lophotoxin (bipinatin), tubocurarine, decamethonium, α-cobratoxin,
Contains epibatidine, acetylcholine, choline, nicotine, carbachol, serotonin, or GABA. The structure of these molecules, for example to improve the affinity and / or specificity of the drug, or for example to render the drug acting on another target non-reactive with a particular ligand-gated ion channel. Can be used in conjunction with the structure of target ligand binding domain crystals to model compounds and elucidate side chains, functional groups, etc. that may be added, deleted or modified.

In a preferred embodiment for use according to the invention, the ligand-gated ion channel is a nicotinic acetylcholine receptor and the mediated and related disorders are Tourette's syndrome, Alzheimer's disease, nicotine addiction, or schizophrenia. is there.

As previously described herein, it can be shown that water-soluble ligand-binding proteins are present in mollusks and are very similar to the ligand-binding domain of higher mammalian ligand-gated ion channels. This is the first time for me. Similar ligand-binding proteins to other mollusc species or mollusks (Mollusca)
, Protostomia, Coelomata, Bilateri
a), Authentic metazoa (Eumetazoa), metazoa (Metazoa), fungi / metazoa (Fung)
It is expected to exist even in the lineages of the i / Metazoa group. Therefore, the present invention also
It relates to the use of ligands of ligand-gated ion channels to identify and isolate water soluble ligand binding proteins from such species, preferably molluscs. Preferably, the ligand used for protein isolation is α-bungarotoxin. Water-soluble ligand binding proteins available from these organisms as well as derivatives that can be made according to the disclosures present herein are also subject matter of the present invention.

Furthermore, the crystal structure of the nicotinic binding site was elucidated for the first time. This crystal structure indicates that mollusc AChBP is a homologue of the LGIC superfamily ligand binding domain. The crystal structure reveals extensive data on Ig topology, binding site positions at subunit interfaces, MIR positions, and nicotinic ligand binding residues. Importantly, the crystal structure provides important new information about the correct folding and placement of the nicotinic ligand binding site in three dimensions. This indicates the presence of a second pocket, which is recognized by EM analysis. Furthermore, it reveals the arrangement of subunits by showing the relative positions of the major and complementary parts of the ligand binding site. This explains the role of LGIC superfamily conserved residues in stabilizing the monomeric structure by forming a hydrophobic core and packing secondary structural elements, how the pentamers are assembled, and the pentamers. To clarify how weak the boundary surface is preserved between LGICs.

This structure can be used in numerous drug design studies targeting the LGIC superfamily. General structural knowledge about its folding is GABA,
It could be applied in the field of serotonin (5HT ₃ ) and glycine receptors. This helps to understand their ligand binding properties, and thus, for example, anti-emetic drugs targeting the 5HT ₃ receptor or mood-definin targeting the GABA receptor.
g drug) can be affected. However, the availability of three-dimensional delineation of nicotinic ligand binding sites is particularly relevant to the design of new drugs for Alzheimer's disease, epilepsy, and smoking addiction, which have neuronal nicotinic receptors as targets.

Many embodiments and examples include acetylcholine binding protein (AChBP) of the invention.
Of the invention, and embodiments generally described herein relate preferably to the nicotinic acetylcholine receptor (nAChR), more preferably to the α subunit, most preferably to the α7 subunit. However, it should be understood that all aspects apply equally to other water-soluble ligand binding proteins, generally the ligand-gated ion channels described herein. For example, to model new ligands for the acetylcholine receptor, preferably those with inhibitory or stimulatory effects on the acetylcholine receptor, AChBP
Can be used. Similarly, it is possible to identify and model new ligands for other ligand-gated ion channels, including glycine, GABA, and serotonin receptors, that have inhibitory effects. Such ligands may interfere, for example, with the correct assembly of ligand-gated ion channels. Preferably, such ligands prevent the correct assembly of specific subtypes of ligand-gated ion channels. On the other hand, it is possible to identify and model ligand-gated ion channels, preferably ligands that facilitate the correct assembly of specific subtypes of ligand-gated ion channels. As mentioned above, the method of the invention also allows modeling of inhibitors for non-specific binding sites of ligand-gated ion channels.

Furthermore, the assembly behavior has been modified to enhance the binding site similarity to acetylcholine receptor subtypes at the primary binding site, and in general to specific ligand-gated ion channels in changes in activity and conformation. Mutants and chimeras of AChBP can be predicted and engineered to enhance similarities with and modify ligand binding behavior. Taking into account the closest relationship between AChBP and the acetylcholine receptor, it is particularly preferred to create variants and chimeras that have increased binding site similarity to the acetylcholine receptor subtype at the secondary binding site. However, the prediction and production of variants and chimeras that have increased binding site similarity with other ligand-gated ion channel subtypes at the primary or secondary binding sites are also contemplated.

These and other aspects are disclosed and encompassed by the description and examples of the invention. Further literature on any one of the antibodies, methods, uses and compounds used according to the invention can be retrieved from public libraries and databases, for example using electronic devices. For example, http: //www.ncbi.nlm.nih.
A public database "Medline" available on the Internet such as gov / PubMed / medline.html can be used. For example, http: // ww
w.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/, http://www.fmi.ch/biolog
y / research Additional databases and addresses such as tools.html, http://www.tigr.org/ are well known to those skilled in the art, which can also be found, for example, at http: //www.lycos.
It can be obtained using .com. An overview of patent information in biotechnology and a search of related sources of patent information useful for retrospective and current status studies is given in Berks, TIBTECH 12 (1994), 352-364.

The present disclosure can best be understood in conjunction with the accompanying drawings, which are incorporated herein by reference. Furthermore, a better understanding of the invention and its many advantages will be gained from the following examples, which are given by way of illustration and not intended to be limiting.

Except as indicated in the examples, all recombinant DNA techniques were performed according to Sambrook et al. (1
989), "Molecular Cloning: A Laborato
ry Manual), Cold Spring Harbor Laboratory Press, NY or Ausbel et al. (1994) “Current Protocols in Molec
regular Biology) ”,“ Current Protocols ”, Volumes 1 and 2 of the present invention. Standard materials and methods for plant molecule research are available in BIOS Scientific Publications Ltd (UK) and Bl.
Chloe (RD), jointly published by ackwell Scientific Publications (UK)
． D. Croy) “Plant Molecular Biology L
abfase) ”(1993).

EXAMPLES Example 1: Isolation of Limnaea AChBP from CNS, Determination of Mass and N-Terminal Protein Sequencing Isolation : 80 limnaea CNS, 1 ug / ml aprotinin, 10 ug / ml benzamidine, 0.5 ug / ml
Lysis buffer (PBS [1 containing 24ug / ml pefabloc)
6mM Na ₂ HPO ₄ , 4mM NaH ₂ PO ₄ (pH7.4); 150mM NaCl] 0.5% Nonidet
P-40; 0.1% Triton, 0.2% tween-20). CNS lysates were clarified by centrifugation 3 times at 12,000 xg for 5 minutes. Streptavidin-coated magnetic beads (Dynal, Oslo) 5 mg, biotin-conjugated α-bungarotoxin (4ug) (Molecular Probes, Oxford, U
Saturated with K). These beads were washed with PBS to remove excess α-bungarotoxin, then added to clear CNS lysates and incubated for 1 hour. Following this, the beads were washed 3 times with PBS to remove unbound protein.
A control reaction was performed without α-bungarotoxin. 10 μl PBS containing 10-4 M nicotine
The protein bound to α-bungarotoxin was eluted for 1 hour in.

Mass determination : Eluents were separated on a microcolumn LC system similar to that previously described (Hsieh et al .; Anal. Chem. 70; 1998; 1847-1852). 20 μl / using a commercially available syringe pump (Perkin Elmer / ABI, model 140B)
A minute flow rate was delivered to the column. After loading the sample into the column, the flow rate was dropped to 10 μl / min. Then, in 1 minute, the eluent was switched from 0.2% acetic acid to 0.2% acetic acid / 60% acetonitrile. Electrospray mass spectra in MS mode were obtained on a Micromass Q-TOF quadrupole time-of-flight mass spectrometer equipped with a Z-spray atmospheric pressure ion source.

Protein Sequence Analysis : For sequence analysis, α-bungarotoxin binding protein was extracted using the same procedure, followed by SDS-PAGE and Western blotting on PDVF membrane. Sequence analysis was performed by Edman degradation of the protein (apparent MW) blotted at 24 kDa using a protein sequencer (ABI, Perkin Elmer).

Example 2 Cloning of Limnair AChBP cDNA Sequence: PCR and Screening of cDNA Library PCR Cloning : Amino acid sequence of residues 1-10 of AChBP Degenerate oligonucleotide based on (SEQ ID NO: 10) (Including BamHI restriction site) was synthesized and used with the primers on the IZAPIIλ vector. PCR is a DNA thermal cycler (PerkinElmecetas)
rCetus), CT) at 94 ° C., 20 sec; 53 ° C., 30 sec; In a reaction volume of 100 μl containing
Performed on S IZAP II cDNA library. The amplified cDNA was digested with BamHI and EcoRI, separated on an agarose gel and the ~ 900 bp product cloned and sequenced.

Library screening : Approximately 20,000 clones of the amplified λZAP II CNS cDNA library were transformed into 10 ⁵ pfu / 400c.
Seeded at a density of m ² and charged with a nylon membrane (Boehringer Mannheim, Ge
rmany). The AChBP PCR product was used as a randomly primed probe and labeled with [α ³² P] dATP (specific activity> 10 ⁹ cpm / mg). Replace the membrane with 6 x SSC (
1 x SSC: 0.15 M NaCl and 0.015 M sodium citrate), 0.2% SDS, 5 x Denhardt, and 10 ug / ml herring sperm DNA hybridized at 65 ° C for 18 hours.
The filters were washed with 0.2 x SSC, 0.2% SDS at 65 ° C for 30 minutes and autoradiographed. Four individual cDNA clones were excised in vivo and sequenced using the dideoxy chain termination method in both directions. L-AChBP T1 and L-
AChBP Two types of sequences were obtained, called T2. "SMART" Simple Modular Archite
The signal sequence was determined using the ctur Research Tool (V3.1); Schultz (Sc
hultz) et al., Proc. natl. Acad. Sci. USA 95 (1998), 5857-5864 and Nuclei
c Acids Res. 28 (2000), 231-234. L-AChBP T1 (SEQ ID NO: 2)
In the case of, the prediction could be confirmed experimentally.

Example 3: Limnaea AChBP Related Sequences: Cloning of Brynus transcutatus cDNA Total RNA was isolated from Brynus cranial ganglia (CNS) and reverse transcribed into hexanucleotide-primed cDNA. Limna Air AChBP Two degenerate oligonucleotides directed to the T1 sequence, the forward primer: (SEQ ID NO: 12), reverse primer: (SEQ ID NO: 13) was used to amplify the sequence related to AChBP. PCR was performed using standard conditions of 45 cycles (94 ° C, 20 seconds; 54 ° C, 30 seconds; 72 ° C, 1 minute) for each primer 1
50 pmole was used for CNS cDNA equivalent to one animal. Amplified cDNA is EcoRI /
It was digested with HindIII, cloned into EcoRI / HindIII digested pBluescript and sequenced. The ORF of the obtained sequence is related to Limnaea AChBP in sequence, and B-AChBP_
It showed a Brinnus AChBP called T1. Brynus brain cD using the same hybridization protocol described for the cloning of Limnaea cDNA.
This partial cDNA was used to screen the NA library for B-AChB
P T1 and B-AChBP Two cDNA clones encoding T2 were generated. The cDNA was sequenced in both directions.

Example 4: L-AChBP-T1 and T2 and B-AChBP in yeast Pichia pastoris
-Production of T1 and T2, and functional characterization Production of recombinant AChBP : L-AChBP as a recombinant protein in Pichia pastoris expression system (Pichia Expression Kit version 3.0, Invitrogen) T1 and T2
And B-AChBP The DNA sequences encoding the mature forms of these proteins (see sequence files) were cloned into the pPIC9 expression vector (Invitrogen) to produce T1 and T2. (Pfu-taq DNA polymerase (Stratagene) to avoid introducing errors into the sequence by PCR.
L) -AChBP T1, T2 and B-AChBP The mature sequences of T1 and T2 were PCR amplified and restriction sites were added to the primers to allow for rapid pPIC9 compatible cloning. Mature AChBP L-AChBP The amplified sequence of T1 was inserted into pPIC9 with EcoRI, but L-AChB
P T2 and B-AChBP T1 and T2 were XhoI / EcoRI inserted into pPIC9 (alpha mating factor cleavage site was completely reconstructed after XhoI digestion).

Additional C-Terminal His Tag (L-AChBP In the case of T1, Constructs with or without (SEQ ID NO: 15)) were made for each of the AChBP (sub) types. The AChBP / pPIC9 construct was amplified in E. coli DH5αF, isolated and purified using the plasmid Maxi Kit (Qiagen).
The engineered cleavage site at the N-terminus of the amino acid sequence causes four additional amino acids (EA
EA, SEQ ID NO: 16) precedes the N-terminus of the original mature protein. Prior to transfection into Pichia pastoris, the construct was linearized (see supplier manual for protocol; Pichia Expression Kit version 3.0).
, Invitrogen), followed by phenol / chloroform extraction and ethanol precipitation. About 5 μg of each linear construct was transformed into freshly prepared electrocompetent Pichia pastoris cells and plated on MD plates. (
See supplier's manual for protocol; Pichia Expression
Kit version 3.0, Invitrogen Corporation). Electrocompetent Pichia pastoris cells were obtained according to the protocol provided by Invitrogen. The plates were incubated at 30 ° C. until the appearance of Pichia colonies and then analyzed for the presence of the correct insert by PCR amplification (see supplier manual for protocol; Pichia Expre
ssion Kit version 3.0, Invitrogen). Colonies containing homologous recombination with the Pichia genome and having the AChBP sequence were grown in 25 ml BMGY for 1-2 days (30 ° C .; spin at 250 rpm), after which the cells were centrifuged (10 min, 1500 g), B cell pellet
Resuspended in 10 ml MMY. Proliferation (30 ° C, 250rpm) continued for 4 days (3rd to 6th day)
, AChBP expression during this period, 100% methanol (1% of total culture volume) every 24 hours
Was induced by the addition of On day 7, the culture was centrifuged (15 minutes; 2000g; 4 ° C)
The medium was collected. A small portion of the collection medium was analyzed using SDS polyacrylamide gel electrophoresis to measure AChBP expression levels in various cultures (see supplier's manual; Pichia Expression Kit version 3.0, Invitrogen). Cultures that produced the highest levels of AChBP expression were selected and stored as glycerol stocks.

Recombinant AChBPs containing a C-terminal His-tag were isolated and purified from Pichia pastoris medium using a Talon metal affinity resin (following the protocol described in the user manual; Clontech laboratories In
c.)). After that, protein concentration was analyzed using SDS polyacrylamide gel electrophoresis and reference marker protein. Recombinant in Balb-C mice
L-AChBP T1 and B-AChBP A polyclonal antibody against the T1 protein was successfully produced. Immune sera were obtained without protein cross-linking.

Binding Properties of AChBP : First, the binding curve of α-bungarotoxin to His-tagged AChBP was measured, and 3
An affinity of .5 nM was calculated. Α-bungarotoxin was then used in a competitive binding assay to bind several types of ligand-gated ion channel ligands to His-tagged AChBP, namely ACh, serotonin, GABA, glycine, and glutamate. Tested. Both ACh and serotonin are 4.2 each
It competed with α-bungarotoxin binding with an IC 50 of mM and 269 mM. GABA, glycine,
And glutamate did not compete for binding with α-bungarotoxin. Therefore, as predicted by primary sequence and subunit structure as well, AC
The ligand binding properties of hBP were similar to those of nAChR.

The ligand binding properties of AChBP were investigated in more detail, this time using various agonists and antagonists of AChR in a second series of competitive binding assays. nAC
Nicotine, the classical agonist of hR, is a high affinity ligand for His-tagged AChBP (IC50 98nM). Epibatidine, a high affinity agonist of nAChR, also binds His-tagged AChBP with high affinity (IC50 1.4 nM). This affinity is significantly higher than the 58 pM affinity of epibatidine reported for nAChR (Badio, Mol
． Pharmacol. 45 (1994), 563-569). Other cholinergic agents bind with lower affinity, eg, decamethonium, carbachol, and choline bind with IC50s of 4.1 μM, 43 μM, and 190 μM, respectively. A summary of the affinity is shown in Table 2.

[0144]

[Table 2]

Representative nAChR antagonists (eg, tubocurarine and α-bungarotoxin)
Competitive binding has a high affinity for His-tagged AChBP with IC50 of 93 nM and 2.6 nM, respectively. The cholinergic antagonist succinylcholine is a His-tagged AChBP
It has a very low affinity for (IC50 7.9 mM). Interestingly, muscarinic receptor antagonists (eg, muscarinic allosteric modulator galamine (IC50 39nM) and muscarinic antagonist atropine (IC50
5.3 mM)) also binds His-tagged AChBP with relatively high affinity. Physostigmine, a known acetylcholinesterase blocker and nAChR antagonist, binds His-tagged AChBP with an IC50 of 1.3 mM.

Finally, a synthetic form of coral phototoxin, bipinatin-B, was tested against AChBP (Groebe and Abramson, J. Biol. C).
hem, 270 (1995), 281-286). Bipinatin-B is a common nAChR blocker,
It is known to covalently bind to Tyr-190 of the α subunit (Abramson, J
． Biol. Chem. 263 (1988), 18568-18573). His-tagged AChBP was incubated with toxin to increase protein mass (430.1 Da was 431 Da bipinatin
-Very consistent with the calculated mass of B). From this, this toxin is also His-tagged AChBP
It can be seen that it binds to Tyr-184.

Example 5: Expression and Purification of Recombinant AChBP for Crystallization Using Invitrogen's AOX1 Gene Expression System, AChBP from Limnea Stagnaris in Pichia pastoris GS115 Strain A large amount of T1 protein (AChBP) was expressed. The media and methods used for AChBP expression are also described in Invitrogen's Manual Pichia Expression Kit. Transformants were grown overnight at 30 ° C. in YPD medium for long-term storage.

[0148] YPD or yeast extract peptone dextrose medium   1% yeast extract (Difco)   2% peptone (Difco)   2% dextrose (glucose) (Merck)

Cells were harvested and suspended in YPD medium containing 15% glycerol at a final OD600 of -50. Freeze cells in a dry ice / ethanol bath and use a freezer (Revco
)) And stored at -80 ° C. Usually, AChBP expression was started by plating cells from a glycerol stock on MD plates.

[0150] MD or minimum dextrose medium   1.34% YNB (yeast nitrogen base w / o amino acid) (Difco)   4 × 10-5% d-Biotin (Sigma)   1% dextrose   Add 15g agar (difco) for plate

Place plates in incubator (Heraeus) for 3-4 days 30
Store at ℃. Pick a single colony from the plate, containing 25 ml of BMGY medium 15
A 0 ml baffled flask (Nalgene) was inoculated.

[0152] BMGY or buffered glycerol complex medium   1% yeast extract   2% peptone   100 mM potassium phosphate (pH 6.0) (Merck)   1.34% YNB   4 × 10-5% d-biotin   1% glycerol (Merck)

The culture was placed on a shaker (New Brunswick) and grown overnight at 250 rpm and 30 ° C. rotation. The next day, 12.5 ml of culture was inoculated into 225 ml of BMGY medium in a 1000 ml baffled flask. A larger number of flasks (usually 16) were used to increase the yield of expressed AChBP. The flask was placed on a shaker and the starting culture was spun at 250 rpm at 30 ° C. After 2 days, this starting culture was
Centrifugation was performed at room temperature for 15 minutes at 500 rpm (Sorvall RC3B +, rotor H-6000A). 2 to increase cell mass for larger scale protein production
Cell pellets from individual starting culture flasks were pooled together and resuspended in 200 ml BMMY medium containing 1% (w / v) casamino acid.

[0154] BMMY, buffered methanol complex medium + 1% casamino acid   1% yeast extract   2% peptone   100 mM potassium phosphate (pH 6.0)   1.34% YNB   4 × 10-5% d-biotin   0.5% methanol (Merck)   1% casamino acid (Difco)

[0155]   The culture was returned to the shaker (250 rpm, 30 ° C) and induced for another 4 days. 24 hours a day
Methanol in the culture medium by adding 1% (v / v) methanol to the culture.
The concentration was kept constant. After 4 days, 100 ml of culture was harvested and freshed with 1% casamino acid.
The original volume was readjusted to 200 ml by adding different BMMY medium. Remaining culture
Articles were induced for an additional 4 days. Collect the collected culture at 4000 rpm (Soval RC3B +, low
H-6000A) for 15 minutes and the cell pellet was discarded. First left
The supernatant is filtered with a 0.22 μm filter (Millipore) to remove cells
) Through a 30kDa cut-off filter (Waters / Millipore (Wate
rs / Millipore)) Minitan device (Waters / Millipore)
Was concentrated using. Both filtration and concentration were done at 4 ° C. Finally, the first two
In order to remove the debris left after the step, centrifugation was performed at 16000 rpm (so
Global RC5C, rotor SS-34). The final volume of the concentrated sample is approximately 80 ml and the 15 kDa
2 × 5l (20mM Tris [p] using a Tooff dialysis membrane (Spectra / Por)
H8.0], 150 mM NaCl, and 0.02% NaN₃) Against 4) overnight at 4 ° C. Dialysis tank
Use the anion exchange column (POROS 50 HQ, Pharmacia,
The column volume was 8 ml). Use loading buffer and use a maximum of approximately 15 column volumes.
After the first wash step, 30 column volumes of a salt gradient of 150 mM to 1000 mM NaCl were run. Both
20 mM Tris (pH8.0) and 0.02% NaN₃Included. The target peak is about
Elution was performed with 300 mM NaCl (conductivity range 16-24 mS / cm). The existence of AChBP is Bio-Rad
Bio-Rad Protein Assay (Bio-Rad)
And SDS-PAGE, pooled fractions of interest, and sent Centriprep (Centri
prep) and 30 kDa cut-off membrane (Amicon)
. Concentrate sample (5 ml volume) with 20 mM Tris (pH 8.0), 150 mM NaCl, and 0.02% NaN ₃ Gel filtration column (Superdex 200 HR 16/60,
Lumasia, column volume 120 ml). Protein starts from 60ml and is 71m
Elution was performed in the range of 1 and the peak was about 66 ml. The final purification step of the protein was
Performed on an on-exchange column (MonoQ HR10 / 10, Pharmacia, column volume 6 ml). Ta
The protein is dissolved in the same buffer that was eluted from the gel filtration column and applied to the column.
Loaded The salt gradient used for this column was also used for the POROS 50 HQ column.
Was the same as. Fractions in the conductivity range 25-27.5 mS / cm were pooled together and 50 mM HE
PES (pH 7.0) and 0.02% NaN₃Dialyzed against a buffer containing Protein
Up to about 20 mg / ml using Centricon and 30kDa cut-off membrane (Amicon)
Concentrated. Total yield was approximately 2 mg purified protein per liter expression medium
. The concentrated protein was stored at 4 ° C and used for crystallization experiments and biochemical characterization.
It was From the N-terminal sequencing, the signal sequence encoded by pIC9, preceded by two residues.
Is part of The presence of residues was revealed. The experimentally determined mass is based on the amino acid sequence.
It was determined to be 26544 Da (MALDI), which is about 2 kDa higher than the calculated mass (24649 Da). This difference is
Due to AChBP glycosylation at position Asn66 in the mature sequence, which results in N-glycosylation.
Confirmed by deglycosylation experiment using Dase F (Boehringer)
It has been certified.

Purification of the first harvest was done separately from all harvests. They were pooled together before the last purification step (anion exchange chromatography step on MonoQ column). All the chromatographic columns described above were mounted on a FPLC device (Pharmacia) controlled by a UNICORN device (Pharmacia). FP
All solutions used in the LC instrument were prepared with MilliQ UF + water, filtered through a 0.22 μm filter (Millipore) and degassed.

Example 6: AChBP Crystallization All crystallization experiments were carried out by evaporative diffusion method in hanging drop format using a 12-well tray (Nelipak) and a silicone treated cover slide (Hampton Research). It was The tray was placed in a sandwich box (Semadeni) and stored in a room temperature controlled at 19 ° C. Initial crystallization attempts were made using Hampton Crystal Screens I and II (Hampton Research). Each drop contained 2 μl of protein (50 mM HEPES [pH 7.0] and 0.0
10 mg / ml in 2% NaN ₃ ) as well as 2 μl of reservoir solution was included. Initial screening revealed that AChBP produced crystalline precipitates in the presence of CaCl ₂ salt. A more detailed screen was performed that yielded crystals suitable for X-ray analysis. AChBP
Crystals appeared under the following conditions: 9-11% (w / v) PEG 4000 (Hampton Research),
100mM HEPES (pH7.0), (including 0.3mM zinc acetate as _{additives) 50~200mM CaCl 2 × 6H 2 0} , and 0.02% NaN ₃ or PEG MME 550 10 to 18% at the same conditions. Depending on the protein batch used and the CaCl ₂ concentration, three different crystal forms were found: orthorhombic, tetragonal and monoclinic. The orthorhombic and monoclinic crystal forms are often twins. Orthorhombic rod-shaped crystals appeared (within the first few hours) under high [CaCl ₂ ] as soon as the crystallization experiment was started. The crystal size is 0.05 x
It was 0.05 × 0.15 to 0.25 × 0.25 × 1.0 mm. The crystal diffracts X-rays up to 3Å resolution,
It shows a high mosaic angle (about 05-1.2 °). They had the symmetry of the space group P2 ₁ 2 ₁ 2 ₁ and had lattice constants (cell constants) a = 120.62Å, b = 137.01Å, c = 161.54Å, two pentameric molecules per asymmetric unit. . A tetragonal crystal whose shape is square is
It grew at a lower CaCl ₂ concentration and reached a size of 0.2 × 0.3 × 0.35 mm. The maximum resolution obtained was 2.7Å, and the mosaic angle (0.5 °) was lower. These are space groups
It belongs to the space group P4 _{2 1} 2 and has lattice dimensions a = b = 141.66Å, c = 120.83Å, and one pentameric molecule per asymmetric unit. The precise crystallization conditions of the tetragonal crystal used for the refinement of the crystal structure are as follows: 11.5% (w / v) PEG4000, 100 mM HEPES (p
H7.0), 150 mM CaCl ₂ , and 0.02% NaN ₃ . Third monoclinic P2 ₁ in crystalline form,
The structure is very similar to the orthorhombic crystal and the lattice dimensions are a = 121.1Å, b ＝ 162.1Å, c = 1.
39.4Å, β = 90.13 °, containing 4 pentameric molecules per asymmetric unit. This crystal yielded lower resolution data (approximately 3.3Å resolution). All three crystal forms
It was used to determine the structure of AChBP.

The resolution limit of diffraction was greatly influenced by the size of the crystal. The largest crystals weakly diffracted to a resolution of about 4Å when exposed to a conventional rotating anode X-ray source. Therefore, the use of synchrotron radiation was important for structure determination. The crystals had to be cryoprotected to counteract the damage caused by high intensity synchrotron radiation. Cryoprotection of AChBP crystals was performed in multiple stages. The first step involved stabilization of the crystals by the addition of 2 μl of mother liquor (equilibrium reservoir solution) to the droplets containing the crystals. After 5 minutes, 3 μl of stabilizing solution was added to the drops. Usually, the stabilizing solution contained a slightly higher concentration (1-5%) of the components of the initial crystallization buffer. Glycerol (Merck) was added as a protective agent and the concentration was increased stepwise from 0% to 30% (v / v). For example, the starting solution is 15% PEG4000,100mM HEPES (pH7.0), comprises 150 mM CaCl _2, and 0.02% NaN _3, the final solution, in addition to the components mentioned above 30% (v / v) glycerol Inclusive. AChBP crystals do not tolerate a sharp increase in glycerol concentration and therefore a gentle but more time consuming approach must be taken. Change the solution around the crystal stepwise (usually increase glycerol concentration 5%)
The crystals must be equilibrated for at least 5 minutes at each glycerol concentration. Once the crystals were in equilibrium in a stabilizing solution containing 30% glycerol, they were rapidly cooled with liquid nitrogen or a cryo-stream. In all three space groups, AChBP forms a decameric structure with perfect 52 symmetry, where 2
The five pentamers are in contact with each other via a calcium binding site "above" the α1 helix. This binding site (Asp2 and Asp5 from the two monomers) is LGIC
Not preserved in the family. In the tetragonal space group, twice the decamer coincided with the crystallographic doubling, which is the pseudo-centrosymmetric behavior of phase at low resolution (pseud
ocentrosymmetric behavior). In solution, the AChBP protein acts as a pentamer.

Those skilled in the art will appreciate that the crystallization conditions described above can be varied. Such variations may be used alone or in combination and may range from 1 mg / ml to 30
mg / ml protein (selectively in complex with ligand) final concentration; A
All combinations of ChPB / ligand to precipitant ratios; use of citrate concentrations of 0 mM to 200 mM; DTT concentrations of 0 mM to 10 mM; any concentration of β-mercaptoethanol; 5.5 to 9.5
PH range; 5% to 25% (w / v) PEG concentration; 2000 to 8000 PEG weight; 5 to 500 mM HEPE
S concentration; use of TRIS or other solution in place of HEPES, and any concentration or type of surfactant; any other type of precipitant; any other buffer; any of -50 ° C to 30 ° C Temperature, as well as crystallization of AChBP or its complex by batch, liquid bridge, or dialysis methods using these conditions or variations thereof.

Example 7: Structure determination The crystal structure was determined by multiwave anomalous disper for Pb derivative.
sion (MAD) method, but non-crystallographic symmetry (NCS) averaging was required to obtain an interpretable electron density. Native data and collection of heavy atom derivatives are available in Grenoble (ESRF / BM14
And ID14) and synchrotron beamlines in Hamburg (DESY / BW7A, BW7B and X11). AChBP orthorhombic crystal, 5 mM trimethyllead acetate (
It was immersed in a stabilizing solution containing trimethylleadacetate (MePb) for 5 days. The data set is run through four different wavelengths (0.9492Å, 0.8610Å, 0.9507Å, and 0.9499Å).
), Integrates the data, and collects DENZO / SCALEPACK (Otwinowski
ski) and Minor (1997) "Processing of X-ray diffraction data collected in oscillation mo
de) ”,“ Method in Enzymology ”, Volume 276: Macromole
cular Crystallography, Part A. Carter (CW Carter) and Suite (
R. M. Sweet) (New York: Academic Press), pp. 307-326). Program SOLVE (Terwilliger (1997) "SOLVE: An automated structure solution for MAD and MIR
MIR) ", version 1.16) discovered five Pb sites located at the interface between two pentamers. SHARP (La Fortelle et al. (1997) "Advance in MIR and MAD phasing: Maximum-likelihood refinement i
na graphical environment, with SHARP) ", Proceedings of the CCP4 study
weekend) was used to calculate the phase. The average figure of merit (FOM) for the four wavelengths was 0.45. 5 times NCS operator
or) search and optimization programs NCS6D and IMP (Kleyweg
Wegt) and Jones (1999) "Software for handling macromolecular envelopes", Ac
ta Crystallo., D55, 941-944). A refined NCS operator and DM (Cowtan (1994) “DM: An automated procedure for phase improvement by density
modification) '', Joint CCP4 and ESF-EACBM Newsletter on Protein Crystal
Interpretable electron density maps were obtained by 10-fold averaging with density correction according to lography 31, 34-38). However, some parts of the pentamer have not yet been clearly defined. Therefore, a second MAD experiment was performed on monoclinic crystals immersed in 10 mM MePb for 5 days. Data were collected at Pb peak (0.9479Å) and remote (0.9498Å) wavelengths for only two wavelengths. MOSFLM (Leslie (1992) "Recent changes to the MOSFLM package for processing film and ima" describes the processing of two acquired data sets.
ge plate data) '', Joint CCP4 and ESF-EACBM Newsletter on Protein Crysta
llography, Number 26), and the data is SCALA (“CCP4. CCP4 suite: programs for prote
in crystallography) ”, Acta Crystallog. D50, 760-763). 10 Pb sites were identified using Solve. The Pb parameters were refined and the data collected at the Pb absorption peaks were used to single anomalous dispersio
n) Phase was calculated by SHARP in (SAD) format. The NCS operator required for 20x averaging was found by NCS6D and improved by IMP. 20 times averaging and density correction by program DM further improved the electron density. Initial model tracing and sequence assignment using program O (Jones et al., 1991)
It was performed based on 20 times averaged electron density. However, some of the molecules have not been clearly defined. Multi-crystal averaging with DMMULTI (Korton, 1994) using the amplitudes of tetragonal, orthorhombic, and native datasets and experimental phases of orthorhombic and monoclinic MAD experiments. ) Was performed to further improve the electron density. The parts that were initially missing were clearly clarified and a complete model could be built. Using tetragonal native data up to 2.7 Å resolution and without the contribution of experimental phase, the program CNS for maximum likelihood targets (Brunger et al. (1998) Acta Crystallogr. D 54, 905-921) initially Refined atomic model. The refinement is 5x NCS restraint, overall anisotropic B factor, and bulk solvent correction (bulk solv).
ent correction). The 5-fold NCS constraint was published for parts of the pentamer that clearly do not follow 5-fold symmetry. The latest model is a well-ordered AChBP pentamer consisting of 1035 residues, 14 well-organized solvent molecules, 5 Ca ²⁺ ions, 5 Cl ^- ions, and 5 Hepes molecules. Solvent molecules, and 5 HEPES
Contains molecules. The following residues were not fully defined in electron density:
-8 to 0 position (part of the signal sequence of α-adapted S. cerevisiae, which is not originally found in AChBP, SEQ ID NO: 21), 125-135, 155-165, 186-191, and 206-210.

Electron density can be detected at the ligand binding site of AChBP. It is believed that the HEPES molecule may be responsible for this extra electron density based on its chemical properties. HEPES or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid contains quaternary ammonium ions similar to ligands such as acetylcholine (ACh) and d-tubocurarine. It has been proposed that ACh binding is mediated by cation-π interactions involving the π system of aromatic residues present at the binding sites of N ⁺ and nicotinic acetylcholine receptors. Without wishing to be bound by theory, it is suggested according to the observations of the present invention that the observed HEPES molecule mimics ligand binding similar to that of natural ligands such as ACh at the ligand binding site.

Example 8: A more detailed description of the structure determined in Example 7 As described in the previous example, using weak Pb MAD data in two crystal forms, A
The crystal structure of ChBP was clarified. Electron density maps show cross-crystal averaging of three crystal forms, each having 20, 10, and 5 copies of monomer in the asymmetric unit.
averaging) substantially improved (Table 3).

[0163]

[Table 3] Data collection statistical table

The structure is 2.7Å in the space group P4 ₂ 2 ₁ 2 with one AChBP pentamer in the asymmetric unit.
Refined in. Therefore, native data (X11), as well as the Pb-1 dataset at the EMBL / DESY synchrotron in Hamburg (BW7A) and the Pb-2 dataset at ESRF in Grenoble (BM14) were collected (Table 3). Data to DENZO
/ SCALEPACK (Otowinowski and Minnow, Methods Enzymol. 276, 307-3
26 1997) (native) or MOSFLM (Leslie, Acta Crystallogr. D. Biol
． Crystallogr. 55, 1696 to 1702,1999) / SCALA (CCP4) (Pb-1, Pb-2). The Pb site located at the interface of the two pentamers is SOLVE (Turbilger, Acta Cryst
allogr. 55, 849-861, 1999) found in both MAD sets and the heavy atom parameters SHARP (Lafortele et al., Methods Enzymol. 276, 472-494, 1997).
Optimized by. NCS Operators NCS6D and IMP (Clay Hugt and Jones, SERC Daresbury Laboratory, Warrington, pages 59-66, 1994).
Discovered and refined in. DM-multi (Korton, Joint CCP4 and ESF-EACBM N
ewsletter on Protein Crystallography, 31, 34-38, 1994) Polycrystal averaging, monoclinic, orthorhombic, and native (tetragonal) dataset amplitudes, and orthorhombic and monoclinic The experimental phase of the crystalline MAD experiment was used. The model was built at O (Jones et al., Acta Crystallogr. A47, 110-119, 1991), 2.7.
Å Program CNS (Brunger) for tetragonal data up to resolution
Et al., Acta Crystallogr. D54, 905-921, 1998). Refinements included partial 5-fold NCS constraint, total anisotropy B-factor, and bulk solvent correction. The special double cysteines Cys187-Cys188 formed obvious disulfide bridges. Due to the limited resolution, this was refined with standard parameters. The final model is AChB
It contains 1025 residues of the P pentamer, 5 HEPES molecules, 10 Ca ²⁺ ions, and 15 water molecules. The entire AChBP pentamer is well organized, except for the N-terminal 7 residues (part of the signal sequence) and the last 5 C-terminal residues. Furthermore, the sugar residues bound to HEPES, loop regions 155-160, and residue Asn66 are not fully degraded in electron density. From the ideal shape of bond distance and bond angle, r. m. The s deviations are 0.01Å and 1.6 °, respectively. Sequence alignments were calculated by CLUSTALX (Thompson et al., Nucleic. Acids. Res. 25, 4876-4882, 1997) and corresponding figures by Espript (Goue et al., Bioinformatics. 15, 305-308, 1999). did. 2-5 show the program MOLSCR1PT (Kraulis, PJ, J.
Appl. Cryst. 24, 946-950, 1991), BOBSCRIPT (Esnouf, Acta
Crystallogr. D55, 938-940, 1999), and RASTER3D (Merritt)
And Bacon, Methods Enzymol. 277, 505 to 524, 1997). The refinement was performed using a partial 5-fold NCS constraint, and an R _{factor of} 26.4% (R _free = 30%) was obtained.

AChBP pentamer : When viewed along the five-fold axis, the AChBP homopentamer resembles a model of a windmill with petaloid monomers (Fig. 6a). Viewed perpendicular to the 5x axis, it has a discoidal appearance (Fig. 6b). The overall size of the pentamer is ~ 80x80x62Å and the diameter of the central hole is ~ 18Å. These dimensions are in good agreement with the N-terminal domain EM data of Torpedo nAChR (Miyazawa et al., J. Mol. Biol. 28).
8, 765-786, 1999). The only subunit contact in the AChBP pentamer is the dimer interface, each of which has two contact surfaces, one called the plus side and one the minus side. We have five equivalent interfaces AB, BC, CD, DE.
And as an example of EA (Fig. 6), mention the A (plus) -B (minus) interface.

AChBP Monomers : Each AChBP monomer is an asymmetrically shaped, single domain protein with a size of ˜50 × 21 × 27Å (FIG. 12a). This helix N-terminus beta, consisting of ten beta strands of the core to form two short 3 ₁₀ helix, and beta sandwich. The order of β-strands is consistent with a modified immunoglobulin (Ig) topology with extra β-hairpins (f′-f ″) and extra strands (b ′) (FIG. 12b) (Bork et al., J. Chem. Mol. Biol. 242, 309-320, 1994. These additional strands introduce two so-called "Greek key" folding motifs. Ig-based structural predictions (Le Novere et al., 1999; Collinger et al., Biophys. J. 76, 2329-2345, 1999) are in good agreement with the AChBP structure, but with the presence of an extra β-strand (Fig. 12b). Therefore, the position of the binding site was omitted. Classic I
Compared to the g-fold, the AChBP β-strands are considerably twisted and the β-sheets rotate counter to each other, resulting in two distinct hydrophobic cores. Therefore, this three-dimensional fold does not resemble other Ig-like proteins and is compared to protein databases (Holm and Sander, Nucleic. Acids. Res. 25, 231-234, 1).
997) did not yield a significant match with any known structure.

Localization of functional regions : In the AChBP structure, the positions of pairs of regions important for receptor function can be determined. In muscle-type nAChR, the major immunogen region comprising residues of α ₁ 67~α ₁ 76 (MIR) is
Acts as an epitope of myasthenia gravis, an autoimmune disease (Tzartos
s) et al., Mol. Neurobiol. 5, 1-29, 1991). MIR related area in AChBP (65 ~ 7
(Residue at position 2) does not show sequence homology with the α ₁ subunit, but its position in loop L3 above the pentamer in a highly accessible position is similar to the predicted accessibility of this region. Match. This is also in line with the EM study of placing the MIR at the receptor end to the membrane (Beroukhim and Unwin, Neuron.
15, 323-331, 1995).

On each AChBP monomer, there is a large cavity accessible from the central pore of the pentamer ring. This cavity is surrounded by β-strands (β3, β4, β5, and β5 ′) at the entrance (FIG. 12a), does not charge to it, and is predominantly hydrophobic. This region probably corresponds to the tunnel surrounded by the twisted β-strands observed in the Torpedo receptor α ₁ subunit with a resolution of 4.6Å (Miyazawa et al., J. Mol. Biol. 288, 765-786). , 1999). However, this cavity is not in contact with another large pocket observed at each interface between subunits. These latter pockets are lined with residues shown to be involved in ligand binding at nAChR (Arias, Neurochem. Int. 36,
595-645, 2000; Collinger et al., Annu. Rev. Pharmacol. Toxicol. 40, 431-45
8, 2000). They are buried away from the solvent and are located close to the outside of the pentamer ring. When viewed perpendicular to the 5-fold axis, they are located approximately equatorial approximately 30 Å away from the C-terminus (Fig. 6b) and are labeled (Fernando Valenzuela).
ando Valenzuela) et al., Biophys. J. 66, 674-682, 1994) and EM studies (Annwin, J. Mol. Biol. 229, 1101-1124, 1993), which is consistent with the predicted position of the torpedo receptor ligand binding site.

Ligand Binding Sites : Each ligand binding site is found in a cleft formed by a series of loops from the major face of one subunit and a series of β-strands from the complementary faces of adjacent subunits. This is a large cavity filled with a series of loops from the major side and β-strands from the complementary side (Figure 13). The major side, which is on the plus side of the AB boundary surface, is "loop A" (TyrA89), "loop B" (TrpA143).
, A145), and "loop C" (TyrA185, double cysteines A187-A188, and
TyrA192) (Fig. 13c). The complementary part of this binding site is provided by monomer B, "loop D" (TrpB53, GlnB55), "loop E" (ArgB104, Val
B106, LeuB112, and MetB114), and "Loop F" (TyrB164) (Fig. 13d).
Made of In this pocket, four of the aromatic residues (TyrA89, TyrA185, TyA89
rB164, and TrpB53) form the lower half of the cavity. The wall of the pocket is Tyr
A192, TrpA143, A145 Met B114 main chain, GlnB55 side chain, and double cysteine (
CysA187 to CysA188). The hydrophobic parts of Arg104, ValB106, and LeuB112 form the upper pocket (Fig. 13a).

All residues in the pocket have been successfully identified by photoaffinity labeling and mutagenesis studies (Arias, Neurochem. Int. 36, 595-645, 2000;
Collinger et al., Annu. Rev. Pharmacol. Toxicol. 40, 2000). The side chain of HisA145 is located away from the cavity, but its main chain is involved. One residue, TrpA82 (α ₁ Trp86), identified by labeling studies (Galzi et al., J. Biol. Che.
m. 265, 10430-10437, 1990; Dennis et al., Biochemistry 27, 2346-2.
357, 1988) is involved in the formation of the hydrophobic core and is located away from the pocket, thus
It is not involved in ligand binding. In other respects, the AChBP ligand binding site is nAC
Fully corroborates available biochemical and mutational data for hR.

However, this structure shows for the first time how these residues are in relation to each other and thus provides a valuable tool for the drug design described in the above description of the invention.

All observed residues except the "Loop F" TyrB164 residue are conserved among known nicotinic ligand binding subunits. Although the "loop F" region has a special conformation, it is relatively weakly degraded and its rigorous analysis is difficult. The "loop F" region is structurally stabilized by a calcium binding site formed in the AspB161, AspB175, and B176 backbones. This Ca ²⁺ ion is structurally important for TyrB164 orientation and therefore may be important for proper ligand binding. This finding is supported by labeling studies on the muscle / torpedo subunit, which show that residues homologous to AspB161, γAsp174 / δAsp180 play a role in ligand binding (Czajkowski et al., Proc. Natl.
Acad. Sci. USA. 90, 6285-6289, 1993; Kzazykowski and Karin (Ka
rlin), J. Biol. Chem. 270, 3160-3164, 1995; Martin et al., J.
． Biol. Chem. 271, 13497-13503, 1996). In addition, a calcium binding site that enhances response to agonist binding is found in the homologous region of the neuronal α ₇ receptor (residue range 1
61-172) (Gargi et al., EMBO J. 15, 5824-5832, 1996).
). The "loop F" region has low sequence conservation in the nicotinic family (
Figure 11), in other superfamily members, they probably have different conformations even to the extent that they join the two sheets to form a β-strand into a single β-barrel. Presumably, such changes would lead to changes in affinity, for example by changing the size of the ligand binding site or its approach.

The most likely access route to the ligand binding site involves a double cysteine.
From above or below Loop C "(Fig. 13a). This region fills the pocket away from the solvent and thus prevents access from the outside. Access from the central hole has been suggested in the literature (Miyazawa, J. Mol. Biol. 288, 765-786, 1999).
), This requires significant structural relocation at the interface, which reduces the likelihood of access.

Ligand binding : Surprisingly, the experimentally obtained transverse crystal averaged electron densities (FIG. 13b) were characterized by a bulky electron density stacking on Trp143 at each ligand binding site. It's been found. Upon examination, we have determined that this includes HEPES (N-2 hydroxyethylpiperazine, which contains a positively charged quaternary ammonium group and therefore has some similarities to known nicotinic receptor ligands. -N'-2-ethanesulfonic acid) buffer molecule. This EC50 is 100 mM, which indicates that HEPES binding is possible under crystallization conditions (100-150 mM). The HEPES molecule does not make specific contact with proteins, but is stacked on Trp143 by its quaternary ammonium and undergoes cation-π interactions as expected for nicotinic agents (Dougherty, Science 271, Science 271, 163-168, 1996) (
Figure 13b). However, due to the limited resolution and possible low occupancy of this data, some conditions should be taken into account in the exact orientation of the HEPES molecule.

It has been suggested that the ligand binding site of nAChR may resemble the binding site of acetylcholinesterase (AChE) (Changeux and Edelstein, Neuron 21, 959-980). , 1998). The size of the binding site is about the same in AChBP and AChE, but the observed arrangement of aromatic residues is quite different. However, the quaternary ammonium stack of HEPES is similar to the quaternary ammonium stack of decamezonium on the Trp84 residue of AChE, as long as it is refined by the current AChBP structure (Harel ( Har
el) et al., Proc. Natl. Acad. Sci. USA. 90, 9031 to 9035, 1993).

Subunit Arrangement : From the position of the ligand binding site, conclusions can be drawn about the relative arrangement of the subunits at the torpedo and muscle receptors. When you look at the membrane,
It has been suggested that the α ₁ γ and α ₁ δ interfaces occur in a clockwise α ₁ γα ₁ δβ ₁ configuration (Machold et al., Eur. J. Biochem. 234, 427-430, 199).
Five). Such a clockwise arrangement is inconsistent with the structure determined by the present invention. This is because the relative orientation of the major binding site and its complementary partner is counterclockwise when looking towards the “lower” (membrane) side of the pentamer (FIG. 6).

Pentameric interface : The subunit interface on the plus side is completely loop region (L1, L2, L4, L5, L7
, L8, and L10), but the negative side represents secondary structural elements predominantly to the interface (α1, β1, β2, β3, β5, β6, and L9) (FIG. 14). Several residues are important for both ligand binding and pentamer formation. The interface has predominantly uncharged features containing only one branching salt bridge (GluA149-ArgB3 and Arg104) and fills a significant surface area (2700Å ² ). Not only AChBP but also between each other,
Most interesting are interface residues that lack the conservation of these particular residues across the superfamily (Figure 11). These changes are accompanied by significant changes in characteristics, including a change from hydrophobic to charged. Even if the residues are conserved in any particular sub-unit, except for the only exception of the ligand binding site, even in the same subunit as (alpha ₇ homopentamer, muscle alpha ₁ [delta] or _{One of} the expected pairs is not found (even at contacts such as α ₁ γ or neurons α ₄ β ₂ ). However, a high degree of structural conservation determines the involvement of the same topological region at these contacts in all family members (Fig. 14b). This indicates that shape complementarity must play a major role in determining the conservation of pentameric structure. This also shows that different combinations of subunits have different interfaces, which produces precise allosteric contact variations and movements in different subclasses of these ion channels.

Ligand-gated ion channels : Because the residues that form the interface are between the lowest conserved regions of the domain (FIG. 11),
Lack of conservation of interface residues appears to be a common feature in the LGIC superfamily. Apparently, pentamer formation does not impose in this case very strict evolutionary requirements. However, there is clear sequence conservation within the superfamily (Figure 11), and it is interesting to analyze this against the structure.

[0179]   Conserved hydrophobic residues can be classified into three clusters in the AChBP monomer structure.
And (Fig. 15). Like other proteins with Ig-like folds, AChBP
The β-sheet packing is mainly due to hydrophobicity and less
To some extent facilitated by electrostatic interactions. The first cluster is the main skeleton of the monomer
Involved in the packing of N-terminal helix α1
Residues at positions 71, 81, 105, and 111. Second cluster is 20th
, 27th, 29th, 31st, 58th, 82nd, 84th, 86th, 140th, 150th, 152nd, and 1st
It contains the residue at position 95 and is located in the upper half of the β core region. 3rd cluster, 33rd
, 35, 38, 41, 48, 52, 125, 138, 171, 173, 199, and
And residues 201 to 201 and are located at the bottom of the structure (Fig. 15). To super family
Highly conserved non-hydrophobic residues are Asp60, Asp85, Asn90, and Gly.
There is only 109. Asp60 and Gly109 connect the β3, β5, β6, and β2 strands.
Is involved in stabilizing the turn of the Greek key motif that connects, where Asp60 is a small 3 _Ten Stabilizing the N-terminus of the helix, Gly109 allows tight turn formation. Asp85
And the conserved residues of Asn90 are involved in β-sheet packing. Asp85 is highly protected
It forms hydrogen bonds with the existing Ser142 and Thr144, and the residue of Asn90 is at Ser122 or Ser122.
And an absolutely conserved disulfide bond near the main chain oxygens of Arg137 and
Allows disulfide bond formation at positions (123-136). This disulfide bond
, A β-sheet that binds two β-sheets in an Ig-like protein
(Tyrosine cornerstone) "(Hemmingsen et al., Protein Sci. 3
, 1927 to 1937, 1994) and is topologically equivalent. This is a torpedo receptor
The Cys128-Cys142 binding is associated with maintenance of subunit conformational stability (mi
Mishina et al., Nature 313, 364-369, 1985) and the complete nAChR assembly (group
Green and Wanamaker, J.C. Neurosci. 18, 5555-5
564, 1998). Observed overall structure
Due to the high conservation, it is clear that all LGIC N-terminal domains have the same three-dimensional structure.
It's mild.

In contrast to the residues mentioned above, the Cys loop is a highly conserved hydrophobic region in the LGIC family, but shows entirely different characteristics in AChBP (FIG. 11).

In AChBP, this loop is hydrophilic and is found on the underside of the protein (membrane side), at the dimer interface. This position and its hydrophobicity in the LGIC family means that this loop can interact with the receptor or the transmembrane domain, a function not present in the membrane or AChBP.

Since all ligand-gated ion channels have essentially the same function of opening membrane pores, the conserved regions of proteins are likely to determine this function. This also indicates that the pentameric interface is unlikely to play a major role in channel opening. It is possible that the conserved Cys loop is directly involved in the transmission of this kind of information to the LGIC membrane part. Another option is that large structural changes in the β-sheet region play a role in channel opening. Indeed, the movement observed at 9Å upon binding of the agent to Torpedo nAChR (Annwin, Nature 373, 37-43).
, 1995) is in good agreement with this idea. According to the present invention, twisted β
The sandwich is observed with two distinct hydrophobic cores, and it is fully possible for these cores to move relative to each other upon ligand binding. The effects of such movements are then regulated by different subunit interfaces at different subtypes of receptors, allowing complex specificities in neuronal signaling.

Example 9: Ligand Binding Crystallization Studies AChBP was co-crystallized in complex with α-bungarotoxin (αBTX, Sigma). The stability of the complex has been investigated before crystallization experiments. It has been found possible to purify stable complexes of AChBP and αBTX using gel filtration chromatography (Superdex 200 HR 10/30, Pharmacia, column volume 24 ml). A gel filtration run was performed using 20 mM Tris (pH 8.0), 150 mM NaCl, and 0.02% NaN ₃ . The stability of the complex was also confirmed by native PAGE. Crystallization experiments were based on the same set of conditions found to work well with AChBP alone; see Example 6. A small screen was initiated with different precipitant concentrations and various AChBP: αBTX concentrations. Very small crystals show 10-12% PEG 40
Appeared under conditions containing 00, 100 mM HEPES (pH 7.0), 20-80 mM CaCl ₂ , and 0.02% NaN ₃ . The best-looking crystals grew under the conditions described above when AChBP: αBTX was mixed in a 1:10 molar ratio. To see if the complex actually crystallized, the crystals were washed extensively, dissolved in denaturing buffer and examined by SDS-PAGE. SDS-PAGE clearly showed that the crystals contained both proteins.

In addition, a number of small ligands were bound to AChBP in immersion experiments. As these ligands, B-bipinatin (a synthetic analogue of lophotoxin), acetylcholine (ACh, sigma), d-tubocurarine chloride (sigma), carbamylcholine chloride (CCh, sigma), galantamine hydrobromide (sigma), Includes epibatidine (Sigma), and nicotine (Sigma). The dipping solution was replaced with the stabilizing solution (Example
6) with lysing ligand (ligand was usually dissolved in 20 mM HEPES [pH 7.0]). The ligand concentration used was dependent on its binding constant determined by ligand binding studies. The immersion time varied depending on the ligand used. After the soaking step, the crystals were rapidly cooled in liquid nitrogen.

Example 10: Generation of human α7 nAChR / AChBP chimera nAChR subunits and chimeric proteins of AChBP can be used as tools in the development of novel nAChR subtype-specific ligands. As a first step in the development of these tools, a chimeric protein was designed and constructed where a portion of human α7 nAChR was ligated to AChBP. Earlier work on the molecular determinants of ligand binding by α7 nAChR further adds to "loops A, B,
Three amino acid domains have been identified that make up the primary part of the ligand binding site, referred to as "and C". Within each of the three loops there are amino acid residues that are believed to interact directly with the ligand. AC based on sequence conservation of nAChR and AChBP
The locations of the three possible ligand binding loops of hBP have been precisely identified according to the invention as follows: Loop A, Trp101->Tyr108; Loop B, Trp162->His164; Loop C, Tyr204-> Tyr211. The constructed chimeric protein has one (A, B or C) or multiple (A and B, A and C, B and C, and A and B and C) of the ligand binding loops of AChBP corresponding to the corresponding human α7. It replaces the nAChR sequence.

A two-step polymerase chain reaction (PCR) was used to replace the loop A domain of AChBP with the corresponding domain of human α7 nAChR. In the first stage, two separate P
CR amplification (94 ° C .; 30 sec, 58 ° C .; 30 sec, and 72 ° C .; 60 sec for 35 cycles) yielded each of the two types of chimeric constructs. AChBP cDNA (wild type) is used as a template, and the outer primer is located immediately before the start codon. Or the outer primer located just before the stop codon 2 types of inner primer (SEQ ID NO: 19) and It was used in combination with (SEQ ID NO: 20). Inner primer is introduced α7
It contained a 5'tag sequence encoding the nAChR domain. Therefore, the two generated chimeric PCR products share a common tag that contains part of the α7 nAChR subunit.
In the second step, the two PCR products from the first round were pooled and subjected to PCR amplification (94 ° C; 30 sec, 54 ° C; 3 min, and 72 ° C; 90 sec) in the absence of primers. Made 5 rounds. This allowed the two chimeras to anneal to each other with the common α7 nAChR tag. 2 pcs of outer primer then added, 25 pcs
An additional 35 cycles of R amplification (94 ° C; 30s, 58 ° C; 30s, and 72 ° C; 90s) were performed to obtain the final chimeric construct. All PCR amplifications were done with the hot start method and with PFU DNA polymerase (Invitrogen). The Loop A AChBP / α7 chimera was cloned into the His-tag containing yeast expression vector pPIC9 (Invitrogen) using the XhoI / EcoRI restriction sites in the outer primers. Confirmation of the construct was performed by DNA sequencing. Expression of the chimeric construct was performed according to Invitrogen's Pichia pastoris protein expression protocol.

As described in the Examples and Descriptions, the present invention is for screening mollusc-derived water-soluble ligand-binding proteins and analogs of ligand-gated ion channels, their crystals, and ligands of ligand-gated ion channels. To provide their use of. In particular, water soluble ligand binding proteins have been identified that are capable of forming multimers and crystallizing. A crystal structure of one of these proteins, acetylcholine binding protein (AChBP), is provided, which is used to create a 3D model of the extracellular ligand-binding domain of the ligand-gated ion channel, and thus these It can be used to screen for drugs that act on ion channels. Further provided is a chimeric protein capable of binding a ligand of a ligand-dependent receptor, comprising at least an AChBP amino acid determining the solubility of AChBP at the same position as AChBP, and further comprising an amino acid determining binding to the ligand. To be done.

It will be appreciated that the invention may be practiced otherwise than as described in detail in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above disclosure and are therefore within the scope of the following claims.

All disclosures of each document (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) cited in the background, description, and examples of the invention are hereby incorporated by reference. Incorporated in the description. In addition, the sequence listing is incorporated herein by reference.

[0190]

[Table 1] Structural coordinates of AChBP "Atomic type" means the element whose coordinates were measured. The first letter in the column defines the element. “Residue” means an amino acid in the AChBP protein sequence, using standard three-letter abbreviations well known in the art. "#" Means the residue number. "X, Y, Z" defines the position of the atom in the three-dimensional space of the measured element crystallographically. "OCC" is an occupancy volume. "B" is a temperature factor that is a measure of the movement of an atom around its center.

[Sequence list]

[Brief description of drawings]

FIG. 1. Clustal X (1.8) multiple sequence alignment of AChBP amino acid sequences. The AChBP alignment is ClustalX 1.8 "(Thompson et al., Nuc
leic Acids Research 24 (1997), 4876-4882. The next alignment is "Genedoc" version 2.5.000 (Nicholas et al. (1997) "
Genedoc, a tool for editing and adding multiple sequence alignments (Gen
edoc a tool for editing and annotating multiple sequence alignments) ''
Was further processed with. Identical amino acids are indicated by " ^* ", equivalent amino acids are indicated by ":" and similar amino acids are indicated by ".". The glycosylation site is Asn66 in L-AChBP and Asn21 and 26 in B-AChBP in the amino acid sequences of mature AChBP SEQ ID NOs: 2 and 4 and 6 and 8, respectively.

FIG. 2. Hydrophobicity plot of mature AChBP amino acid sequence. B-AChBP and L-AChB
The P hydrophobicity plot is based on Kyte and Doolite (J. Mol. Bi.
ol. 157 (1982), 105-132) according to the method described in "Protein Sequence Analysis (Prote
in sequence analyzes) ". 2A: L-AChBP T1 (SEQ ID NO: 2), 2
B: L-AChBP T2 (SEQ ID NO: 4), 2C: B-AChBP T1 (SEQ ID NO: 6), 2D: B-AChBP T2 (SEQ ID NO: 8).

FIG. 3: AChBP amino acid sequence and ligand-dependent receptors nAChR-α7 and GABA _A R
Clus with the amino acid sequences of the ligand binding domains of -β1, 5-HT3R, and GlyR-α1
tal X (1.8) multiple sequence alignment. Sequence alignment and processing was performed as described in Figure 1. The accession numbers of the amino acid sequences used for the alignment are as follows: human α1: human α7: Y08420; human 5HT3: CAA06442.
Human GlyR α1: S12382; human GABA _A b1: NP 000797. The corresponding rat sequence (ratnAChRa7) that does not show identical results but shows substantially similar results. Q05941, rat5HT3
R P35563, ratGABARb1 P15431, ratGlyRa1 Similar sequence alignments can be performed using p24524).

FIG. 4: Clustal X (1.8) multiple sequence alignment of AChBP and nAChR amino acid sequences. Sequence alignment and processing was performed as described in Figure 1. The accession numbers of the amino acid sequences used for the alignment are as follows: human α1: ACHUA1; human α2: AAG23253; human α3: A53956; human α4: P43681; human α5: P30532; human α6: Q15825; human α7. : Y08420; human α9
: CAB65091. The corresponding rat sequence (ratnAChRa7) that does not show identical results but shows substantially similar results. Q05941, rnAChRa9 P43144, rnAChR2 P1238, rnAChRa3 P047
57, rnAChRa4 Similar sequence alignments can be performed using P09483).

FIG. 5: Clustal of AChBP amino acid sequence and nAChR α1 and 7 amino acid sequences
X (1.8) multiple sequence alignment. Sequence alignment and processing was performed as described in Figure 1. The accession numbers of the amino acid sequences used for the alignment are as follows: human α1: ACHUA1; human α7: Y08420. The corresponding rat sequence ratnAChRa7, which does not show identical results but shows substantially similar results Similar sequence alignments can be performed using Q05941.

FIG. 6 shows the pentameric structure of AChBP. a In this schematic diagram, each monomer has a different color tone. Subunits are displayed counterclockwise, with AB, BC, CD, DE, and EA forming plus and minus interface sides, the major and complementary ligand binding sites (represented by spheres and bars, respectively). ) Has. b Display the AChBP pentamer perpendicular to the 5-fold axis. Equatorially located ligand binding sites (indicated by spheres and bars) are highlighted only at the interface between A (light) and B (dark).

FIG. 7. AChBP monomer. Ribbon display of AChBP monomer. Secondary structure runs from the N-terminus (top) to the C-terminus (bottom). Monomers are displayed towards the center of the pentamer. In nAChR, the top corresponds to the N-terminus of the ligand binding domain facing the synaptic cleft, while the C-terminus enters the membrane at the bottom and is followed by the transmembrane domain. AChBP monomers are mainly assembled from β-strands, except for the N-terminal α-helix. It contains 14 β-strands arranged in two antiparallel β-sheets, with an immunoglobulin topology. However, in contrast to classical immunoglobulin folding, the β-sheets of AChBP rotate in opposite directions,
Create a small pocket as you can see in Figure 6.

FIG. 8. Ligand binding site at the dimer interface. Ribbon display of two adjacent AChBP monomers. Monomer A is shown in grey, Monomer B is shown in dark grey. The ligand binding site is located at the interface between the two monomers. As predicted for nAChR,
The acetylcholine binding site in AChBP lies at the interface between two adjacent subunits. Similar to the model proposed for nAChR, the ligand binding site is asymmetric and is mainly formed by aromatic residues. Residues from mature AChBP monomer A (TyrA89, TrpA143, TyrA185, CysA187, CysA188, and TyrA192) form the major component, while residues from TrpB53 from monomer B form the ligand binding site. Produces a complementary portion. Similar to the homomeric α7 neuron receptor, the AChBP pentamer has a 5
There are three identical ligand binding sites.

FIG. 9. Ligand binding site. A three-dimensional view showing the ligand binding site of AChBP on the interface between two monomers. Mature AChBP Monomer A (TyrA89, TrpA143, TyrA185, CysA
187, CysA188, and TyrA192) form the major component, whereas
Residues of TrpB53 from Monomer B, along with additional Arg104, LeuB112, and MetB114 residues, form the complementary portion of the ligand binding site. Similar to the homomeric α7 neuron receptor, the AChBP pentamer has five identical ligand binding sites.

FIG. 10: Multiple sequence alignment of AChBP amino acid sequences with an indication of secondary structure and solvent accessibility obtained from the crystal structure. Alignment of four mollusc AChBP sequences with secondary structure and solvent accessibility of Lymnea stagnalis AChBP-1 revealed by crystal structure. This figure shows a DSSP (Kabsch and Sander, Biopolymers. 22 (1983), 2577-2637.
), ESPript (Gouet et al., Bioinformatics. 15 (1999), 305).
~ 308). ESPript default (blue A> 0.4, cyan 0.1 <A <0.
4, according to white A <0.1), solvent accessibility is shown below the alignment, with white being the most buried and dark blue being the most exposed.

FIG. 11: Sequence alignment of AChBP and LGIC. LGIC alignment
Only the N-terminal domain of the subunit is shown and is based on a multiple sequence alignment of 92 full length LGIC sequences. The abbreviations H and Tca used refer to human and Torpedo californica. According to FIG. 12a, the secondary structural elements (α: α helix, β: β strand, η: 3 ₁₀ helix) are shown above the sequence. AChBP has 23% sequence identity with the ligand binding domain of human α ₇ . Show residues stored in LGIC (bold, gray background). The beginning and end of the Cys loop are indicated by " ^* ". The nicotinic receptor ligand binding residues on the major and complement sides are shown.

FIG. 12: Outline of AChBP monomer structure. a AChBP as seen from the outside of the pentamer ring
Stereoscopic display of monomers. Disulfide bridges are shown as spheres and bars. In the complete ion channel, the N-terminus faces the synaptic cleft, while the C-terminus enters the membrane at the bottom and continues to the first transmembrane domain. b Topological diagram of AChBP monomer. For comparison with the Ig fold, the strands are labeled a to g and show additional strands (b ') and hairpins (f' to f "). In this structure, the strands precede each strand. L1-L10 loops (or turns) with the same numbers
And are indicated by β1 to β10. The β5 strand is disrupted by the inner loop L5 ′ (β5-β5 ′) and β6 also has a small disruption, but is shown continuously (see FIG. 11). The exact beginning and end of the strand is
It may change slightly with increasing resolution, but the topology seen here is
Highly conserved across the LGIC family.

FIG. 13. Ligand binding site. a major "loop" A (TyrA89 / α ₁ Tyr93),
"Loop" B (TrpA143 / α ₁ Trp149), and "loop" C (TyrA185 / α ₁ Tyr190,
CysA187 / α ₁ Cys192, CysA188 / α ₁ Cys193, TyrA192 / α ₁ Tyr198), as well as the complementary “loop” D (TrpB53 / γTrp55, GlnB55 / γGlu57), “loop” E (ArgB104 / γLe).
Stereogram of the ligand binding site in the representation of spheres and bars showing the contribution of u109, ValB106 / γTyr111, LeuB112 / γTyr117, MetB114 / γLeu119), and “loop” F (TyrB164). b Three-dimensional view of an electron density map showing HEPES buffer molecules at the ligand binding site. This experimental density (outlined at 1σ) was obtained from transverse crystal averaging. c Position of the main ligand binding residue in the monomer. d Position of the complementary ligand binding residue in the monomer (same orientation as in Figure 6b).

FIG. 14: Dimer interface. a Three-dimensional view of the dimer interface. Boundary residues (spheres and bars) are shown in the secondary structure schematic. This figure shows the plus side of subunit A and the minus side of subunit B. b Dimer interface interaction. Due to the low conservation of these interfaces (Fig. 11), the actual interactions are not conserved at any LGIC interfaces, but it is likely that these topological regions form interfaces at all receptors. Please note.

Figure 15: Preservation in the LGIC superfamily. Conserved residues are shown at the top, middle, and bottom of the monomer, respectively, as viewed from the central pore. Conserved hydrophilic residues are shown in dark shade. Conserved residues are shown as viewed from the central hole. Hydrophobic cluster I: 6th, 10th, 63
Residues at positions 65, 71, 81, 105, 111; Cluster II: 20, 27, 29, 31, 58, 82, 84, 86, 140, Residues at positions 150, 152, 195; Cluster III: 33, 35, 38, 41, 48, 52, 125, 138, 171, 173, 199, 201 Residue of. Conserved hydrophilic residues: Asp60, Asp85, Asn90, Gly109, Cys12
3, Cys136, Lys203. Conserved residues in the ligand binding site: 106th, 145th, and 192nd positions. These three residues and Lys203 are the only conserved residues with no structural role in the monomer. Note how few conserved residues are on the surface.
Within the LGIC family, Cys loop residues are also highly conserved; see bottom left.

[Procedure amendment]

[Submission date] January 10, 2003 (2003.1.10)

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0190

[Correction method] Change

[Contents of correction]

[0190]

[Table 1] Structural coordinates of AChBP "Atomic type" means the element whose coordinates were measured. The first letter in the column defines the element. “Residue” means an amino acid in the AChBP protein sequence, using standard three-letter abbreviations well known in the art. "#" Means the residue number. "X, Y, Z" defines the position of the atom in the three-dimensional space of the measured element crystallographically. "OCC" is an occupancy volume. "B" is a temperature factor that is a measure of the movement of an atom around its center.

[Sequence list]                         SEQUENCE LISTING <110> Stichting Voor De Technische Wetenschappen <120> Water-soluble ligand-binding proteins and analogs of       ligand-gated ion channels, crystals thereof and their       use for screening ligands of ligand-gated ion channels <140> JP 2001-558097 <141> 2001-02-09 <150> EP 00200443.0 <151> 2000-02-10 <150> EP 00203810.7 <151> 2000-10-31 <160> 21 <170> PatentIn Ver. 2.1 <210> 1 <211> 690 <212> DNA <213> Lymnaea stagnalis <220> <221> CDS <222> (1) .. (687) <220> <221> mat_peptide <222> (58) .. (687) <400> 1 atg cgt cga aac att ttc tgc ctt gct tgt ctc tgg atc gtg caa gcg 48 Met Arg Arg Asn Ile Phe Cys Leu Ala Cys Leu Trp Ile Val Gln Ala                 -15 -10 -5 tgt cta agc ttg gac cgg gca gac atc ttg tac aac ata cgt cag aca 96 Cys Leu Ser Leu Asp Arg Ala Asp Ile Leu Tyr Asn Ile Arg Gln Thr          -1 1 5 10 tcg aga ccg gat gtg att ccc aca cag cga gat cgc cca gtg gcg gtg 144 Ser Arg Pro Asp Val Ile Pro Thr Gln Arg Asp Arg Pro Val Ala Val      15 20 25 tcc gtc tct ttg aag ttc atc aac atc ttg gaa gtg aat gaa ata acc 192 Ser Val Ser Leu Lys Phe Ile Asn Ile Leu Glu Val Asn Glu Ile Thr  30 35 40 45 aat gaa gtg gac gtg gtc ttt tgg cag cag acg aca tgg tcg gac agg 240 Asn Glu Val Asp Val Val Phe Trp Gln Gln Thr Thr Trp Ser Asp Arg                  50 55 60 acc ctc gcc tgg aac agt tct cac tca cca gat cag gtt tcc gtg cca 288 Thr Leu Ala Trp Asn Ser Ser His Ser Pro Asp Gln Val Ser Val Pro              65 70 75 ata agc tct ttg tgg gtg cct gac ctc gct gca tac aac gcc atc tcg 336 Ile Ser Ser Leu Trp Val Pro Asp Leu Ala Ala Tyr Asn Ala Ile Ser          80 85 90 aaa cct gaa gtc ctt aca ccg caa ctg gcc agg gtc gta tcc gat ggt 384 Lys Pro Glu Val Leu Thr Pro Gln Leu Ala Arg Val Val Ser Asp Gly      95 100 105 gaa gtg ctg tac atg ccg agt atc cgc cag cgg ttc tcc tgc gat gta 432 Glu Val Leu Tyr Met Pro Ser Ile Arg Gln Arg Phe Ser Cys Asp Val 110 115 120 125 tcg ggt gtc gat acg gag tcc ggt gct aca tgt cgg atc aaa att ggt 480 Ser Gly Val Asp Thr Glu Ser Gly Ala Thr Cys Arg Ile Lys Ile Gly                 130 135 140 tcc tgg acc cac cac agt aga gag att tct gta gat ccc acg aca gaa 528 Ser Trp Thr His His Ser Arg Glu Ile Ser Val Asp Pro Thr Thr Glu             145 150 155 aat agt gat gat tct gaa tac ttc tcc caa tac tct cgc ttt gaa atc 576 Asn Ser Asp Asp Ser Glu Tyr Phe Ser Gln Tyr Ser Arg Phe Glu Ile         160 165 170 ttg gac gtc aca cag aag aag aac tcg gtt acc tac tct tgc tgt ccg 624 Leu Asp Val Thr Gln Lys Lys Asn Ser Val Thr Tyr Ser Cys Cys Pro     175 180 185 gag gca tac gag gac gtt gaa gtg agt ctc aat ttc cgg aag aag gga 672 Glu Ala Tyr Glu Asp Val Glu Val Ser Leu Asn Phe Arg Lys Lys Gly 190 195 200 205 cgc tcc gaa att ctt tag 690 Arg Ser Glu Ile Leu                 210 <210> 2 <211> 229 <212> PRT <213> Lymnaea stagnalis <400> 2 Met Arg Arg Asn Ile Phe Cys Leu Ala Cys Leu Trp Ile Val Gln Ala                 -15 -10 -5 Cys Leu Ser Leu Asp Arg Ala Asp Ile Leu Tyr Asn Ile Arg Gln Thr          -1 1 5 10 Ser Arg Pro Asp Val Ile Pro Thr Gln Arg Asp Arg Pro Val Ala Val      15 20 25 Ser Val Ser Leu Lys Phe Ile Asn Ile Leu Glu Val Asn Glu Ile Thr  30 35 40 45 Asn Glu Val Asp Val Val Phe Trp Gln Gln Thr Thr Trp Ser Asp Arg                  50 55 60 Thr Leu Ala Trp Asn Ser Ser His Ser Pro Asp Gln Val Ser Val Pro              65 70 75 Ile Ser Ser Leu Trp Val Pro Asp Leu Ala Ala Tyr Asn Ala Ile Ser          80 85 90 Lys Pro Glu Val Leu Thr Pro Gln Leu Ala Arg Val Val Ser Asp Gly      95 100 105 Glu Val Leu Tyr Met Pro Ser Ile Arg Gln Arg Phe Ser Cys Asp Val 110 115 120 125 Ser Gly Val Asp Thr Glu Ser Gly Ala Thr Cys Arg Ile Lys Ile Gly                 130 135 140 Ser Trp Thr His His Ser Arg Glu Ile Ser Val Asp Pro Thr Thr Glu             145 150 155 Asn Ser Asp Asp Ser Glu Tyr Phe Ser Gln Tyr Ser Arg Phe Glu Ile         160 165 170 Leu Asp Val Thr Gln Lys Lys Asn Ser Val Thr Tyr Ser Cys Cys Pro     175 180 185 Glu Ala Tyr Glu Asp Val Glu Val Ser Leu Asn Phe Arg Lys Lys Gly 190 195 200 205 Arg Ser Glu Ile Leu                 210 <210> 3 <211> 690 <212> DNA <213> Lymnaea stagnalis <220> <221> CDS <222> (1) .. (687) <220> <221> mat_peptide <222> (58) .. (687) <400> 3 atg cgt cga aac att ttc tgc ctt gct tgt ctc tgg atc gtg caa ggg 48 Met Arg Arg Asn Ile Phe Cys Leu Ala Cys Leu Trp Ile Val Gln Gly                 -15 -10 -5 tgt cta agc ttg gac cgg gca gac atc ttg tac aac ata cgt cag aca 96 Cys Leu Ser Leu Asp Arg Ala Asp Ile Leu Tyr Asn Ile Arg Gln Thr          -1 1 5 10 tcg aga ccg gat gtg att ccc aca cag cga gat cgc cca gtg gcg gtg 144 Ser Arg Pro Asp Val Ile Pro Thr Gln Arg Asp Arg Pro Val Ala Val      15 20 25 tcc gtc tct ttg aag ttc atc aac atc ttg gaa gtg aat gaa ata acc 192 Ser Val Ser Leu Lys Phe Ile Asn Ile Leu Glu Val Asn Glu Ile Thr  30 35 40 45 aat gaa gtg gac gtg gtc ttt tgg cag cag acg aca tgg tcg gac agg 240 Asn Glu Val Asp Val Val Phe Trp Gln Gln Thr Thr Trp Ser Asp Arg                  50 55 60 acc ctc gcc tgg aac agt tct cac tca cca gat cag gtt tcc gtg cca 288 Thr Leu Ala Trp Asn Ser Ser His Ser Pro Asp Gln Val Ser Val Pro              65 70 75 ata agc tct ttg tgg gtg cct gac ctc gct gca tac aac gcc atc tcg 336 Ile Ser Ser Leu Trp Val Pro Asp Leu Ala Ala Tyr Asn Ala Ile Ser          80 85 90 aaa cct gaa gtc ctt aca ccg caa ctg gcc agg gtc gta tcc gat ggt 384 Lys Pro Glu Val Leu Thr Pro Gln Leu Ala Arg Val Val Ser Asp Gly      95 100 105 gaa gtg ctg tac atg ccg agt atc cgc cag cgg ttc tcc tgc gat gta 432 Glu Val Leu Tyr Met Pro Ser Ile Arg Gln Arg Phe Ser Cys Asp Val 110 115 120 125 tcg ggt gtc gat acg gag tcc ggt gct acg tgt cgg atc aaa att ggt 480 Ser Gly Val Asp Thr Glu Ser Gly Ala Thr Cys Arg Ile Lys Ile Gly                 130 135 140 tcc tgg acc cac cac agt gga gag att tct gta gat ccc acg aca gaa 528 Ser Trp Thr His His Ser Gly Glu Ile Ser Val Asp Pro Thr Thr Glu             145 150 155 aat agt gat gat tct gaa tac ttc tcc caa tac tct cgc ttt gaa atc 576 Asn Ser Asp Asp Ser Glu Tyr Phe Ser Gln Tyr Ser Arg Phe Glu Ile         160 165 170 ttg gac gtc aca cag aag aag aac tcg gtt atc tac tct tgc tgt ccg 624 Leu Asp Val Thr Gln Lys Lys Asn Ser Val Ile Tyr Ser Cys Cys Pro     175 180 185 gag gca tac gag gac gtt gaa gtg agt ctc aat ttc cgg aag aag gga 672 Glu Ala Tyr Glu Asp Val Glu Val Ser Leu Asn Phe Arg Lys Lys Gly 190 195 200 205 cgc tcc gaa att ctt tag 690 Arg Ser Glu Ile Leu                 210 <210> 4 <211> 229 <212> PRT <213> Lymnaea stagnalis <400> 4 Met Arg Arg Asn Ile Phe Cys Leu Ala Cys Leu Trp Ile Val Gln Gly                 -15 -10 -5 Cys Leu Ser Leu Asp Arg Ala Asp Ile Leu Tyr Asn Ile Arg Gln Thr          -1 1 5 10 Ser Arg Pro Asp Val Ile Pro Thr Gln Arg Asp Arg Pro Val Ala Val      15 20 25 Ser Val Ser Leu Lys Phe Ile Asn Ile Leu Glu Val Asn Glu Ile Thr  30 35 40 45 Asn Glu Val Asp Val Val Phe Trp Gln Gln Thr Thr Trp Ser Asp Arg                  50 55 60 Thr Leu Ala Trp Asn Ser Ser His Ser Pro Asp Gln Val Ser Val Pro              65 70 75 Ile Ser Ser Leu Trp Val Pro Asp Leu Ala Ala Tyr Asn Ala Ile Ser          80 85 90 Lys Pro Glu Val Leu Thr Pro Gln Leu Ala Arg Val Val Ser Asp Gly      95 100 105 Glu Val Leu Tyr Met Pro Ser Ile Arg Gln Arg Phe Ser Cys Asp Val 110 115 120 125 Ser Gly Val Asp Thr Glu Ser Gly Ala Thr Cys Arg Ile Lys Ile Gly                 130 135 140 Ser Trp Thr His His Ser Gly Glu Ile Ser Val Asp Pro Thr Thr Glu             145 150 155 Asn Ser Asp Asp Ser Glu Tyr Phe Ser Gln Tyr Ser Arg Phe Glu Ile         160 165 170 Leu Asp Val Thr Gln Lys Lys Asn Ser Val Ile Tyr Ser Cys Cys Pro     175 180 185 Glu Ala Tyr Glu Asp Val Glu Val Ser Leu Asn Phe Arg Lys Lys Gly 190 195 200 205 Arg Ser Glu Ile Leu                 210 <210> 5 <211> 675 <212> DNA <213> Bulinus truncatus <220> <221> CDS <222> (1) .. (672) <220> <221> mat_peptide <222> (64) .. (672) <400> 5 atg gct gaa cta cga agg atc att ctt ctg cta tgt act att gcc ttt 48 Met Ala Glu Leu Arg Arg Ile Ile Leu Leu Leu Cys Thr Ile Ala Phe     -20 -15 -10 cat gtt tcc cat gga caa ata aga tgg acg ctg ctg aat cag atc acc 96 His Val Ser His Gly Gln Ile Arg Trp Thr Leu Leu Asn Gln Ile Thr  -5 -1 1 5 10 ggt gaa tct gac gtc att ccg ctg tct aac aac acg ccc ctg aat gtg 144 Gly Glu Ser Asp Val Ile Pro Leu Ser Asn Asn Thr Pro Leu Asn Val              15 20 25 tcg ctg aat ttt aag ctg atg aat atc gta gag gcg gac aca gaa aaa 192 Ser Leu Asn Phe Lys Leu Met Asn Ile Val Glu Ala Asp Thr Glu Lys          30 35 40 gat caa gtg gag gtc gtg ctg tgg aca cag gct agc tgg aaa gtg ccg 240 Asp Gln Val Glu Val Val Leu Trp Thr Gln Ala Ser Trp Lys Val Pro      45 50 55 tat tac agc tca ctg ctg tcc tct agc agt tta gac cag gtg agc tta 288 Tyr Tyr Ser Ser Leu Leu Ser Ser Ser Ser Leu Asp Gln Val Ser Leu  60 65 70 75 cca gtc agc aaa atg tgg acc cca gac ctt tct ttc tac aac gcc atc 336 Pro Val Ser Lys Met Trp Thr Pro Asp Leu Ser Phe Tyr Asn Ala Ile                  80 85 90 gct gca ccc gag ttg ctc tcc gca gac cgc gtg gtg gtc tct aag gac 384 Ala Ala Pro Glu Leu Leu Ser Ala Asp Arg Val Val Val Ser Lys Asp              95 100 105 ggg agc gtc att tac gtc ccc agc cag agg gtc cgt ttc acc tgc gac 432 Gly Ser Val Ile Tyr Val Pro Ser Gln Arg Val Arg Phe Thr Cys Asp         110 115 120 ctt att aat gtc gac acg gag ccg gga gcc acc tgt cgc atc aaa gtc 480 Leu Ile Asn Val Asp Thr Glu Pro Gly Ala Thr Cys Arg Ile Lys Val     125 130 135 gga tcc tgg acc cac gac aac aaa cag ttc gcc ctg atc acc ggg gag 528 Gly Ser Trp Thr His Asp Asn Lys Gln Phe Ala Leu Ile Thr Gly Glu 140 145 150 155 gag ggg gtg gtg aat att gca gag tac ttc gac agc cca aag ttt gac 576 Glu Gly Val Val Asn Ile Ala Glu Tyr Phe Asp Ser Pro Lys Phe Asp                 160 165 170 ctt ttg agt gcc aca cag agt ctg aat cgc aag aag tac agc tgt tgc 624 Leu Leu Ser Ala Thr Gln Ser Leu Asn Arg Lys Lys Tyr Ser Cys Cys             175 180 185 gag aat atg tat gat gac att gaa att acc ttt gca ttc aga aag aag 672 Glu Asn Met Tyr Asp Asp Ile Glu Ile Thr Phe Ala Phe Arg Lys Lys         190 195 200 taa 675 <210> 6 <211> 224 <212> PRT <213> Bulinus truncatus <400> 6 Met Ala Glu Leu Arg Arg Ile Ile Leu Leu Leu Cys Thr Ile Ala Phe     -20 -15 -10 His Val Ser His Gly Gln Ile Arg Trp Thr Leu Leu Asn Gln Ile Thr  -5 -1 1 5 10 Gly Glu Ser Asp Val Ile Pro Leu Ser Asn Asn Thr Pro Leu Asn Val              15 20 25 Ser Leu Asn Phe Lys Leu Met Asn Ile Val Glu Ala Asp Thr Glu Lys          30 35 40 Asp Gln Val Glu Val Val Leu Trp Thr Gln Ala Ser Trp Lys Val Pro      45 50 55 Tyr Tyr Ser Ser Leu Leu Ser Ser Ser Ser Leu Asp Gln Val Ser Leu  60 65 70 75 Pro Val Ser Lys Met Trp Thr Pro Asp Leu Ser Phe Tyr Asn Ala Ile                  80 85 90 Ala Ala Pro Glu Leu Leu Ser Ala Asp Arg Val Val Val Ser Lys Asp              95 100 105 Gly Ser Val Ile Tyr Val Pro Ser Gln Arg Val Arg Phe Thr Cys Asp         110 115 120 Leu Ile Asn Val Asp Thr Glu Pro Gly Ala Thr Cys Arg Ile Lys Val     125 130 135 Gly Ser Trp Thr His Asp Asn Lys Gln Phe Ala Leu Ile Thr Gly Glu 140 145 150 155 Glu Gly Val Val Asn Ile Ala Glu Tyr Phe Asp Ser Pro Lys Phe Asp                 160 165 170 Leu Leu Ser Ala Thr Gln Ser Leu Asn Arg Lys Lys Tyr Ser Cys Cys             175 180 185 Glu Asn Met Tyr Asp Asp Ile Glu Ile Thr Phe Ala Phe Arg Lys Lys         190 195 200 <210> 7 <211> 675 <212> DNA <213> Bulinus truncatus <220> <221> CDS <222> (1) .. (672) <220> <221> mat_peptide <222> (64) .. (672) <400> 7 atg gct gaa cta cga ggg atc att ctt ctg cta tgt act att gcc ttt 48 Met Ala Glu Leu Arg Gly Ile Ile Leu Leu Leu Cys Thr Ile Ala Phe     -20 -15 -10 cat gtt tcc cat gga caa ata aga tgg acg ctg ctg aat cag atc acc 96 His Val Ser His Gly Gln Ile Arg Trp Thr Leu Leu Asn Gln Ile Thr  -5 -1 1 5 10 ggt gaa tct gac gtc att ccg ctg tct aac aac acg cca ctg aat gtg 144 Gly Glu Ser Asp Val Ile Pro Leu Ser Asn Asn Thr Pro Leu Asn Val              15 20 25 tcg ctg aat ttt aag ctg atg aat atc tta gag gcg gac aca gag aaa 192 Ser Leu Asn Phe Lys Leu Met Asn Ile Leu Glu Ala Asp Thr Glu Lys          30 35 40 gat caa gtg gag gtc gtg ctg tgg aca cag gct agc tgg aaa gtg ccg 240 Asp Gln Val Glu Val Val Leu Trp Thr Gln Ala Ser Trp Lys Val Pro      45 50 55 tat tac agc tca ctg ctg tcc tct agc agt tta gac cag gtg agc tta 288 Tyr Tyr Ser Ser Leu Leu Ser Ser Ser Ser Leu Asp Gln Val Ser Leu  60 65 70 75 cca gcc agc aaa atg tgg acc cca gac ctt tct ttc tat aac gcc atc 336 Pro Ala Ser Lys Met Trp Thr Pro Asp Leu Ser Phe Tyr Asn Ala Ile                  80 85 90 gct gca ccc gag ttg ctc tcc aca gac cgc gtg gtg gtc tct aag gac 384 Ala Ala Pro Glu Leu Leu Ser Thr Asp Arg Val Val Val Ser Lys Asp              95 100 105 ggg agc gtc att tac gtg ccc agc cag agg gtc cgt ttc acc tgc gac 432 Gly Ser Val Ile Tyr Val Pro Ser Gln Arg Val Arg Phe Thr Cys Asp         110 115 120 ctt att aat gtg gac acg gag ccg gga gcc acc tgt cgc atc aaa gtc 480 Leu Ile Asn Val Asp Thr Glu Pro Gly Ala Thr Cys Arg Ile Lys Val     125 130 135 gga tcc tgg acc ttc gac aac aaa cag ctc gcc ctg atc acc ggg gag 528 Gly Ser Trp Thr Phe Asp Asn Lys Gln Leu Ala Leu Ile Thr Gly Glu 140 145 150 155 gag ggg gtg gtg aat att gca gag tac ttc gac agc cca aag tac gac 576 Glu Gly Val Val Asn Ile Ala Glu Tyr Phe Asp Ser Pro Lys Tyr Asp                 160 165 170 ctt ttg agt gcc aca cag agt ctg aat cgc aag aag tac aga tgt tgc 624 Leu Leu Ser Ala Thr Gln Ser Leu Asn Arg Lys Lys Tyr Arg Cys Cys             175 180 185 gag aat atg tat gaa gac att gaa att acc ttt gca ttc aga aag aag 672 Glu Asn Met Tyr Glu Asp Ile Glu Ile Thr Phe Ala Phe Arg Lys Lys         190 195 200 taa 675 <210> 8 <211> 224 <212> PRT <213> Bulinus truncatus <400> 8 Met Ala Glu Leu Arg Gly Ile Ile Leu Leu Leu Cys Thr Ile Ala Phe     -20 -15 -10 His Val Ser His Gly Gln Ile Arg Trp Thr Leu Leu Asn Gln Ile Thr  -5 -1 1 5 10 Gly Glu Ser Asp Val Ile Pro Leu Ser Asn Asn Thr Pro Leu Asn Val              15 20 25 Ser Leu Asn Phe Lys Leu Met Asn Ile Leu Glu Ala Asp Thr Glu Lys          30 35 40 Asp Gln Val Glu Val Val Leu Trp Thr Gln Ala Ser Trp Lys Val Pro      45 50 55 Tyr Tyr Ser Ser Leu Leu Ser Ser Ser Ser Leu Asp Gln Val Ser Leu  60 65 70 75 Pro Ala Ser Lys Met Trp Thr Pro Asp Leu Ser Phe Tyr Asn Ala Ile                  80 85 90 Ala Ala Pro Glu Leu Leu Ser Thr Asp Arg Val Val Val Ser Lys Asp              95 100 105 Gly Ser Val Ile Tyr Val Pro Ser Gln Arg Val Arg Phe Thr Cys Asp         110 115 120 Leu Ile Asn Val Asp Thr Glu Pro Gly Ala Thr Cys Arg Ile Lys Val     125 130 135 Gly Ser Trp Thr Phe Asp Asn Lys Gln Leu Ala Leu Ile Thr Gly Glu 140 145 150 155 Glu Gly Val Val Asn Ile Ala Glu Tyr Phe Asp Ser Pro Lys Tyr Asp                 160 165 170 Leu Leu Ser Ala Thr Gln Ser Leu Asn Arg Lys Lys Tyr Arg Cys Cys             175 180 185 Glu Asn Met Tyr Glu Asp Ile Glu Ile Thr Phe Ala Phe Arg Lys Lys         190 195 200 <210> 9 <211> 502 <212> PRT <213> Homo sapiens <220> <221> DOMAIN <222> (1) .. (235) <400> 9 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu   1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu              20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser          35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp      50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met  65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly                  85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile             100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr         115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly     130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp                 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro             180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg         195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val     210 215 220 Thr Met Arg Arg Arg Thr Leu Tyr Tyr Gly Leu Asn Leu Leu Ile Pro 225 230 235 240 Cys Val Leu Ile Ser Ala Leu Ala Leu Leu Val Phe Leu Leu Pro Ala                 245 250 255 Asp Ser Gly Glu Lys Ile Ser Leu Gly Ile Thr Val Leu Leu Ser Leu             260 265 270 Thr Val Phe Met Leu Leu Val Ala Glu Ile Met Pro Ala Thr Ser Asp         275 280 285 Ser Val Pro Leu Ile Ala Gln Tyr Phe Ala Ser Thr Met Ile Ile Val     290 295 300 Gly Leu Ser Val Val Val Thr Val Ile Val Leu Gln Tyr His His His 305 310 315 320 Asp Pro Asp Gly Gly Lys Met Pro Lys Trp Thr Arg Val Ile Leu Leu                 325 330 335 Asn Trp Cys Ala Trp Phe Leu Arg Met Lys Arg Pro Gly Glu Asp Lys             340 345 350 Val Arg Pro Ala Cys Gln His Lys Gln Arg Arg Cys Ser Leu Ala Ser         355 360 365 Val Glu Met Ser Ala Val Ala Pro Pro Pro Ala Ser Asn Gly Asn Leu     370 375 380 Leu Tyr Ile Gly Phe Arg Gly Leu Asp Gly Val His Cys Val Pro Thr 385 390 395 400 Pro Asp Ser Gly Val Val Cys Gly Arg Met Ala Cys Ser Pro Thr His                 405 410 415 Asp Glu His Leu Leu His Gly Gly Gln Pro Pro Glu Gly Asp Pro Asp             420 425 430 Leu Ala Lys Ile Leu Glu Glu Val Arg Tyr Ile Ala Asn Arg Phe Arg         435 440 445 Cys Gln Asp Glu Ser Glu Ala Val Cys Ser Glu Trp Lys Phe Ala Ala     450 455 460 Cys Val Val Asp Arg Leu Cys Leu Met Ala Phe Ser Val Phe Thr Ile 465 470 475 480 Ile Cys Thr Ile Gly Ile Leu Met Ser Ala Pro Asn Phe Val Glu Ala                 485 490 495 Val Ser Lys Asp Phe Ala             500 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: N-terminus of       mature LAChBP1 <400> 10 Leu Asp Arg Ala Asp Ile Leu Tyr Asn Ile   1 5 10 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotides encoding N-terminal peptide of       LAChBP1 <220> <221> modified_base <222> (13) <223> i <220> <221> modified_base <222> (16) <223> a, t, g or c <220> <221> modified_base <222> (25) <223> a, t, g or c <400> 11 cggatccgay mgngcngaya thytntayaa ya 32 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer1 useful       for cloning cDNA encoding LAChBP (optionally with       (Primer2) <220> <221> modified_base <222> (14) <223> i <220> <221> modified_base <222> (20) <223> i <220> <221> modified_base <222> (23) <223> any base <220> <221> modified_base <222> (26) <223> any base <220> <221> modified_base <222> (29) <223> any base <400> 12 gcgaattcga yacngarwsn ggngcnacnt g 31 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer2       useful for cloning cDNA encoding LAChBP       (optionally with Primer1) <220> <221> modified_base <222> (20) <223> i <220> <221> modified_base <222> (26) <223> any base <400> 13 gcgaagcttc rtcytcrtan gcytcngcrc arc 33 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: His-tag <400> 14 Ser Arg Gly His His His His His His   1 5 <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: His-tag <400> 15 Glu Phe Lys Asp Asp Asp Asp Asp Lys His His His His His His   1 5 10 <210> 16 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Additonal       amino acids at the N-terminus of mature LAChBP due       to alpha-mating factor cleavage site <400> 16 Glu Ala Glu Ala   1 <210> 17 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer useful       for generating LAChBP / alpha7 nAChR chimera <400> 17 gcgctcgaga aaagagaggc tgaagctttg gaccgggcag acatctt 47 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer useful       for generating LAChBP / alpha7 nACHR chimera <400> 18 cgcgaattca agaatttcgg agcgtccctt 30 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer useful       for generating LAChBP / alpha7 nACHR chimera <400> 19 gtggaaacca gacattctcc tctacaacgc catctcgaaa cc 42 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer useful       for generating LAChBP / alpha7 nACHR chimera <400> 20 gaggagaatg tctggtttcc acaaagagct tattggcac 39 <210> 21 <211> 8 <212> PRT <213> S. cerevisiae <400> 21 Glu Ala Glu Ala Tyr Val Glu Phe   1 5

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 31/4045 A61K 31/4439 4C084 31/4439 39/00 H 4C085 38/00 39/395 D 4C086 39 / 00 N 4C206 39/395 45/00 4H045 A61P 25/18 45/00 25/28 A61P 25/18 25/34 25/28 43/00 111 25/34 123 43/00 111 C07K 14/705 123 16/28 C07K 14/705 19/00 16/28 C12N 1/15 ZTD 19/00 1/19 C12N 1/15 ZTD 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 D G01N 33/15 33/566 33/50 C12N 15/00 ZNAA 33/53 5/00 A 33/566 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, E, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU , CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU , ZA, ZW F-term (reference) 2G045 AA40 DA36 FB03 4B024 AA01 AA11 BA63 CA04 CA09 DA12 EA04 FA01 GA11 HA01 HA12 4B063 QA08 QQ05 QQ13 QQ20 QQ79 QQ91 QQ96 QR55 QR74 QR80 QS34 QS38 4B064 AG20 CA01 CA06 CA19 CC24 DA01 4B065 AA72X AA77X AA90Y AB01 AC14 BA02 CA24 CA44 CA46 4C084 AA01 AA02 AA07 AA16 BA01 BA02 BA08 BA22 CA47 DC50 NA14 NA15 ZA162 ZA182 ZC392 ZC412 4C085 AA03 AA13 AA14 BB11 CC22 CC23 MY01 FA45 A04 A01A45 A04 A45A45 A0107 NA14 ZA16 ZA18 ZC39 ZC41 4H045 AA10 AA11 AA20 AA30 BA10 CA50 DA50 DA75 EA40 EA50 FA72 FA73 FA74

Claims (80)

[Claims] 1. A mollusc-derived water-soluble protein capable of binding a ligand of a ligand-dependent receptor. 2. The protein according to claim 1, wherein the ligand is acetylcholine, γ-aminobutyric acid (GABA), glycine, or serotonin. 3. The protein according to claim 2, which is an acetylcholine binding protein (AChBP). 4. The protein according to any one of claims 1 to 3, which is capable of forming multimers. 5. Pulmonata species, preferably Basalmatophores.
a) The protein according to any one of claims 1 to 4, which is derived from a species. 6. An amino acid sequence selected from the group consisting of:
The protein according to any one of 1 to 5: (a) The amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, or 8 or a functional equivalent thereof, or at least 5 contiguous sequences thereof. Amino acid fragments;
(B) An amino acid sequence having at least 30% amino acid identity with the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, or 8. 7. A ligand-dependent receptor ligand capable of binding and comprising at least 5 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, or 8. , And / or preferably at least 10 ⁻⁷ M
A water-soluble ligand-binding protein that can be detected by a monoclonal antibody or a polyclonal antibody that recognizes the protein according to any one of claims 1 to 6 with a binding affinity of. 8. A water-soluble protein capable of binding a ligand of a ligand-dependent receptor, including: (a) the same position as in the water-soluble protein according to any one of claims 1 to 6, Or at the corresponding position, at least the amino acid of the protein that determines the solubility of the protein; and (b) at least 4 amino acids that determine the binding to the ligand. 9. The protein according to claim 7 or 8, which is capable of forming multimers. 10. The protein according to any one of claims 7 to 9, which comprises 200 to 240 amino acids. 11. The ligand is acetylcholine, nicotine, rhotoxin (
lophotoxin), d-tubocurarine, carbamylcholine, galantamine, or epibatidine, The protein according to any one of claims 7 to 10. 12. The ligand-dependent receptor is an arthropod (preferably an insect),
The protein according to any one of claims 1 to 11, which is derived from a plant (preferably a higher plant, most preferably a seed plant) or a vertebrate (preferably a mammal, most preferably a human). 13. The protein according to any one of claims 7 to 12, wherein the ligand-dependent receptor is a nicotinic acetylcholine receptor. 14. The solubility determining amino acid is at the same position as AChBP having the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6 or 8 and is preferably solvent accessible. The protein according to any one of claims 7 to 13, wherein the protein comprises the region in the crystal structure shown in Fig. 10. 15. An amino acid sequence having at least 40% amino acid identity with the amino acid sequence of positions 20 to 223 of any one of SEQ ID NOs: 2, 4, 6, or 8.
The protein according to any one of claims 7 to 14, wherein the amino acid that binds the ligand is replaced with the corresponding amino acid of the ligand-dependent receptor. 16. The amino acid (a) which determines solubility is 20 to 44 of SEQ ID NO: 2.
Position, 73-81st, 86-92th, 112-120th, 135-152th, 166-189th, 196-20th, 2nd
Hydrophilic amino acids (Asp, Glu, from the sequence at positions 09-213 and / or 219-227)
Arg, Lys), The protein according to any one of claims 7 to 15. 17. The amino acid (a) which determines the solubility is the amino acids Asp (36), Asp (68), Glu (115), Arg (137), Asp (143) and Asp (148) of SEQ ID NO: 2. ,
Includes Glu (150), Arg (167), Arg (189), and Glu (215), Glu may be exchanged for Asp (and vice versa), and Arg may be exchanged for Lys (reverse) Also in the case of), the protein according to claim 16. 18. Amino acid Cys (142), Thr (149), Ala (15 of SEQ ID NO: 2
3), Thr (154), Cys (155), Arg (156), Ile (157) and / or Lys (158
The protein according to any one of claims 7 to 17, further comprising: 19. Amino acid (b) Pro (39), Trp (77), Trp of SEQ ID NO: 2
8. Including (101), Pro (103), Asp (194), and / or Ser (161).
18. The protein according to any one of to 17. 20. The amino acid sequence of positions 165-169 and / or 200-203 of SEQ ID NO: 2 has been replaced with the corresponding sequence of the ligand-dependent receptor.
9. The protein according to any one of 9. 21. An acetylcholine receptor ligand capable of binding,
SEQ ID NO: 2 Trp (101) to Tyr (T08), Trp (162) to His (164), and Tyr (
The protein according to any one of claims 7 to 20, wherein at least one of the amino acid sequences of 204) to Tyr (211) is exchanged with the corresponding sequence of the acetylcholine receptor. 22. A method of making a water soluble ligand dependent receptor or a corresponding ligand binding domain, or a method of improving the water solubility and crystallinity of such a receptor or domain, comprising: According to the manner of determining the water solubility of the protein according to any one of Items 1 to 21, or substituting, adding, deleting, or modifying at least one amino acid at the position corresponding to the amino acid that contributes to water solubility. A method comprising altering the amino acid sequence of the extracellular domain of a ligand dependent receptor. 23. The method of claim 22, wherein the ligand-dependent receptor is defined as in any one of claims 1-21. 24. At least one amino acid is changed to a corresponding amino acid or equivalent amino acid of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, or 8 and preferably determines solubility. 24. The method of claim 22 or 23, wherein the amino acid comprises a solvent accessible region in the crystal structure of Figure 10. 25. The method of any one of claims 22-24, wherein the Cys123-Cys136 loop of SEQ ID NO: 2 is inserted in the corresponding region of the ligand-binding domain of the ligand-dependent receptor. 26. The method of any one of claims 22-25, further comprising the step of: (a) transfecting a polynucleotide comprising a nucleotide sequence encoding an altered amino acid sequence, the polynucleotide Culturing a host cell capable of expressing C .; and, optionally, (b) recovering the water-soluble ligand-dependent receptor or the corresponding ligand-binding domain from the culture. 27. A water soluble ligand dependent receptor or ligand binding domain obtainable by the method according to any one of claims 22 to 26. 28. The protein of any one of claims 1-21 or 27, further comprising a spacer sequence that binds to the carrier body. 29. A fusion protein comprising a water soluble ligand binding protein according to any one of claims 1-21, 27 or 28, or a binding fragment thereof and a fragment of a ligand dependent receptor. 30. A dimer or pentamer comprising at least one monomer containing the protein according to any one of claims 1 to 21 or 27 to 29. 31. A ligand-gated ion channel comprising the protein according to any one of claims 1-21 or 27-29, or the dimer or pentamer according to claim 30. 32. Encodes the protein of any one of claims 1-21 or 27-29, the dimer or pentamer of claim 30, or the ligand-gated ion channel of claim 31. , One or more polynucleotides. 33. The polynucleotide of claim 32, which comprises: (a) having at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, 5, or 7. A nucleotide sequence, or a degenerate sequence thereof; or (b) a nucleotide sequence capable of hybridizing to the nucleotide sequence of (a) under stringent hybridization conditions. 34. The polynucleotide of claim 32 or 33 operably linked to a heterologous expression control sequence that enables expression in prokaryotic or eukaryotic cells. 35. One or more vectors comprising the polynucleotide according to any one of claims 32-34. 36. A host cell that has been genetically engineered with the polynucleotide of any one of claims 32-34 or the vector of claim 35. 37. An epitope of at least 5 consecutive amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6 or 8 and / or preferably a binding affinity of at least 10 ^-7 M. 7. An antigen comprising an epitope that can be detected by a monoclonal antibody or a polyclonal antibody that recognizes the protein according to any one of claims 1 to 6. 38. A protein according to any one of claims 1-21 or 27-29, a dimer or pentamer according to claim 30, a ligand-gated ion channel according to claim 31, or a claim. An antibody that specifically recognizes the antigen described in 37. 39. An oligonucleotide probe having at least 15 contiguous nucleotides of the polynucleotide of any one of claims 32-34, or comprising a nucleotide sequence encoding the antigen of claim 37. 40. The protein according to any one of claims 1 to 21 or 27 to 29, the dimer or pentamer according to claim 30, the ligand-gated ion channel according to claim 31, and the claim 32. 35. The polynucleotide according to any one of claims 34 to 35,
The vector of claim 36, the host cell of claim 36, the antigen of claim 37, the antibody of claim 38, or the oligonucleotide probe of claim 39; and, optionally,
A composition comprising suitable means for detection or for conducting a ligand-receptor binding assay. 41. A method of identifying an agonist / activator or antagonist / inhibitor of a ligand-dependent receptor, comprising the steps of: (a) any one of claims 1-21 or 27-29. The water-soluble ligand binding protein according to claim, the dimer or pentamer according to claim 30, the ligand-gated ion channel according to claim 31, or a cell expressing the protein, and a compound to be screened, Contacting a ligand-binding protein under conditions that bind the compound in the presence of a component capable of producing a detectable signal in response to ligand binding; and (b) a signal resulting from the binding activity of the ligand-binding protein. Presence / absence and absence / decrease of the signal, respectively. Stages showing activators and antagonists / inhibitors. 42. A crystal according to any one of claims 1-21 or 27-29, a dimer or pentamer according to claim 30 or a crystal of a ligand-gated ion channel according to claim 31. 43. The protein according to any one of claims 1-21 or 27-29, the dimer or pentamer according to claim 30, or the ligand-gated ion channel according to claim 31, and a ligand. A crystal of a protein-ligand complex containing. 44. The crystal of claim 43, wherein the ligand comprises an N-alkylated hydroxyalkyl and / or a quaternary ammonium ion. 45. The ligand is 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), B-bippinatin, rhotoxin, d-
44. The crystal of claim 43, which comprises tubocurarine, carbamylcholine, galantamine, epibatidine, or α-bungarotoxin. 46. Efficiently for determination of atomic coordinates of a protein or protein-ligand complex up to a resolution of more than 5.0Å, preferably more than 4.0Å.
46. A crystal according to any one of claims 42 to 45, which diffracts rays. 47. The protein has an amino acid sequence of amino acids 20 to 223 of SEQ ID NO: 2, or an amino acid sequence that differs only by conservative substitutions from amino acids 20 to 223 of SEQ ID NO: 2. The crystal according to any one of claims 42 to 46. 48. The crystal of claim 47, wherein the ligand is HEPES. 49. (1) P2 ₁ 2 ₁ 2 ₁ space group, and a = 120.6Å, b = 137.0Å
, And unit cell dimensions of c = 161.5Å; (2) P4 2 2 1 2 space group, and a = b = 1
Unit cell dimensions of 41.6Å and c = 120.8Å; or (3) P2 ₁ space group, and a
= 121.1Å, b = 162.1Å, c = 139.4Å, β = 90.1 ° with unit cell size,
The crystal according to claim 46. 50. The protein has a secondary structural element comprising an α-helix and an antiparallel β-sheet as shown in FIGS. 7, 10, 11 and / or 12.
50. The crystal according to any one of to 49. 51. The crystal according to any one of claims 42 to 50, which has a three-dimensional structure as defined by the atomic coordinates shown in Table 1. 52. A crystal according to any one of claims 42 to 51, which has a binding cavity as shown in Figures 6, 8, 9 and / or 13. 53. A method of using the crystals of any one of claims 42-52 in a drug screening assay, comprising the steps of: (a) using a determined three-dimensional structure for the crystals. Selecting potential ligands by performing structural drug design, the selection being carried out in conjunction with computer modeling; optionally, (b) potential ligand and ligand dependence in an in vitro or in vivo assay. Contacting with a ligand binding domain of a receptor; and (c) detecting binding of a potential ligand to the ligand binding domain. 54. The method of claim 53, wherein the ligand-dependent receptor is a nicotinic acetylcholine receptor. 55. The method of claim 53 or 54, further comprising the following steps:
(D) The water-soluble ligand-binding protein according to any one of claims 1 to 21 or 27 to 29, the dimer or pentamer according to claim 30, the ligand-gated ion channel and latency of claim 31. It is a step of forming a supplemental crystal of the protein-ligand complex by co-crystallizing or immersing a crystal with an effective drug, and the crystal is more than 5.0 Å, preferably more than 4.0 Å, Preferably
Efficiently diffracting X-rays to determine the atomic coordinates of the protein-ligand complex up to a resolution of more than 3Å; The step of selecting a candidate drug by designing a drug with a structure using the determined three-dimensional structure, and the selection is performed together with computer modeling; (g) the candidate drug and ligand dependence Contacting cells expressing the receptor; and (h) detecting a cellular response, where the candidate drug is a drug if the cellular response is altered compared to cells not contacting the candidate compound. The stage to be identified. 56. The water-soluble ligand binding protein according to any one of claims 1 to 21 or 27 to 29, the dimer or pentamer according to claim 30, and the ligand-gated ion channel according to claim 31. And determining the three-dimensional structure of a crystal containing a protein-ligand complex formed by the ligand of the ligand-dependent receptor,
The initial step, performed prior to step (a), that efficiently diffracts X-rays for the determination of atomic coordinates of protein-ligand complexes up to a resolution of more than 5.0Å, preferably more than 4.0Å, for crystals. 56. The method of any one of claims 53-55, further comprising. 57. A method of growing a protein-ligand complex crystal comprising the steps of: (a) a water soluble ligand binding protein according to any one of claims 1-21 or 27-29. 32. A step of contacting the dimer or pentamer according to claim 30, or the ligand-gated ion channel according to claim 31 with a ligand of a ligand-gated receptor, wherein the water-soluble ligand-binding protein comprises a ligand and a protein-ligand complex. Forming a body; and (b) growing a crystal of the protein-ligand complex, wherein the crystal is 5.0
Efficiently diffracting X-rays for the determination of atomic coordinates of protein-ligand complexes to a resolution above Å, preferably above 4.0Å. 58. The method according to claim 42, wherein the compound to be screened is in a solution.
53. A drug screening assay comprising the steps of immersing the crystal of any one of 52 and detecting binding of the compound to a ligand binding protein. 59. The method of claim 57 or 58, wherein the ligand comprises an alkylated nitrogen and / or a quaternary ammonium ion. 60. A method of increasing or decreasing the affinity of a drug for a ligand dependent receptor, comprising the steps of: (a) Determined for the crystal of any of claims 42-52. And (b) interacting with the binding site or non-specific binding site of the protein's binding cavity in the crystal, which is a step of structurally designing the drug by using the three-dimensional structure Modifying the drug to alter or eliminate some of the drug suspected of being. 61. The method of claim 60, wherein step (a) further comprises the steps of the method of any one of claims 53-59. 62. The method according to claim 60 or 61, further comprising the following additional steps after step (b): (c) using the modified drug of step (b) of step (a). Repeating the method used to perform structural drug design. 63. Computational evaluation of a chemical entity for binding to a ligand binding site or a non-specific binding site of a ligand binding protein using the structural coordinates of a water soluble ligand binding protein crystal, including the coordinates in Table 1. Methods of drug design, including steps: 64. The method of any one of claims 53-63, wherein the identified drug interferes with or promotes correct assembly of ligand-gated ion channels. 65. The method of any one of claims 53-63, wherein the identified drug binds to a non-specific binding site of a ligand-gated ion channel. 66. The method of any one of claims 53-65, comprising synthesizing a therapeutically effective amount of the drug. 67. A drug produced by the method according to claim 66 or a prodrug thereof. 68. A drug according to claim 67 which interacts with a ligand dependent receptor comprising the pentamer according to claim 30 having monomers A to E, of the mature protein of SEQ ID NO: 2. 15-21st, 44-47th, 85-87th, 91-94th, 122-124th, 143-146
One or more primary contact regions of the monomer (residues from A that contact B) defined by the amino acid residues at positions 149, 185-187 and / or SEQ ID NO: 2.
3rd to 4th, 7th to 8th, 11th, 37th to 39th, 53rd, 75th to 77th, 96 to 10th of the mature protein of
One or more complementary contact regions (residues from B in contact with A (contact with E) of another monomer defined by amino acid residues at positions 4, 114-118, and 163-170. The same as the residue in A))), or the contact region shown in Figure 14, or the corresponding contact region of the ligand-gated ion channel monomer. 69. The drug of claim 68, wherein the ligand-gated ion channel is a nicotinic acetylcholine receptor and the order of monomers is αγαβδ. 70. A nucleotide sequence of the polynucleotide according to any one of claims 32 to 34, the protein according to any one of claims 1 to 21 or 27 to 29, the dimer according to claim 30 or A computer readable medium comprising a pentamer, or the amino acid sequence of the ligand-gated ion channel of claim 31, or the structural coordinates of the crystal of any one of claims 40-50. 71. An apparatus comprising the computer-readable medium of claim 70. 72. Use of a computer readable medium according to claim 70 or a device according to claim 71 for modeling antagonists / inhibitors or agonists / activators of a ligand dependent receptor. 73. Use of the crystal according to any one of claims 42 to 52 or its structural coordinates as a template for modeling the 3D structure of a ligand-gated ion channel. 74. A polynucleotide according to any one of claims 32-34, any one of claims 1-21 or 27-29 for screening or profiling putative ligands for a ligand dependent receptor. Claimed protein, claim 30
The dimer or pentamer according to claim 31, the ligand-gated ion channel according to claim 31,
The vector according to claim 35, the host cell according to claim 36, the antigen according to claim 37, the antibody according to claim 38, the oligonucleotide probe according to claim 39, and any one of claims 42 to 52. 67. Use of crystals or a method according to any one of claims 53 to 66. 75. Any one of claims 53-66 for preparing a pharmaceutical composition for treating a disorder mediated by a ligand-gated ion channel or a disorder associated with a ligand-gated ion channel. Use of antagonists / inhibitors or agonists / activators identified by the method described. 76. The antagonist / inhibitor is the protein according to any one of claims 1-21 or 27-29, the antigen according to claim 37, the antibody according to claim 38, or The protein according to any one of 1 to 21 or 27 to 29, 3.
76. The use according to claim 75, which is obtained from the antigen according to claim 7, the antibody according to claim 38, or the toxin of a ligand-gated ion channel. 77. The agent / activator is a protein according to any one of claims 1-21 or 27-29, an antigen according to claim 37, an antibody according to claim 38, or The protein according to any one of claims 1 to 21 or 27 to 29, the antigen according to claim 37, is obtained from the antibody according to claim 38, or epibatidine, acetylcholine, choline, nicotine, carbachol, 76. Use according to claim 75, which is obtained from serotonin or GABA. 78. The ligand-gated ion channel is a nicotinic acetylcholine receptor and the mediated disorder or related disorders is Tourette's syndrome,
78. Use according to any of claims 75 to 77, which is Alzheimer's disease, nicotine addiction, or schizophrenia. 79. Use of a ligand of a ligand-dependent receptor to identify and isolate a mollusc-derived water-soluble ligand binding protein. 80. The ligand is α-bungarotoxin. Use according to claim 79.