WO2000037500A1 - Three dimensional structure of a sterile alpha motif domain - Google Patents

Abstract

The invention relates to the three dimensional structure of sterile alpha motif (Sam) domain. The atomic coordinates that define the structure and any compounds bound to the structure enable the determination of the three dimensional structures of SAM domains with unknown structure, and the identification of modulators of a SAM domain.

Description

TITLE: Three Dimensional Structure of a Sterile Alpha Motif Domain FIELD OF THE INVENTION

The invention relates to the three dimensional structure of a sterile alpha motif (Sam) domain. The atomic coordinates that define the structure and any compounds bound to the structure enable the determination of homologues, the three dimensional structures of polypeptides with unknown structure, and the identification of modulators of a SAM domain. BACKGROUND OF THE INVENTION

The Eph family of receptor tyrosine kinases have been implicated in the control of axon guidance [Henkemeyer, 1996; Orioli, 1996], cell migration [Krull, 1997], patterning of the nervous system [Xu, 1996] and angiogenesis [Wang, 1998], and are activated by clustering into dimers or tetramers [Stein, 1998]. However, the cell-surface ligands for Eph receptors (ephrins) apparently lack an intrinsic ability to induce receptor oligomerization [Lackmann, 1997]. Factors that influence receptor aggregation include the pre-clustering of ephrins [Davis, 1994], the homotypic interaction between the extracellular domains of two receptor chains [Lackmann, 1998], and the binding of PDZ domain containing proteins to the receptor's C-terminus [Hock, 1998].

All Eph receptors have a Sterile Alpha Motif (SAM) domain within their cytoplasmic regions. The SAM domain was identified as a conserved sequence present in a small set of yeast sexual differentiation proteins referred to as the Sterile Alpha Mating factors [Ponting, 1995; Schultz, 1997]. In ETS family transcription factors this sequence has also been termed the Pointed domain [Klambt, 1993]. The domain is found in a variety of proteins, many of which contain catalytic domains or recognized protein interaction domains. SAM domains are almost always located at a protein's N- or C-terminus. A highly conserved SAM domain is located in the cytoplasmic region of Eph receptors (approx. 50 % identity over 14 family members), C-terminal to the catalytic domain and followed by only 5 residues that form a potential PDZ domain binding site [Hock, 1998]. Amongst receptor tyrosine kinases, the presence of a cytoplasmic module other than the protein kinase domain is unique to Eph receptors.

The SAM domain can function as a protein interaction module through an ability to homo- and hetero-dimerize with other SAM domains [Jousset, 1997; Peterson, 1997; Tu, 1997; Kyba, 1998]. This dimerizing property elicits oncogenic activation of chimeric proteins arising from translocation of the SAM domain of TEL to coding regions of the βPDGF receptor [Golub, 1994); Abl [Golub, 1996], and JAK2 protein kinases [Lacronique, 1997] or the AML1 transcription factor [Golub, 1995]. A functional role in mediating homo and hetero-typic dimerization has been shown for SAM domains in the transcription factor TEL [Jousset, 1997], members of the polycomb group of transcriptional repressors (RAE28, Scm and ph) [Peterson, 1997], the protein kinase Byr2p [Tu, 1997], and the α and β isoforms of the liprin scaffolding proteins [Serra-Pages, 1998]. SUMMARY OF THE INVENTION

Broadly stated, the present invention relates to the three-dimensional structure of one or more SAM domains. The three-dimensional structures may be complexed with one or more compounds. The defined boundaries and properties of the structures and any of the compounds bound to it are pertinent to methods for determining the three-dimensional structures of polypeptides with unknown structure, and to methods that identify modulators of SAM domain function. These modulators are potentially useful as therapeutics for diseases, including (but not limited to) cell proliferative diseases, such as cancer, angiogenesis, atherosclerosis, and arthritis, and diseases associated with the nervous system.

Broadly stated the present invention relates to a crystalline form of a polypeptide corresponding to one or more SAM domains, preferably one or more SAM domains of an Eph receptor, preferably of EphA. The crystalline form may comprise one or more heavy metal atoms, or at least one compound. In a preferred embodiment, a unit cell of the crystalline form of the invention has dimensions of about a=b= 77.14 ± .03 angstroms, c= 24.3 ± .04 angstroms.

The invention also relates to a method of forming a crystalline form of the invention comprising

(a) mixing a volume of a SAM domain with a reservoir solution; and

(b) incubating the mixture obtained in step (a) over the reservoir solution in a closed container under conditions suitable for crystallization.

The invention also features a method of determining three dimensional structures of polypeptides with unknown structure comprising the step of applying the structural atomic coordinates of a crystalline form of one or more SAM domains of the invention.

Methods are also provided for identifying a potential modulator of a SAM domain function preferably a SAM domain of an Eph receptor function by docking a computer representation of a structure of a compound with a computer representation of a structure of one or more SAM domains of the invention preferably a SAM domain of an Eph receptor that is defined by the atomic structural coordinates described herein. In an embodiment the method comprises the following steps:

(a) docking a computer representation of a compound from a computer data base with a computer representation of a selected site on a SAM domain, preferably a SAM domain of an Eph receptor,to obtain a complex;

(b) determining a conformation of the complex with a favourable geometric fit and favourable complementary interactions; and

(c) identifying compounds that best fit the selected site as potential modulators of SAM domain function.

In another embodiment the method comprises the following steps:

(a) modifying a computer representation of a compound complexed with a selected site on a SAM domain, preferably a SAM domain of an Eph receptor, by deleting or adding a chemical group or groups; (b) determining a conformation of the complex with a favourable geometric fit and favourable complementary interactions; and (c) identifying a compound that best fits the selected site as a potential modulator of a SAM domain. In still another embodiment the method comprises the following steps: (a) selecting a computer representation of a compound complexed with a selected site on a SAM domain, preferably a SAM domain of an Eph receptor; and

(b) searching for molecules in a data base that are similar to the compound using a searching computer program, or replacing portions of the compound with similar chemical structures from a data base using a compound building computer program. The invention also features a potential modulator of a function of a SAM domain preferably a SAM domain of an Eph receptor identified by the methods of the invention, and a method of treating a disease associated with a SAM domain preferably a SAM domain of an Eph receptor with inappropriate activity in a cellular organism, comprising:

(a) administering a modulator identified using the methods of the invention in an acceptable pharmaceutical preparation; and

(b) activating or inhibiting a SAM domain function to treat the disease. The invention also provides peptides that mediate SAM domain function. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:

Figure 1A shows a sequence alignment of SAM domains from selected proteins (SEQ. ID. NOS. 1 to 21);

Figure IB shows a selection of multi-domain proteins containing SAM domain (S); Figure 2A is a ribbons depiction of the SAM homo-dimer viewed down the twofold symmetry axis;

Figure 2B is a ribbons depiction of the SAM homo-dimer viewed perpendicular to the symmetry axis;

Figure 2C is a ribbons stereo view highlighting the dimer interface region; Figure 3 A is a molecular surface and worm representation of the SAM homodimer;

Figure 3B is a molecular surface and worm representation of the SAM homodimer; and

Figure 4 is a gel filtration elution profile of wild type and single or double site mutants of the EphA4 receptor SAM domain.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS:

Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Current Protocols in Molecular Biology (Ansubel) for definitions and terms of the art.

Abbreviations for amino acid residues are the standard 3-letter and/or 1 -letter codes used in the art to refer to one of the 20 common L-amino acids. Likewise abbreviations for nucleic acids are the standard codes used in the art.

The term "crystalline form" in the context of the invention, is a crystal formed from an aqueous solution comprising a purified polypeptide comprising one or more SAM domains, preferably a SAM domain of an Eph receptor. A crystalline form of a SAM domain is characterized as being capable of diffracting x-rays in a pattern defined by one of the crystal forms depicted in Blundel et al 1976, Protein Crystallography, Academic Press. A crystalline form may include a crystal structure in association with one or more heavy-metal atoms i.e. a derivative crystal, or a crystal structure in association with one or more compounds i.e. a co-crystal. The term "association" refers to a condition of proximity between a chemical entity or compound or portions or fragments thereof, and a SAM domain or portions or fragments thereof. The association may be non-covalent i.e. where the juxtaposition is energetically favored by for example, hydrogen-bonding, van der Waals, or electrostatic or hydrophobic ineractions, or it may be covalent. The term "heavy-metal atoms" refers to an atom that is a transition element, a lanthanide metal, or an actinide metal. Lanthanide metals include elements with atomic numbers between 57 and 71, inclusive. Actinide metals include elements with atomic numbers between 89 and 103, inclusive.

The term "Eph receptor" refers to a subfamily of closely related transmembrane receptor tyrosine kinases related to Eph, a receptor named for its expression in an erythropoietin-producing human hepatocellular carcinomas cell line. The receptors contain cell adhesion-like domains on their extracellular surface. The Eph subfamily receptor tyrosine kinases are more specifically characterised as encoding a structurally related cysteine rich extracellular domain containing a single immunoglobulin (Ig)-like loop near the N-terminus and two fibronectin III (FN HI) repeats adjacent to the plasma membrane. The Eph receptors are divided into two groups based on the relatedness of their extracellular domain sequences. The grouping also corresponds to the ability of the receptors to bind preferentially to the ephrin-A or ephrin-B proteins. The group that includes receptors interacting preferentially with ephrin A proteins is called EphA and includes EphAl (also known as Eph and Esk), EphA2 (also known as Eck, Myk2, Sek2), EphA3 (also known as Cek4, Mek4, Hek, Tyro4, Hek4), EphA4 (also known as Sek, Sekl, Cek8, Hek8, Tyrol), EphA5 (also known as Ehkl, Bsk, Cek7, Hek7, and Rek7), EphA6 (Ehk2, and Hekl2) EphA7 (also known as Mdkl, Hekl 1, Ehk3, Ebk, Cekl 1), and EphA8 (also known as Eek, Hek3). The group that includes receptors interacting preferentially with ephrin B proteins is called Eph B and includes EphBl (also known as Elk, Cek6, Net, Hek6), EphB2 (also known as Cek5, Nuk, Erk, Qek5, Tyro5, Sek3, hek5, Drt), EphB3 (also known as CeklO, Hek2, Mdk5, Tyro6, and Sek4), EphB4 (also known as Htk, Mykl, Tyrol 1, Mdk2), EphB5 (also known as Cek9, Hek9), and EphB6 (also known as Mep). "Ephrin" refers to a class of ligands which are anchored to the cell membrane through a transmembrane domain, and bind to the extracellular domain of an Eph receptor, facilitating dimerization and autophosphorylation of the receptor and autophosphorylation of the ligand. The ephrins which are targeted in the methods of the invention are those that bind to and activate (i.e. phosphorylate) an EphA or an EphB receptor, preferably an EphA receptor. The ephrin-A ligands (GPI-anchored ligands) are ephrin-A (also known as B61, LERKl, EFL-1), ephrin- A2 (also known as LERK6, Elfl, mCek7-L, cElfl), ephrin-A3 (also known as LERK3, Ehkl-L, and EFL-2), ephrin-A4 (also known as LERK4, EFL-4, mLERK4), ephrin-A5 (AL1, LERK7, EFL-5, mALl, [rLERK.7], RAGS), and the ephrin-B ligands (transmembrane ligands) are ephrin-B 1 (also known as LEKR2, ELK-L, EFL-3, Cek5-L, Stral, [LERK2]), ephrin-B2 (also known as LERK5, HTK-L, NLERK1, Elf2, Htk-L), and ephrin-B3 (also known as LERK8, ELK-L3, NLERK2, EFL-6, Elβ, [rELK-L3]).

The term "SAM domain" refers to a region known as the Sterile Alpha Motif (SAM) domain within the cytoplasmic regions of all Eph receptors (Figure IB), and in other proteins such as TEL [Jousset, 1997], members of the polycomb group of transcriptional repressors (RAE28, Scm and ph) [Peterson, 1997], the protein kinase Byr2p [Tu, 1997], the and β isoforms of the liprin scaffolding proteins [Serra-Pages, 1998], and tankyrase (Smith, S. et al, Science 282: 1484-1487, 1998, Acession AF082556). The SAM domain was identified as a conserved sequence present in a small set of yeast sexual differentiation proteins referred to as the Sterile Alpha Mating factors [Ponting, 1995; Schultz, 1997]. In ETS family transcription factors this sequence has also been termed the Pointed domain [Klambt, 1993]. Extensive database searching and sequence alignment analysis (Figure 1A) reveals that this domain is found in a variety of proteins, many of which contain catalytic domains or recognized protein interaction domains (Figure IB). SAM domains are almost always located at a protein's N- or C-terminus. A highly conserved SAM domain is located in the cytoplasmic region of Eph receptors (approximately 50 % identity over 14 family members), C-terminal to the catalytic domain and followed by only 5 residues that form a potential PDZ domain binding site [Hock, 1998]. The term also includes amino acid sequences having substantial sequence identity to a SAM domain, a mutant, or a subunit of a SAM domain. Preferably the SAM domain is an "Eph SAM domain" i.e. a SAM domain of an Eph receptor. "SAM domain structure" or "SAM domain three dimensional structure" refers to the three dimensional structure of a purified polypeptide comprising one or more SAM domains, preferably a crystalline form.

As applied to polypeptides, the term " substantial sequence identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. For example, the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to effect the properties of a protein. Examples include glutamine for asparagine or glutamic acid for aspartic acid. The term "mutant" refers to a polypeptide that is obtained by replacing at least one amino acid residue in a native SAM domain with a different amino acid residue. Mutation can be accomplished by adding and/or deleting amino acid residues within the native SAM domain. A mutant may or may not be functional.

The term "function" refers to the ability of a modulator to enhance or inhibit the association between a SAM domain and a compound.

The term "atomic structural coordinates" as used herein refers to a data set that defines the three dimensional structure of a molecule or molecules (e.g. unit cell axial lengths, space group). Structural coordinates can be slightly modified and still render nearly identical three dimensional structures. A measure of a unique set of structural coordinates is the root-mean-square deviation of the resulting structure. Structural coordinates that render three dimensional structures that deviate from one another by a root-mean-square deviation of less than 1.5 A may be viewed by a person of ordinary skill in the art as identical. Structural coordinates for a SAM domain are in Table 2.

The term "unit cell" refers to the smallest and simplest volume element (i.e. parallelpiped- shaped block) of a crystal that is completely representative of the unit of pattern of the crystal. The unit cell axial lengths are represented by a, b, and c where a = x axis, b = y axis, and c = z axis. Those of skill in the art understand that a set of atomic coordinates determined by X-ray crystallography is not without standard error.

The term "space group" refers to the symmetry of a unit cell. In a space group designation the capital letter indicates the lattice type and the other symbols represent symmetry operations that can be carried out on the unit cell without changing its appearance.

The term "purified" in reference to a polypeptide, does not require absolute purity such as a homogenous preparation rather it represents an indication that the sequence is relatively purer than in the natural environment. Generally, a purified polypeptide is substantially free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated, preferably at a functionally significant level for example at least 85% pure, more preferably at least 95% pure, most preferably at least 99% pure. A skilled artisan can purify a polypeptide comprising a SAM domain using standard techniques for protein purification. A substantially pure polypeptide comprising a Sam domain will yield a single major band on a non-reducing polyacrylamide gel. The purity of the SAM domain polypeptide can also be determined by amino-terminal amino acid sequence analysis. Three Dimensional Structure of SAM Domain

The present invention provides a purified SAM domain three dimensional structure. In an embodiment the structure is a crystalline form. A SAM domain structure may comprise one or more SAM domains in a unit cell, preferably two, three or four SAM domains. In a preferred embodiment, a SAM domain is arranged in a crystallline manner in a space group P6 so as to form a unit cell of dimensions a=b= 77.14 angstroms, c= 24.37 angstroms and which effectively diffracts X-rays for determination of the atomic coordinates of the SAM domain to a resolution of about 2.9 angstroms.

The 3-dimensional structure of a preferred SAM domain of the invention is shown in Figures 2 and 3.

A crystalline form includes native crystals, derivative crystals, and co-crystals. The native crystals generally comprise substantially pure polypeptides comprising one or more SAM domains in crystalline form. It is understood that the crystalline form is not limited to naturally occurring or native SAM domains but includes mutants of native SAM domains obtained by replacing at least one amino acid residue in a native SAM domain with a different amino acid residue or by adding or deleting amino acid residues within the native polypeptide, and having substantially the same three dimensional structure as the native SAM domain from which the mutant is derived i.e. having a set of atomic structural coordinates that have a root mean square deviation of less than or equal to about 2A when superimposed with the atomic structure coordinates of the native SAM domain from which the mutant is derived when at least 50% to 100% of the atoms of the native SAM domain are included in the superimposition. It should be noted that the mutants contemplated herein need not exhibit SAM domain activity.

The derivative crystals of the invention generally comprise a crystalline SAM domain in covalent association with one or more heavy metal atoms. The SAM domain may correspond to a native or mutated SAM domain. Heavy metal atoms useful for providing derivative crystals include by way of example, and not limitation gold, mercury, etc.

The invention features a crystalline form of a SAM domain in association with one or more compounds. The association may be covalent or non-covalent. These types of crystalline forms are referred to herein as co-crystals. The compound may be any organic molecule, and it may modulate the function of a SAM domain by for example inhibiting or enhancing its function, or it may be an analogue of a SAM domain. It is preferred that the geometry of the compound and the interactions formed between the compound and the SAM domain provide high affinity binding between the two molecules. High affinity binding is preferably governed by a dissociation equilibrium constant on the order of 10^'6 or less. Method for Preparing Crystal Forms of SAM Domain

The invention also features a method for creating the crystalline SAM domain structures described herein. The method may utilize a polypeptide comprising a SAM domain described herein to form a crystal. A polypeptide used in the method may be chemically synthesized in whole or in part using techniques that are well-known in the art. Alternatively, methods are well known to the skilled artisan to construct expression vectors containing the native or mutated SAM domain coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See for example the techniques described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.

Crystals are grown from an aqueous solution containing the purified and concentrated SAM domain polypeptide by a variety of conventional processes. These processes include batch, liquid, bridge, dialysis, vapor diffusion, and hanging drop methods. (See for example, McPherson, 1982 John Wiley, New York; McPherson, 1990, Eur. J. Biochem. 189: 1-23; Webber. 1991, Adv. Protein Chem. 41 :1-36). Generally, the native crystals of the invention are grown by adding precipitants to the concentrated solution of the SAM domain polypeptide. The precipitants are added at a concentration just below that necessary to precipitate the protein. Water is removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases. In an embodiment of the invention, the method generally comprises the steps of (a) mixing a volume of polypeptide solution with a reservoir solution; and

(b) incubating the mixture obtained in step (a) over the reservoir solution in a closed container, under conditions suitable for crystallization.

For crystals of the invention, it has been found that hanging drops containing about 1 μl of SAM domain polypeptide (50-150 mg/ml, preferably 100 mg/ml, in 5-2-mM, preferably 7mM Hepes pH 5.5 to 9, preferably 7.5) and equal volumes of reservoir buffer (50-150 mM, preferably lOOmM cacodylate pH 5.5 to 7.5, preferably 6.5; 5-10% preferably 7% (w/v) PEG 8000; and 10-30%, preferably 20% (v/v) ethylene glycol) suspended overnight at room temperature provide crystals suitable for high resolution X-ray structure determination. It will be appreciated that the above- described crystallization conditions can be varied and such variations can be used alone or in combination. For example other buffer solutions such as Tris-HCL buffer may be used.

Derivative crystals of the invention can be obtained by soaking native crystals in a solution containing salts of heavy metal atoms. Co-crystals of the invention can be obtained by soaking a native crystal in a solution containing a compound that binds the SAM domain, or they can be obtained by co-crystallizing the SAM domain polypeptide in the presence of one or more compounds that bind to the SAM domain.

Once the crystal is grown it can be placed in a glass capillary tube and mounted onto a holding device connected to an X-ray generator and an X-ray detection device. Collection of X-ray diffraction patterns are well documented by those skilled in the art (See for example, Ducruix and Geige, 1992, IRL Press, Oxford, England). A beam of X-rays enter the crystal and diffract from the crystal. An X-ray detection device can be utilized to record the diffraction patterns emanating from the crystal. Suitable devices include the Marr 345 imaging plate detector system with an RU200 rotating anode generator.

Methods for obtaining the three dimensional structure of the crystalline form of a molecule or complex are described herein and known to those skilled in the art (see Ducruix and Geige, supra). Generally, the unit cell dimensions and orientation in the crystal can be determined from the spacing between the diffraction emissions as well as the patterns made from the emissions. The symmetry of the unit cell in the crystal is also determined. Each diffraction pattern emission is characterized as a vector and the data collected at this stage determines the amplitude of each vector. The phases of the vectors may be determined by the isomorphous replacement method where heavy atoms soaked into the crystal are used as reference points in the X-ray analysis (see for example, Otwinowski, 1991, Daresbury, United Kingdom, 80-86). The phases of the vectors may also be determined by molecular replacement (see for example, Naraza, 1994, Proteins 11:281-296). The amplitudes and phases of vectors from the crystalline form of an Eph SAM domain, preferably an EphA4 SAM domain, determined in accordance with these methods can be used to analyze other crystalline SAM domains.

The unit cell dimensions and symmetry, and vector amplitude and phase information can be used in a Fourier transform function to calculate the electron density in the unit cell i.e. to generate an experimental electron density map. This may be accomplished used the PHASES package (Furey,

1990). Amino acid sequence structures are fit to the experimental electron density map (ie. model building) using computer programs (e.g. Jones, TA. et al, Acta Crystallogr A47, 100-119, 1991) to calculate a theoretical electron density map. The theoretical and experimental electron density maps can be compared and the agreement between the maps can be described by a parameter referred to as R-factor. A high degree of overlap in the maps is represented by a low value R-factor. The R-factor can be minimized by using computer programs that refine the theoretical electron density map. For example, the XPLOR program, developed by Brunger (1992, Nature 355:472-475) can be used for model refinement.

A three dimensional structure of the molecule or complex may be described by atoms that fit the theoretical electron density characterized by a minimum R value. Files can be created for the structure that defines each atom by coordinates in three dimensions. Identification of Homologues

The knowledge of the three dimensional structure of a SAM domain, in particular the EphA4 SAM domain, enables one skilled in the art to identify homologues. This is achieved by searches of three-dimensional databases. Since structural folds are conserved to a greater extent than sequence, one may identify homologues with very little sequence similarity. Programs that provide this type of database searching are known in the art and include Dali. The structural coordinates of a protein structure are submitted and the program performs a multiple structural alignment with proteins in the protein data bank. Methods for Determining Three Dimensional Structures The structure coordinates of a SAM domain structure described herein can be used as a model for determining the three dimensional structures of additional native or mutated SAM domains with unknown structure, as well as the structures of co-crystals of SAM domains with compounds such as modulators (e.g. agonists or antagonists). The structure coordinates and models of a SAM domain three dimensional structure can also be used to determine solution-based structures of native or mutant SAM domains.

Three dimensional structure may be determined by applying the structural coordinates of a SAM domain structure to other data such as an amino acid sequence, X-ray crystallographic diffraction data, or nuclear magnetic resonance (NMR) data. Homology modeling, molecular replacement, and nuclear magnetic resonance methods using these other data sets are described below.

Homology modeling (also known as comparative modeling or knowledge-based modeling) methods develop a three dimensional model from a polypeptide sequence based on the structures of known proteins. In the present invention the method utilizes a computer representation of the three dimensional structure of a SAM domain, preferably the EphA SAM domain, more preferably the EphA4 SAM domain, or a complex of same, a computer representation of the amino acid sequence of a polypeptide with an unknown structure, and standard computer representations of the structures of amino acids. The method in particular comprises the steps of; (a) identifying structurally conserved and variable regions in the known structure; (b) aligning the amino acid sequences of the known structure and unknown structure (c) generating coordinates of main chain atoms and side chain atoms in structurally conserved and variable regions of the unknown structure based on the coordinates of the known structure thereby obtaining a homology model; and (d) refining the homology model to obtain a three dimensional structure for the unknown structure. This method is well known to those skilled in the art (Greer, 1985, Sceince 228, 1055; Bundell et al 1988, Eur. J. Biochem. 172, 513; Knighton et al., 1992, Science 258:130-135, http://biochem.vt.edu/courses/modeling/homology.htn). Computer programs that can be used in homology modeling are Quanta and the Homology module in the Insight II modeling package distributed by Molecular Simulations Inc, or MODELLER (Rockefeller University, www.iucr.ac.uk/sinris-top/logical/prg-modeller.html). In step (a) of the homology modeling method, the known SAM domain structure (e.g. structure of the EphA4 SAM domain) is examined to identify the structurally conserved regions (SCRs) from which an average structure, or framework, can be constructed for these regions of the protein. Variable regions (VRs), in which known structures may differ in conformation, also must be identified. SCRs generally correspond to the elements of secondary structure, such as alpha-helices (the four α-helices in the EphA4 SAM domain) and beta-sheets, and to ligand- and substrate-binding sites. The VRs usually lie on the surface of the proteins and form the loops where the main chain turns.

Many methods are available for sequence alignment of known structures and unknown structure. Sequence alignments generally are based on the dynamic programming algorithm of Needleman and Wunsch [J. Mol. Biol. 48: 442-453, 1970]. Current methods include FASTA, Smith- Waterman, and BLASTP, with the BLASTP method differing from the other two in not allowing gaps. Scoring of alignments typically involves construction of a 20x20 matrix in which identical amino acids and those of similar character (i.e., conservative substitutions) may be scored higher than those of different character. Substitution schemes which may be used to score alignments include the scoring matrices PAM (Dayhoff et al., Meth. Enzymol. 91 : 524-545, 1983), and BLOSUM (Henikoff and Henikoff, Proc. Nat. Acad. Sci. USA 89: 10915-' 0919, 1992), and the matrices based on alignments derived from three-dimensional structures including that of Johnson and Overington (JO matrices) (J. Mol. Biol. 233: 716-738, 1993).

Alignment based solely on sequence may be used, though other structural features also may be taken into account. In Quanta, multiple sequence alignment algorithms are available that may be used when aligning a sequence of the unknown with the known structures. Four scoring systems (i.e. sequence homology, secondary structure homology, residue accessibility homology, CA-CA distance homology) are available, each of which may be evaluated during an alignment so that relative statistical weights may be assigned. When generating coordinates for the unknown structure, main chain atoms and side chain atoms, both in SCRs and VRs need to be modeled. A variety of approaches known to those skilled in the art may be used to assign coordinates to the unknown. In particular, the coordinates of the main chain atoms of SCRs will be transferred to the unknown structure. VRs correspond most often to the loops on the surface of the polypeptide and if a loop in the known structure is a good model for the unknown, then the main chain coordinates of the known structure may be copied. Side chain coordinates of SCRs and VRs are copied if the residue type in the unknown is identical to or very similar to that in the known structure. For other side chain coordinates, a side chain rotamer library may be used to define the side chain coordinates. When a good model for a loop cannot be found fragment databases may be searched for loops in other proteins that may provide a suitable model for - l i the unknown. If desired, the loop may then be subjected to conformational searching to identify low energy conformers if desired.

Once a homology model has been generated it should be analyzed to determine its correctness. A computer program available to assist in this analysis is the Protein Health module in Quanta which provides a variety of tests. Other programs that provide structure analysis along with output include PROCHECK and 3D-Profiler [Luthy R. et al, Nature 356: 83-85, 1992; and Bowie, J.U. et al, Science 253: 164-170, 1991]. Once any irregularities have been resolved, the entire structure may be further refined. Refinement may consist of energy minimization with restraints, especially for the SCRs. Restraints may be gradually removed for subsequent minimizations. Molecular dynamics may also be applied in conjunction with energy minimization.

Molecular replacement involves applying X-ray diffraction data of a known structure to the incomplete X-ray crystallographic data set of a polypeptide of unknown structure. The method can be used to define the phases describing the X-ray diffraction data of a polypeptide of unknown structure when only the amplitudes are known. Commonly used computer software packages for molecular replacement are X-PLOR (Brunger 1992, Nature 355: 472-475), AMoRE (Navaza, 1994, Acta Crystallogr. A50:157-163), the CCP4 package (Collaborative Computational Project, Number 4, "The CCP4 Suite: Programs for Protein Crystallography", Acta Cryst., Vol. D50, pp. 760-763, 1994), and the MERLOT package (P.M.D. Fitzgerald, J. Appl. Cryst., Vol. 21, pp. 273-278, 1988). It is preferable that the resulting structure not exhibit a root-mean-square deviation of more than 3 A. The objective of molecular replacement is to align positions of atoms in the unit cell by matching electron diffraction data from two crystals. Molecular replacement computer programs generally involve the following steps: (1) determining the number of molecules in the unit cell and defining the angles between them; (2) rotating the diffraction data to define the orientation of the molecules in the unit cell; (3) translating the electron density in three dimensions to correctly position the molecules in the unit cell; (4) determining the amplitudes and phases of the X-ray diffraction data and calculating an R-factor calculated from the reference data set and from the new data wherein an R-factor between 30-50% indicates that the orientations of the atoms in the unit cell have been reasonably determined by the method; and (5) optionally decreasing the R-factor to about 20% by refining the new electron density map using iterative refinement techniques known to those skilled in the art.

In an embodiment of the invention, a method is provided for determining three dimensional structures of polypeptides with unknown structure by applying the structural coordinates of a SAM domain structure to an incomplete X-ray crystallographic data set for a polypeptide of unknown structure, and determining a low energy conformation of the resulting structure. The structural coordinates of a SAM domain structure may be applied to nuclear magnetic resonance (NMR) data to determine the three dimensional structures of polypeptides. (See for example, Wuthrich, 1986, John Wiley and Sons, New York: 176-199; Pflugrath et al., 1986, J. Molecular Biology 189: 383-386; Kline et al., 1986 J. Molecular Biology 189:377-382). While the secondary structure of a polypeptide may often be determined by NMR data, the spatial connections between individual pieces of secondary structure are not as readily determined. The structural coordinates of a polypeptide defined by X-ray crystallography can guide the NMR spectroscopist to an understanding of the spatial interactions between secondary structural elements in a polypeptide of related structure. Information on spatial interactions between secondary structural elements can greatly simplify Nuclear Overhauser Effect (NOE) data from two-dimensional NMR experiments. In addition, applying the structural coordinates after the determination of secondary structure by NMR techniques simplifies the assignment of NOE's relating to particular amino acids in the polypeptide sequence and does not greatly bias the NMR analysis of polypeptide structure.

In an embodiment, the invention relates to a method of determining three dimensional structures of polypeptides with unknown structures by applying the structural coordinates of a SAM domain structure to nuclear magnetic resonance (NMR) data of the unknown structure. This method comprises the steps of: (a) determining the secondary structure of an unknown structure using NMR data; and (b) simplifying the assignment of through-space interactions of amino acids. The term " through-space interactions" defines the orientation of the secondary structural elements in the three dimensional structure and the distances between amino acids from different portions of the amino acid sequence. The term "assignment" defines a method of analyzing NMR data and identifying which amino acids give rise to signals in the NMR spectrum. Identification of Potential Modulators of SAM Domains

Modulators of a SAM domain may be designed and identified that may modify the inappropriate activity of a SAM domain involved in a clinical disorder. The rational design and identification of modulators of SAM domains can be accomplished by utilizing the atomic structural coordinates that define a SAM domain's three dimensional structure.

Modulators may include substances that bind to or mimic the residues of a SAM domain that are required for dimerization of SAM domains. For example, a substance that binds to or mimics the interface residues of an EphA SAM domain (e.g. Val 913, Val 914, Met 972, Met 976, Met 979, Val 944, and Leu 940), or the proximal residues of an EphA SAM domain (e.g. He 959 to Lys) may modify inappropriate activity of a SAM domain involved in a clinical disorder.

Structure-based modulator design identification methods are powerful techniques that can involve searches of computer databases containing a variety of potential modulators and chemical functional groups. (See Kuntz et al., 1994, Ace. Chem. Res. 27:117; Guida, 1994, Current Opinion in Struc. Biol. 4: 777; and Colman, 1994, Current Opinion in Struc. Biol. 4: 868, for reviews of structure-based drug design and identification;and Kuntz et al 1982, J. Mol. Biol. 162:269; Kuntz et al., 1994, Ace. Chem. Res. 27: 117; Meng et al., 1992, J. Compt. Chem. 13: 505; Bohm, 1994, J. Comp. Aided Molec. Design 8: 623 for methods of structure-based modulator design). The SAM domain three dimensional structure described herein, and the three dimensional structures of other polypeptides determined by the homology modeling, molecular replacement, and NMR techniques described herein can also be applied to modulator design and identification methods. Modulators of SAM domains may be identified by docking the computer representation of compounds from a database of molecules. Databases which may be used include ACD (Molecular Designs Limited), NCI (National Cancer Institute), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited), Maybridge (Maybridge Chemical Company Ltd), Aldrich (Aldrich Chemical Company), DOCK (University of California in San Francisco), and the Directory of Natural Products (Chapman & Hall). Computer programs such as CONCORD (Tripos Associates) or DB-Converter (Molecular Simulations Limited) can be used to convert a data set represented in two dimensions to one represented in three dimensions. Generally, the computer programs comprise the following steps:

(a) docking the structure of a compound into an active-site of a polypeptide (e.g.. EphA4 SAM domain) using the computer program, or by interactively moving the compound into the active-site;

(b) characterizing the geometry and the complementary interactions formed between the atoms of the active-site and the compound; and optionally

(c) searching libraries for molecular fragments which can fit into the empty space between the compound and active site and can be linked to the compound; and

(d) linking the fragments found in (c) to the compound and evaluating the new modified compound. "Docking" refers to a process of placing a compound in close proximity with an active site of a polypeptide (e.g.. an Eph SAM domain), or a process of finding low energy conformations of a compound/polypeptide complex (e.g. compound Eph SAM domain).

Examples of other computer programs that may be used for structure-based modulator design are CAVEAT (Bartlett et al., 1989, in "Chemical and Biological Problems in Molecular Recognition", Roberts, S.M. Ley, S.V.; Campbell, N.M. eds; Royal Society of Chemistry: Cambridge, pp 182-196); FLOG (Miller et al., 1994, J. Comp. Aided Molec. Design 8:153); PRO Modulator (Clark et al., 1995 J. Comp. Aided Molec. Design 9:13); MCSS (Miranker and Karplus, 1991, Proteins: Structure, Fuction, and Genetics 8:195); and, GRID (Goodford, 1985, J. Med. Chem. 28:849). In an embodiment of the invention, a method is provided for identifying potential modulators of SAM domain function. The method utilizes the structural coordinates of a SAM domain three dimensional structure. The method comprises the steps of (a) removing a computer representation of a SAM domain structure, preferably an Eph SAM domain structure, more preferably an EphA4 SAM domain structure, and docking a computer representation of a compound from a computer data base with a computer representation of the active site of the SAM domain; (b) determining a conformation of the complex with a favourable geometric fit or favorable complementary interactions; and (c) identifying compounds that best fit the SAM domain active-site as potential modulators of SAM domain function. The initial SAM domain structure may or may not have compounds bound to it. A favourable geometric fit occurs when the surface areas of a compound in a compound-SAM domain complex is in close proximity with the surface area of the active-site of the SAM domain without forming unfavorable interactions. A favourable complementary interaction occurs where a compound in a compound-SAM domain complex interacts by hydrophobic, aromatic, ionic, or hydrogen donating and accepting forces, with the active-site of a SAM domain without forming unfavorable interactions. Unfavourable interactions may be steric hindrance between atoms in the compound and atoms in the SAM active-site.

In another embodiment, potential modulators are identified utilizing a three dimensional structure of a SAM domain with or without compounds bound to it. The method comprises the steps of (a) modifying a computer representation of a SAM domain (e.g. an Eph SAM domain) having one or more compounds bound to it, where the computer representations of the compound or compounds and SAM domain are defined by atomic structural coordinates; (b) determining a conformation of the complex with a favorable geometric fit and favorable complementary interactions; and (c) identifying the compounds that best fit the SAM active site as potential modulators. A computer representation may be modified by deleting or adding a chemical group or groups. Computer representations of the chemical groups can be selected from a computer database.

Another way of identifying potential modulators is to modify an existing modulator in the polypeptide active-site. The computer representation of modulators can be modified within the computer representation of a SAM domain active-site. This technique is described in detail in Molecular Simulations User Manual, 1995 in LUDI. The computer representation of a modulator may be modified by deleting a chemical group or groups, or by adding a chemical group or groups. After each modification to a compound, the atoms of the modified compound and active-site can be shifted in conformation and the distance between the modulator and the active site atoms may be scored on the basis of geometric fit and favourable complementary interactions between the molecules. Compounds with favourable scores are potential modulators. Compounds designed by modulator building or modulator searching computer programs may be screened to identify potential modulators. Examples of such computer programs include programs in the Molecular Simulations Package (Catalyst), ISIS/HOST, ISIS/BASE, and ISIS/DRAW (Molecular Designs Limited), and UNITY (Tripos Associates). A building program may be used to replace computer representations of chemical groups in a compound complexed with a SAM domain with groups from a computer data base. A searching program may be used to search computer representations of compounds from a computer database that have similar three dimensional structures and similar chemical groups as a compound that binds to a SAM domain. The programs may be operated on the structure of the active-site of the three dimensional structure of an Eph SAM domain, preferably an EphA4 SAM domain. A typical program may comprise the following steps:

(a) mapping chemical features of the compound such as by hydrogen bond donors or acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or negatively ionizable sites;

(b) adding geometric constraints to selected mapped features; (c) searching data bases with the model generated in (b). In an embodiment of the invention a method of identifying potential modulators of a SAM domain, preferably an Eph SAM domain, more preferably an EphA SAM domain, is provided using the three dimensional conformation of the SAM domain in various modulator construction or modulator searching computer programs on compounds complexed with the SAM domain. The method comprises the steps of (a) removing a computer representation of one or more compounds complexed with a SAM domain; (b) (i) searching a data base for a compound with a similar geometric structure or similar chemical groups to the removed compounds using a computer program that searches computer representations of compounds from a database that have similar three dimensional structures and similar chemical groups, or (ii) replacing portions of the compounds complexed with the SAM domain with similar chemical structures (i.e. nearly identical shape and volume) from a database using a compound construction computer program that replaces computer representations of chemical groups with groups from a computer database, where the representations of the compounds are defined by structural coordinates. Potential modulators of SAM domains identified using the above-described methods may be prepared using methods described in standard reference sources utilized by those skilled in the art. For example, organic compounds may be prepared by organic synthetic methods described in references such as March, 1994 Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, New York, McGraw Hill. Cellular assays, as well as animal model assays in vivo, may be used to test the activity of a potential modulator of a SAM domain as well as diagnose a disease associated with inappropriate SAM domain activity. In vivo assays are also useful for testing the bioactivity of a potential modulator designed by the methods of the invention.

The invention also relates to a potential modulator identified by the methods of the invention. Peptides

The invention provides peptide molecules that modulate SAM domain function. The molecules are derived from the interface residues necessary for dimer formation. For example, peptides of the invention include the amino acids Val 913, Val 914, Met 972, Met 976, Met 979, Val 944, and Leu 940 of the EphA4 SAM domain. Other proteins containing sequences corresponding to the sequences necessary for dimer formation of a SAM domain may be identified with a protein homology search, for example by searching available databases such as GenBank or SwissProt and various search algorithms and/or programs may be used including FASTA, BLAST (available as a part of the GCG sequence analysis package, University of Wisconsin, Madison, Wis.), or ENTREZ (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD).

In accordance with an embodiment of the invention, specific peptides are contemplated that mediate SAM domain function comprising VVSV (SEQ ID. NO. 21), SAVVSV (SEQ ID. N0.22), FSAVV (SEQ ID. NO.23 ), FSAVVSV (SEQ ID. NO. 24), FSAVVSVGD (SEQ ID. NO. 25), VVSVGDWL (SEQ ID. NO. 26), FNTV (SEQ ID. NO. 27), FNTVDE (SEQ ID. NO. 28), FNTVDEWL (SEQ ID. NO. 29), TSFNTVDEWL (SEQ ID. NO. 30), TSFNTV (SEQ ID. NO. 31), YTSFNTV (SEQ ID. NO. 32), RSEV (SEQ ID. NO. 33), RSEVLG (SEQ ID. NO. 34), RSEVLGWD (SEQ ID. NO. 35), VPFRSEV (SEQ ID. NO. 36), and VPFRSEVLGW (SEQ ID. NO. 37).

In accordance with another embodiment of the invention, specific peptides are contemplated that mediate SAM domain function. In particular, a peptide of the formula I is provided which mediates SAM domain function:

wherein X and X r-6 represent 0 to 70, preferably 0 to 50 amino acids, more preferably 2 to 20 amino acids, and X¹ represents Leu, Phe, Asp, Ala, Glu, or Gly, preferably Leu or Gly, X² represents Glu, Asp, Ser, He, Ala, Arg, Lys, and Gin, preferably Glu or Asp, X³ represents Ala, Val, Glu, Phe, Ser, He, Met, Leu, His, Gin, Arg, or Asp preferably Ala, Val, or Phe, X⁴ is Val, Leu, Met, Phe, and He, preferably Val or Leu, or Phe, X⁵ is Val, Ser, Leu, Asp, Ala, Pro, Asn, Lys, or Cys, preferably Val or Ser.

In an embodiment of the present invention a peptide of the formula I is provided: wherein X represents TT, ID, TS, DD, GYTT (SEQ ID. NO. 38), AAGYTT (SEQ ID. NO. 39), FTAAGYTT (SEQ ID. NO. 40), DNFTAAGYTT (SEQ ID. NO. 41), or YKDNFTAAGYTT (SEQ ID. NO. 42). In another embodiment X⁶ represents HM, HMSQ (SEQ ID. NO. 43), HMSQD (SEQ ID. NO. 44), HMSQDD (SEQ ID. NO. 45), HMSQDDLA (SEQ ID. NO. 46), QMMM (SEQ ID. NO. 47), QMMMED (SEQ ID. NO. 48), QMMMEDLL (SEQ ID. NO. 49), DITE (SEQ ID. NO. 50), DITEED (SEQ ID. NO. 51), DITEEDL (SEQ ID. NO. 52), NLTE (SEQ ID. NO. 53), NLTEND (SEQ ID. NO. 54), NLTENDI (SEQ ID. NO. 55). Preferred peptides of the formula I include the following: X-LEAVV-X⁶, X-FDVVS-X⁶, X-

LEFLS-X⁶, X-GARFL-X⁶, LEAVV (SEQ ID. NO. 56), TTLEAVV (SEQ ID. NO. 57), LEAVVHM (SEQ ID. NO. 58), LEAVVHMSQ (SEQ ID. NO. 59), LEAVVHMSQD (SEQ ID. NO. 60), LEAVVHMSQDDL (SEQ ID. NO. 61), LEAVVHMSQDDLAR (SEQ ID. NO. 62), TTLEAVVHMS (SEQ ID. NO. 63), TTLEAVVHMSQD (SEQ ID. NO. 64), TTLEAVVHMSQDDL (SEQ ID. NO. 65), TTLEAVVHMSQDDLAR (SEQ ID. NO. 66), GYTTLEAVV (SEQ ID. NO. 67), GYTTLEAVVHMS (SEQ ID. NO. 68), GYTTLEAVVHMSQD (SEQ ID. NO. 69), GYTTLEAVVHMSQDDL (SEQ ID. NO. 70), GYTTLEAVVHMSQDDLAR (SEQ ID. NO. 71), FDVVS (SEQ ID. NO. 72), FDVVSQ (SEQ ID. NO. 73), FDVVSQMM (SEQ ID. NO. 74), FDVVSQMMME (SEQ ID. NO. 75), FDVVSQMMMEDIL (SEQ ID. NO. 76), TSFDVVS (SEQ ID. NO. 77), TSFDVVSQ (SEQ ID. NO. 78), TSFDVVSQMM (SEQ ID. NO. 79), TSFDVVSQMMME (SEQ ID. NO. 80), TSFDVVSQMMMEDIL (SEQ ID. NO. 81), LEFLS (SEQ ID. NO. 82), LEFLSD (SEQ ID. NO. 83), LEFLSDIT (SEQ ID. NO. 84), LEFLSDITEE (SEQ ID. NO. 85), LEFLSDITEEDL (SEQ ID. NO. 86), DDLEFLS (SEQ ID. NO. 87), GWDDLEFLS (SEQ ID. NO. 88), DDLEFLSD (SEQ ID. NO. 89), DDLEFLSDIT (SEQ ID. NO. 90), DDLEFLSDITEE (SEQ ID. NO. 91), DDLEFLSDITEEDL (SEQ ID. NO. 92), GARFL (SEQ ID. NO. 93), GARFLN (SEQ ID. NO. 94), GARFLNLT (SEQ ID. NO. 95), GARFLNLTEN (SEQ ID. NO. 96), and IDGARFL (SEQ ID. NO. 97).

In accordance with another embodiment of the invention, specific peptides are contemplated that mediate SAM domain function. In particular, a peptide of the formula II is provided which mediates SAM domain function:

X⁷-X⁸-X⁹-X¹⁰-Xⁿ-X¹²-X¹³-X¹⁴-X^I5-X¹⁶ II

wherein X⁷ and X¹⁶ represent 0 to 70, preferably 0 to 50 amino acids, more preferably 2 to 20 amino acids, and X⁸ represents Met, He, Ser, Leu, Asn, Phe, or Val, preferably Met, X⁹ represents Arg, Ser, Lys, Met, Leu, Glu, Gin, or Asn, preferably Gin or Arg, X¹⁰ represents Thr, Ala, Arg, Leu, Ser, Glu, Asp, Met, Lys, Gin, or Gly, preferably Thr, Ala, or Glu, X¹¹ represents Gin, Ser, Glu, Leu, Phe, Asp, Thr, Arg, preferably Gin or Arg, X¹² represents Met, Ala, He, Asn, Ser, Arg, Thr, Pro, Leu, Gin, Val, Lys, preferably Met or Arg, X¹³ represents Gin, Asn, Pro, Ser, Tyr, Glu, Leu, Arg, or Lys, preferably Gin, Asn, or Arg, X¹⁴ represents Gin, Ala, Pro, Asp, Leu, Lys, He, Glu, Arg, or Asn, preferably Gin or He, and X¹⁵ represents Met, He, Val, His, Ser, Arg, Lys, Phe, Cys, Glu, Tyr, Ala, He, Tip, or Leu.

In an embodiment of the present invention a peptide of the formula II is provided: wherein X⁷ represents QA, QV, NK, SVQA (SEQ ID. NO. 98), LSSVQA (SEQ ID. NO. 99), ILSSVQA (SEQ ID. NO. 100), NKILSSVQA (SEQ ID. NO. 101), HQNKILSSVQA (SEQ ID. NO. 102), THQNKILSSVQA (SEQ ID. NO. 103), ENIK (SEQ ID. NO. 104), SQEINK (SEQ ID. NO. 105), KLSQEINK (SEQ ID. NO. 106), ILNSIQV (SEQ ID. NO. 107), or NSIQV (SEQ ID. NO. 108). In another embodiment X⁷ is HG, QS, HGRM (SEQ ID. NO. 109), HGRMVP (SEQ ID. NO. 110), QSVEV (SEQ ID. NO. 111), or TRKP (SEQ ID. NO. 112). Preferred peptides of the formula II include the following: X⁷-MRTQMQQM-X¹⁶, X⁷-

MRAQMNQI-X¹⁶, X⁷-NEERRSIF-X¹⁶, MRTQMQQM (SEQ ID. NO. 113), QAMRTQMQQM (SEQ ID. NO. 1 14), SVQAMRTQMQQM (SEQ ID. NO. 115), LSSVQAMRTQMQQM (SEQ ID. NO. 116), ILSSVQAMRTQMQQM (SEQ ID. NO. 117), MRTQMQQMHG (SEQ ID. NO. 118), MRTQMQQMHGRM (SEQ ID. NO. 119), MRTQMQQMHGRMVPV (SEQ ID. NO. 120), NEERRSIF (SEQ ID. NO. 121), INKNEERRSIF (SEQ ID. NO. 122), NEERRSIFTRKP (SEQ ID. NO. 123). MRAQMNQI (SEQ ID. NO. 124), MRAQMNQIQS (SEQ ID. NO. 125), MRAQMNQIQSVEV (SEQ ID. NO. 126).

All of the peptides of the invention, as well as molecules substantially homologous, complementary or otherwise functionally or structurally equivalent to these peptides may be used for purposes of the present invention. In addition to full-length peptides of the invention, truncations of the peptides are contemplated in the present invention. Truncated peptides may comprise peptides of about 7 to 10 amino acid residues

The truncated peptides may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy- carbonyl (PMOC) group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end. The truncated peptides may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end.

The peptides of the invention may also include analogs of a peptide of the invention and/or truncations of the peptide, which may include, but are not limited to a peptide of the invention containing one or more amino acid insertions, additions, or deletions, or both. Analogs of the peptide of the invention exhibit the activity characteristic of the peptide e.g. interference with SAM domain dimer formation, and may further possess additional advantageous features such as increased bioavailability, stability, or reduced host immune recognition. One or more amino acid insertions may be introduced into a peptide of the invention. Amino acid insertions may consist of a single amino acid residue or sequential amino acids.

One or more amino acids, preferably one to five amino acids, may be added to the right or left termini of a peptide of the invention. Deletions may consist of the removal of one or more amino acids, or discrete portions from the peptide sequence. The deleted amino acids may or may not be contiguous. The lower limit length of the resulting analog with a deletion mutation is about 7 amino acids.

The invention also includes a peptide conjugated with a selected protein, or a selectable marker (see below) to produce fusion proteins.

The peptides of the invention may be prepared using recombinant DNA methods. Accordingly, nucleic acid molecules which encode a peptide of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the peptide. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses so long as the vector is compatible with the host cell used. The expression vectors contain a nucleic acid molecule encoding a peptide of the invention and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Suitable regulatory sequences may be obtained from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may also be incorporated into the expression vector. The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, β- galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes that encode a fusion portion which provides increased expression of the recombinant peptide; increased solubility of the recombinant peptide; and or aid in the purification of the recombinant peptide by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be inserted in the recombinant peptide to allow separation of the recombinant peptide from the fusion portion after purification of the fusion protein. Examples of fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.

Recombinant expression vectors may be introduced into host cells to produce a transformant host cell. Transformant host cells include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to include the introduction of nucleic acid (e.g. a vector) into a cell by one of many techniques known in the art. For example, prokaryotic cells can be transformed with nucleic acid by electroporation or calcium- chloride mediated transformation. Nucleic acid can be introduced into mammalian cells using conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells may be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the peptides of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).

The peptides of the invention may be tyrosine phosphorylated using the method described in Reedijk et al. (The EMBO Journal 11(4): 1365, 1992). For example, tyrosine phosphorylation may be induced by infecting bacteria harbouring a plasmid containing a nucleotide sequence encoding a peptide of the invention, with a λgtl 1 bacteriophage encoding the cytoplasmic domain of the Elk tyrosine kinase as a LacZ-Elk fusion. Bacteria containing the plasmid and bacteriophage as a lysogen are isolated. Following induction of the lysogen, the expressed peptide becomes phosphorylated by the Elk tyrosine kinase.

The peptides of the invention may be synthesized by conventional techniques. For example, the peptides may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J.D. Young, Solid Phase Peptide Synthesis, 2^nd Ed., Pierce Chemical Co., Rockford III. (1984) and G. Barany and R.B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky, Principles fo Peptide Synthesis, Springer- Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biologu, suprs, Vol 1, for classical solution synthesis). By way of example, the peptides may be synthesized using 9-fluorenyl methoxycarbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphotyrosine as the N- fluorenylmethoxy-carbonyl-O-dimethyl phosphono-L-tyrosine derivative.

N-terminal or C-terminal fusion proteins comprising a peptide of the invention conjugated with other molecules may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the peptide, and the sequence of a selected protein or selectable marker with a desired biological function. The resultant fusion proteins contain the peptide fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc. Cyclic derivatives of the peptides of the invention are also part of the present invention.

Cyclization may allow the peptide to assume a more favorable conformation for association with molecules in complexes of the invention. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The side chains of Tyr and Asn may be linked to form cyclic peptides. The components that form the bonds may be side chains of amino acids, non-amino acid components or a combination of the two. In an embodiment of the invention, cyclic peptides are contemplated that have a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.

It may be desirable to produce a cyclic peptide that is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations. Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of the proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.

Peptides of the invention may be developed using a biological expression system. The use of these systems allows the production of large libraries of random peptide sequences and the screening of these libraries for peptide sequences that interact with particular amino acid residues. Libraries may be produced by cloning synthetic DNA that encodes random peptide sequences into appropriate expression vectors, (see Christian et al 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404; Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries may also be constructed by concurrent synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).

Peptides of the invention may be used to identify lead compounds for drug development. The structure of the peptides described herein can be readily determined by a number of methods such as NMR and X-ray crystallography. A comparison of the structures of peptides similar in sequence, but differing in the biological activities they elicit in target molecules can provide information about the structure-activity relationship of the target. Information obtained from the examination of structure-activity relationships can be used to design either modified peptides, or other small molecules or lead compounds which can be tested for predicted properties as related to the target molecule. The activity of the lead compounds can be evaluated using assays similar to those described herein.

Information about structure-activity relationships may also be obtained from co- crystallization studies. In these studies, a peptide with a desired activity is crystallized in association with a target molecule i.e. SAM domain, and the X-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison may be used to design compounds expected to possess desired activities.

The peptides of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids. The peptides of the invention may be used to prepare antibodies. Conventional methods can be used to prepare the antibodies.

The peptides and antibodies specific for the peptides of the invention may be labelled using conventional methods with various enzymes, fluorescent materials, luminescent materials and radioactive materials. Suitable enzymes, fluorescent materials, luminescent materials, and radioactive material are well known to the skilled artisan. Antibodies and labeled antibodies specific for the peptides of the invention may be used to screen for proteins containing SAM domains. Computer modelling techniques known in the art may also be used to observe the interaction of a peptide of the invention, and truncations and analogs thereof with a SAM domain (for example, Homology Insight II and Discovery available from BioSym/Molecular Simulations, San Diego, California, U.S.A.). If computer modelling indicates a strong interaction, the peptide can be synthesized and tested for its ability to interfere with SAM domain dimer formation. Compositions and Methods of Treatment

A purified three dimensional SAM domain structure of the invention, the peptides of the invention, and the modulators identified using the methods of the invention may be used to modify the inappropriate activity of a SAM domain involved in a clinical disorder. They may be used in the treatment and diagnosis of disorders associated with aberrant T cell signaling and to modulate telomere function. In particular, they may be useful in methods for therapy of cellular senescence and immortalization controlled by telomere length and telomerase activity, and as selective immunosuppressants (e.g. in organ transplantation). They may also be useful in the treatment of cancers, such as melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiocarcinoma, squamous cell carcinoma, CLL, and cancers of the pancreas, breast, brain, prostate, bladder, thyroid, ovary, uterus, testis, kidney, stomach, colon and rectum, particularly leukemia including B-cell leukemia, T-cell leukemia, null-cell leukemia, myelogenous leukemia, and lymphocytic leukemia,

Further, the three dimensional SAM domain structure of the invention, the peptides of the invention, and the modulators identified using the methods of the invention may be used to modulate the biological activity of an Eph receptor or Eph ligand in a cell, including inhibiting or enhancing signal transduction activities of the receptor or ligand, and in particular modulating a pathway in a cell regulated by the ligand or receptor, particularly those pathways involved in neuronal development, axonal migration, pathfinding and regeneration. The three dimensional SAM domain structure of the invention, the peptides of the invention, and modulators identified using the methods of the invention will be useful as pharmaceuticals to modulate axonogenesis, nerve cell interactions and regeneration, to treat conditions such as neurodegenerative diseases and conditions involving trauma and injury to the nervous system, for example Alzheimer's disease, Parkinson's disease, Huntington's disease, demyelinating diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, bacterial and viral infections of the nervous system, deficiency diseases, such as Wernicke's disease and nutritional polyneuropathy, progressive supranuclear palsy, Shy Drager's syndrome, multistem degeneration and olivo ponto cerebellar atrophy, peripheral nerve damage, and trauma and ischemia resulting from stroke.

The present invention thus provides a method for treating cancer (e.g. leukemia), and disorders associated with T cell signaling, modulating telomere function, or affecting neuronal development or regeneration, in a subject comprising administering to a subject an effective amount of a three dimensional SAM domain structure of the invention, a peptide of the invention, or a modulator identified using the methods of the invention. The invention also contemplates a method for stimulating or inhibiting axonogenesis in a subject comprising administering to a subject an effective amount of a three dimensional SAM domain structure of the invention, a peptide of the invention, or a modulator identified using the methods of the invention.

The invention still further relates to a pharmaceutical composition which comprises a purified three dimensional SAM domain structure of the invention, a peptide of the invention, or a modulator identified using the methods of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical compositions may be used to stimulate or inhibit neuronal development, regeneration and axonal migration associated with neurodegenerative conditions, and conditions involving trauma and injury to the nervous system. They may also be used to treat cancer and disorders associated with T cell signaling, and modulate telomere function. The compositions of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the protein to be administered in which any toxic effects are outweighed by the therapeutic effects of the protein. The term subject is intended to include mammals and includes humans, dogs, cats, mice, rats, and transgenic species thereof. Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a three dimensional SAM domain structure of the invention, peptides of the invention, or modulators of the invention may vary according to factors such as the condition, age, sex, and weight of the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The active compound may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or intracerebral administration. In particular embodiments, pharmaceutical compositions of the invention are administered directly to the peripheral or central nervous system, for example by administration intracerebrally.

A pharmaceutical composition of the invention can be administered to a subject in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as microporous or solid beads or liposomes. The term "pharmaceutically acceptable carrier" as used herein is intended to include diluents such as saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27). The active compound may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Depending on the route of administration, the active compound may be coated to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. The pharmaceutical compositions may be administered locally to stimulate axonogenesis and pathfinding, for example the compositions may be administered in areas of local nerve injury or in areas where normal nerve pathway development has not occurred. The pharmaceutical compositions may also be placed in a specific orientation or alignment along a presumptive pathway to stimulate axon pathfinding along that line, for example the pharmaceutical compositions may be incorporated on microcarriers laid down along the pathway. In particular, the pharmaceutical compositions of the invention may be used to stimulate formation of connections between areas of the brain, such as between the two hemispheres or between the thalamus and ventral midbrain. The pharmaceutical compositions may be used to stimulate formation of the medial tract of the anterior commissure or the habenular interpeduncle.

Therapeutic administration of polypeptides may also be accomplished using gene therapy, A nucleic acid including a promoter operatively linked to a heterologous polypeptide may be used to produce high-level expression of the polypeptide in cells transfected with the nucleic acid. DNA or isolated nucleic acids may be introduced into cells of a subject by conventional nucleic acid delivery systems. Suitable delivery systems include liposomes, naked DNA, and receptor-mediated delivery systems, and viral vectors such as retroviruses, herpes viruses, and adenoviruses.

The following non-limiting example is illustrative of the present invention:

EXAMPLE The following methods were used to determine the crystal structure of the SAM domain of the Eph receptor isoform A4.

Protein expression, mutagenesis and purification: The SAM domain of the Eph receptor isoform A4 (residues 890 to 981) was expressed in E. coli as a GST fusion protein using the pGEX-2T vector (Pharmacia). The Quickchange kit (Stratagene) was used to generate site directed mutants for dimerization analysis and for heavy atom phasing. Protein was purified by affinity chromatography using glutathione Sepharose beads (Pharmacia). Bound protein was eluted by cleavage with thrombin. After concentrating to 10 mM, protein was applied to a Superdex 75 gel filtration column (Pharmacia) for final purification and characterization.

Crystallization and data collection: Hanging drops containing lμl of 100 mg/ml native or mutant (Glu 941 Cs) protein in 7mM Hepes pH 7.5 were mixed with equal volumes of reservoir buffer containing 100 mM cacodylate pH 6.5, 7% (w/v) PEG 8000, and 20% (v/v) ethylene glycol. Rod like crystals of approximate dimensions 0.05 x 0.05 x 0.2 mm were obtained overnight. The crystals contain one molecule of the EphA4 SAM domain per asymmetric unit, and belong to the space group P6₄, (a = b = 77.14 A, c = 24.37 A). The solution dimer corresponds to a crystallographic dimer generated from the asymetric unit by a two fold rotation parallel to the unique crystal axis. Crystals were cryo-protected in reservoir buffer emiched to 20% (w/v) PEG 8000 and 20% (v/v) ethylene glycol prior to stream freezing. Heavy atom derivatives were prepared by soaking crystals overnight in 1-10 mM heavy atom solution prepared in cryo-protection buffer. Native and derivative diffraction data were collected on frozen crystals (108°K) using a Marr 345 imaging plate detector system with an RU200 rotating anode generator (Table 1). Data processing and reduction was carried out with the HKL, DENZO, and SCALEPACK programs.

Single isomorphous replacement (SIR) protein phases were calculated using lead derivative data collected on two separate protein crystals. The heavy atom site was identified by the Patterson search program HASSP [Terwilliger, 1987]. A Glu 941 to Cys site directed mutant of the EphA4 SAM domain construct was employed for mercury derivatization. The heavy atom position of the mercury derivative data, which was collected on three separate crystals, was identified by difference Fourier synthesis. Multiple isomorphous replacement and anomalous scattering (MIRAS) phases, using only the lead derivative anomalous signal, were calculated and iterative rounds of automatic solvent boundary determination/density modification were performed using the PHASES package [Furey, 1990]. The resultant experimental electron density map allowed for the complete tracing of the SAM domain backbone structure. Model building and Refinement: Model building was performed using O [Jones, 1991]. A starting model comprising approximately 65% of the total structure was refined using XPLOR [Brunger, 1992]. Bulk solvent correction was applied during refinement and simulated annealing protocols were employed. The remaining structure was built into 2F₀-F_C electron density maps generated with XPLOR. The final refinement statistics are shown in Table 1. The first 20 residues of the SAM domain construct are disordered (residues 890 to 909) and have not been modeled. No amino acid residues occupy disallowed regions of the Ramachandran plot and 94 % occupy the most favored regions. Results:

The X-ray crystal structure of the SAM domain from the EphA4 receptor tyrosine kinase (Table 1 and 2) was determined. The boundaries of the structure were defined by limited proteolysis and mass-spectrometry. Overall, the structure of the homodimer is oblong and arises from the association of two 'lobster claw' shaped subunits. Each subunit possesses a globular fold consisting of an N-terminal extended strand segment, followed by four short α helices (αl to α4) and one long C-terminal helix α5 (Figure 2A, 2B, and 2C). The N- and the C-termini are located on one side of the subunit fold, similar to other protein interaction modules with signaling function (SH3, SH2, PH domains etc.) [Kuriyan, 1997]. However, in contrast to these other domains, the termini compose the functional end of the molecule rather than lying opposite to the ligand-binding surface. As shown in Figure 3A and 3B, the N-terminal strand region and the C-terminal helix α5 extend from the subunit core and interdigitate in a pincer like manner with the termini of a second subunit, to form an elaborate dimer interface. In addition to the N- and C-terminal regions, α-helices αl and α3 contribute side chains to the dimer interface.

The N-terminal strands cross in an anti-parallel manner and project the side chains of Ala 912, Val 913, Val 914 and Phe 910 downward to form one mandible of the 'lobster claw' shaped subunit. The C-terminal helices α5 also cross in an anti-parallel manner with each α-helix projecting the side chains of Met 972, Met 976, and Met 979, upwards to form the second mandible. Together these side chains compose a hydrophobic core that is fully continuous with those of the individual subunits. Residues bridging the subunit and interface cores include Tip 919, Ala 922 and He 923 from helix αl and Leu 940 and Val 944 from helix α3. Complementing these hydrophobic interactions, the conserved side chain of arginine 973 forms intermolecular electrostatic interactions with the free carboxylate of glycine 981 and a stabilizing charge/helix dipole interaction with the C-terminus of helix α5 (Figure 2C). Additional polar residues located at or in close proximity to the dimer interface include His 980, Gin 975, His 945, Gin 977, Glu 941 and Ser 911.

In order to identify determinants of dimerization and to test that the crystallographic dimer model reflects the solution structure of the EphA4 SAM domain, SAM domain residues, either singly or in combination, were substituted and the behaviour of these mutants was tested using size exclusion chromatography (Figure 4). In agreement with predictions from the crystal structure, mutations involving the interface residues Val 913, Val 914, Met 972, Met 976, Met 979, Val 944, and Leu 940 abolished dimer formation. In contrast, mutation of Val 969 to Ala, which comprises part of the second hydrophobic surface region (Figure 3 A and 3B), did not affect dimerization while mutation of the proximal residue He 959 to Lys, appeared to disrupt the integrity of the subunit fold. Additionally, mutation of the surface exposed residues Glu 941, Asp 949, and Ser 968 to cysteine, did not disrupt SAM domain dimerization. In summary, the mutagenesis results are consistent with and support the notion that the SAM domain dimer observed in the crystal structure represents a mechanism through which the SAM domain associates in solution. To investigate whether the dimer model for the Eph receptor SAM domain has more general relevance for SAM domain containing proteins, the predicted locations of residues that are required for the dimerization of SAM domains on other polypeptides were examined. When mutations that map to conserved features of the subunit core and therefore are likely to disrupt the subunit fold are eliminated, a number of informative mutations stand out. For example, the homo- and hetero-typic dimerization of the Polycomb family of transcriptional repressors ph, RAE28 and Scm, is abolished by mutation of two residues predicted to map to the dimer interface [Kyba, 1998]. These residues, He 62 and Tip 1 of the ph SAM domain, correspond to the N-terminal strand residue Phe 910 and the α5 helix residue Met 972, respectively, of the EphA4 SAM domain. Both residues are highly conserved amongst the SAM domains and yet are unlikely to affect the individual subunit fold. The mutation of the latter residue (Met 972 to Lys) in the EphA4 SAM domain yields a compact monomer structure (Figure 4). In addition, the hetero-dimerization of the SAM domain containing proteins Byr2p and Ste4p is disrupted by the substitution of Arg 69 with cysteine[Tu, 1997 #25]. This mutation maps to the interface residue Gin 977 of the EphA4 SAM dimer, and is located at the crossing site of the two α5 helices. Taken together, these observations indicate that the dimer structure of the EphA4 SAM domain may reflect a more general mode of SAM domain dimerization.

The crystallographic model for SAM domain dimerization is attractive for a number of reasons. Firstly, in the case of the Eph receptors, the linkers between the SAM and the catalytic domains is short (5 residues of poorly conserved sequence) so that the N-termini of the dimer would have to be oriented in the same direction and in close proximity if the kinase domains of clustered receptors were to be juxtaposed. The structure shows this to be the case. Secondly, the mechanism of dimerization revealed by the structure could account for the observation that the SAM domain is found at either terminus of signaling proteins. Because the N- and C-terminal ends of the SAM domain compose the dimer interface, the insertion of a SAM domain at an internal site in a polypeptide chain would sterically restrict access to a second SAM domain, especially if the host sequence was itself structured. The solutions to this dilemma would be to place a SAM domain at the end of a protein (as is usually observed), or to surround it with long linker sequences. In this regard the SAM domain differs from modules such as SH2 and SH3 domains, which can readily be located at internal positions in a polypeptide chain since the ligand-binding site is located opposite to the location of the N- and C-termini [Kuriyan, 1997]. Thirdly, in the case of the liprins we have noted three adjacent SAM domains in a region previously shown to mediate liprin hetero-dimerization [Serra-Pages, 1998]. Because the C-termini of the dimerized SAM domain are in close proximity, on the opposite side from the N-termini, a configuration of stacked SAM domains can be readily envisioned. SAM dimerization may contribute to receptor oligomerization and activation by bringing catalytic elements into proximity for autophosphorylation. The SAM domain may have a direct inhibitory interaction with the kinase domain that can be competed away by dimerization. Alternatively SAM domain mediated dimerization might maintain opposing catalytic domains in a mutually inaccessible, and thus repressed state. The Eph SAM domains might also recruit signaling partners through heteromeric SAM-SAM interactions, or through specific recognition of cytoplasmic proteins by the Eph SAM dimer.

SAM dimerization might be constitutive, but controlled through co-operative or antagonistic interactions with other clustering forces. Dimerization could potentially be controlled by modifications such as tyrosine phosphorylation, and indeed a residue within the SAM domain of the EphBl receptor can become tyrosine phosphorylated in vivo [Stein, 1996]. Finally, the five residues that lie C-terminal to the Eph SAM domain represent a potential binding site for PDZ domain proteins[Hock, 1998],which might influence the organization of the SAM domain.

The structure of the EphA4 domain reveals a novel mechanism through which modular domains control protein-protein interactions. Since SAM domains are found in cell surface receptors, cytoplasmic signaling proteins, and transcriptional activators and repressors, as well as chimeric human oncoproteins, these results have general implications for understanding the formation of complexes involved in normal and oncogenic signal transduction.

Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. All modifications coming within the scope of the following claims are claimed.

All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Detailed Description of the Drawings

Figure 1A shows a sequence alignment of SAM domains from selected proteins. Secondary structure is indicated for the SAM domain from the EphA4 receptor tyrosine kinase. Residue numbers for the start of each SAM domain are shown on the left and Genebank accession numbers on the right. Conserved hydrophobic residues are colored green, acidic residues red, basic residues blue, polar residues orange and glycines are colored pink. Residues at the dimer interface shown in Figure 2C are indicated (•). Liprin αl contains 3 SAM domains designated SI, S2 and S3.

Figure IB shows a selection of multi-domain proteins containing SAM domain (S) is shown.

Domains listed include, tyrosine or serine/threonine kinase catalytic domains, myosin-like domain, F- actin binding domain (F-actin BD), PDZ domain, SH2 domain, inositol phosphatase catalytic domain

(inositol p' tase), GTPase activating domain (GAP), DNA-binding domain (DNA-BD) and a transmembrane region (TM).

Figure 2A, 2B, and 2C. Ribbons depiction of the SAM homo-dimer viewed (Figure 2A) down the twofold symmetry axis and (Figure 2B) perpendicular to the symmetry axis. The dimer subunits are coloured red and blue and α-helices are labeled. (Figure 2C) Ribbons stereo view highlighting the dimer interface region. Aromatic, aliphatic, methionine, histidine and arginine interacting side chains are coloured light blue, green, yellow, orange, and blue (see Figure 1A for residue identification). All ribbon diagrams were generated using RIBBONS [Carson, 1991].

Figure 3A, B. Molecular surface and worm representations of the SAM homodimer. The molecular surface of one subunit is shown with hydrophobic (Met, Val, Leu, He, Phe,), basic (Arg, Lys) and acidic (Glu, Asp) side chains coloured green, blue and red, respectively. The two perspectives differ by a 90° rotation about the vertical axis. In Figure 3B the twofold rotation axis relating the two subunits of the dimer is shown. The buried surface area of the dimer interface is 1923 A. All molecular surfaces were generated using GRASP [Nicholls, 1991]. Figure 4. Gel filtration elution profile of wild type and single or double site mutants of the

EρhA4 receptor SAM domain. Chromatograms correspond to the loading of equivalent concentrations (10 mM) and total volumes (100 μl) of protein on a Superdex-75 gel filtration column (24 ml bed volume). The column was calibrated using Pharmacia low molecular weight standards.

Table 1 . Data Collection, Structure Determination and Refinement Statistics

Resolution Reflections Completeness Rsym* Rderiv + Sites Phasing Power® F0M& (A) total / unique (%) (%) (%) (#) (2.9A) (2.9A)

Native Dβta 2.0 1 7787 / 5663 97.5(91.7) 4(30.1 ) (l/sig > -3)

MIR analysis Pb Acetate 1 2.3 15572 / 3761 98.0 (96.0) 5.9 (23.0) 17.60 2.1 0.41 Pb Acetate 2 2.8 20533 / 21 13 98.0 (99.0) 7.1 (19.5) 19.00 1.8 0.37

Hg Acetate 1 2.6 9358 / 2569 95.6 (85.5) 6.8 (25.9) 1 1.60 1.2 0.31 Hg Acetate 2 2.9 9683 / 1857 95.9 (90.9) 9.4 (25.9) 16.20 1 .8 0.36 Hg Acetate 3 2.9 5667 / 1928 98.2 (97.0) 10.0 (28.7) 13.60 1 .7 0.37

MIRAS combined (Pb anomalous only) 0.74

Reflections Completeness Rfactor Rfreetf

Refinement er

30-2.0A l/sig > 0 5498 94.6 (86.1 ) 22.9 27.8

30-2.0A l/sig > 2 5194 89.4 (72.5) 21.7 27.3

RMSD from ideal

Bond Length (A) 0.01 total nonhydrogen atoms 561

Bond Angle !⁰) 1 .38 water molecules 53

Ave. B factor (A) 22.3 (33.2 for solvent)

RMSD in B factor (A) 2.01

*R_{sym =} 100 X ∑_h ∑i \l_Ki - <l_h>\ l∑_h ∑i ∑_h

"Free Rf_aC{0r was calculated with 5% of the data.

©Phasing Power is RMS (|F_n |/E) where the subscript h' represents heavy-atom' and E is the residual lack of closure.

^&FOM (mean figure of merit) = < | ΣP(α) e'^α/ ΣP(α) t>, where α is the phase and P(α) is the phase probability distribution.

Table 2

REMARK Eph A4 SAM dom

ATOM 1 CB PHE 910 29 .722 33 .942 12 .780 1 .00 34 .97

ATOM 2 CG PHE 910 28 .445 34 .752 12 .839 1 .00 31 .67

ATOM 3 CD1 PHE 910 27 .612 34 .694 13 .956 1, .00 30 .66

ATOM 4 CD2 PHE 910 28 .067 35 .563 11 .771 1 .00 31 .49

ATOM 5 CE1 PHE 910 26 .424 35 .428 14 .007 1, .00 28, .32

ATOM 6 CE2 PHE 910 26 .881 36 .301 11 .812 1 .00 28 .34

ATOM 7 CZ PHE 910 26 .059 36 .230 12 .931 1, .00 28, .11

ATOM 8 C PHE 910 28 .471 32 .190 11 .520 1, .00 38 .36

ATOM 9 O PHE 910 28 .797 ' 32 .277 10 .336 1, .00 39, .23

ATOM 10 HT1 PHE 910 31 .128 32 .065 11 .446 1, .00 10 .00

ATOM 11 HT2 PHE 910 30 .595 30, .703 12 .275 1, .00 10, .00

ATOM 12 N PHE 910 30 .779 31 .717 12 .369 1, .00 37, .87

ATOM 13 HT3 PHE 910 31 .459 31, .917 13 .127 1. .00 10, .00

ATOM 14 CA PHE 910 29 .489 32 .432 12 .628 1, .00 37, .16

ATOM 15 N SER 911 27 .236 31, .915 11 .923 1. .00 37, .36

ATOM 16 H SER 911 27 .001 31, .871 12 .873 1. .00 0, .00

ATOM 17 CA SER 911 26 .138 31, .684 10 .994 1. .00 36. .43

ATOM 18 CB SER 911 25, .532 30, .288 11, .233 1. .00 40. .15

ATOM 19 OG SER 911 24. .503 30, .015 10 .300 1. .00 45. .27

ATOM 20 HG SER 911 24, .111 29. .153 10, .464 1. .00 0. .00

ATOM 21 C SER 911 25, .075 32, .757 11, .224 1. .00 32. .88

ATOM 22 O SER 911 24, .780 33. .107 12, .365 1. .00 30. .65

ATOM 23 N ALA 912 24, .515 33. .274 10, .130 1. .00 29. .12

ATOM 24 H ALA 912 24, .829 32. .997 9, .247 1. .00 10. .00

ATOM 25 CA ALA 912 23, .491 34, ,300 10, .214 1. .00 27. .10

ATOM 26 CB ALA 912 23. .525 35. ,190 8, .991 1. .00 28. .25

ATOM 27 C ALA 912 22, .091 33. .725 10, .416 1. .00 25. .69

ATOM 28 O ALA 912 21, .124 34. .480 10. ,538 1. .00 25. .68

ATOM 29 N VAL 913 21, .968 32. .395 10, .409 1. .00 25. .64

ATOM 30 H VAL 913 22. .774 31. .852 10. .334 1. .00 10. .00

ATOM 31 CA VAL 913 20, .667 31. .749 10, .602 1. .00 25. .94

ATOM 32 CB VAL 913 20. .410 30. .608 9, .572 1. .00 27. .20

ATOM 33 CGI VAL 913 19, .853 31. .180 8. .278 1. ,00 28. .69

ATOM 34 CG2 VAL 913 21. .683 29. .825 9. .304 1. .00 28. .91

ATOM 35 C VAL 913 20. .519 31. .194 12. .013 1. .00 24. .45

ATOM 36 O VAL 913 21. .515 30. .851 12. .659 1. .00 24. .44

ATOM 37 N VAL 914 19. .277 31. .120 12. .486 1. ,00 22. .36

ATOM 38 H VAL 914 18. .546 31. .390 11. .893 1. .00 10. .00

ATOM 39 CA VAL 914 18. .990 30. .618 13. .826 1. ,00 23. .06

ATOM 40 CB VAL 914 17. .612 31. .113 14. .354 1. ,00 24. .08

ATOM 41 CGI VAL 914 17. .600 32. .633 14. .442 1. ,00 24. ,61

ATOM 42 CG2 VAL 914 16. .479 30. .620 13. .456 1. ,00 24. .73

ATOM 43 C VAL 914 19. .050 29. .096 13. .891 1. ,00 23. ,11

ATOM 44 O VAL 914 19. .208 28. ,427 12. .867 1. ,00 22. .16

ATOM 45 N SER 915 18. .928 28. ,561 15. .104 1. ,00 24. .03

ATOM 46 H SER 915 18. .786 29. ,134 15. .882 1. ,00 10. .00

ATOM 47 CA SER 915 18. .986 27. ,119 15. .340 1. 00 24. ,05

ATOM 48 CB SER 915 19. .224 26. .857 16. .827 1. ,00 23. ,17

ATOM 49 OG SER 915 18. ,136 27. ,348 17. .596 1. 00 24. ,34

ATOM 50 HG SER 915 17. .398 26. ,778 17. .408 1. 00 10. .00

ATOM 51 C SER 915 17. ,722 26. ,379 14. .905 1. 00 23. ,08

ATOM 52 O SER 915 16. ,632 26. 961 14. .867 1. 00 21. 72

ATOM 53 N VAL 916 17. ,879 25. ,083 14. ,636 1. 00 22. ,36

ATOM 54 H VAL 916 18. ,769 24. 688 14. ,711 1. 00 10. .00 ATOM 55 CA VAL 916 16.771 24.222 14.240 1.00 21.66

ATOM 56 CB VAL 916 17 .243 22 .776 13 .978 1 .00 20 .59

ATOM 57 CGI VAL 916 16 .069 21 .892 13 .601 1 .00 20 .62

ATOM 58 CG2 VAL 916 18 .279 22 .760 12 .880 1 .00 18 .09

ATOM 59 C VAL 916 15 .758 24 .206 15 .380 1 .00 21 .55

ATOM 60 O VAL 916 14 .552 24 .235 15 .145 1 .00 22 .25

ATOM 61 N GLY 917 16 .265 24 .201 16 .613 1 .00 21 .67

ATOM 62 H GLY 917 17 .236 24 .168 16 .755 1 .00 10 .00

ATOM 63 CA GLY 917 15 .408 24 .200 17 .788 1 .00 21 .14

ATOM 64 C GLY 917 14 .484 25 .406 17 .849 1 .00 21 .35

ATOM 65 O GLY 917 13 .297 25 .271 18 .159 1 .00 22 .38

ATOM 66 N ASP 918 15 .025 26 .582 17 .535 1 .00 21 .54

ATOM 67 H ASP 918 15 .955 26 .594 17 .261 1 .00 10 .00

ATOM 68 CA ASP 918 14 .254 27 .823 17 .544 1 .00 20 .67

ATOM 69 CB ASP 918 15 .165 29 .027 17 .298 1 .00 24 .65

ATOM 70 CG ASP 918 16 .073 29 .322 18 .471 1 .00 28 .02

ATOM 71 OD1 ASP 918 17 .203 29 .794 18 .235 1 .00 30 .66

ATOM 72 OD2 ASP 918 15 .656 29 .089 19 .626 1 .00 30 .44

ATOM 73 C ASP 918 13 .170 27 .788 16 .482 1, .00 17 .85

ATOM 74 O ASP 918 12 .042 28 .225 16, .716 1, .00 16 .82

ATOM 75 N TRP 919 13 .536 27 .296 15 .305 1, .00 15 .94

ATOM 76 H TRP 919 14 .465 26 .996 15, .186 1. .00 10, .00

ATOM 77 CA TRP 919 12 .608 27 .180 14, .191 1, .00 14 .95

ATOM 78 CB TRP 919 13, .360 26, .724 12, .931 1. .00 15, .34

ATOM 79 CG TRP 919 12 .486 26 .125 11, .871 1. .00 13 .90

ATOM 80 CD2 TRP 919 12, .470 24, .754 11, .453 1. .00 13, .38

ATOM 81 CE2 TRP 919 11 .460 24, .627 10, .478 1. .00 11. .80

ATOM 82 CE3 TRP 919 13, .212 23, .622 11. .813 1. .00 13, .78

ATOM 83 CD1 TRP 919 11. .519 26. .757 11. .149 1. .00 13. .86

ATOM 84 NE1 TRP 919 10, .895 25, .862 10. .312 1. .00 13. .06

ATOM 85 HE1 TRP 919 10. .171 26. .089 9. ,693 1. .00 10. .00

ATOM 86 CZ2 TRP 919 11. .167 23. .410 9. .856 1. .00 12. .61

ATOM 87 CZ3 TRP 919 12. .920 22. .408 11. .193 1. .00 14. .43

ATOM 88 CH2 TRP 919 11. .905 22. .315 10. .226 1. .00 11. .85

ATOM 89 C TRP 919 11. .473 26. .213 14. .540 1. .00 13. .78

ATOM 90 O TRP 919 10. .303 26. .526 14. .332 1. .00 14. .59

ATOM 91 N LEU 920 11. .819 25. .053 15. .093 1. .00 13. .35

ATOM 92 H LEU 920 12. .765 24. .865 15. .260 1. ,00 10. .00

ATOM 93 CA LEU 920 10. .820 24. .058 15. .468 1. ,00 12. .53

ATOM 94 CB LEU 920 11. .493 22. .771 15. ,954 1. ,00 10. .48

ATOM 95 CG LEU 920 12. .110 21. .896 14. .853 1. ,00 11. ,20

ATOM 96 CD1 LEU 920 12. .821 20. .705 15. ,470 1. ,00 12. .10

ATOM 97 CD2 LEU 920 11. .036 21. .425 13. .879 1. ,00 10. .00

ATOM 98 C LEU 920 9. .836 24. .581 16. ,511 1. 00 12. ,61

ATOM 99 O LEU 920 8. .635 24. .334 16. ,407 1. ,00 13. ,77

ATOM 100 N GLN 921 10. ,331 25. ,303 17. 512 1. 00 13. ,95

ATOM 101 H GLN 921 11. ,298 25. ,464 17. 569 1. 00 10. ,00

ATOM 102 CA GLN 921 9. ,447 25. ,848 18. 538 1. 00 16. ,28

ATOM 103 CB GLN 921 10. 247 26. 450 19. 694 1. 00 16. 54

ATOM 104 CG GLN 921 9. 368 26. ,933 20. 840 1. 00 16. ,62

ATOM 105 CD GLN 921 10. 165 27. 457 22. 006 1. 00 16. 33

ATOM 106 OE1 GLN 921 11. 072 26. .794 22. 500 1. 00 16. 95

ATOM 107 NE2 GLN 921 9. 820 28. 650 22. 467 1. 00 18. 37

ATOM 108 HE21 GLN 921 9. 081 29. 120 22. 029 1. 00 10. 00

ATOM 109 HE22 GLN 921 10. 307 29. 002 23. 245 1. 00 10. 00

ATOM 110 C GLN 921 8. 504 26. 899 17. 948 1. 00 15. 94

ATOM 111 O GLN 921 7. 327 26. 953 18. 294 1. 00 17. 35

ATOM 112 N ALA 922 9. 024 27. 698 17. 021 1. 00 17. 97

ATOM 113 H ALA 922 9. 965 27. 577 16. 767 1. 00 10. 00 ATOM 114 CA ALA 922 8.260 28.750 16.363 1.00 16.54

ATOM 115 CB ALA 922 9 .178 29 .591 15 .504 1 .00 16 .52

ATOM 116 C ALA 922 7 .107 28 .207 15 .525 1 .00 17 .54

ATOM 117 O ALA 922 6 .082 28 .875 15 .364 1 .00 16 .79

ATOM 118 N ILE 923 7 .287 27 .015 14 .962 1 .00 16 .97

ATOM 119 H ILE 923 8 .139 26 .545 15 .085 1 .00 10 .00

ATOM 120 CA ILE 923 6 .239 26 .400 14 .153 1 .00 16 .57

ATOM 121 CB ILE 923 6 .775 25 .894 12 .783 1 .00 17 .14

ATOM 122 CG2 ILE 923 7 .410 27 .054 12 .011 1 .00 14 .50

ATOM 123 CGI ILE 923 7 .782 24 .756 12 .971 1 .00 13 .94

ATOM 124 CD1 ILE 923 8 .047 23 .979 11 .704 1. .00 13 .69

ATOM 125 C ILE 923 5 .530 25 .272 14 .912 1 .00 18, .05

ATOM 126 O ILE 923 4 .846 24 .439 14 .319 1 .00 18 .28

ATOM 127 N LYS 924 5 .710 25 .264 16 .230 1, .00 19, .42

ATOM 128 H LYS 924 6 .267 25 .957 16 .639 1. .00 10 .00

ATOM 129 CA LYS 924 5 .100 24 .287 17 .130 1, .00 20, .32

ATOM 130 CB LYS 924 3 .612 24 .605 17 .319 1, .00 23, .93

ATOM 131 CG LYS 924 3 .384 25 .983 17 .936 1. .00 28, .19

ATOM 132 CD LYS 924 1. .925 26, .241 18, .251 1, .00 34, .63

ATOM 133 CE LYS 924 1 .746 27 .585 18 .949 1. .00 38, .73

ATOM 134 NZ LYS 924 0, .313 27, .854 19, .284 1. .00 44, .05

ATOM 135 HZ1 LYS 924 -0 .035 27 .120 19 .931 1. .00 10, .00

ATOM 136 HZ2 LYS 924 -0, .257 27, .845 18, .412 1, .00 10. .00

ATOM 137 HZ3 LYS 924 0. .227 28, .791 19. .730 1. .00 10. .00

ATOM 138 C LYS 924 5, .326 22, .816 16, .781 1. .00 18. .87

ATOM 139 O LYS 924 4, .485 21, .956 17, .054 1. .00 17. .67

ATOM 140 N MET 925 6, .497 22, .530 16, .218 1. .00 18. .00

ATOM 141 H MET 925 7. .115 23. .269 16, .046 1. .00 10. .00

ATOM 142 CA MET 925 6. .878 21. .168 15, .848 1. .00 16. .42

ATOM 143 CB MET 925 7. .227 21. .093 14. .358 1. .00 15. .95

ATOM 144 CG MET 925 6. .033 21. .236 13. .427 1. .00 17. .12

ATOM 145 SD MET 925 4. .947 19. .804 13. .475 1. .00 18. .04

ATOM 146 CE MET 925 3. .547 20. .453 14. .365 1. .00 22. .39

ATOM 147 C MET 925 8. .080 20. .731 16. .680 1. .00 15. .49

ATOM 148 O MET 925 8. .801 19. .813 16. .300 1. .00 15. .05

ATOM 149 N ASP 926 8. .265 21. .364 17. .834 1. .00 15. .42

ATOM 150 H ASP 926 7. .628 22. .064 18. .079 1. .00 10. .00

ATOM 151 CA ASP 926 9. .387 21. .053 18. .716 1. .00 16. .61

ATOM 152 CB ASP 926 9. .600 22. .158 19. .768 1. .00 18. .40

ATOM 153 CG ASP 926 8. .342 22. .492 20. .550 1. .00 22. .45

ATOM 154 OD1 ASP 926 8. .366 22. .335 21. .785 1. .00 29. .54

ATOM 155 OD2 ASP 926 7. .343 22. .944 19. .951 1. .00 23. .78

ATOM 156 C ASP 926 9. .349 19. .660 19. ,351 1. ,00 17. ,08

ATOM 157 O ASP 926 10. .315 19. .229 19. .987 1. .00 17. .30

ATOM 158 N ARG 927 8. .260 18. .932 19. .126 1. .00 15. ,97

ATOM 159 H ARG 927 7. .519 19. .305 18. .614 1. .00 10. .00

ATOM 160 CA ARG 927 8. .140 17. .576 19. .645 1. ,00 14. ,49

ATOM 161 CB ARG 927 6. .691 17. .083 19. .528 1. .00 13. ,98

ATOM 162 CG ARG 927 6. .249 16. .700 18. .111 1. ,00 13. ,40

ATOM 163 CD ARG 927 4. ,780 16. ,271 18. .058 1. ,00 15. ,27

ATOM 164 NE ARG 927 3. .884 17. .421 18. .158 1. ,00 19. ,72

ATOM 165 HE ARG 927 4. .290 18. .300 18. ,276 1. 00 10. ,00

ATOM 166 CZ ARG 927 2. .557 17. .356 18. ,111 1. ,00 18. ,27

ATOM 167 NH1 ARG 927 1. ,946 16. ,188 17. ,973 1. 00 14. ,35

ATOM 168 HHll ARG 927 2. .481 15. .348 17. .913 1. ,00 0. ,00

ATOM 169 HH12 ARG 927 0. .946 16. .148 17. ,941 1. .00 0. ,00

ATOM 170 NH2 ARG 927 1. .840 18. .472 18. ,178 1. 00 17. 89

ATOM 171 HH21 ARG 927 2. ,306 19. ,353 18. ,264 1. ,00 0. ,00

ATOM 172 HH22 ARG 927 0. ,840 18. 431 18. ,143 1. 00 0. 00 ATOM 173 C ARG 927 9.072 16.682 18.822 1.00 14.29

ATOM 174 O ARG 927 9 .361 15 .547 19 .204 1, .00 14 .14

ATOM 175 N TYR 928 9 .536 17 .219 17 .691 1 .00 14 .12

ATOM 176 H TYR 928 9 .268 18 .120 17 .426 1 .00 10 .00

ATOM 177 CA TYR 928 10 .431 16 .522 16 .770 1, .00 13 .74

ATOM 178 CB TYR 928 9 .938 16 .706 15 .332 1 .00 11 .65

ATOM 179 CG TYR 928 8 .610 16 .029 15 .057 1 .00 12 .93

ATOM 180 CD1 TYR 928 7 .501 16 .774 14 .659 1, .00 10 .58

ATOM 181 CE1 TYR 928 6 .288 16 .171 14 .386 1, .00 10 .49

ATOM 182 CD2 TYR 928 8 .463 14 .645 15 .181 1, .00 12 .08

ATOM 183 CE2 TYR 928 7 .236 14 .020 14 .907 1, .00 12 .39

ATOM 184 CZ TYR 928 6 .159 14 .797 14. .509 1, .00 10, .20

ATOM 185 OH TYR 928 4, .945 14, .241 14. .204 1. .00 11, .30

ATOM 186 HH TYR 928 4 .612 15 .000 13 .786 1, .00 10 .00

ATOM 187 C TYR 928 11 .904 16, .934 16, .857 1, .00 14, .27

ATOM 188 O TYR 928 12, .714 16, .495 16, .043 1. .00 15, .38

ATOM 189 N LYS 929 12, .255 17, .749 17, .849 1. .00 16. .73

ATOM 190 H LYS 929 11, .563 18 .045 18, .482 1. .00 10, .00

ATOM 191 CA LYS 929 13, .633 18, .208 18, .031 1, .00 17, .93

ATOM 192 CB LYS 929 13, .804 18, .863 19, .402 1. .00 19, .71

ATOM 193 CG LYS 929 13. .604 20, .361 19. .404 1. .00 27. .71

ATOM 194 CD LYS 929 13. .928 20, .967 20, .765 1. .00 32. .21

ATOM 195 CE LYS 929 14, .009 22, .491 20, .690 1. .00 32. .72

ATOM 196 NZ LYS 929 12. .749 23, .124 20. .175 1. .00 31. .10

ATOM 197 HZ1 LYS 929 11. .938 22. .865 20. .776 1. .00 10. .00

ATOM 198 HZ2 LYS 929 12. .566 22, .800 19. .207 1. .00 10. .00

ATOM 199 HZ3 LYS 929 12. .856 24, .158 20. .192 1. .00 10. .00

ATOM 200 C LYS 929 14. .653 17. .084 17. .892 1. .00 19. .40

ATOM 201 O LYS 929 15. .566 17. .149 17. .064 1. .00 18. .56

ATOM 202 N ASP 930 14. .460 16, .040 18. .691 1. .00 20. .41

ATOM 203 H ASP 930 13. .694 16, .063 19. .294 1. .00 10. .00

ATOM 204 CA ASP 930 15. .347 14. .887 18. .702 1. .00 20. .42

ATOM 205 CB ASP 930 14. .958 13, .932 19. .836 1. .00 23. .30

ATOM 206 CG ASP 930 15. .210 14. .523 21. .214 1. .00 25. .30

ATOM 207 OD1 ASP 930 14. .734 13. .926 22. .201 1. .00 27. .38

ATOM 208 OD2 ASP 930 15. .885 15. .575 21. .317 1. .00 26. .70

ATOM 209 C ASP 930 15. .390 14. .144 17. .374 1. .00 17. .86

ATOM 210 O ASP 930 16. .417 13. .561 17. .031 1. .00 17. .51

ATOM 211 N ASN 931 14. .282 14. .171 16. .635 1. .00 14. .65

ATOM 212 H ASN 931 13. ,530 14. .700 16. .955 1. .00 10. .00

ATOM 213 CA ASN 931 14. .200 13. .504 15. .335 1. .00 13. .52

ATOM 214 CB ASN 931 12. .788 13. .625 14. .753 1. .00 13. .46

ATOM 215 CG ASN 931 11. .742 12. .911 15. .590 1. .00 12. .15

ATOM 216 OD1 ASN 931 11. .114 11. .957 15. .138 1. .00 15. .43

ATOM 217 ND2 ASN 931 11. ,531 13. .386 16. .810 1. .00 10. .00

ATOM 218 HD21 ASN 931 12. .030 14. .158 17. .132 1. .00 0. .00

ATOM 219 HD22 ASN 931 10. .842 12. .912 17. .313 1. ,00 0. .00

ATOM 220 C ASN 931 15. .220 14. .106 14. ,363 1. ,00 14. .47

ATOM 221 O ASN 931 15. .917 13. .382 13. .654 1. .00 13. .19

ATOM 222 N PHE 932 15. .319 15. .431 14. .350 1. .00 14. .74

ATOM 223 H PHE 932 14. .747 15. .968 14. .943 1. ,00 10. .00

ATOM 224 CA PHE 932 16. ,266 16. .119 13. ,478 1. ,00 16. .13

ATOM 225 CB PHE 932 15. .963 17. .621 13. .441 1. .00 13. .55

ATOM 226 CG PHE 932 14. .757 17. .965 12. ,615 1. ,00 13. .40

ATOM 227 CD1 PHE 932 13. ,489 17. .942 13. .174 1. ,00 12. .04

ATOM 228 CD2 PHE 932 14. ,888 18. .271 11. .269 1. 00 12. .73

ATOM 229 CE1 PHE 932 12. .370 18. .212 12. .408 1. ,00 11. .44

ATOM 230 CE2 PHE 932 13. ,771 18. .543 10. ,493 1. ,00 12. .55

ATOM 231 CZ PHE 932 12. ,511 18. .512 11. ,066 1. .00 12. .81 ATOM 232 C PHE 932 17.708 15.875 13.914 1.00 17.19

ATOM 233 O PHE 932 18 .544 15 .470 13 .109 1 .00 18 .79

ATOM 234 N THR 933 17 .968 16 .059 15 .205 1 .00 18 .93

ATOM 235 H THR 933 17 .242 16 .348 15 .800 1 .00 10 .00

ATOM 236 CA THR 933 19 .293 15 .878 15 .779 1 .00 19 .46

ATOM 237 CB THR 933 19 .269 16 .179 17 .294 1 .00 20 .06

ATOM 238 OGl THR 933 18 .741 17 .494 17 .509 1 .00 23 .65

ATOM 239 HGl THR 933 19 .273 18 .153 17 .052 1 .00 10 .00

ATOM 240 CG2 THR 933 20 .666 16 .100 17 .891 1 .00 22 .07

ATOM 241 C THR 933 19 .860 14 .476 15 .553 1 .00 19 .94

ATOM 242 O THR 933 20 .987 14 .323 15 .078 1 .00 20 .57

ATOM 243 N ALA 934 19 .068 13 .457 15 .871 1 .00 19 .21

ATOM 244 H ALA 934 18 .162 13 .633 16 .194 1, .00 10 .00

ATOM 245 CA ALA 934 19 .504 12 .073 15 .722 1 .00 17 .60

ATOM 246 CB ALA 934 18 .426 11 .120 16, .217 1 .00 15 .43

ATOM 247 C ALA 934 19, .910 11, .717 14, .301 1, .00 16, .03

ATOM 248 O ALA 934 20 .743 10 .837 14, .100 1 .00 17 .80

ATOM 249 N ALA 935 19, .335 12 .408 13, .321 1, .00 16, .14

ATOM 250 H ALA 935 18, .692 13. .119 13. .520 1. .00 10, .00

ATOM 251 CA ALA 935 19, .645 12 .144 11, .920 1, .00 14, .43

ATOM 252 CB ALA 935 18, .381 12. .177 11, .094 1, .00 14, .61

ATOM 253 C ALA 935 20. .692 13. .091 11. .337 1. .00 13, .86

ATOM 254 O ALA 935 20, .924 13. .101 10, .127 1, .00 15, .04

ATOM 255 N GLY 936 21. .328 13, .878 12. .197 1, .00 13. .66

ATOM 256 H GLY 936 21. .122 13, .837 13, .153 1, .00 10, .00

ATOM 257 CA GLY 936 22. .354 14, .796 11. .736 1. .00 16. .69

ATOM 258 C GLY 936 21. .878 16. .152 11. .247 1. .00 17. .44

ATOM 259 O GLY 936 22. .689 16, .954 10. .787 1. .00 18. .41

ATOM 260 N TYR 937 20. .573 16. .412 11. .312 1. .00 19. .20

ATOM 261 H TYR 937 19. .964 15. .731 11. .666 1. .00 10. .00

ATOM 262 CA TYR 937 20. .034 17. .705 10. .883 1. .00 17. .59

ATOM 263 CB TYR 937 18. .572 17. .581 10. .469 1. .00 14. .83

ATOM 264 CG TYR 937 18. .399 16. .809 9. .194 1. .00 15. .36

ATOM 265 CD1 TYR 937 18. .107 15. .452 9. .220 1. .00 15. .74

ATOM 266 CE1 TYR 937 17. .966 14. .726 8. .048 1. .00 16. .34

ATOM 267 CD2 TYR 937 18. .551 17. .427 7. .960 1. .00 14. .36

ATOM 268 CE2 TYR 937 18. .412 16. .710 6. .779 1. .00 17. .13

ATOM 269 CZ TYR 937 18. .120 15. .360 6. .831 1. .00 15. .80

ATOM 270 OH TYR 937 17. ,962 14. .662 5. .665 1. ,00 17. .66

ATOM 271 HH TYR 937 18. .108 15. .268 4. .961 1. .00 10. .00

ATOM 272 C TYR 937 20. .171 18. .685 12. ,030 1. .00 17. .88

ATOM 273 O TYR 937 19. .223 18. .919 12. .783 1. .00 18. . 66

ATOM 274 N THR 938 21. .369 19. .242 12. .163 1. .00 19. .08

ATOM 275 H THR 938 22. ,094 18. .997 11. ,546 1. .00 10. .00

ATOM 276 CA THR 938 21. ,668 20. .182 13. .230 1. .00 20. .43

ATOM 277 CB THR 938 23. ,015 19. .845 13. ,891 1. ,00 20. .49

ATOM 278 OGl THR 938 24. ,024 19. ,724 12. ,880 1. ,00 23. ,01

ATOM 279 HGl THR 938 24. ,865 19. .550 13. .311 1. ,00 10. .00

ATOM 280 CG2 THR 938 22. ,912 18. ,538 14. .666 1. ,00 21. ,47

ATOM 281 C THR 938 21. 666 21. ,643 12. 816 1. 00 20. ,47

ATOM 282 O THR 938 21. ,740 22. ,524 13. ,672 1. ,00 22. ,23

ATOM 283 N THR 939 21. 610 21. ,906 11. 515 1. 00 19. ,49

ATOM 284 H THR 939 21. 592 21. 174 10. 869 1. 00 10. 00

ATOM 285 CA THR 939 21. 588 23. ,282 11. 027 1. 00 22. ,03

ATOM 286 CB THR 939 22. 919 23. 679 10. 306 1. 00 21. ,88

ATOM 287 OGl THR 939 23. 138 22. 832 9. 170 1. 00 23. 38

ATOM 288 HGl THR 939 22. 419 22. 887 8. 550 1. 00 10. 00

ATOM 289 CG2 THR 939 24. 112 23. 567 11. 255 1. 00 22. 58

ATOM 290 C THR 939 20. 414 23. 478 10. 072 1. 00 22. ,29 ATOM 291 O THR 939 19.983 22.533 9.403 1.00 23.11

ATOM 292 N LEU 940 19 .892 24 .699 10 .016 1 .00 20 .97

ATOM 293 H LEU 940 20 .257 25 .408 10 .589 1 .00 10 .00

ATOM 294 CA LEU 940 18 .778 25 .008 9 .127 1 .00 21 .45

ATOM 295 CB LEU 940 18 .231 26 .411 9 .396 1 .00 21 .73

ATOM 296 CG LEU 940 17 .245 26 .592 10 .553 1 .00 20 .62

ATOM 297 CD1 LEU 940 16 .837 28 .057 10 .656 1 .00 19 .11

ATOM 298 CD2 LEU 940 16 .015 25 .721 10 .330 1 .00 22 .59

ATOM 299 C LEU 940 19 .172 24 .869 7 .656 1 .00 21 .08

ATOM 300 O LEU 940 18 .313 24 .702 6 .796 1 .00 19 .68

ATOM 301 N GLU 941 20 .470 24 .926 7 .371 1 .00 22 .89

ATOM 302 H GLU 941 21 .105 25 .087 8 .093 1 .00 10 .00

ATOM 303 CA GLU 941 20 .956 24 .782 5 .998 1 .00 24 .55

ATOM 304 CB GLU 941 22 .467 25 .046 5 .931 1 .00 29 .29

ATOM 305 CG GLU 941 22 .890 26 .397 6 .509 1 .00 39 .55

ATOM 306 CD GLU 941 24 .371 26 .712 6 .317 1 .00 45. .17

ATOM 307 OE1 GLU 941 24 .698 27 .902 6 .088 1 .00 47. .24

ATOM 308 OE2 GLU 941 25 .205 25 .780 6 .406 1, .00 46 .83

ATOM 309 C GLU 941 20. .640 23 .367 5 .506 1, .00 23, .01

ATOM 310 O GLU 941 20. .234 23 .169 4, .361 1, .00 23, .35

ATOM 311 N ALA 942 20, .799 22 .393 6 .397 1, .00 20, .90

ATOM 312 H ALA 942 21, .090 22 .606 7, .301 1, .00 10, .00

ATOM 313 CA ALA 942 20, .531 20 .998 6, .080 1, .00 19, .22

ATOM 314 CB ALA 942 21, .183 20 .096 7, .115 1, .00 18, .33

ATOM 315 C ALA 942 19. .026 20. .740 6. .016 1. .00 19. .76

ATOM 316 O ALA 942 18. .547 20, .018 5. .140 1. .00 19. .96

ATOM 317 N VAL 943 18. .283 21, .357 6. .934 1. .00 18. .87

ATOM 318 H VAL 943 18. .728 21, .923 7. .601 1. .00 10. .00

ATOM 319 CA VAL 943 16. .831 21, .203 6. .991 1. .00 16. .47

ATOM 320 CB VAL 943 16. .233 21, .956 8. .210 1. .00 14. .10

ATOM 321 CGI VAL 943 14. ,724 21, .972 8. .130 1. .00 13. .60

ATOM 322 CG2 VAL 943 16. .665 21, .297 9. .509 1. .00 13. .04

ATOM 323 C VAL 943 16. ,165 21. .715 5. .718 1. .00 17. .56

ATOM 324 O VAL 943 15. .303 21. .054 5. .142 1. .00 18. .81

ATOM 325 N VAL 944 16. .611 22. .874 5. .256 1. .00 18. .22

ATOM 326 H VAL 944 17. .343 23. .321 5. .728 1. .00 10. .00

ATOM 327 CA VAL 944 16. .056 23. .511 4. .076 1. .00 19. .55

ATOM 328 CB VAL 944 16. .627 24. .961 3. .944 1. .00 20. .45

ATOM 329 CGI VAL 944 17. .814 25. .028 2. .986 1. .00 22. .10

ATOM 330 CG2 VAL 944 15. .537 25. .919 3. .577 1. .00 20. ,47

ATOM 331 C VAL 944 16. .193 22. .694 2. .779 1. .00 21. .31

ATOM 332 O VAL 944 15. .579 23. .019 1. .760 1. .00 21. .60

ATOM 333 N HIS 945 16. .983 21. .626 2. .815 1. .00 22. .23

ATOM 334 H HIS 945 17. .461 21. .383 3. .636 1. .00 10. .00

ATOM 335 CA HIS 945 17. .148 20. .776 1. .638 1. .00 24. .47

ATOM 336 CB HIS 945 18. 627 20. .625 1. ,270 1. 00 29. .45

ATOM 337 CG HIS 945 19. 258 21. ,897 0. ,787 1. .00 35. 62

ATOM 338 CD2 HIS 945 20. 466 22. ,447 1. 041 1. 00 37. .57

ATOM 339 ND1 HIS 945 18. 596 22. ,785 -0. 036 1. 00 39. .22

ATOM 340 HD1 HIS 945 17. 684 22. .691 -0. ,399 1. .00 10. .00

ATOM 341 CE1 HIS 945 19. 370 23. ,830 -0. 263 1. 00 39. .31

ATOM 342 NE2 HIS 945 20. 509 23. ,653 0. 379 1. 00 40. 84

ATOM 343 HE2 HIS 945 21. 255 24. ,292 0. 385 1. 00 10. 00

ATOM 344 C HIS 945 16. 493 19. ,405 1. 813 1. 00 23. 50

ATOM 345 O HIS 945 16. 784 18. ,468 1. 068 1. 00 25. 20

ATOM 346 N MET 946 15. 606 19. ,294 2. 794 1. 00 19. 74

ATOM 347 H MET 946 15. 384 20. ,079 3. 341 1. 00 10. 00

ATOM 348 CA MET 946 14. 908 18. 045 3. 049 1. 00 18. 63

ATOM 349 CB MET 946 14. 359 18. ,014 4. 473 1. 00 17. 73 ATOM 350 CG MET 946 15.406 17.951 5.545 1.00 19.84

ATOM 351 SD MET 946 14 .648 17 .941 7 .167 1 .00 21 .77

ATOM 352 CE MET 946 14 .493 16 .171 7 .450 1 .00 18 .09

ATOM 353 C MET 946 13 .745 17 .867 2 .089 1 .00 18 .12

ATOM 354 O MET 946 13 .234 18 .834 1 .523 1 .00 18 .77

ATOM 355 N SER 947 13 .327 16 .617 1 .927 1 .00 17 .33

ATOM 356 H SER 947 13 .780 15 .896 2 .405 1 .00 10 .00

ATOM 357 CA SER 947 12 .198 16 .267 1 .078 1 .00 15 .98

ATOM 358 CB SER 947 12 .641 15 .297 •0 .022 1 .00 16 .41

ATOM 359 OG SER 947 13 .144 14 .084 0 .523 1 .00 14 .22

ATOM 360 HG SER 947 12 .472 13 .637 1 .029 1 .00 10 .00

ATOM 361 C SER 947 11 .193 15 .585 1 .999 1 .00 15 .60

ATOM 362 O SER 947 11 .484 15 .367 3 .182 1, .00 15 .48

ATOM 363 N GLN 948 10 .033 15 .221 1 .461 1 .00 15 .65

ATOM 364 H GLN 948 9 .852 15 .411 0 .521 1, .00 10 .00

ATOM 365 CA GLN 948 9 .011 14 .543 2 .251 1. .00 16 .60

ATOM 366 CB GLN 948 7 .764 14 .268 1 .395 1, .00 21 .94

ATOM 367 CG GLN 948 6 .567 13 .726 2, .190 1. .00 27, .32

ATOM 368 CD GLN 948 5. .542 13 .007 1 .323 1, .00 31 .25

ATOM 369 OE1 GLN 948 5, .303 13 .382 0, .175 1. .00 34 .83

ATOM 370 NE2 GLN 948 4, .941 11 .958 1, .868 1, .00 32 .46

ATOM 371 HE21 GLN 948 5, .180 11 .719 2, .787 1, .00 10, .00

ATOM 372 HE22 GLN 948 4, .291 11. .486 1, .312 1. .00 10, .00

ATOM 373 C GLN 948 9, .583 13 .216 2, .768 1. .00 15, .21

ATOM 374 O GLN 948 9, .406 12, .864 3. .936 1. .00 12, .66

ATOM 375 N ASP 949 10, .304 12 .511 1, .900 1. .00 13, .65

ATOM 376 H ASP 949 10, .406 12, .861 0, .994 1. .00 10. .00

ATOM 377 CA ASP 949 10. .901 11, .223 2. .251 1. ,00 14. .81

ATOM 378 CB ASP 949 11, .553 10, .574 1. .022 1. ,00 15, .09

ATOM 379 CG ASP 949 12. .016 9, .153 1. .286 1. ,00 17, .60

ATOM 380 OD1 ASP 949 13. .236 8. .913 1. .211 1. ,00 18. .94

ATOM 381 OD2 ASP 949 11. .167 8, .276 1. .564 1. .00 19. .58

ATOM 382 C ASP 949 11. .905 11. .352 3. .386 1. .00 13. .01

ATOM 383 O ASP 949 12. .030 10. .449 4, .205 1. .00 14. .17

ATOM 384 N ASP 950 12. .608 12. .480 3. .438 1. .00 13. .51

ATOM 385 H ASP 950 12. ,479 13. .181 2. ,775 1. ,00 10. .00

ATOM 386 CA ASP 950 13. .580 12. .717 4, .501 1. .00 13. .81

ATOM 387 CB ASP 950 14. .390 13. .990 4. .234 1. ,00 14. .10

ATOM 388 CG ASP 950 15. .410 13. .813 3. .120 1. .00 14. .34

ATOM 389 OD1 ASP 950 15. .924 12. .691 2. .941 1. ,00 16. .13

ATOM 390 OD2 ASP 950 15. .706 14. ,798 2. ,422 1. .00 16. .24

ATOM 391 C ASP 950 12. .861 12, .830 5. .838 1. .00 13. .61

ATOM 392 O ASP 950 13. .312 12. .272 6. ,834 1. .00 14. .29

ATOM 393 N LEU 951 11. .721 13. .521 5. .843 1. .00 12. .11

ATOM 394 H LEU 951 11. .404 13. .921 5. ,005 1. .00 10. .00

ATOM 395 CA LEU 951 10. .923 13. .702 7. ,054 1. ,00 12. ,52

ATOM 396 CB LEU 951 9. .802 14. .722 6. ,815 1. ,00 11. .55

ATOM 397 CG LEU 951 10. .264 16. .155 6. ,522 1. 00 13. .10

ATOM 398 CD1 LEU 951 9. ,085 17. .016 6. ,102 1. ,00 11. .51

ATOM 399 CD2 LEU 951 10. ,956 16. .743 7. ,740 1. 00 10. ,00

ATOM 400 C LEU 951 10. ,345 12. ,376 7. ,551 1. 00 11. ,50

ATOM 401 O LEU 951 10. .320 12. .122 8. ,755 1. 00 12. ,61

ATOM 402 N ALA 952 9. 887 11. ,532 6. 628 1. 00 11. ,25

ATOM 403 H ALA 952 9. ,920 11. ,800 5. ,686 1. 00 10. ,00

ATOM 404 CA ALA 952 9. ,341 10. ,229 6. 991 1. 00 11. ,84

ATOM 405 CB ALA 952 8. .741 9. .544 5. 161 1. 00 10. ,00

ATOM 406 C ALA 952 10. .455 9. .363 7. 598 1. 00 13. ,43

ATOM 407 O ALA 952 10. 278 8. .750 8. 654 1. 00 14. 76

ATOM 408 N ARG 953 11. 614 9. ,361 6. 944 1. 00 12. ,41 ATOM 409 H ARG 953 11.707 9.895 6.128 1.00 10.00

ATOM 410 CA ARG 953 12 .776 8 .591 7 .390 1 .00 15 .00

ATOM 411 CB ARG 953 13 .921 8 .763 6 .381 1 .00 15 .69

ATOM 412 CG ARG 953 15 .223 8 .077 6 .762 1 .00 22 .48

ATOM 413 CD ARG 953 16 .299 8 .246 5 .686 1 .00 24 .87

ATOM 414 NE ARG 953 16 .604 9 .651 5 .409 1 .00 25 .58

ATOM 415 HE ARG 953 16 .130 10 .085 4 .673 1 .00 10 .00

ATOM 416 CZ ARG 953 17 .475 10 .387 6 .095 1 .00 25 .21

ATOM 417 NH1 ARG 953 18 .146 9 .863 7 .115 1 .00 24 .46

ATOM 418 HHll ARG 953 17 .997 8 .911 7 .378 1. .00 0 .00

ATOM 419 HH12 ARG 953 18 .796 10 .425 7 .626 1. .00 0 .00

ATOM 420 NH2 ARG 953 17 .668 11 .656 5 .765 1 .00 22 .85

ATOM 421 HH21 ARG 953 17 .157 12 .052 5 .000 1. .00 0 .00

ATOM 422 HH22 ARG 953 18 .321 12 .214 6 .276 1, .00 0 .00

ATOM 423 C ARG 953 13 .225 9 .028 8 .786 1. .00 15 .66

ATOM 424 O ARG 953 13 .771 8 .237 9 .551 1. .00 16 .37

ATOM 425 N ILE 954 12 .947 10 .285 9 .115 1. .00 16 .54

ATOM 426 H ILE 954 12 .499 10 .847 8 .451 1. .00 10 .00

ATOM 427 CA ILE 954 13 .303 10 .891 10, .396 1. .00 15, .90

ATOM 428 CB ILE 954 13 .518 12 .437 10 .194 1. .00 18 .15

ATOM 429 CG2 ILE 954 12 .974 13 .269 11, .338 1. .00 20 .90

ATOM 430 CGI ILE 954 14 .997 12 .722 9, .975 1. .00 18, .92

ATOM 431 CD1 ILE 954 15, .609 11, .919 8, .863 1. .00 23. .36

ATOM 432 C ILE 954 12, .305 10, .573 11, .519 1. .00 14. .18

ATOM 433 O ILE 954 12 .613 10, .743 12, .700 1. .00 12. .65

ATOM 434 N GLY 955 11, .135 10, .065 11, .153 1. .00 13. .39

ATOM 435 H GLY 955 10, .942 9, .909 10. .205 1. .00 10. .00

ATOM 436 CA GLY 955 10, .136 9. .736 12. .154 1. .00 14. .64

ATOM 437 C GLY 955 8, .901 10, .616 12, .114 1. .00 14. .46

ATOM 438 O GLY 955 7. .993 10, .462 12. .930 1. .00 17. .04

ATOM 439 N ILE 956 8, .862 11. .558 11. .181 1. .00 14. .53

ATOM 440 H ILE 956 9. .610 11. .653 10. .555 1. .00 10. .00

ATOM 441 CA ILE 956 7, .710 12. .441 11. .055 1. .00 15. .41

ATOM 442 CB ILE 956 8, .142 13. .883 10. .676 1. .00 14. .68

ATOM 443 CG2 ILE 956 6. .925 14. .817 10. .645 1. .00 13. .49

ATOM 444 CGI ILE 956 9. .186 14. .384 11. .683 1. .00 14. .55

ATOM 445 CD1 ILE 956 9, .793 15. .712 11. .340 1. ,00 14. .38

ATOM 446 C ILE 956 6. .819 11. .829 9. .977 1. .00 14. .93

ATOM 447 O ILE 956 6. .954 12. .128 8. .789 1. ,00 15. .07

ATOM 448 N THR 957 5. .937 10. .934 10. .408 1. ,00 16. .86

ATOM 449 H THR 957 5. .929 10. .728 11. .366 1. ,00 10. .00

ATOM 450 CA THR 957 5. .031 10. .231 9. .506 1. ,00 17. .30

ATOM 451 CB THR 957 4. .822 8. .779 9. .960 1. .00 17. .04

ATOM 452 OGl THR 957 4. .334 8. .770 11. .308 1. .00 19. .91

ATOM 453 HGl THR 957 4. .987 9. .124 11. ,917 1. 00 10. .00

ATOM 454 CG2 THR 957 6. ,134 8. .005 9. .887 1. ,00 18. .15

ATOM 455 C THR 957 3. .671 10. .890 9. .336 1. ,00 16. .02

ATOM 456 O THR 957 2. .987 10. .638 8. ,349 1. 00 16. .25

ATOM 457 N ALA 958 3. .269 11. .710 10. ,304 1. 00 16. ,24

ATOM 458 H ALA 958 3. .857 11. .836 11. .074 1. ,00 10. .00

ATOM 459 CA ALA 958 1. .982 12. .406 10. ,223 1. 00 15. ,66

ATOM 460 CB ALA 958 1. .680 13. ,140 11. ,536 1. 00 10. ,94

ATOM 461 C ALA 958 2. ,063 13. ,399 9. 067 1. 00 14. ,30

ATOM 462 O ALA 958 2. .830 14. .365 9. .128 1. 00 14. .94

ATOM 463 N ILE 959 1. ,282 13. ,156 8. .019 1. 00 14. ,35

ATOM 464 H ILE 959 0. ,709 12. ,360 8. 024 1. 00 10. ,00

ATOM 465 CA ILE 959 1. ,286 14. ,017 6. 838 1. 00 16. ,17

ATOM 466 CB ILE 959 0. 286 13. 521 5. 769 1. 00 17. ,13

ATOM 467 CG2 ILE 959 0. ,351 14. ,410 4. 532 1. 00 17. ,99 ATOM 468 CGI ILE 959 0.622 12.078 5.371 1.00 20.81

ATOM 469 CD1 ILE 959 -0 .355 11 .449 4 .383 1 .00 21 .87

ATOM 470 C ILE 959 1 .042 15 .493 7 .163 1 .00 16 .99

ATOM 471 O ILE 959 1 .661 16 .374 6 .564 1 .00 18 .92

ATOM 472 N THR 960 0 .179 15 .765 8 .137 1 .00 16 .32

ATOM 473 H THR 960 •0 .303 15 .030 8 .563 1 .00 10 .00

ATOM 474 CA THR 960 •0 .103 17 .142 8 .534 1 .00 16 .86

ATOM 475 CB THR 960 •1 .165 17 .190 9 .660 1 .00 18 .03

ATOM 476 OGl THR 960 •2 .384 16 .597 9 .197 1 .00 18 .57

ATOM 477 HGl THR 960 ^■3 .070 16 .619 9 .863 1 .00 10 .00

ATOM 478 CG2 THR 960 ^■1 .438 18 .618 10 .082 1 .00 18 .31

ATOM 479 C THR 960 1 .180 17 .824 9 .026 1 .00 16 .90

ATOM 480 O THR 960 1 .465 18 .974 8 .683 1 .00 16 .92

ATOM 481 N HIS 961 1 .955 17 .105 9 .830 1, .00 16, .15

ATOM 482 H HIS 961 1 .713 16 .175 10 .007 1 .00 10 .00

ATOM 483 CA HIS 961 3 .197 17 .646 10 .364 1. .00 15, .68

ATOM 484 CB HIS 961 3, .689 16, .789 11 .532 1. .00 15, .19

ATOM 485 CG HIS 961 2 .758 16 .807 12 .706 1, .00 14, .80

ATOM 486 CD2 HIS 961 1, .715 17, .624 12 .994 1, .00 14, .08

ATOM 487 ND1 HIS 961 2, .834 15, .909 13 .747 1. .00 14. .93

ATOM 488 HD1 HIS 961 3, .301 15, .070 13 .827 1. .00 10, .00

ATOM 489 CE1 HIS 961 1, .886 16, .172 14 .625 1. .00 16. .16

ATOM 490 NE2 HIS 961 1, .191 17 .207 14 .192 1. .00 14, .72

ATOM 491 HE2 HIS 961 0, .422 17, .616 14, .676 1. .00 10. .00

ATOM 492 C HIS 961 4. .248 17, .788 9, .268 1. .00 15. .94

ATOM 493 O HIS 961 5, .020 18, .750 9 .272 1. .00 14. .39

ATOM 494 N GLN 962 4. .242 16, .853 8, .316 1. .00 16. .90

ATOM 495 H GLN 962 3. .608 16. .106 8, .356 1. .00 10. .00

ATOM 496 CA GLN 962 5. .167 16, .895 7, .185 1. .00 16. .19

ATOM 497 CB GLN 962 4. .959 15. .694 6, .254 1. .00 15. .77

ATOM 498 CG GLN 962 5. .490 14. .367 6, .780 1. ,00 16. .87

ATOM 499 CD GLN 962 5. .269 13. .218 5, .802 1. .00 17. .54

ATOM 500 OE1 GLN 962 4. .677 13. .396 4. .737 1. .00 18. .11

ATOM 501 NE2 GLN 962 5. .743 12. .036 6. .163 1. .00 17. .65

ATOM 502 HE21 GLN 962 6. .214 11, .964 7. .022 1. .00 10. .00

ATOM 503 HE22 GLN 962 5. .602 11. .288 5. .549 1. ,00 10. .00

ATOM 504 C GLN 962 4. .903 18, .173 6. .401 1. .00 15. .74

ATOM 505 O GLN 962 5. .832 18. .899 6. .059 1. ,00 15. .23

ATOM 506 N ASN 963 3. .625 18. .463 6. .168 1. .00 15. .23

ATOM 507 H ASN 963 2. .932 17. .852 6. .489 1. .00 10. .00

ATOM 508 CA ASN 963 3. .232 19. .649 5. .415 1. ,00 16. ,00

ATOM 509 CB ASN 963 1. . 161 19. ,562 4. .981 1. ,00 16. ,74

ATOM 510 CG ASN 963 1. ,567 18. ,605 3. .821 1. ,00 20. ,62

ATOM 511 OD1 ASN 963 2. ,379 18. ,560 2. .895 1. 00 24. ,90

ATOM 512 ND2 ASN 963 0. ,510 17. .811 3. .879 1. ,00 22. ,06

ATOM 513 HD21 ASN 963 0. ,085 17. .835 4. .647 1. 00 0. 00

ATOM 514 HD22 ASN 963 0. ,384 17. .209 3. .108 1. ,00 0. ,00

ATOM 515 C ASN 963 3. ,513 20. ,955 6. .129 1. 00 14. ,80

ATOM 516 O ASN 963 3. 907 21. ,925 5. ,495 1. 00 13. 94

ATOM 517 N LYS 964 3. ,344 20. ,978 7. .448 1. 00 15. ,34

ATOM 518 H LYS 964 3. 023 20. ,172 7. ,901 1. 00 10. 00

ATOM 519 CA LYS 964 3. 618 22. 191 8. ,208 1. 00 16. 38

ATOM 520 CB LYS 964 3. 254 22. ,013 9. ,686 1. 00 16. 89

ATOM 521 CG LYS 964 3. 415 23. 289 10. ,501 1. 00 20. 93

ATOM 522 CD LYS 964 2. 888 23. ,125 11. .911 1. 00 28. 63

ATOM 523 CE LYS 964 1. 917 24. 244 12. .280 1. 00 31. 73

ATOM 524 NZ LYS 964 2. 519 25. 607 12. 214 1. 00 33. 69

ATOM 525 HZ1 LYS 964 2. 843 25. 813 11. .248 1. 00 10. 00

ATOM 526 HZ2 LYS 964 3. 314 25. 670 12. 878 1. 00 10. 00 ATOM 527 HZ3 LYS 964 1.798 26.313 12.479 1.00 10.00

ATOM 528 C LYS 964 5 .103 22 .514 8 .078 1 .00 16 .37

ATOM 529 O LYS 964 5 .487 23 .662 7 .844 1 .00 17 .92

ATOM 530 N ILE 965 5 .930 21 .481 8 .199 1 .00 16 .65

ATOM 531 H ILE 965 5 .562 20 .586 8 .353 1 .00 10 .00

ATOM 532 CA ILE 965 7 .374 21 .629 8 .099 1 .00 14 .00

ATOM 533 CB ILE 965 8 .088 20 .346 8 .560 1 .00 12 .06

ATOM 534 CG2 ILE 965 9 .560 20 .369 8 .151 1 .00 11 .41

ATOM 535 CGI ILE 965 7 .934 20 .202 10 .076 1 .00 10 .00

ATOM 536 CD1 ILE 965 8 .302 18 .849 10 .602 1, .00 10 .00

ATOM 537 C ILE 965 7 .800 22 .012 6 .687 1, .00 13, .95

ATOM 538 O ILE 965 8 .574 22 .951 6 .506 1, .00 15 .38

ATOM 539 N LEU 966 7 .274 21 .310 5 .691 1, .00 13, .46

ATOM 540 H LEU 966 6 .650 20 .584 5 .886 1, .00 10 .00

ATOM 541 CA LEU 966 7 .609 21 .605 4 .304 1, .00 15, .13

ATOM 542 CB LEU 966 7 .023 20, .542 3 .372 1. .00 15, .28

ATOM 543 CG LEU 966 7 .738 19 .187 3 .463 1. .00 14, .45

ATOM 544 CD1 LEU 966 6 .910 18, .105 2 .791 1. .00 14, .55

ATOM 545 CD2 LEU 966 9 .123 19 .285 2 .841 1. .00 12, .40

ATOM 546 C LEU 966 7 .173 23, .015 3 .886 1. .00 15. .31

ATOM 547 O LEU 966 7, .909 23, .712 3, .181 1. .00 13. .98

ATOM 548 N SER 967 6 .000 23, .446 4 .345 1. .00 15, .58

ATOM 549 H SER 967 5, .435 22, .863 4, .889 1. .00 10. .00

ATOM 550 CA SER 967 5 .506 24, .780 4 .029 1. .00 15. .48

ATOM 551 CB SER 967 4, .073 24, .974 4, .537 1. .00 16. .99

ATOM 552 OG SER 967 3, .142 24. .292 3, .715 1. ,00 19. .67

ATOM 553 HG SER 967 2, .272 24. .346 4, .118 1. ,00 10, .00

ATOM 554 C SER 967 6, .427 25. .824 4, .643 1. .00 16. .18

ATOM 555 O SER 967 6, .725 26. .839 4, .017 1. .00 16, .88

ATOM 556 N SER 968 6, .892 25. .554 5, .859 1. .00 16. ,46

ATOM 557 H SER 968 6. .624 24. .721 6. .303 1. ,00 10. .00

ATOM 558 CA SER 968 7. .789 26. .459 6, .570 1. .00 17. .49

ATOM 559 CB SER 968 8. ,024 25. .947 7^'. .995 1. ,00 17. .79

ATOM 560 OG SER 968 8. .766 26. .871 8, .774 1. ,00 16. .89

ATOM 561 HG SER 968 8. .248 27. .676 8. .828 1. ,00 10. .00

ATOM 562 C SER 968 9. .114 26. .562 5. .815 1. ,00 18. .08

ATOM 563 O SER 968 9. .661 27. .654 5. .650 1. ,00 18. .78

ATOM 564 N VAL 969 9. .621 25. .418 5. .360 1. ,00 17. .77

ATOM 565 H VAL 969 9. .145 24. .581 5. .543 1. .00 10. .00

ATOM 566 CA VAL 969 10. .863 25. .362 4. .598 1. ,00 16. .32

ATOM 567 CB VAL 969 11. .201 23. .892 4. .189 1. 00 15. ,01

ATOM 568 CGI VAL 969 12. .232 23. .855 3. .065 1. ,00 14. ,69

ATOM 569 CG2 VAL 969 11. ,729 23. .123 5. .390 1. 00 12. ,25

ATOM 570 C VAL 969 10. .720 26. .256 3. .360 1. ,00 16. .88

ATOM 571 O VAL 969 11. .597 27. .072 3. .071 1. 00 18. .31

ATOM 572 N GLN 970 9. .590 26. .140 2. .666 1. 00 17. .34

ATOM 573 H GLN 970 8. .915 25. .497 2. .971 1. 00 10. ,00

ATOM 574 CA GLN 970 9. .334 26. .945 1. .475 1. 00 18. ,13

ATOM 575 CB GLN 970 7. .977 26. .588 0. .859 1. 00 17. ,14

ATOM 576 CG GLN 970 7. .886 25. ,155 0. .350 1. 00 18. ,59

ATOM 577 CD GLN 970 6. ,520 24. 809 -0. ,215 1. 00 20. 55

ATOM 578 OE1 GLN 970 6. ,417 24. ,141 -1. .237 1. 00 23. ,07

ATOM 579 NE2 GLN 970 5. ,466 25. 251 0. ,453 1. 00 21. 52

ATOM 580 HE21 GLN 970 5. ,590 25. ,769 1. .273 1. 00 10. ,00

ATOM 581 HE22 GLN 970 4. ,591 25. 025 0. ,074 1. 00 10. 00

ATOM 582 C GLN 970 9. .391 28. 442 1. 792 1. 00 19. 00

ATOM 583 O GLN 970 9. ,998 29. 216 1. ,047 1. 00 19. 59

ATOM 584 N ALA 971 8. 793 28. 832 2. ,915 1. 00 18. 42

ATOM 585 H ALA 971 8. ,345 28. 158 3. ,469 1. 00 10. 00 ATOM 586 CA ALA 971 8.772 30.227 3.351 1.00 18.65

ATOM 587 CB ALA 971 7 .797 30 .397 4 .510 1 .00 16 .83

ATOM 588 C ALA 971 10 .160 30 .733 3 .748 1 .00 19 .41

ATOM 589 O ALA 971 10 .512 31 .884 3 .477 1 .00 20 .57

ATOM 590 N MET 972 10 .945 29 .880 4 .399 1 .00 20 .08

ATOM 591 H MET 972 10 .613 28 .976 4 .582 1 .00 10 .00

ATOM 592 CA MET 972 12 .283 30 .263 4 .819 1 .00 20 .97

ATOM 593 CB MET 972 12 .876 29 .248 5 .788 1 .00 23 .16

ATOM 594 CG MET 972 12 .260 29 .289 7 .174 1 .00 25 .68

ATOM 595 SD MET 972 13 .275 28 .393 8 .360 1 .00 29 .72

ATOM 596 CE MET 972 13 .134 26 .696 7 .733 1 .00 28 .75

ATOM 597 C MET 972 13 .199 30 .431 3 .620 1 .00 23, .17

ATOM 598 O MET 972 14 .072 31 .297 3 .624 1 .00 22 .87

ATOM 599 N ARG 973 13 .002 29 .603 2 .597 1, .00 22, .37

ATOM 600 H ARG 973 12 .308 28 .911 2 .661 1 .00 10 .00

ATOM 601 CA ARG 973 13, .803 29 .690 1 .382 1, .00 22, .73

ATOM 602 CB ARG 973 13, .513 28 .507 0 .458 1, .00 19. .60

ATOM 603 CG ARG 973 14 .116 27 .209 0 .929 1, .00 18. .92

ATOM 604 CD ARG 973 13, .681 26 .058 0 .056 1, .00 19. .57

ATOM 605 NE ARG 973 13, .960 26 .318 -1 .353 1, .00 23, .42

ATOM 606 HE ARG 973 13, .341 26 .895 -1, .845 1, .00 10. .00

ATOM 607 CZ ARG 973 14 .994 25 .817 -2 .020 1, .00 22. .59

ATOM 608 NH1 ARG 973 15, .862 25, .015 -1 .414 1, .00 23. .50

ATOM 609 HHll ARG 973 15. .747 24, .775 -0, .452 1. .00 0. .00

ATOM 610 HH12 ARG 973 16. .628 24, .635 -1, .935 1. .00 0. .00

ATOM 611 NH2 ARG 973 15. .167 26, .135 -3, .295 1. .00 22. .09

ATOM 612 HH21 ARG 973 14. .515 26, .744 -3, .751 1. .00 0. .00

ATOM 613 HH22 ARG 973 15. .933 25, .750 -3, .811 1. .00 0. .00

ATOM 614 C ARG 973 13. .507 31, .010 0, .670 1. .00 24. .33

ATOM 615 O ARG 973 14. .432 31, .730 0, .282 1. .00 22. .76

ATOM 616 N THR 974 12. .220 31. .332 0. .527 1. .00 26. .94

ATOM 617 H THR 974 11. .515 30. .729 0. .852 1. .00 10, .00

ATOM 618 CA THR 974 11. .795 32. .576 -0. .113 1. .00 31. ,31

ATOM 619 CB THR 974 10. .264 32, .669 -0. .193 1. .00 30. .78

ATOM 620 OGl THR 974 9. .750 31. .491 -0. .822 1. .00 31. .97

ATOM 621 HGl THR 974 10. .092 31. .425 -1. .714 1. .00 10. ,00

ATOM 622 CG2 THR 974 9. .842 33. .884 -1. .005 1. .00 32. .02

ATOM 623 C THR 974 12. .325 33. .783 0, .671 1. .00 35. .46

ATOM 624 O THR 974 12. .720 34. .792 0. .081 1. .00 36. .00

ATOM 625 N GLN 975 12. .364 33. .655 1. .996 1. .00 39. .13

ATOM 626 H GLN 975 12. .031 32. .830 2. .414 1. .00 10. .00

ATOM 627 CA GLN 975 12. .858 34. .715 2. .869 1. .00 42. .57

ATOM 628 CB GLN 975 12. ,586 34. .359 4. .334 1. ,00 45. ,95

ATOM 629 CG GLN 975 12. .793 35. .519 5. .300 1. .00 51. .94

ATOM 630 CD GLN 975 12. .891 35. .080 6. .751 1. ,00 54. ,33

ATOM 631 OE1 GLN 975 12. .227 34. .130 7. .180 1. .00 54. .56

ATOM 632 NE2 GLN 975 13. ,728 35. .773 7. .517 1. ,00 55. ,74

ATOM 633 HE21 GLN 975 14. .227 36. .509 7. .105 1. .00 10. ,00

ATOM 634 HE22 GLN 975 13. ,811 35. .543 8. .462 1. ,00 10. ,00

ATOM 635 C GLN 975 14. 361 34. .917 2. .660 1. 00 42. 58

ATOM 636 O GLN 975 14. ,862 36. .043 2. .706 1. ,00 43. ,50

ATOM 637 N MET 976 15. 072 33. .818 2. .424 1. 00 42. 59

ATOM 638 H MET 976 14. ,606 32. .954 2. .412 1. ,00 10. ,00

ATOM 639 CA MET 976 16. ,513 33. .860 2. .204 1. 00 42. 79

ATOM 640 CB MET 976 17. ,129 32. .480 2. ,416 1. 00 41. 79

ATOM 641 CG MET 976 16. 916 31. ,925 3. ,810 1. 00 42. 17

ATOM 642 SD MET 976 17. 517 33. 018 5. 102 1. 00 44. 85

ATOM 643 CE MET 976 16. 022 33. ,352 6. 019 1. 00 42. 12

ATOM 644 C MET 976 16. 892 34. 408 0. 827 1. 00 43. 71 ATOM 645 O MET 976 18.016 34.871 0.631 1.00 44.60

ATOM 646 N GLN 977 15 .975 34 .327 -0 .136 1 .00 43 .94

ATOM 647 H GLN 977 15 .105 33 .913 0 .057 1 .00 10 .00

ATOM 648 CA GLN 977 16 .235 34 .859 -1 .472 1 .00 45 .08

ATOM 649 CB GLN 977 15 .122 34 .482 -2 .449 1 .00 46 .20

ATOM 650 CG GLN 977 15 .070 33 .029 -2 .832 1 .00 49 .33

ATOM 651 CD GLN 977 14 .291 32 .813 -4 .112 1 .00 52 .53

ATOM 652 OE1 GLN 977 14 .742 33 .196 -5 .195 1 .00 52 .71

ATOM 653 NE2 GLN 977 13 .119 32 .198 -4 .000 1 .00 53 .68

ATOM 654 HE21 GLN 977 12 .811 31 .913 -3 .116 1 .00 10 .00

ATOM 655 HE22 GLN 977 12 .622 32 .067 -4 .836 1 .00 10 .00

ATOM 656 C GLN 977 16 .282 36 .375 -1 .370 1 .00 45, .82

ATOM 657 O GLN 977 17 .046 37 .037 -2 .070 1 .00 45 .80

ATOM 658 N GLN 978 15 .453 36 .903 -0 .475 1, .00 47, .73

ATOM 659 H GLN 978 14 .872 36 .302 0 .037 1 .00 10 .00

ATOM 660 CA GLN 978 15 .337 38 .333 -0 .224 1, .00 48. .95

ATOM 661 CB GLN 978 14 .007 38 .606 0 .482 1 .00 50, .40

ATOM 662 CG GLN 978 12 .800 38 .062 -0 .288 1, .00 51, .80

ATOM 663 CD GLN 978 11 .535 37 .947 0 .556 1, .00 54, .30

ATOM 664 OE1 GLN 978 10, .439 37, .770 0 .021 1. .00 53, .56

ATOM 665 NE2 GLN 978 11 .683 38 .018 1 .877 1, .00 54, .22

ATOM 666 HE21 GLN 978 12, .559 38, .123 2, .294 1. .00 10, .00

ATOM 667 HE22 GLN 978 10, .843 37 .960 2 .381 1, .00 10, .00

ATOM 668 C GLN 978 16, .511 38, .860 0, .604 1. .00 49. .37

ATOM 669 O GLN 978 16. ,656 40. .068 0, .792 1. ,00 51. .47

ATOM 670 N MET 979 17. .361 37. .949 1, .070 1. .00 49. .20

ATOM 671 H MET 979 17. .205 37. .003 0. .891 1. .00 10, .00

ATOM 672 CA MET 979 18. .532 38. .309 1, .863 1. .00 49. .49

ATOM 673 CB MET 979 18. .939 37. .138 2. .767 1. .00 50. .98

ATOM 674 CG MET 979 19. .641 37. .532 4. .064 1. .00 52. .98

ATOM 675 SD MET 979 18. .533 38. .331 5. .249 1. .00 56. .67

ATOM 676 CE MET 979 17. .092 37. .225 5. .208 1. .00 55. .34

ATOM 677 C MET 979 19. .702 38. .672 0. .941 1. .00 48. .66

ATOM 678 O MET 979 20. .842 38. .788 1. .392 1. .00 48. ,62

ATOM 679 N HIS 980 19. ,424 38. .802 -0. .356 1. .00 46. .50

ATOM 680 H HIS 980 18. .519 38. .655 -0. .702 1. .00 10. .00

ATOM 681 CA HIS 980 20. .454 39. .155 -1. .319 1. ,00 44. .17

ATOM 682 CB HIS 980 21. .468 38. .018 -1. .487 1. .00 39. .73

ATOM 683 CG HIS 980 20. .883 36. .743 -2. .002 1. .00 37. .33

ATOM 684 CD2 HIS 980 20. .507 35. .611 -1. .360 1. .00 34. .61

ATOM 685 ND1 HIS 980 20. .667 36. .504 -3. .343 1. .00 34. ,47

ATOM 686 HD1 HIS 980 20. ,768 37. .181 -4. .053 1. ,00 10. ,00

ATOM 687 CE1 HIS 980 20. .192 35. .286 -3. .505 1. ,00 30. ,99

ATOM 688 NE2 HIS 980 20. .087 34. .722 -2. .315 1. 00 32. ,54

ATOM 689 HE2 HIS 980 19. .880 33. .801 -2. .092 1. ,00 10. ,00

ATOM 690 C HIS 980 19. .910 39. ,599 -2. .676 1. 00 45. 06

ATOM 691 O HIS 980 20. .126 38. ,942 -3. .695 1. ,00 46. ,18

ATOM 692 N GLY 981 19. .171 40. ,703 -2. .668 1. 00 46. 12

ATOM 693 H GLY 981 18. ,996 41. .168 -1. .838 1. ,00 10. ,00

ATOM 694 CA GLY 981 18. 623 41. .249 -3. .900 1. 00 46. 12

ATOM 695 C GLY 981 19. ,611 42. .222 -4. ,526 1. 00 46. ,17

ATOM 696 O GLY 981 19. 297 42. .809 -5. ,583 1. 00 45. 93

ATOM 697 OT GLY 981 20. ,710 42. ,404 -3. ,954 1. 00 47. ,24

ATOM 698 OH2 TIP3 1 5. 348 20. ,105 18. ,757 1. 00 13. 76

SOLV

ATOM 699 OH2 TIP3 2 • 1 . 607 18 . 597 5 . 643 1 . 00 10 . 67

SOLV

ATOM 700 OH2 TIP3 3 11 . 575 6 .309 4 . 064 1 . 00 22 .41

SOLV ATOM 701 OH2 TIP3 4 -1.390 17.519 15.624 1.00 26.01 SOLV ATOM 702 OH2 TIP3 5 10 .169 12 .836 -1 .287 1 .00 21 .06 SOLV ATOM 703 OH2 TIP3 6 22 .240 14 .887 7 .379 1 .00 23 .85 SOLV ATOM 704 OH2 TIP3 7 4 .944 28 .525 2 .399 1 .00 23 .14 SOLV ATOM 705 OH2 TIP3 8 9 .075 16 .556 -1 .049 1 .00 20 .96 SOLV ATOM 706 OH2 TIP3 9 20 .954 26 .572 12 .360 1 .00 24 .79 SOLV ATOM 707 OH2 TIP3 10 11 .237 23 .923 22 .404 1, .00 26 .34 SOLV ATOM 708 OH2 TIP3 12 2 .003 11 .736 0 .983 1, .00 32 .92 SOLV ATOM 709 OH2 TIP3 13 22, .341 26 .794 8, .789 1. .00 30, .68 SOLV ATOM 710 OH2 TIP3 14 20, .229 8 .193 9, .431 1. .00 32, .28 SOLV ATOM 711 OH2 TIP3 15 9, .002 9 .880 15, .816 1. .00 43, .59 SOLV ATOM 712 OH2 TIP3 16 -1, .539 23, .366 11. .757 1. .00 34. .88 SOLV ATOM 713 OH2 TIP3 18 17, .039 21 .091 18, .454 1. .00 24, .01 SOLV ATOM 714 OH2 TIP3 19 1, .972 14, .588 0, .930 1. .00 37, .49 SOLV ATOM 715 OH2 TIP3 20 2, .846 13, .524 16, .999 1. .00 38, .34 SOLV ATOM 716 OH2 TIP3 21 23. .328 11, .454 15. .152 1. .00 32. .30 SOLV ATOM 717 OH2 TIP3 22 20. .264 11. .491 3. .039 1. .00 37. .02 SOLV ATOM 718 OH2 TIP3 23 -1. .623 13. .755 9. .215 1. .00 24. .03 SOLV ATOM 719 OH2 TIP3 24 7. .117 7. .426 3. .341 1. .00 39. .59 SOLV ATOM 720 OH2 TIP3 25 12. .549 12. .170 21. .440 1. ,00 39. .58 SOLV ATOM 721 OH2 TIP3 26 18. .509 15. ,812 3. .112 1. ,00 19. .84 SOLV ATOM 722 OH2 TIP3 27 -1. .105 20. .234 15. .360 1. ,00 54. .40 SOLV ATOM 723 OH2 TIP3 28 13. ,308 28. .872 -3. ,398 1. 00 31. .59 SOLV ATOM 724 OH2 TIP3 29 20. .963 24. .454 14. .891 1. ,00 29. .47 SOLV ATOM 725 OH2 TIP3 30 11. .976 15. .552 20. .454 1. ,00 22. .32 SOLV ATOM 726 OH2 TIP3 31 15. .358 9. .999 2. .685 1. 00 25. .88 SOLV ATOM 727 OH2 TIP3 32 7. ,138 31. .193 -0. ,433 1. 00 34. ,94 SOLV ATOM 728 OH2 TIP3 33 18. ,565 19. ,866 15. ,827 1. 00 31. ,14 SOLV ATOM 729 OH2 TIP3 35 10. ,191 11. ,998 19. ,068 1. 00 36. ,35 SOLV ATOM 730 OH2 TIP3 36 -1.668 14.793 13.334 1.00 32.50 SOLV ATOM 731 OH2 TIP3 37 24 .133 32 .437 15 .070 1 .00 58 .18 SOLV ATOM 732 OH2 TIP3 38 8 .142 29 .716 8 .516 1 .00 38 .02 SOLV ATOM 733 OH2 TIP3 39 16 .767 15 .194 -0 .104 1 .00 22 .13 SOLV ATOM 734 OH2 TIP3 40 -0 .649 26 .330 15 .687 1 .00 40 .04 SOLV ATOM 735 OH2 TIP3 41 20 .560 21 .578 16 .917 1, .00 41 .45 SOLV ATOM 736 OH2 TIP3 42 24 .262 18, .640 8, .488 1, .00 46 .62 SOLV ATOM 737 OH2 TIP3 43 8, .129 9, .379 1, .097 1. .00 38, .14 SOLV ATOM 738 OH2 TIP3 44 4, .176 17. .058 22, .386 1. .00 43, .87 SOLV ATOM 739 OH2 TIP3 45 4, .329 26, .311 8. .281 1. .00 32, .81 SOLV ATOM 740 OH2 TIP3 46 19, .760 18, .168 3, .397 1. .00 26, .43 SOLV ATOM 741 OH2 TIP3 47 4, .718 28. .777 6, .740 1. .00 27, .69 SOLV ATOM 742 OH2 TIP3 48 7. .659 9. .629 -3. .363 1. .00 41. .77 SOLV ATOM 743 OH2 TIP3 49 2. .827 14. .980 21. .015 1. .00 38. .06 SOLV ATOM 744 OH2 TIP3 50 5. .873 25. .576 -4. .108 1. .00 58. .77 SOLV ATOM 745 OH2 TIP3 51 22. .281 25. .625 17. .414 1. ,00 46, .99 SOLV ATOM 746 OH2 TIP3 53 8. .311 41. .084 2. .721 1. ,00 41. .45 SOLV ATOM 747 OH2 TIP3 54 23. .900 11. .535 10. .030 1. ,00 28. .69 SOLV ATOM 748 OH2 TIP3 55 23. .435 27. ,423 11. ,489 1. 00 40. .75 SOLV ATOM 749 OH2 TIP3 56 16. ,616 38. ,557 -4. ,772 1. 00 41. .46 SOLV ATOM 750 OH2 TIP3 57 10. ,916 5. ,891 -0. ,754 1. 00 26. .82 SOLV END

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

1. Schultz, J., Ponting, C.P., Hofmann, K. & Bork, P. SAM as a protein interaction domain involved in developmental regulation Protein Sci 6, 249-53 (1997). 2. Jousset, C, et al. A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein Embo 16, 69-82 (1997).

3. Peterson, A.J., et al. A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions Mol Cell Biol 17, 6683-92 (1997). 4. Tu, H., Barr, M., Dong, D.L. & Wigler, M. Multiple regulatory domains on the Byr2 protein kinase Mol Cell Biol 17, 5876-87 (1997).

5. Kyba, M. & Brock, H.W. The SAM domain of polyhomeotic, RAE28, and scm mediates specific interactions through conserved residues Dev Genet 22, 74-84 (1998).

6. Golub, T.R., Barker, G.F., Lovett, M. & Gilliland, D.G. Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation

Cell 77, 307-16 (1994).

7. Golub, T.R., et al. Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia Mol Cell Biol 1 , 4107- 16 ( 1996).

8. Lacronique, V., et al. A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia Science 278, 1309-12 (1997).

9. Golub, T.R., et al. Fusion of the TEL gene on 12pl3 to the AML1 gene on 21q22 in acute lymphoblastic leukemia Proc Natl Acad Sci USA 92, 4917-21 (1995).

10. Henkemeyer, M., et al. Nuk controls pathfinding of commissural axons in the mammalian central nervous system Cell 86, 35-46 (1996). 11. Orioli, D., Henkemeyer, M., Lemke, G., Klein, R. & Pawson, T. Sek4 and Nuk receptors cooperate in guidance of commissural axons and in palate formation EmboJ 15, 6035-49 (1996).

12. Krull, C.E., et al. Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration Curr Biol 7, 571-80 (1997).

13. Xu, Q., Alldus, G., Macdonald, R., Wilkinson, D.G. & Holder, N. Function of the Eph- related kinase rtkl in patterning of the zebrafish forebrain Nature 381, 319-22 (1996).

14. Wang, H.U., Chen, Z.F. & Anderson, D.J. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4 Cell 93, 741-53 (1998).

15. Stein, E., et al. Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses Genes Dev 12, 667-78 (1998).

16. Lackmann, M., et al. Ligand for EPH-related kinase (LERK) 7 is the preferred high affinity ligand for the HEK receptor J Biol Chem 272, 16521-30 (1997).

17. Davis, S., et al. Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity Science 266, 816-9 (1994). 18. Lackmann, M., et al. Distinct subdomains of the EphA3 receptor mediate ligand binding and receptor dimerization J Biol Chem 273, 20228-37 (1998).

19. Hock, B., et al. PDZ-domain-mediated interaction of the eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor Proc Natl Acad Sci USA 95, 9779-84 (1998).

20. Ponting, C.P. SAM: a novel motif in yeast sterile and Drosophila polyhomeotic proteins Protein Sci 4, 1928-30 (1995).

21. Klambt, C. The Drosophila gene pointed encodes two ETS-like proteins which are involved in the development of the midline glial cells Development 117, 163-76 (1993). 22. Serra-Pages, C, Medley, Q.G., Tang, M., Hart, A. & Streuli, M. Liprins, a family of LAR transmembrane protein-tyrosine phosphatase- interacting proteins J Biol Chem 273, 15611-20 (1998).

23. Kuriyan, J. & Cowburn, D. Modular Peptide Binding Domains Annu. Rev. Biophys. Biomol. Struct. 26, 259-288 (1997).

24. Stein, E., Cerretti, D.P. & Daniel, T.O. Ligand activation of ELK receptor tyrosine kinase promotes its association with GrblO and Grb2 in vascular endothelial cells J Biol Chem 111, 23588-

93 (1996).

25. Terwilliger, T.C., Kim, S.H. & Eisenberg, D. Generalized method of determining heavy- atom positions using the difference Patterson function Acta Crytallogr. Sect. A 43, 1-5 (1987).

26. Furey, W. & Swaminathan, S. PHASES in American Crystallographic Association Meeting Abstracts 13 (1990).

27. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models Acta Crystallogr. A47, 110-119 (1991).

28. Brunger, A.T. (Yale University, New Haven, CT, 1992). 29. Carson, M. Ribbons 2.0 Journal of applied Crystallography 24, 958 (1991).

30. Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons Proteins: Struct. Fund, and Genetics 11, 281-296 (1991).

Claims

WE CLAIM:

I . A purified three dimensional structure of a polypeptide corresponding to one or more SAM domains.

2. A three dimensional structure as claimed in claim 1, wherein the SAM domain is a SAM domain of an Eph receptor.

3. A three dimensional structure as claimed in claim 2 wherein the Eph receptor is EphA.

4. A three dimensional structure as claimed in claim 1 complexed with one or more compounds.

5. A three dimensional structure as claimed in claim 1 comprising one or more heavy metal atoms.

6. A purified crystalline form of a polypeptide corresponding to one or more SAM domains.

7. A crystalline form as claimed in claim 6 having dimensions of about a=b= 77.14 ± .03 angstroms, c= 24.3 ± .04 angstroms.

8. A crystalline form as claimed in claim 7 having the co-ordinates set out in Table 2.

9. A method of forming a crystalline form as claimed in claim 6 comprising (a) mixing a volume of a SAM domain with a reservoir solution; and

(b) incubating the mixture obtained in step (a) over the reservoir solution in a closed container under conditions suitable for crystallization.

10. A method of determining three dimensional structures of polypeptides with SAM domains of unknown structure comprising the step of applying the structural atomic coordinates of a three dimensional structure as claimed in claim 1 or a crystalline form as claimed in claim 7 or 8.

I I . A method for identifying a potential modulator of a SAM domain of an Eph receptor function comprising docking a computer representation of a structure of a compound with a computer representation of a structure of one or more SAM domains of an Eph receptor that is defined by the atomic structural coordinates of the three dimensional structure as claimed in claim 2 or a crystalline form as claimed in claim 7 or 8.

12. A method as claimed in claim 1 1 comprising the following steps:

(a) docking a computer representation of a compound from a computer data base with a computer representation of a selected site on a three dimensional structure of a SAM domain of an Eph receptor as claimed in claim 2 or a crystalline form as claimed in claim 7 or 8 to obtain a complex;

(b) determining a conformation of the complex with a favourable geometric fit and favourable complementary interactions; and

(c) identifying compounds that best fit the selected site as potential modulators of SAM domain function.

13. A method as claimed in claim 11, comprising the following steps:

(a) modifying a computer representation of a compound complexed with a selected site on a three dimensional structure of a SAM domain of an Eph receptor as claimed in claim 2 or a crystalline form as claimed in claim 7 or 8, by deleting or adding a chemical group or groups; (b) determining a conformation of the complex with a favourable geometric fit and favourable complementary interactions; and

(c) identifying a compound that best fits the selected site as a potential modulator of a SAM domain.

14. A method as claimed in claim 11 comprising the following steps:

(a) selecting a computer representation of a compound complexed with a selected site on a three dimensional structure of a SAM domain of an Eph receptor as claimed in claim 2 or a crystalline form as claimed in claim 7 or 8; and

(b) searching for molecules in a data base that are similar to the compound using a searching computer program, or replacing portions of the compound with similar chemical structures from a data base using a compound building computer program.

15. A potential modulator of a function of a SAM domain of an Eph receptor identified by a method as claimed in any one of claims 11 to 14.

16. A method of treating a disease associated with a SAM domain of an Eph receptor with inappropriate activity in a cellular organism, comprising:

(a) administering a crystalline form of a polypeptide as claimed in claim 6 or a modulator identified using a method as claimed in any one of claims 11 to 14, in an acceptable pharmaceutical preparation; and

(b) activating or inhibiting a SAM domain function to treat the disease.

17. A method as claimed in claim 16 wherein the disease is a cell proliferative disease or disease associated with the nervous system.

18. A peptide of the formula I which mediates SAM domain function:

wherein X and X⁶ represent 0 to 70, preferably 0 to 50 amino acids, more preferably 2 to 20 amino acids, and X¹ represents Leu, Phe, Asp, Ala, Glu, or Gly, preferably Leu or Gly, X² represents Glu, Asp, Ser, He, Ala, Arg, Lys, and Gin, preferably Glu or Asp, X³ represents Ala, Val, Glu, Phe, Ser, He, Met, Leu, His, Gin, Arg, or Asp preferably Ala, Val, or Phe, X⁴ is Val, Leu, Met, Phe, and He, preferably Val or Leu, or Phe, X⁵ is Val, Ser, Leu, Asp, Ala, Pro, Asn,

Lys, or Cys, preferably Val or Ser.

19. A peptide as claimed in claim 18 wherein X represents TT, ID, TS, DD, GYTT (SEQ ID. NO. 38), AAGYTT (SEQ ID. NO. 39), FTAAGYTT (SEQ ID. NO. 40), DNFTAAGYTT (SEQ ID. NO. 41), or YKDNFTAAGYTT (SEQ ID. NO. 42).

20. A peptide as claimed in claim 18 wherein X⁶ represents HM, HMSQ (SEQ ID. NO. 43), HMSQD (SEQ ID. NO. 44), HMSQDD (SEQ ID. NO. 45), HMSQDDLA (SEQ ID. NO. 46), QMMM (SEQ ID. NO. 47), QMMMED (SEQ ID. NO. 48), QMMMEDLL (SEQ ID. NO. 49), DITE (SEQ ID. NO. 50), DITEED (SEQ ID. NO. 51), DITEEDL (SEQ ID. NO. 52), NLTE (SEQ ID. NO. 53), NLTEND (SEQ ID. NO. 54), or NLTENDI (SEQ ID. NO. 55).

21. A peptide of the formula I as claimed in claim 18 which is LEAVV (SEQ ID. NO. 56), TTLEAVV (SEQ ID. NO. 57), LEAVVHM (SEQ ID. NO. 58), LEAVVHMSQ (SEQ ID. NO. 59), LEAVVHMSQD (SEQ ID. NO. 60), LEAVVHMSQDDL (SEQ ID. NO. 61), LEAVVHMSQDDLAR (SEQ ID. NO. 62), TTLEAVVHMS (SEQ ID. NO. 63), TTLEAVVHMSQD (SEQ ID. NO. 64), TTLEAVVHMSQDDL (SEQ ID. NO. 65),

TTLEAVVHMSQDDLAR (SEQ ID. NO. 66), GYTTLEAVV (SEQ ID. NO. 67), GYTTLEAVVHMS (SEQ ID. NO. 68), GYTTLEAVVHMSQD (SEQ ID. NO. 69), GYTTLEAVVHMSQDDL (SEQ ID. NO. 70), GYTTLEAVVHMSQDDLAR (SEQ ID. NO. 71), FDVVS (SEQ ID. NO. 72), FDVVSQ (SEQ ID. NO. 73), FDVVSQMM (SEQ ID. NO. 74), FDVVSQMMME (SEQ ID. NO. 75), FDVVSQMMMEDIL (SEQ ID. NO. 76), TSFDVVS

(SEQ ID. NO. 77), TSFDVVSQ (SEQ ID. NO. 78), TSFDVVSQMM (SEQ ID. NO. 79), TSFDVVSQMMME (SEQ ID. NO. 80), TSFDVVSQMMMEDIL (SEQ ID. NO. 81), LEFLS (SEQ ID. NO. 82), LEFLSD (SEQ ID. NO. 83), LEFLSDIT (SEQ ID. NO. 84), LEFLSDITEE (SEQ ID. NO. 85), LEFLSDITEEDL (SEQ ID. NO. 86), DDLEFLS (SEQ ID. NO. 87), GWDDLEFLS (SEQ ID. NO. 88), DDLEFLSD (SEQ ID. NO. 89), DDLEFLSDIT (SEQ ID.

NO. 90), DDLEFLSDITEE (SEQ ID. NO. 91), DDLEFLSDITEEDL (SEQ ID. NO. 92), GARFL (SEQ ID. NO. 93), GARFLN (SEQ ID. NO. 94), GARFLNLT (SEQ ID. NO. 95), GARFLNLTEN (SEQ ID. NO. 96), and IDGARFL (SEQ ID. NO. 97).

22. A peptide of the formula II which mediates SAM domain function:

X⁷ ^X⁹ C¹ X^{1 ,}-X¹Vx^I3-X¹⁴-X^I3-X¹' II

wherein X⁷ and X¹⁶ represent 0 to 70, preferably 0 to 50 amino acids, more preferably 2 to 20 amino acids, and X⁸ represents Met, He, Ser, Leu, Asn, Phe, or Val, preferably Met, X⁹ represents Arg, Ser, Lys, Met, Leu, Glu, Gin, or Asn, preferably Gin or Arg, X¹⁰ represents Thr, Ala, Arg,

Leu, Ser, Glu, Asp, Met, Lys, Gin, or Gly, preferably Thr, Ala, or Glu, X^u represents Gin, Ser, Glu, Leu, Phe, Asp, Thr, Arg, preferably Gin or Arg, X¹² represents Met, Ala, He, Asn, Ser, Arg, Thr, Pro, Leu, Gin, Val, Lys, preferably Met or Arg, X¹³ represents Gin, Asn, Pro, Ser, Tyr, Glu, Leu, Arg, or Lys, preferably Gin, Asn, or Arg, X¹⁴ represents Gin, Ala, Pro, Asp, Leu, Lys, He, Glu, Arg, or Asn, preferably Gin or He, and X¹⁵ represents Met, He, Val, His, Ser, Arg, Lys, Phe,

Cys, Glu, Tyr, Ala, He, Tip, or Leu.

23. A peptide of the formula II as claimed in claim 22 wherein X⁷ represents QA, QV, NK, SVQA (SEQ ID. NO. 98), LSSVQA (SEQ ID. NO. 99), ILSSVQA (SEQ ID. NO. 100), NKILSSVQA (SEQ ID. NO. 101), HQNKILSSVQA (SEQ ID. NO. 102), THQNKILSSVQA (SEQ ID. NO. 103), ENIK (SEQ ID. NO. 104), SQEINK (SEQ ID. NO. 105), KLSQEINK (SEQ ID. NO.

106), ILNSIQV (SEQ ID. NO. 107), or NSIQV (SEQ ID. NO. 108).

24. A peptide of the formula II as claimed in claim 22 wherein X¹⁶ is HG, QS, HGRM (SEQ ID. NO. 109), HGRMVP (SEQ ID. NO. 110), QSVEV (SEQ ID. NO. 111), or TRKP (SEQ ID. NO. 112).

25. A peptide of the formula II as claimed in claim 22 which is MRTQMQQM (SEQ ID. NO. 113), QAMRTQMQQM (SEQ ID. NO. 114), SVQAMRTQMQQM (SEQ ID. NO. 115), LSSVQAMRTQMQQM (SEQ ID. NO. 116), ILSSVQAMRTQMQQM (SEQ ID. NO. 117), MRTQMQQMHG (SEQ ID. NO. 1 18), MRTQMQQMHGRM (SEQ ID. NO. 119), MRTQMQQMHGRMVPV (SEQ ID. NO. 120), NEERRSIF (SEQ ID. NO. 121),

INKNEERRSIF (SEQ ID. NO. 122), NEERRSIFTRKP (SEQ ID. NO. 123). MRAQMNQI (SEQ ID. NO. 124), MRAQMNQIQS (SEQ ID. NO. 125), MRAQMNQIQSVEV (SEQ ID. NO. 126).

26. A peptide which mediates SAM domain function comprising VVSV (SEQ ID. NO. 21), SAVVSV (SEQ ID. N0.22), FSAVV (SEQ ID. N0.23 ), FSAVVSV (SEQ ID. NO. 24),

FSAVVSVGD (SEQ ID. NO. 25), VVSVGDWL (SEQ ID. NO. 26), FNTV (SEQ ID. NO. 27), FNTVDE (SEQ ID. NO. 28), FNTVDEWL (SEQ ID. NO. 29), TSFNTVDEWL (SEQ ID. NO. 30), TSFNTV (SEQ ID. NO. 31), YTSFNTV (SEQ ID. NO. 32), RSEV (SEQ ID. NO. 33), RSEVLG (SEQ ID. NO. 34), RSEVLGVD (SEQ ID. NO. 35), VPFRSEV (SEQ ID. NO. 36), and VPFRSEVLGW (SEQ ID. NO. 37).

27. A pharmaceutical composition comprising a peptide as claimed in any one of claims 18 to 26 and a pharmaceutically acceptable carrier, diluent or excipient.