AU767092B2 - Crystalline FRAP complex - Google Patents

Description

Q:\OpEjb\tobr6337%9m mp.div.pagel d-20/l1/00 -la- Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

(ORIGINAL)

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Name of Applicant: Comell Research Foundation, Inc.

Actual Inventors: Jon C. CLARDY and Jungwon CHOI Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia Invention Title: "Crystalline FRAP complex" Details of Parent Application No: 74684/96 The following statement is a full description of this invention, including the best method of performing it known to us: Crystalline FRAP Complex Copyright Notice A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

Field of the Invention The invention relates to a complex, in crystalline form, of two proteins, FKBP12 and the FRB domain of FRAP, in association with rapamycin, a small organic molecule to which the proteins bind. The crystalline form of this ternary complex is particularly useful for the determination of the three-dimensional structure of the complex at the atomic level. The three dimensional structure provides information useful for the design of pharmaceutical 15 compositions which inhibit the biological function of proteins such as FRAP which contain an FRB domain, particularly those biological functions mediated by molecular interactions involving rapamycin or other compounds capable of binding to an FRB domain.

Background Rapamycin (sometimes called sirolimus) was first described in 1975 as an antifungal agent isolated from Streptomyces hygroscopicus (Vezina, 1975; Sehgal, 1975). In 1987, the structurally related compound FK506 (sometimes called tacrolimus) was characterized as a potent immunosuppressive agent (Tanaka, 1987), and shortly thereafter, rapamycin was also shown to have potent immunosuppressive activity. In spite of rapamycin's immunosuppressive 25 activity and structural similarity to FK506, the two compounds suppress the immune response in completely different ways (Schreiber, 1992). FK506 inhibits the T cell receptor (TCR) signal and prevents activation of a resting helper T cell. Rapamycin inhibits the autocrine signaling pathway involving interleukin-2 (IL-2) and the IL-2 receptor (IL-2R). These latter signals commit the cell to a program of cell division by communicating with the components of the cell cycle machinery necessary for DNA replication.

Both FK506 and rapamycin are potentially useful in the treatment of human disease.

FK506 has been approved by the FDA for use in treating the rejection of transplanted organs. A similar use has been envisioned for rapamycin, and its demonstrated activity in organ transplantation and autoimmune animal models indicate a high clinical potential. Rapamycin has been shown to have antitumor activity against B16 melanocarcinoma, colon 26 tumor, EM ependymoblastoma, CD8F1 mammary and colon 38 murine tumors (Sehgal, 1993). Rapamycin has also shown immunosuppressive activity in assays to measure prevention of development of autoimmune adjuvant arthritis, experimental allergic encephalomyelitis and autoimmune uveoretinitis in the rat (Sehgal, 1993).

The biological activity and structural novelty of both rapamycin and FK506 led to a search for their cellular target(s), and the target of both compounds was identified as the plentiful cytoplasmic protein FKBP12 (for FK506 binding protein) of 12 kDa molecular mass.

Since FK506 and rapamycin bound to the same target (Kd of 0.4 and 0.2 nM, respectively) and affected different pathways, a new function was attributed to the FKBP12-ligand complex.

This new function arises from the ability of FKBP12-FK506 and FKBP12-rapamycin complexes, but not the individual components, to bind to and inhibit still other protein targets. The FKBP12-FK506 complex inhibits the phosphatase activity of calcineurin, a crucial component of the TCR pathway. Calcineurin is a serine/threonine phosphatase also called PP2B. The FKBP12-rapamycin complex inhibits the IL-2R signal by binding to a large (289kDa) protein named FRAP in humans (Brown et al, 1994) or RAFT in rats (Sabatini et al, 1994; Chiu et al, 1994).

SThe structural basis for the tight binding of FK506 and rapamycin by FKBP12 has been investigated by both X-ray diffraction and NMR techniques (Clardy, 1995). In particular, high 15 resolution X-ray structures are available for FKBP12-FK506 (1.4 A resolution) and FKBP12rapamycin (1.7 A resolution) (Van Duyne et al, 1991; Van Duyne et al, 1991a; Van Duyne et al, 1993). These structures reveal, among other things, the fold of FKBP12, the atomic details of the hydrophobic binding pocket, and the details of how FK506 and rapamycin interact with the binding pocket. A structural analysis of the complex formed between FKBP12-FK506calcineurin is also available (Griffith et al, 1995). That structure reveals how the portion of S FK506 not involved in binding FKBP12 interacts with calcineurin and inhibits its phosphatase activity.

The biochemical characterization of FRAP, the target of the FKBP12-rapamycin complex, remains incomplete. The C-terminal domain resembles a phosphatidylinositol (PI) kinase, but to 25 date no PI or protein kinase activity has been convincingly demonstrated. FRAP (RAFT, TOR) are members of a rapidly growing and important family of proteins that have been identified only recently (Zakian, 1995). ATM, TEL1, DNA-PK and MEC1 are some of the recently characterized members of this family of PIK-related kinases. (See Keith, 1995). ATM (for ataxia telngiectasia mutant) is responsible for a human autosomal hereditary disease characterized by cerebellar degeneration, progressive mental retardation, uneven gait, dilation of blood vessels, immune deficiencies, premature aging and a hundredfold increase in cancer susceptibility (Zakian, 1995). Persons who are heterozygous in ATM are believed to be at elevated risk for cancer. Mutations to TEL1 lead to abnormally short telomeres, and in conjunction with other mutations can lead to sensitivity to X-rays, UV radiation and hydroxyurea. DNA-PK is, as the name suggests, a DNA-dependent protein kinase that recognizes damaged DNA, and human cells without DNA-PK activity are radiation sensitive and repair deficient. MEC1 is required for both S-M and G2-M checkpoint progression as well as for meiotic recombination in yeast. Thus MEC1 is arguably the master checkpoint gene in yeast.

FRAP is a large protein (2549 amino acid residues), and only a small fraction can be involved in recognizing the FKBP12-rapamycin complex. Fortunately all of these residues are in one domain, and this domain, which is called the FKBP12-rapamycin binding (FRB) domain, is the protein used in this invention. It was identified through tryptic digests of FRAP and independently produced as an 11 kDa soluble protein (Chen et al, 1995) Unfortunately, until now, three-dimensional structural details of the association of FKBP12-rapamycin with the FRB domain of FRAP have remained completely unknown. In the absence of such three-dimensional structural details, it has been impossible to design compounds based on that structure which would be capable of mimicking rapamycin's binding to the FRB domain. We have now obtained crystals of that ternary complex and have determined its three dimensional structure. With this information, it is now possible for the first time to rationally design compounds capable of binding to an FRB domain and mimicking the pharmacological activity of rapamycin. Such mimics may be used in place of rapamycin as immunosuppressive agents or in other pharmacological applications.

Summary of the Invention This invention centers on the FRB domain of human FRAP and begins with obtaining crystals of human FKBP12-rapamycin-FRB of sufficient quality to determine the three dimensional (tertiary) structure of the complex by X-ray diffraction methods.

In considering our work, it should be appreciated that obtaining protein crystals in any case is a somewhat unpredictable art, especially in cases in which the practitioner lacks the guidance of prior successes in preparing and/or crystalizing any closely related proteins.

Obtaining our first crystals of the ternary complex was therefore itself an unexpected result. In addition, our data represents the first detailed information available on the three dimensional 25 structure of FRAP or of any of the PIK-related kinases and revealed an unpredicted array of surface features.

Our results are useful in a number of applications. As previously mentioned, the atomic details of how the FKBP12-rapamycin complex interacts with the FRB domain is essential for the structure-based design of rapamycin analogs. As noted above, rapamycin has several promising clinical indications, and improved rapamycin analogs would be useful therapeutic agents. This structure can be used as an essential starting point in predicting, via homology modeling, the structures of related proteins which contain homologous FRB domains, including other members of the PIK-related kinase family.

Furthermore, the structure shows-in atomic detail-how a small organic molecule, rapamycin, can be used to hold two proteins, FKBP12 and FRB, in cose proximity. As such, this structure contains important lessons for the design of heterodimerizing agents.

Thus, the knowledge obtained concerning the FRB of FRAP can be used to model the tertiary structure of related proteins. By way of example, the structure of renin has been modeled using the tertiary structure of endothiapepsin as a starting point for the derivation.

Model building of cercarial elastase and tophozoite cysteine protease were each built from known serine and cysteine proteases that have less than 35% sequence identity. The resultant models were used to design inhibitors in the low micromolar range. (Proc. Natl. Acad. Sci. 1993, 3583). Furthermore, alternative methods of tertiary structure determination that do not rely on X-ray diffraction techniques and thus do not require crystallization of the protein, such as NMR techniques, are simplified if a model of the structure is available for refinement using the additional data gathered by the alternative technique. Thus, knowledge of the tertiary structure of the FRB region of FRAP provides a significant window to the structure of other proteins containing a homologous FRB domain, including the other PIK-related kinases.

Accordingly, one object of this invention is to provide a composition, in crystalline form, comprising a protein containing an FRB domain. The protein may have a bound ligand or may be part of a complex with a second protein molecule and a shared ligand. For instance, the crystalline composition may contain a complex containing a first protein having a peptide sequence derived or selected from that of an FKBP12 protein, human FKBP12; a second protein having a peptide sequence derived or selected from that of an FRB domain of a PIKrelated kinase family member, e.g. the FRB domain of human FRAP; and a ligand such as rapamycin which is capable of binding to both proteins to form a ternary complex. Such a crystalline composition may contain one or more heavy atoms, one or more lead, mercury, gold and/or selenium atoms. Such a heavy atom derivative may be obtained, for example, by expressing a gene encoding the protein of interest under conditions permitting the incorporation of one or more heavy atom labels as in the incorporation of selenomethionine), reacting the protein with a reagent capable of linking a heavy atom to the protein trimethyl lead acetate) or soaking a substance containing a heavy atom into the crystals.

Preferred crystalline compositions of this invention are capable of diffracting x-rays to a 25 resolution of better than about 3.5 A, and more preferably to a resolution of 2.7 A or better, and are useful for determining the three-dimensional structure of the material. (The smaller the number of angstroms, the better the resolution.) Crystalline compositions of this invention specifically include those in which the crystals are characterized by the structural coordinates of the FRB protein set forth in the accompanying Appendix I or characterized by coordinates having a root mean square deviation therefrom, with respect to backbone atoms of amino acids listed in Appendix I, of 1.5 A or less.

Furthermore, our crystalline compositions include crystals characterized by the structural coordinates of both the FRB and FKBP12 proteins set forth in Appendix I, optionally including a molecule of rapamycin as defined structurally by the accompanying coordinates therefor.

Structural coordinates of a crystalline composition of this invention may be stored in a machine-readable form on a machine-readable storage medium, e.g. a computer hard drive, diskette, DAT tape, etc., for display as a three-dimensional shape or for other uses involving computer-assisted manipulation of, or computation based on, the structural coordinates or the three-dimensional structures they define. For example, data defining the three dimensional structure of a composition of this invention or a portion thereof containing an FRB domaincontaining protein of the PIK-related kinase family, or portions or structurally similar homologues of such proteins, may be stored in a machine-readable storage medium, and may be displayed as a graphical three-dimensional representation of the protein structure, typically using a computer capable of reading the data from said storage medium and programmed with instructions for creating the representation from such data. This invention thus encompasses a machine, such as a computer, having a memory which contains data representing the structural coordinates of a crystalline composition of this invention, e.g. the coordinates set forth in Appendix I, together with additional optional data and instructions for manipulating such data. Such data may be used for a variety of purposes, such as the elucidation of other related structures and drug discovery.

A first set of such machine readable data may be combined with a second set of machinereadable data using a machine programmed with instructions for using the first data set and the second data set to determine at least a portion of the coordinates corresponding to the second 15 set of machine-readable data. For instance, the first set of data may comprise a Fourier transform of at least a portion of the coordinates for the complex set forth in Appendix I, while the second data set may comprise X-ray diffraction data of a molecule or molecular complex.

More specifically, one of the objects of this invention is to provide three-dimensional structural information on the FRB domain of FRAP, of other members of the PIK-related kinase family which containg homologous FRB domains, and of homologs or variants thereof, preferably in association with a bound ligand or bound ligand:protein complex (such as FKBP12-rapamycin). To that end, we provide for the use of the structural coordinates of a crystalline composition of this invention, or portions thereof, to solve, e.g. by molecular replacement, the three dimensional structure of a crystalline form of another such protein, S 25 protein:ligand complex, or protein:ligand:protein complex. Doing so involves obtaining x-ray diffraction data for crystals of the protein or complex for which one wishes to determine the three dimensional structure. Then, one determines the three-dimensional structure of that protein or complex by analyzing the x-ray diffraction data using molecular replacement techniques with reference to the previous structural coordinates. As described in US Patent No.

5,353,236, for instance, molecular replacement uses a molecule having a known structure as a starting point to model the structure of an unknown crystalline sample. This technique is based on the principle that two molecules which have similar structures, orientations and positions in the unit cell diffract similarly. Molecular replacement involves positioning the known structure in the unit cell in the same location and orientation as the unknown structure. Once positioned, the atoms of the known structure in the unit cell are used to calculate the structure factors that would result from a hypothetical diffraction experiment. This involves rotating the known structure in the six dimensions (three angular and three spatial dimensions) until alignment of the known structure with the experimental data is achieved. This approximate structure can be fine-tuned to yield a more accurate and often higher resolution structure using various refinement techniques. For instance, the resultant model for the structure defined by the experimental data may be subjected to rigid body refinement in which the model is subjected to limited additional rotation in the six dimensions yielding positioning shifts of under about The refined model may then be further refined using other known refinement methods.

For example, one may use molecular replacement to exploit a set of coordinates such as set forth in Appendix I to determine the structure of a crystalline co-complex of the FRB domain, FKBP12 and a ligand other than rapamycin. Likewise one may use that same approach to determine the three dimensional structure of a complex of FKBP12, rapamycin and a proteincontaining a modified FRAP FRB domain or an FRB domain from a homolog of FRAP.

Another object of the invention is to provide a method for determining the threedimensional structure of a protein containing an FRB domain, or a complex of the protein with a ligand therefor, using homology modeling techniques and structural coordinates for a composition of this invention. Homology modeling involves constructing a model of an ""unknown structure using structural coordinates of one or more related proteins, protein 15 domains and/or subdomains. Homology modeling may be conducted by fitting common or °homologous portions of the protein or peptide whose three dimensional structure is to be solved •to the three dimensional structure of homologous structural elements. Homology modeling can include rebuilding part or all of a three dimensional structure with replacement of amino acids (or other components) by those of the related structure to be solved. The structural coordinates obtained for the related protein or complex may be stored, displayed, manipulated and otherwise used in like fashion as those for the ternary complex of FKBP12-rapamycin-FRB set forth in Appendix I.

Crystalline compositions of this invention thus provide a starting material, and their three dimensional structure coordinates a point of reference, for use in solving the three-dimensional S 25 structure of other proteins containing an FRB domain homologous to that of FRAP, as well as complexes containing such a protein. Sequence similarity may be determined using any conventional similarity matrix. (See e.g. Dayhoff,1979; Greer, 1981; and Gonnet, 1992). Proteins containing at least one FRB domain having at least 15% peptide sequence identity or similarity with respect to our FRB, as determined by any of the approaches described above, are considered FRAP homologs for the purpose of this disclosure.

By way of further example, the three dimensional structure defined by the machine readable data for the FRB domain (with or without the FKBP12 component) may be computationally evaluated for its ability to associate with various chemical entities. The term "chemical entity", as used herein, refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds or complexes.

For instance, a first set of machine-readable data defining the 3-D structure of FRAP or a FRAP homolog, or a portion or complex thereof, is combined with a second set of machinereadable data defining the structure of a chemical entity or moiety of interest using a machine programmed with instructions for evaluating the ability of the chemical entity or moiety to associate with the FRAP or FRAP homolog protein or portion or complex thereof and/or the location and/or orientation of such association. Such methods provide insight into the location, orientation and energetics of association of protein surfaces with such chemical entities.

Chemical entities that are capable of mimicking rapamycin's ability to associate with FRAP or a FRAP homolog should share part or all of rapamycin's pharmacologic activities, e.g.

immunosuppressive activity, but may be designed for more convenient or economical preparation, improved pharmacokinetics, reduced side effects, etc. Such chemical entities therefore include potential drug candidates.

The three dimensional structure defined by the data may be displayed in a graphical format permitting visual inspection of the structure, as well as visual inspection of the association of the protein component(s) with rapamycin or other chemical entities.

Alternatively, more quantitative or computational methods may be used. For example, one method of this invention for evaluating the ability of a chemical entity to associate with any of the molecules or molecular complexes set forth herein comprises the steps of: employing 15 computational means to perform a fitting operation between the chemical entity and a binding pocket or other surface feature of the molecule or molecular complex; and analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding pocket.

This invention further provides for the use of the structural coordinates of a crystalline 20 composition of this invention, or portions thereof, to identify reactive amino acids, such as cysteine residues, within the three-dimensional structure, preferably within or adjacent to a ligand binding site; to generate and visualize a molecular surface, such as a water-accessible surface or a surface comprising the space-filling van der Waals surface of all atoms; to calculate and visualize the size and shape of surface features of the protein or complex, ligand binding pockets; to locate potential H-bond donors and acceptors within the three-dimensional structure, preferably within or adjacent to a ligand binding site; to calculate regions of hydrophobicity and hydrophilicity within the three-dimensional structure, preferably within or adjacent to a ligand binding site; and to calculate and visualize regions on or adjacent to the protein surface of favorable interaction energies with respect to selected functional groups of interest amino, hydroxyl, carboxyl, methylene, alkyl, alkenyl, aromatic carbon, aromatic rings, heteroaromatic rings, etc.). One may use the foregoing approaches for characterizing the FRB domain-containing protein and its interactions with moieties of potential ligands to design or select compounds capable of specific covalent attachment to reactive amino acids cysteine) and to design or select compounds of complementary characteristics size, shape, charge, hydrophobicity/hydrophilicity, ability to participate in hydrogen bonding, etc.) to surface features of the protein, a set of which may be preselected. Using the structural coordinates, one may also predict or calculate the orientation, binding constant or relative affinity of a given ligand to the protein in the complexed state, and use that information to design or select compounds of improved affinity.

In such cases, the structural coordinates of the FRAP or FRAP homolog protein, or portion or complex thereof, are entered in machine readable form into a machine programmed with instructions for carrying out the desired operation and containing any necessary additional data, e.g. data defining structural and/or functional characteristics of a potential ligand or moiety thereof, defining molecular characteristics of the various amino acids, etc.

One method of this invention provides for selecting from a database of chemical structures a compound capable of binding to FRAP or a FRAP homolog. The method starts with structural coordinates of a crystalline composition of the invention, coordinates defining the three dimensional structure of FRAP or a FRAP homolog or a portion thereof or a complex thereof. Points associated with that three dimensional structure are characterized with respect to the favorability of interactions with one or more functional groups. A database of chemical structures is then searched for candidate compounds containing one or more functional groups disposed for favorable interaction with the protein based on the prior characterization. Compounds having structures which best fit the points of favorable 15 interaction with the three dimensional structure are thus identified.

S.

It is often preferred, although not required, that such searching be conducted with the aid of a computer. In that case a first set of machine-readable data defining the 3D structure of a FRAP or FRAP homolog protein, or a portion or protein-ligand complex thereof, is combined "with a second set of machine readable data defining one or more moieties or functional groups of interest, using a machine programmed with instructions for identifying preferred locations for •favorable interaction between the functional group(s) and atoms of the protein. A third set of data, i.e. data defining the location(s) of favorable interaction between protein and functional group(s) is so generated. That third set of data is then combined with a fourth set of data defining the 3D structures of one or more chemical entities using a machine programmed with 25 instructions for identifying chemical entities containing functional groups so disposed as to best fit the locations of their respective favorable interaction with the protein.

Compounds having the structures selected or designed by any of the foregoing means may be tested for their ability to bind to FRAP or a FRAP homolog, inhibit the binding of FRAP or a FRAP homolog to a natural or non-natural ligand therefor FKBP12-rapamycin, in the case of FRAP), and/or inhibit a biological function mediated by FRAP or the FRAP homolog.

This invention also permits methods for designing a compound capable of binding to a FRAP or FRAP homolog based on the three dimensional structure of bound rapamycin. One such method involves graphically displaying a three-dimensional representation based on coordinates defining the three-dimensional structure of a FRAP or FRAP homolog protein or a portion thereof complexed with a ligand such as the FKBP12:rapamycin complex. Interactions between portions of ligand and protein are characterized in order to identify candidate moieties of the ligand for replacement. One or more portions of the ligand which interact with the protein may be replaced with substitute moieties selected from a knowledge base of one or more candidate substitute moieties, and/or moieties may be added to the ligand to permit additional interactions with the protein. Compounds first identified by any of the methods described herein are also encompassed by this invention.

Brief Description of the Drawings FIG. 1 depicts a computer system.

FIG. 2 depicts storage media of this invention.

FIG. 3 depicts a ribbon diagram of the three dimensional structure of the FKBP12:rapamycin:FRB domain complex, as defined by the coordinates of Appendix I.

Detailed Description of the Invention Despite the key role played by the FKBP12:rapamycin:FRAP complex in the IL-2/IL-2R signaling pathway, and despite the growing appreciation of the biological importance of the PIK-related kinase family, nothing was known of the three-dimensional architecture by which the FRB domain of FRAP (or of any FRAP homolog) engages the FKBP12:rapamycin complex 15 required for its biological activity. X-ray crystallographic techniques could in principle address such issues. However, notwithstanding the key biological functions mediated by FRAP, there have been no reports disclosing that suitable crystals had been or could be obtained, let alone reports disclosing any x-ray crystallographic data or other information concerning the threedimensional structure of any FRB domain. Even in the event that crystals had been obtained, 20 then-available three-dimensional structural data relating to the FKBP12:rapamycin complex would not have been been sufficient for solving the ternary complex structure, at least in part, because the initial electron density maps wouldn't have permitted the chain of FRB to be traced.

Even if parts of the chain could have been traced, they would not have refined under leastsquares minimization techniques.

Nonetheless, we have succeeded in producing FKBP12 and FRAP FRB proteins, and have obtained crystals of their ternary complex with rapamycin. We have solved the threedimensional structure of the crystalline complex using x-ray diffraction techniques. In view of our successes as disclosed herein, it can now be said that proteins comprising FRB domains can be produced in stable form, purified, and crystallized, and that their three-dimensional structures can be determined, all using materials and methods such as disclosed herein.

As mentioned elsewhere, FRAP is one of a number of PIK-related kinase family members that contain an FRB domain. PIK-related kinase family members share regions of homology including lipid kinase homologous regions, kinase domains and, in at least a number of cases, FRB domains. The presence and boundaries of homologous regions in a protein sequence can be identified by using a computer alignment program that identifies amino acid sequence homology to a known sequence or domain. For example, the FRB domain (amino acids 2015 2114) of FRAP may be used for such analysis, but FRB domains from other proteins such as RAPT or TOR1 or TOR2 can be used as well. The alignment method typically used by such programs is the Needleman-Wunch alignment. See "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins." Needlman, Wunch, C.D. I. Mol.

Biol. 1970, 48 443-453.

We expressed the FRAP FRB domain as a glutathione-S-transferase (GST) fusion protein.

The cDNA encoding residues 2015 2114 from human FRAP (Chen et al, 1995) was cloned into a pGEX vector and expressed in E coli, the resulting fusion protein was recovered and cleaved to yield the FRB protein which was then purified, all as described in detail below.

FKBP12 protein was similarly obtained using a cDNA encoding residues 1 107 from human FKBP12 (Standaert et al, 1990, Nature 346: 671-674..

Other proteins containing an FRB domain may also be used, including larger FRAP fragments containing the FRB and flanking peptide sequence, including up to the entire FRAP protein. Additionally, FRB proteins can be prepared by analogous means containing homologous FRB regions from other proteins, including RAPT, TOR1, TOR2 or other members of the PIK-related kinase family. It should further be appreciated that other expression systems may be readily employed., including, materials and methods for expression in E. coli using 15 T7, maltose-binding protein fusion (MBP), with epitope tags (His6, HA, myc, Flag) included or cleaved off. Baculoviral expression may be used, e.g. using pVL1393 or derivatives, for tFRB domain, fused (or not) to epitope tag or fusion partner such as GST. Conventional materials and methods for expression in mammalian, yeast or other cells may also be used.

Rapamycin may be prepared by known methods or may be obtained from commercial sources. Rapamycin analogs such as disclosed, in Luengo et al, 1995, Chemistry Biology 2(7):471-481, may be used in place of rapamycin, in forming complexes of this invention.

Complex formation, crystallization, X ray diffraction experiments and interpretation of the diffraction data were conducted as described in detail in the Experimental Examples below.

The resulting structural coordinates for a crystalline composition comprising S 25 FKBP12:rapamycin:FRB of FRAP (one molecule of complex per asymmetric unit) are set forth in Protein Database format in Appendix I. Solving the X-ray crystal structure of the ternary complex allowed us to conduct the first three dimensional characterization of an FRB:ligand complex (viewing FKBP12:rapamycin as the "ligand"). The complex, depicted in schematic form in FIG. 3, involves an elaborate array of contacts between the two protein domains and their mutual small molecule ligand. This work reveals the first structural insights into an FRB domain-containing protein.

Structure of the Ternary Complex The ternary complex of FKBP12-rapamycin-FRB has overall dimensions of 60 A x 45 A x 35 A with the rapamycin sandwiched between FKBP12 and FRB. The FKBP12 structure is basically the same as in previously reported binary structures, with a five stranded anti parallel P-sheet and a short a-helix. This binary structure was originally determined in the FKBP12-FK506 complex and later in the FKBP12-rapamycin complex (Van Duyne et al, 1993).

The four helix bundle of FRB does not wrap around the effector site of FKBP12-rapamycin; it just touches the effector FRB-binding) interface of the binary complex with few proteinprotein interactions. All of the interactions between rapamycin and FRB are hydrophobic interactions, and protein-protein interactions between FKBP12 and FRB are limited to the loop and one side chain of the 40s loop of FKBP12 (Table The solvent accessible surface areas of FKBP12 and FRB are 5348 A 2 and 5711 A 2 respectively. Since the solvent accessible surface area of the FKBP12-FRB complex (protein only) is 10342 A 2 binding results in a very modest 6% reduction of solvent accessible surface area. Two long side chains in the 40s loop (Lys44 and Lys47) and three residues in the 80s loop (Thr85, Gly86 and His87) of FKBP12 appear to make crucial contact in the ternary complex. In the FRB site, two residues at the end of al and the al-a2 loop (Arg2042 and Tyr2038) contact the 80s loop of FKBP12, and two residues in helix a4 (Tyr2105 and Asp2102) form direct or water-mediated hydrogen bonds to the 40s loop of FKBP12. The loop-loop interaction between 80s loop (FKBP12) and the al-a2 loop (FRB) and the loop-helix interaction between 40s loop (FKBP12) and helix a4 are the main protein-protein interactions in this ternary complex and thus contribute all of the protein- S 15 protein binding force forming the ternary complex.

.Structure of FRB domain of FRAP The FRB domain of the FRAP forms a typical four helix bundle, which is one of the most common structural motifs in globular proteins. The overall dimensions of this domain are 45 A x 30 A x 30 A. All four helices (termed al-a4) are connected with short underhand loops. The longest helix a3 (residues 2065-2091) has a bend at residue 2074 of 59°. Except for a small bent part of a3 (residues 1065-2073), all four helices have similar lengths (16-19 residues, about A in length). The o2 helix also has a small bend around residues Glu2049, Val2050 and Leu2051 to form a 310-helical turn rather than a normal a-helix. The angle between al and a2 is 220 and the angle between a3 and a4 is 200. The angles between these pairs are in the range of 40-60°, which indicates that this four helix bundle is close to the type interhelical Table 2 Intra-molecular hydrogen bonds and close contacts in the ternary complex Inter-helical interactions in the FRB domain of FRAP Distance (A) His 2055 (a2) Ne2 Tyr 2104 (a4) OH 2.85 Ser 2112 (C His 2028 (al) NE2 terminal) 07 3.23 Close contacts of rapamycin and FRB domain of FRAP Rapamycin FRB domain of FRAP Distance (A) Thr 2098 O 3.13 C27 Ser 2035 Oy 3.39 C51 Ser 2035 Oy 3.38 -11- Interactions of FKBP12 and FRB domain of FRAP FKBP12 FRB domain of FRAP Distance (A) Lys 47 O Tyr 2105 OH 2.56 Thr 85 Oyl Arg 2042 NHI 3.10 Thr 85 Oyl Arg 2042 NH2 2.88 Gly 86 0 Arg 2042 NH2 2.79 His 87 NE2 Tyr 2038 OH via H20 301 His 87 N61 Arg 2042 NH2 via H20 303 Lys 44 NC Asp 2102 061 via H20 310 pattern which is the alternating pattern of parallel and perpendicular helix-helix interactions (Harris et al, 1994). As usual, most of the hydrophobic and aromatic residues are located in the inter-helical interface and most of the hydrophilic residues are in the outside of the bundle, 5 which is exposed to the solvent. Only two strong hydrogen bonds were found for the interhelical interactions (Table 2) and could be key interactions maintaining the overall conformation of the four helix bundle. Helices al and a4, which have an interhelical angle of 440, form a deep cleft on the molecular surface of this domain. This cleft is surrounded by six aromatic side chains forming the 'aromatic pocket' which has exquisite steric complementary for the rapamycin effector domain binding.

Structure of FKBP12-rapamycin The structure of FKBP12 in the ternary complex is basically the same as that in the binary complex of FKBP12-rapamycin or FKBP12-FK506. The protein fold and the architecture of the 15 secondary structure are exactly the same as in the binary complex, and the interaction with rapamycin is also the same as that of the binary complex. The overall r.m.s. deviation between the FKBP12 in the ternary complex and that in the FKBP12-rapamycin complex is 1.14 A (0.49 A for the main chain), and the deviation between FKBP12 in the ternary complex and that in the FKBP12-FK506 complex is 1.11 A (0.48 A for the main chain), which implies that binding of FKBP12:rapamycin to the FRAP FRB domain is not accompanied by significant changes in the conformation of the FRB binding site on FKBP12 or of the effector domain of rapamycin.

Even the 40s loop and 80s loop regions in the FKBP12, that have direct interaction to the FRB domain, are not significantly different in 3D structure from that seen in the binary complexes.

These r.m.s. values were calculated by the rigid-body fitting on the main chain atoms in the FKBP12 using QUANTA. The overlay of FKBP12-FK506 to the ternary complex clearly confirmed the fact that FKBP12-FK506 complex can't bind FRAP as FK506's effector region does not extend enough. The protein-protein interactions by themselves between FKBP12 and FRB are not enough for the formation of a binary complex; rapamycin is essential to mediate the interaction of the two proteins.

-12- FKBP12-rapamycin binding to FRAP While the interactions of rapamycin with FRB are all hydrophobic, rapamycin-FKBP12 interactions employ five hydrogen bonds which are the same found in the binary complex of FKBP12-rapamycin, to govern this interaction. Rapamycin is surrounded by five conserved aromatic residues in FKBP12, which makes the binding pocket for the rapamycin a complete aromatic pocket' along with six aromatic residues in FRB domain. Comparing the sequence of these aromatic residues of FRB domain with other FKBP-rapamycin target proteins, these six aromatic residues are all conserved in RAFT (Sabatini et al, 1994), TOR1, and TOR2 (Stan, et al, 1994)-suggesting that these structural results will be applicable to other members of the PIKrelated kinase family. It is expected that binding domains of these other proteins have a similar structure with FRB domain. For the interaction between rapamycin and FRB domain, two major sites on FRB are considered crucial for rapamycin binding. Ser2035, which is also conserved in other FKBP12-rapamycin target proteins, has cose contact with C27 and C51 of rapamycin (Table The other site is Thr2098 which has a close contact with C50 of rapamycin. C50 of 15 the rapamycin is at the end of C16 methoxy group, which has been a key target for substituted analogs. All of the hydrophobic interactions between rapamycin and FRB including Ser2035 and Thr2098 can be considered as the main force contributing to complete ternary complex.

Mutational studies Ser2035 in FRB has been the major site for the site-directed mutation studies of FRAP (Chen et al, 1995). Those studies revealed that the substitution of this residue to other residues larger than alanine abolish binding affinity toward FKBP12-rapamycin The crystal structure of the ternary complex shows the direct effect of steric hindrance when this position is substituted by longer side chains. It has been suggested that this conserved serine site is a phosphorylation site, and phosphorylation would abrogate binding. By the binding of FKBP12-rapamycin, this serine site, which is open to the solvent when unbound, is protected from phosphorylation and this probably causes the inhibition of the downstream of the signaling pathway.

For rapamycin, C16 has been the main site for substitution in published structure-activity studies (Luengo et al, 1995). The studies of C16 analogs of rapamycin showed that the bulky group substitutions on this position have lower affinity for the FKBP12 binding and lower activity. However some analogs with different stereochemistry or different groups showed retained activity and affinity to FKBP12. Such C-16 substituted analogs could be of therapeutic use.

Applications of the invention This invention encompasses crystalline compositions containing FRAP or a FRAP homolog protein or portion thereof having a region characterized by structural coordinates of the FRB domain set forth in Appendix I, or by coordinates having a root mean square deviation -13- Stherefrom of less than about 1.5 A, preferably less than about 1 A, and even more preferably less than about 0.5 A, with respect to backbone atoms of amino acid residues listed there.

As practitioners in this art will appreciate, various computational analyses may be used to determine the degree of similarity between the three dimensional structure of a given protein (or a portion or complex thereof) and FRAP or a FRAP homolog protein or portion the FRB domain) or complex thereof such as are described herein. Such analyses may be carried out with commercially available software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, MA) version 3.3, and as described in the accompanying User's Guide, Volume 3 pgs. 134 135.

The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in Molecular Similarity to compare structures is divided into four steps: (1) load the structures to be compared; define the atom equivalences in these structures; (3) perform a fitting operation; and analyze the results.

o 15 Each structure is identified by a name. One structure is identified as the target the •°•fixed structure); all remaining structures are working structures moving structures). Since atom equivalency within QUANTA is defined by user input, for the purpose of this invention Swe define equivalent atoms as protein backbone atoms Ca, C and 0) for all conserved :.S'.residues between the two structures being compared and consider only rigid fitting operations.

When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses a least squares fitting algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by

QUANTA.

For the purpose of this invention, any set of structural coordinates of a FRAP or FRAP homolog protein, portion of a FRAP or FRAP homolog protein or molecular complex thereof that has a root mean square deviation of conserved residue backbone atoms CcC, C, O) of less than 1.5 when superimposed-using backbone atoms--on the relevant structural coordinates of a protein or complex of this invention, e.g. the coordinates listed in Appendix I, are considered identical. More preferably, the root mean square deviation is less than 1.OA.

Most preferably, the root mean square deviation is less than The term "root mean square deviation" means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the "root mean square deviation" defines the variation in the backbone of a protein from the backbone of a protein of this invention, such as the FRB of FRAP, as defined by the structural coordinates of Appendix I and described herein.

-14- The term "least squares" refers to a method based on the principle that the best estimate of a value is that in which the sum of the squares of the deviations of observed values is a minimum.

In order to use the structural coordinates generated for a crystalline substance of this invention, e.g. the structural coordinates of the FRB of FRAP set forth in Appendix I, it is often necessary or desirable to display them as, or convert them to, a three-dimensional shape, or to otherwise manipulate them. This is typically accomplished by the use of commercially available software such as a program which is capable of generating three-dimensional graphical representations of molecules or portions thereof from a set of structural coordinates.

By way of illustration, a non-exclusive list of computer programs for viewing or otherwise manipulating protein structures include the following: C C C. C

CC

C*

C

9*

C

C.

C

Midas (Univ. of California, San Francisco) MidasPlus (Univ. of Cal., San Francisco) MOIL (Univeristy of Illinois) Yummie (Yale University) Sybyl (Tripos, Inc.) Insight/Discover (Biosym Technologies) MacroModel (Columbia University) Quanta (Molecular Simulations, Inc.) Cerius (Molecular Simulations, Inc.) Alchemy (Tripos, Inc.) LabVision (Tripos, Inc.) Rasmol (Glaxo Research and Development) Ribbon (University of Alabama) NAOMI (Oxford University) Explorer Eyechem (Silicon Graphics, Inc.) Univision (Cray Research) Molscript (Uppsala University) Chem-3D (Cambridge Scientific) Chain (Baylor College of Medicine) O (Uppsala University) GRASP (Columbia University) X-Plor (Molecular Simulations, Inc.; Yale Univ.) Spartan (Wavefunction, Inc.) Catalyst (Molecular Simulations, Inc.) Molcadd (Tripos, Inc.) VMD (Univ.of Illinois/Beckman Institute) Sculpt (Interactive Simulations, Inc.) Procheck (Brookhaven Natl Laboratory) DGEOM (QCPE) RE_VIEW (Brunel University) Modeller (Birbeck Col., Univ. of London) Xmol (Minnesota Supercomputing Center) Protein Expert (Cambridge Scientific) HyperChem (Hypercube) MD Display (University of Washington)

PKB

(Nat'l Center for Biotech. Info., NIH) ChemX (Chemical Design, Ltd.) Cameleon (Oxford Molecular, Inc.) Iditis (Oxford Molecular, Inc.) C. C C

C

For storage, transfer and use with such programs of structural coordinates for a crystalline substance of this invention, a machine-readable storage medium is provided comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, e.g. a computer loaded with one or more programs of the sort identified above, is capable of displaying a graphical threedimensional representation of any of the molecules or molecular complexes described herein.

Machine-readable storage media comprising a data storage material include conventional computer hard drives, floppy disks, DAT tape, CD-ROM, and other magnetic, magnetooptical, optical, floptical and other media which may be adapted for use with a computer.

Even more preferred is a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structural coordinates of a complex, FRB-containing protein component thereof, or portion thereof, comprising structural coordinates of an FRB domain such as the FRAP FRB coordinates set forth in our attached Appendix I a root mean square deviation from the conserved backbone atoms of the amino acids thereof of not more than 1.5 A. An illustrative embodiment of this aspect of the invention is a conventional 3.5" diskette, DAT tape or hard drive encoded with a data set, preferably in PDB format, comprising the coordinates of our Appendix I. FIG. 3 illustrates a print-out of a graphical three-dimensional representation of such a complex.

15 In another embodiment, the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structural coordinates set forth in Appendix I (or again, a derivative thereof), oand which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structural coordinates corresponding to the second set of machine readable data.

*se FIG. 1 illustrates one version of these embodiments. The depicted system includes a computer A comprising a central processing unit a working memory which may be, 4 RAM (random-access memory) or "core" memory, mass storage memory (such as one or 25 more disk drives or CD-ROM drives), one or more cathode-ray tube display terminals, one or more keyboards, one or more input lines and one or more output lines all of which are interconnected by a conventional bidirectional system bus.

••Input hardware B, coupled to computer A by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line L. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives D. In conjunction with the CRT display terminal, a keyboard may also be used as an input device.

Output hardware, coupled to computer A by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a protein of this invention (or portion thereof) using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.

-16- In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage and accesses to and from working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Examples of such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system of FIG. 1 are included as appropriate throughout the following description of the data storage medium.

FIG. 2A shows a cross section of a magnetic data storage medium 100 which can be encoded with a machine-readable data that can be carried out by a system such as a system of FIG. 1. Medium 100 can be a conventional floppy diskette or hard disk, having a suitable substrate 101, which may be conventional, and a suitable coating 102, which may be conventional, on one or both sides, containing magnetic domains (not visible) whose polarity or orientation can be -altered magnetically. Medium 100 may also have an opening (not shown) for to receiving the spindle of a disk drive or other data storage device 24.

15 The magnetic domains of coating 102 of medium 100 are polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as a system of FIG. 1.

2B shows a cross section of an optically-readable data storage medium 110 which also can be encoded with such machine-readable data, or set of instructions, which can be carried out by a system such as a system of FIG. 1. Medium 110 can be a conventional compact **.disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk whidch is optically readable and magneto-optically writable. Medium 100 preferably has a suitable substrate 111, which may be conventional, and a suitable coating 112, which may be too..conventional, usually of one side of substrate 111.

In the case of CD-ROM, coating 112 is reflective and is impressed with a plurality of pits 113 to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of coating 112. A protective coating 114, which preferably is substantially 1. to transparent, is provided on top of coating 112.

In the case of a magneto-optical disk, coating 112 has no pits 113, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser (not shown). The orientation of the domains can be read by measuring the polarization of laser light reflected from coating 112. The arrangement of the domains encodes the data as described above.

Use of Structure in Drug Discovery The availability of the three-dimensional structure of the ternary complex of FKBP12:rapamycin:FRB of FRAP makes structure-based drug discovery approaches possible.

Structure-based approaches include de Nova molecular design, computer-aided optimization of -17lead molecules, and computer-based selection of candidate drug structures based on structural criteria.

Rapamycin mimetics may be developed from the bound conformation of rapamycin by design, by searching databases for replacements of one or more structural segments of rapamycin, or by enhancement of existing ligand-protein interactions by replacing a component moiety of a ligand with a substitute moiety capable of greater interaction with the target protein, whether through accessible protein contact points or by extrusion of otherwise sequestered waters). Knowledge of the bound conformation of a ligand can suggest avenues for conformational restriction and replacement of atoms and/or bonds of rapamycin. A less biased approach involves computer algorithms for searching databases of three dimensional structures to identify replacements for one or more portions of the ligand. By this method, one can generate compounds for which the bioactive conformation is heavily populated, i.e., compounds which are based on particularly biologically relevant conformations of the ligand.

Algorithms for this purpose are implemented in programs such as Cast-3D (Chemical Abstracts 15 Service), 3DB Unity (Tripos, Inc.), Quest-3D (Cambridge Crystallographic Data Center), and MACCS/ISIS-3D (Molecular Design Limited). These geometric searches can be augmented by steric searching, in which the size and shape requirements of the binding site are used to weed Sout hits that have prohibitive dimensions. Programs that may be used to synchronize the geometric and steric requirements in a search applied to the FRB of FRAP include CAVEAT (P.

Bartlett, University of California, Berkeley), HOOK (MSI), ALADDIN (Daylight Software) and DOCK Kuntz, University of California, San Francisco; see e.g.

http://www.cmpharm.ucsf.edu/kuntz-/kuntz.htm and references cited therein). All of these searching protocols may be used in conjunction with existing corporate databases, the Cambridge Structural Database, or available chemical databases from chemical suppliers.

Characterization of Compounds Compounds designed, selected and/or optimized by methods described above may be evaluated for binding activity with respect to proteins containing one or more FRB domains using various approaches, a number of which are well known in the art. For instance, compounds may be evaluated for activity as competitive inhibitors of the binding of a natural ligand for the FRB, e.g. FKBP12:rapamycin in the case of the FRAP FRB. Competitive inhibition may be determined using any of the numerous available technologies known in the art.

Such compounds may be further evaluated for activity in inhibiting cellular or other biological events mediated by a pathway involving the interaction of interest using a suitable cell-based assay or an animal model. Cell-based assays and animal models suitable for evaluating inhibitory actvity of a compound with respect to a wide variety of cellular and other biological events are known in the art. New assays and models are regularly developed and reported in the scientific literature.

-18- For example, compounds which mimic the binding of rapamycin or FKBP12:rapamycin with respect to FRAP may be evaluated for biological activity in the mouse spelocyte mitogenesis assay or the high-flux yeast-based assay of Luengo et al, supra. A battery of in vivo models may be used to profile the breadth of the compound's immunosuppressive (or other) activity and compare the profile to those of positive controls such as rapamycin itself. Comparisons may also be made to other currently accepted immunosuppressive compounds, e.g. cyclophosphamide, and leflunomide. Initial in vivo screening models include: Delayed type hypersensitivity testing, Allogeneic skin transplantation, and Popliteal lymph node hyperplasia. Compounds demonstrating optimal profiles in the above models are advanced into more sophisticated models designed to confirm immunosuppressive activity in specific therapeutic areas including: Rheumatoid arthritis, Transplantation, Graft vs. host disease, and Asthma.

By way of further illustration, compounds may be evaluated in relevant conventional in vitro and in vivo assays for inhibition of the initiation, maintenance or spread of cancerous growth. See Ishii et al., J. Antibiot. XLII:1877-1878 (1989) (in vitro evaluation of 15 cytotoxic/antitumor activity); Sun et al, US Patent 5,206,249 (issued 27 April 1993)(in vitro evaluation of growth inhibitory activity on cultured leukemia cells); and Sun et al, supra (xenograft models using various human tumor cell lines xenografted into mice, as well as various transgenic animal models).

Single and multiple 5 to 7 days) dose investigative toxicology studies are typically S 20 performed in the efficacy test species using the intended route of administration for the efficacy study.

These investigative toxicology studies are performed to identify maximum tolerated dose, subjective bioavailability from the intraperitoneal or oral routes of administration, and estimation of an initial safety margin. Initial bioavailability and pharmacokinetics (blood clearance) of the compounds may be determined, with standard cold or radioactive assay methods, to assist in defining appropriate dosing regimens for the compounds in the animal models.

Pharmaceutical Compositions and Uses of rapamycin mimetics and other FRAP-binding compounds Compounds which bind to an FRB domain may be used as biological reagents in binding assays as described herein for functional classification of members of the PIK-related kinase family, particularly newly discovered proteins, based on ligand specificity.

Moreover, compounds identified as described above can be used for their immunosuppressive or other pharmacologic activity in place of rapamycin.

A compound selected or identified in accordance with this invention can be formulated into a pharmaceutical composition containing a pharmaceutically acceptable carrier and/or other excipient(s) using conventional materials and means. Such a composition can be administered as an immunosuppresant, for example, to an animal, either human or non-human.

Administration of such composition may be by any conventional route (parenteral, oral, inhalation, and the like) using appropriate formulations as are well known in this art. The -19compound can be employed in admixture with conventional excipients, pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral administration.

Pharmaceutical applications By virtue of its capacity to mimic the interaction of rapamycin with FRAP, a compound identified as described herein may be used in pharmaceutical compositions and methods for treatment or prevention of various diseases and disorders in a mammal in need thereof.

Mammals include rodents such as mice, rats and guinea pigs as well as dogs, cats, horses, cattle, sheep, non-human primates and humans.

The preferred method of such treatment or prevention is by administering to a mammal an effective amount of the compound to prevent, alleviate or cure said disease or disorder. Such effective amounts can be readily determined by evaluating the compounds of this invention in conventional assays well-known in the art, including assays described herein.

15 Therapeutic/rophylactic Administration Pharmaceutical Compositions The invention provides methods of treating, preventing and/or alleviating the symptoms and/or severity of an untoward immune response or other disease or disorder referred to above by administration to a subject of a in an amount effective therefor. The subject will be an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.

Various delivery systems are known and can be used to administer the compound, e.g., encapsulation in liposomes, microparticles, microcapsules, etc. One mode of delivery of interest is via pulmonary administration, as detailed more fully infra. Other methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, 25 subcutaneous, intranasal, epidural and oral routes. The compound may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. For treatment or prophylaxis of nasal, bronchial or pulmonary conditions, preferred routes of administration are oral, nasal or via a bronchial aerosol or nebulizer.

In specific embodiments, it may thus be desirable to administer the compound locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of a skin patch or implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

This invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically (or prophylactically) effective amount of the compound, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder 15 or water free concentrate in a hermetically sealed container such as an ampoule or sachette •indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or o*oo saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Administration to an individual of an effective amount of the compound can also be accomplished topically by administering the compound(s) directly to the affected area of the skin of the individual. For this purpose, the compound is administered or applied in a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, 25 glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.

Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol,.

ethanol polyoxyethylene monolaurate in water, or sodium lauryl sulfate in .i water. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

In addition, in certain instances, it is expected that the compound may be disposed within devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.

Materials and methods for producing the various formulations are well known in the art [see e.g. US Patent Nos. 5,182,293 and 4,837,311 (tablets, capsules and other oral formulations as well as intravenous formulations)].

The effective dose of the compound will typically be in the range of about 0.01 to about mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, administered -21in single or multiple doses. Generally, the compound may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient.

The amount of the compound which will be effective in the treatment or prevention of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The precise dosage level of the compound, as the active component(s), should be determined as in the case of all pharmaceutical treatments, by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; and the use (or not) of concomitant therapies.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the S. 15 invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Pulmonary Administration In one embodiment of this invention, the compound is administered by pulmonary administration, e.g. via aerosolization. This route of administration may be particularly useful for treatment or prophylaxis of bronchial or pulmonary infection or tumors.

Pulmonary administration can be accomplished, for example, using any of various 25 delivery devices known in the art (see Newman, 1984, in Aerosols and the Lung, Clarke and Davia Butterworths, London, England, pp. 197-224; PCT Publication No.

WO 92/16192 dated October 1, 1992; PCT Publication No. WO 91/08760 dated June 27, 1991; NTIS Patent Application 7-504-047 filed April 3, 1990 by Roosdorp and Crystal), including but not limited to nebulizers, metered dose inhalers, and powder inhalers. Various delivery devices are commercially available and can be employed, Ultravent nebulizer (Mallinckrodt,:Inc., St. Louis, Missouri); Acorn II nebulizer (Marquest Medical Products, Englewood, Colorado), Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, North Carolina); Spinhaler powder inhaler (Fisons Corp., Bedford, Massachusetts) or Turbohaler (Astra). Such devices typically entail the use of formulations suitable for dispensing from such a device, in which a propellant material may be present.

Ultrasonic nebulizers tend to be more efficient than jet nebulizers in producing an aerosol of respirable size from a liquid (Smith and Spino, "Pharmacokinetics of Drugs in Cystic Fibrosis," Consensus Conference, Clinical Outcomes for Evaluation of New CF Therapies, Rockville, Maryland, December 10-11, 1992, Cystic Fibrosis Foundation).

A nebulizer may be used to produce aerosol partides, or any of various physiologically acceptable inert gases may be used as an aerosolizing agent. Other components such as physiologically acceptable surfactants glycerides), excipients lactose), carriers, and diluents may also be included.

This invention is not to be limited in scope by the specific embodiments described herein.

Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the the scope of the appended claims.

Various patents, patent applications and publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Experimental Examples I. Protein Preparation S. cDNAs encoding human FKBP12 (Standaert et al, 1990) and the 12-kDa FRAP fragment 15 containing the FRB domain (Chen et.al, 1995) (FRAP12) were subcloned into pGEX-2T (Pharmacia) for the expression of GST-FKBP12 and GST-FRAP12 fusion proteins in E.coli strain BL21. Typically, a 2-liter culture was grown to OD600-0.6 at 30 °C and induced with 0.3 mM IPTG at room temperature for 6 hours. Purification and thrombin cleavage of the fusion proteins were performed according to standard procedures (manual from Pharmacia). After removal of free GST, the samples containing FKBP12 or FRAP12 were concentrated to -10 mL in a 50 mL stir-cell ultraconcentrator (Amicon) with a 3-kDa cutoff filter, and fractionated on a Sephacryl S-100 column (2.5 cm x 85 cm) equilibrated in 10 mM phosphate buffer (pH 7.4) containing 136 mM NaC, 3 mM KC1, 1 mM DTT. Fractions containing pure FKBP12 or :FRAP12 purity judged by SDS-PAGE) were combined and concentrated to -10 mg/mL 25 using a stir-cell ultraconcentrator. The concentrated samples were stored in the same phosphate buffer at 4 °C.

II. Crystallization Structure Determination Crystallization Recombinant human FKBP12 purified from E. coli was used at 10 mg/mL in 10 mM tris- HCI pH 8.0. Rapamycin was dissolved in methanol and mixed with FKBP12 in a 2:1 molar ratio. The mixture was lightly vortexed and stored overnight at 4°C to insure complete complex formation. Purified 12-kDa FRB domain of FRAP at 10 mg/mL in 50 mM tris-HCl pH 8.0 was added to this mixture in a 1:1 (FKBP12-rapamycin complex:FRB domain) molar ratio. This mixture was also lightly vortexed and let sit overnight at 4°C to insure complete complex formation. Crystallization conditions were screened using the hanging drop method, and rectangular rod-shaped crystals were obtained using: 20% PEG 8000, 10% MPD and 10 mM tris-HCl at pH 8.5. For the hanging drop method, drops of 4gL containing 2pL of complex solution and 2uL of reservoir solution were equilibrated against 0.5 mL of the reservoir solution.

-23- Micro-seeding techniques were used to prepare additional crystals. The initial crystals were crushed and diluted to prepare a seed solution that was added to newly prepared drops.

After two weeks, a shower of tiny crystals was obtained. Macro-seeding techniques were then applied to get large crystals suitable for X-ray diffraction. A tiny but well-formed crystal was picked and used as a crystallization seed. After two to three weeks, rectangular rod-shaped crystals with a maximum size of 0.3 x 0.2 x 0.1 mm 3 were obtained, and these crystals were suitable for data collection. The Hg-derivative crystal was obtained by soaking the native crystal in 2 mM HgCl2 solution overnight. All of the crystallization experiments were done at 4 0

C.

Data Collection All data sets were collected at room temperature on a San Diego multiwire area detector system mounted on a Rigaku RU-200 rotating anode X-ray source operating at 50 kV and 150mA. The detector was positioned at a 20-value of -30° with a 544 mm detector-crystal 15 distance for the high resolution data and 120 with a 506 mm detector-crystal distance for the low resolution data. The data collection was performed using an co-scan with an increment of 0.100 for each frame and 40 second exposure time per frame. Crystals belong to the orthorhombic space group P2 1 2 1 2 1 with unit-cell dimension of a=44.63, b=52.14, c=102.53 A and one FKBP12-rapamycin-FRB complex in the asymmetric unit. Hg-derivative crystal data S 20 were collected under the same conditions. For the native data set, the measured intensity data were processed using SCALEPACK (Otwinski et al, 1992) giving 6920 unique reflections out of 43447 measured reflections to 2.7 A resolution (98.5% data coverage) with Rsym of For the Hg-derivative data set, the number of unique reflection was 6884 out of 42681 measured reflections to 2.7 A (98.0% data coverage), with Rsym of 7.1%.

Structure determination The crystal structure of the ternary complex was solved using the molecular replacement (MR) method combined with the single isomorphous replacement with anomalous scattering (SIRAS) method. Initial phases were obtained from the molecular replacement search using the FKBP12-rapamycin complex structure as a search model. The cross rotation search revealed a clear peak at 01=10.80, 02=70.00, 03=309.40 with height/r.m.s. ratio of 12.9 and the translation search also showed a clear peak at x=0.000, y=0.230, z=0.417 with height/r.m.s.

ratio of 10.5. Rigid body refinement resulted in an R factor of 0.449 (10-2.7 All molecular replacement calculations used the X-PLOR program (Brunger, 1990). However, the resulting difference electron density map was noisy and hard to interpret. In order to improve the map quality, an Hg derivative crystal was obtained. These data were compared with the native data to give an Rdiff of 12.7%. Two heavy atom sites were found from the difference Patterson map and were refined using the program PHASES (Furey et al,1990). One Hg is bound to Cys22 of FKBP12 with full occupancy the same Hg site seen in the FKBP12-FK506 complex.

-24- The other heavy atom site is in the middle of FRB domain where it is bound to Cys2085 of FRAP with an occupancy factor of 0.6. Both Patterson-deduced heavy atom positions were validated in the Fo-Fc difference map using Fo of the heavy atom derivative and Fc from the molecular replacement solution. Anomalous dispersion measurements were included in this data set and 16 cycles of a solvent flattening procedure were applied, resulting in a phasing power of 2.76 and mean figure of merit of 0.840. All of these calculations were performed using the program PHASES. The electron density map was calculated using the combined phase from the SIRAS and the molecular replacement solution, which cearly showed four helix bundle architecture of FRB domain of FRAP.

Model Building and refinement The FKBP12-rapamycin part was well defined in the initial electron density map; only minor changes in the backbone of 30s loop and some side chains were enough to fit the model of FKBP12-rapamycin structure to this electron density map. For the FRB domain part, most of a 15 polyalanine chain could be traced for the helix regions in the initial map. After several cycles of the positional refinement using X-PLOR, loop regions could be traced and the amino acid sequence could be assigned. The program CHAIN (Sack, 1988) was used for the model fitting and building the ternary complex. A total of 95 residues were built for the FRB domain of FRAP; three residues in the N-terminal and two residues in the C-terminal of FRB domain had 20 no electron density and were not included. Positional refinement was followed by simulated annealing (slow cooling from 3000K to 300K in 25 K steps, 0.0005 ps per step and 50 total steps were used in the simulation at each temperature) and restrained B-factor refinement. All refinements were done using the X-PLOR package. Solvent molecules were assigned during the iterative positional and B-factor refinement procedure, if they appeared at the 3.5 a level of Fo- 25 Fc map, showed good hydrogen bonding geometry and had a low B-factor (less than 50 A2).

The current structure includes 202 amino acids (107 for FKBP12 and 95 for FRB domain), one rapamycin, and 23 water molecules. The final R factor is 19.3% with an Rfree of 29.9%. The free R-factor is calculated with 10% of the data that were selected at the beginning of the analysis. Crystallographic statistics are summarized in Table 1.

Quality of the coordinates The final coordinates have good geometry and r.m.s. deviations from the ideality are 0.008 A for bond lengths and 1.5° for bond angles. Examined by the program PROCHECK (Laskowski, 1993), the current 2.7 A resolution structure shows that the main-chain and sidechain geometrical parameters are better than expected at this resolution with an overall Gfactor of 0.0. Ramachandran plots of xy, angles showed that 86% of the nonglycine and nonproline residues are in energetically most favored regions. The average temperature factors for total atoms and main-chain atoms are 17.0 and 14.7 A2 respectively. The r.m.s. variation in the B-factor of bonded atoms is 2.5 A2. The Luzzati plot (Luzzati, 1952) indicates that the average coordinate error of this complex structure is between 0.25 and 0.30 A.

Those structural coordinates are set forth in Protein Databank format in Appendix I, below. Such data may be transferred to any desired medium, and formatted as desired, for the practitioner's computer.

This invention encompasses those coordinates as well as any translation or rotation or the like thereof which maintains the internal coordinates, which maintains their intrinsic, internal relationship. Those skilled in the art will appreciate that the coordinates may be subjected to other transformations including, e.g. molecular mechanics calculations such as dynamic simulation, minimization, etc. This invention further encompasses the use of coordinates of the FRB of FRAP, of the ternary complex, or of the corresponding region of FRAP homologs, and in particular, the coordinates set forth in Appendix I, in conducting such transformations (or more extensive transformations such as the generation of alternative conformations), as well as the products of such transformations derivatives of the 15 coordinates).: Table 1 Crystallographic statistics of the ternary complex FKBP12-rapamycin-FRB domain of FRAP Data collection statistics Data Set Resolution No. of reflections Data Rsy(%)* Measured Unique coverage Native 2.7 43447 6920 98.5 7.1 Hg C 1 2 2.7 42681 6884 98.0 7.1 Molecular replacement results Rotation function 01=10.82° 92=70.00 0 e 3 =309.350 Height/r.m.s.=12.90 Translation function x=0.000 y=0.230 z=0.417 Height/r.m.s.=10.5 0 SHeavy atom data statistics (SIRAS) Mean Sites Rdiff(%)t Phasing power 0 figure-of-merit 2 12.7 2.76 0.840 Refinement statistics Resolution Reflections Number of R-factor Rfree R.M.S. deviation (with IF|>30)atoms Bond lengths Bond angles (0) 8-2.7 6206 1727 19.3 29.9 0.008 1.48 *Rsym=r I, where I is the observed intensity and is the average intensity from multiple measurement.

tRdiff= |FpH-FpI /FpH, where Fp and FpH are the amplitudes of native and derivative structure factors, respectively.

OPhasing power=r.m.s. where FH is heavy-atom structure factor amplitude and e is residual lack of closure error.

-26m. Assays Compounds which bind to the FRB of FRAP may be evaluated using materials and methods useful for testing the biological or pharmacological activity of rapamycin analogs. See e.g. Luengo et al, 1995. In addition, the following animal models may be used for further evaluation of such compounds: DELAYED TYPE HYPERSENSITIVITY Mouse abdomens are painted with sensitizing chemicals (sensitization) such as dinitroflourobenzene or oxazalone. Seven days later the ears of sensitized mice are painted (challenge) with a lower concentration of the compound. Antigen processing and presentation, T lymphocyte activation, leukocyte infiltration, humoral mediator release, increased microvascular permeability, and plasma exudation all result from challenge of sensitized mice and lead to edema formation. Edema presents as a two- to three- fold increase in ear thickness within twenty-four hours.

The test compounds or standards can be applied (topical or parenteral) at various times 15 before or after the sensitization or challenge phases. Increased ear thickness is prevented by C several compounds including immunosuppressive agents and steroids. This model is a primary model for contact dermatitis.

ALLOGENEIC SKIN TRANSPLANTATION An allogeneic skin transplant model is used to identify immunosuppressive activity of 20 test compounds. In this model, donor mouse thoracic skin (Balb/c) is surgically grafted onto the *a thorax of recipient mice (C57bl/6). Host rejection of the graft is evidenced by erythema, drying out, and retraction of donor skin. The mean graft survival time is 10 to 11 days, with 80% of the grafts being rejected by 12 days. Active novel immunosuppressive compounds, like existing immunosuppressive compounds, will prolong graft survival.

25 POPLITEAL LYMPH NODE HYPERPLASIA This model directly assesses T lymphocyte proliferation in vivo. Spleen cells, obtained from Balb/c mice, are isolated and administered into the foot pads of C3H mice. Within four days, the popliteal lymph nodes can be removed from the recipient mice and weighed. Other hematological assessments including FACS scanning for T lymphocyte subpopulations may also be performed. Active compounds, like existing immunosuppressive compounds, will inhibit the increase in node mass.

RHEUMATOID ARTHRITIS Several models are available for assessment of anti-arthritic activity, including adjuvantinduced, carageenan-induced, and collagen-induced arthritis in rats and/or mice. Paw pads are injected with one of these agents. Paws increase in volume, and measurements are made between 20 and 30 days later. The ability of test compounds to prevent the induction of paw swelling is tested with daily treatment for 12 consecutive days following the injection of inducing agent. The ability for the test compounds to reverse the progression of the paw swelling is tested by administration of the compound for 12 consecutive days beginning on the -27twelfth day following the injection of inducing agent. Paw swelling measurements are made by water displacement plethysmography. Histology is also an appropriate endpoint for these studies. The MRL/lpr-mouse model, described above, is required for the rheumatoid arthritis indication. This model is a spontaneous autoimmune model that develops rheumatoid arthritis resembling the human condition, including the presence of circulating rheumatoid factor, pannus formation, and bone and cartilage erosion.

SYSTEMIC LUPUS ERYTHEMATOSUS Systemic lupus erythematosus is another autoimmune disease with several animal models.

Several murine strains develop spontaneous SLE. One such strain is MRL/lpr-mice. These mice, over time (20 to 30 weeks) develop auto-antibodies against dsDNA, nuclear antigens, and renal basement membrane. This leads to complement fixation and immune complex formation.

Damage to the kidney becomes apparent with the onset of proteinuria. Many of the other physiologic, hematologic, and immunologic aberrations described below for the CGVHD model are present. Immunosuppressive compounds such as cyclosporin, cydophosphamide, and 15 leflunomide can prevent and reverse the course of disease in this model. Interestingly, these mice also develop pathologies akin to rheumatoid arthritis.

The murine chronic graft versus host disease model (CGVHD, described below) is a model of SLE that contains many of the clinical features of SLE. Activity in this model has been shown to be predictive of activity in the more clinically relevant SLE models.

20 TRANSPLANTATION S Allograft transplantation (skin graft) assay is often used as an initial test of immunosuppressive activity. While this model is useful as a screen, it may be supplemented with assays based on animal transplant models involving transplantation of internal organ (heart, liver, kidney, bone marrow) with use of "clinically acceptable" physiologic endpoints to assess graft survival. Efficacy of test compounds in only a very limited number of these rodent models is required. Following observation of activity in a rodent model, the test compounds are S* typically tested in further animal models canine, porcine or non-human primate). Active compounds decrease acute and chronic rejection and prolong transplant survival.

GRAFT VS. HOST DISEASE Chronic GVHD (CGVHD) can be used to model CD4+-dependent humoral immunity. It is induced in BDF 1 mice (which are progeny of DBA/2 male x C57BL/6 female matings) by administering to them isolated spleen:lymph node cells from DBA/2 mice. This results in: a) disregulation and stimulation of CD4+ T lymphocyte (Lyl+; murine marker) activity due to incompatibilities at MHC II molecules, and b) abnormal T-B lymphocyte cooperation. The resulting pathological state, in many ways, mimics systemic lupus erythematosus (SLE). Several measurable endpoints develop within 14 days; including, circulating anti-host IgG and IgE antibodies, altered T and B lymphocyte proliferation activity measured in vitro, complement utilization, hemagglutination, slow progressive wasting, dermal aberrations, splenomegaly, lymphoid hyperplasia, and proteinuria. Only a few of these endpoints need to be measured.

28 Active compounds are are those which limit T lymphocyte disregulation and abrogate changes in these variables. Many steroids prednisolone), cydosporine, FK-506, cyclophosphamide, and leflunomide are all active in this model and can be used as positive controls.

The acute GVHD model (AGVHD) is also produced in BDF1 mice. In this case, isolated spleen:lymph node cells from C57BL/6 mice are administered. This results in disregulation and stimulation of CD8 T lymphocytes due to incompatibilities in the MHC I molecules. Elevated cytokine levels and donor clonal expansion occurs. Ultimately, donor cytotoxic T lymphocytes and NK cells rapidly reject host tissue and cause relatively rapid death of the recipient. The progression of AGVHD in this model is assessed by measurement of hematologic abnormalities (including T cell number and type), cytokine elevations (TNF, IL-1, IL-2, and/or IL-4), low body weight, hypoyglobulinemia, circulating hematologic characteristics indicative of aplastic anemia (granulocytopenia, thrombocytopenia), ex vivo NK or CTL activity, and host survival.

Active compounds are those which abrogate changes in the variables, and prolong survival over 4 to 6 weeks.

ASTHMA

Asthma offers another opportunity for safe immunosuppressive therapy. Atopic asthmatics halre antibody mediated hypersensitivity and the often occurring late phase reaction is likened to a DTH response. Asthma has only recently been defined as an inflammatory disease (1992). Since then, several publications from prominent asthmatologists demonstrate the presence of activated CD4 and CD8 T lymphocytes in bronchoalveolar lavage fluid and blood of atopic asthmatics. The ratios of these cells changes in asthmatic conditions.

Furthermore, several of the T cell associated cytokines (IL-1, IL-2, IL-4, IL-5, and TNF) are all implicated in clinical and expeimental asthma. Inflammatory events in asthma are now considered to be T lymphocyte driven. Initial clinical trials with inhaled cyclosporin suggest that 25 local immunosuppression can ameliorate airway hyperreactivity the underlying defect in asthma.

The guinea pig model of antigen-induced pulmonary aberrations is used as a model for asthma. These animals are actively sensitized to ovalbumin to generate high circulating titers of anti-ovalbumin antibody with seroconversion to the IgE class, as is the case with atopic asthmatics. Aerosol challenge of sensitized guinea pigs results in measurable eosinophil rich pulmonary infiltrates (approximately a 16-fold increase in eosinophils), pulmonary edema, and mucous plugging of the small airways; all culminating in the expression of the underlying defect in asthma- airway hyperreactivity (approximately a 3 to 4-fold increase in reactivity). Acute S* bronchoconstriction is obviously present and points the aforementioned presence of the pathophysiologic sequelae. Active compounds are those which lessen or abrogate such symptoms.

The above description is meant to illustrate, rather than limit the scope of the invention.

Given the foregoing description, numerous variations in the materials or methods employed in performing the invention will be obvious to one skilled in the art. Any such obvious variation is to be considered within the scope of the invention.

-29- P:\OPER\Ejh\A.odd gp-s\2337969.222.do-I8IOS/3 -29a- Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

*0 *o* *o Full references to literature cited above (by reference to author and year) are provided below: References Brown, E. Albers, M. Shin, T. Ichi ckawa, Keith, C. Lane, W. S. Schreiber, S.

L. Nature 369, 756-758 (1994).

Brunger, A. T. X-PLOR Version 3.1 Manual (Yale Univ. Press, New Haven, CT, 1992) Chen, Zbeng, Brown, E. J. Schreiber, S. L. Proc. Natl. Acad. 56. USA 92, 4947- 4951 (1995).

Chiu, M. Katz, H Berlin, V. Proc. Nati. Acad. Sci. USA 91, 12574-12578 (1994).

Clardy, J. Pro c. Natl. Acad. 56i. USA 92, 56-61 (1995).

Dayhoff, Schwartz, KLM.; Orcutt, Atlas of Protein Sequence and Structure, Suppi. 3,345 (1979) Furey, W. and Swarninathan, S. American Crystallographic Association Mtg. Abstr. Ser. 2 18, 73 (1990) Gonnet, Cohen, Benner, S.A. Science 256, 1443 (1992) Greer, J. Mol. Biol, 153, 1027 (1981) Griffith, J. Kim, J- L, Kim, E. Sintchak, M. Thomson, j. Fitzgibbon, M. JFleming, Caron, P. Hsiao, K. Navia, M. A. Cell 82, 507-522 (1995).

20 Harris, N. Presnell, S- and Cohen, F. E. 1. Mol. biol. 236, 1356-1368 (1994) :::.Keith Schreiber, 1995, Science 270:50-51.

Laskowski, R. A. J. Appi. Cryst. 26, 283-291'(1993) Luengo, J. Yamashita, D. Dunnington, Konialian Beck, Rozamus, L Yen, H., Bossard, M. Levy, M- Hand, Newman-Tarr, Badger, Faucette, Johnson, R.

D'Alessio, K, Porter, 5km, A. Heys, Choi, Kongsaeree, Clardy, and Holt, D. A. Chemistry Biology 2, 471-481 (1995).

**Luzza ti, P. V. Acta Cryst. 5, 802-810 (1952) Otwinski, Z. The SCALEPACK Manual (Howard Hughes Medical Institute, Yale Univ., New Haven, C1, 1992).

Sabatini, D. Erdjument-Bromage, Lul, Tempst, P. Snyder, S. H. Cell 78, 35-43 (1994).

Sack, J. S. 1. Mol. Graphics 6, 224-225 (1988) Schreiber, S. L. Cell 70, 365-368 (1992).

Sehgal, S. Baker, H. Vezina, C. J. Antibiot. 6, 727-732 (1975).

Sehgal, S. N. Ann. N.Y. Acad. 56. 696, 1-8 (1993).

Stan, McLaughlin, M. Cafferkey, Johnson, R. Rosenberg, and Livi, G. P. J.

Biol. Chem. 269, 32027-32030 (1994) Standaert, R. Galat, Verdine, G. L. Schreiber, S. L. Nature 346, 671-674 (1990) 10 Tanaka, Kuroda, Marusawa, Hatanaka, Kino, Goto, T. Hashimoto, M. J. Amer. Chem. Soc. 109, 5031-5033 (1987).

VanDuyne, G. Standaert, R. Schreiber, S. L. Clardy, J. Science 251, 839-842 (1991).

VanDuyne, G. Standaert, R. Schreiber, S. L. Clardy, J. J. Am. Chem. Soc. 113, 7433- 7434 (1991a).

Van Duyne, G. Standaert, R. Karplus, Schreiber, S. L. Clardy, J. J. Mol. Biol. 229, 105-124 (1993).

Vezina, Kudeiski, A. Sehgal, S. N. J. Antibiot. 28, 721-726 (1975).

Zakian, V. A. Cell 82, 685-687 (1995) 31 Appendix I

ATOM

ATM

ATCM

ATOM

ATCM

ATCM

ATOM

ATCM

ATCM

ATOIM

ATM

ATM

ATCM

ATM

ATM

ATM

ATIM

ATCM

ATIM

ATCM

ATOM

AT'M

ATCM

ATOM

ATCM

ATOM

ATM

ATOM

AT'CM

ATOM

ATICM

ATM

ATCM

ATCM

ATOM

ATOM

1 C GLY 2 0 GLY 3 HT1 GLY 4 HT2 GLY 5 N GLY 6 HT3 GLY 7 CA GLY 8 N VAL 9 H VAL 10 CA VAL 11 CB VAL 12 3G1 VAL 13 CG2 VAL 14 C VAL 15 0 VAL 16 N GrN 17 H GUN 18 CA GLN 19 CB GIN 20 OG GLN 21 CD GLN 22 OE1 GLN 23 NE2 GLN 24 HE21 GUN 25 HE22 GrN 26 C GUN 27 0 GN 28 N VAL 29 H VAL 30 CA VAL 31 CB VAL 32 OG1 VAL 33 OG2 VAL 34 C VAL 35 0 VAL 36 N GU 37 H GDUJ 1 4.588 25.968 49.84 1 3.587 26.690 49.93 1 5.460 28.281 50.88 1 5.463 28.482 49.22.

1 5.987 28.058 50.01, 1 6.961 28.429 50.041 1 5.986 26.568 49.84! 2 4.539 24.648 49.684 2 5.366 24.143 49.53S 2 3.311 23.862 49.74E 2 2.889 23.360 48.31E 2 4.114 23.006 47.492 2 1.975 22.155 48.411 2 3.549 22.668 50.692 2 4.576 21.989 50.605 3 2.643 22.482 51.646 3 1.852 23.045 51.649 3 2.789 21.445 52.664 3 2.600 22.065 54.056 3 2.416 21.064 55.181 3 3.718 20.451 55.660 3 4.754 20.581 55.015 3 3.665 19.760 56.792 3 2.812 19.651 57.241 3 4.510 19.373 57.085 3 1.817 20.280 52.454 3 0.608 20.466 52.367 4 2.363 19.082 52.313 4 3.336 19.008 52.381 4 1.540 17.890 52.127 4 2.054 17.030 50.930 4 0.924 16.172 50.364 4 2.630 17.930 49.842 4 1.544 17.037 53.401 4 2.600 16.705 53.947 5 0.363 16.733 53.914 5 -0.430 17.182 53.551 3 1.00 12.34 1 1.00 3.24 1 0.00 0.00 1 0.00 0.00 1 1.00 24.95 3 0.00 0.00 9 1.00 14.30 1 1.00 9.85 0.00 0.00 1 1.00 11.89 1 1.00 9.17 1.00 14.93 1.00 2.00 1.00 15.67 1.00 16.61 1.00 17.91 0.00 0.00 1.00 20.42 1.00 26.51 1.00 34.77 1.00 41.28 1.00 44.41 1.00 42.31 0.00 0.00 0.00 0.00 1.00 17.06 1.00 17.79 1.00 14.50 0.00 0.00 1.00 13.12 1.00 10.68 1.00 7.51 1.00 9.85 1.00 12.15 1.00 15.65 1.00 6.97 0.00 0.00

FKBP

FKBP

FKBP

EKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

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ATCM 38 CA GU 5 0.275 15.856 ATOM 39 CB GLU 5 -0.096 16.664 ATCM 40 CG GLU 5 0.621 17.998 ATM 41 CD GLU 5 0.346 18.726 ATOM 42 OE1 GLU 5 1.188 18.629 ATCM 43 OE2 GLU 5 -0.710 19.385 ATCM 44 C GLU 5 -0.743 14.752 ATCMi 45 0 GLU 5 -1.937 15.023 ARCM 46 N THR 6 -0.271 13.511 ATCM 47 H THR 6 0.666 13.372 ATCM 48 CA THR 6 -1.125 12.365 ATM 49 CB THR 6 -0.337 11.045 ATIM 50 OG1 THR 6 0.881 11.178 ATCM 51 HG1 THR 6 .1.493 10.508 ATM 52 CG2 THR 6 -1.132 9.919 ATM 53 C THR 6 -2.355 12.240 ATCM 54 0 THR 6 -2.281 12.454 ATM 55 N ILE 7 -3.509 12.099 ATCM 56 H ILE 7 -3.506 12.334 ATM 57 CA ILE 7 -4.755 11.709 ATM 58 CB ILE 7 -5.965 12.465 ATM 59 CG2 ILE 7 -7.275 11.841

A

T

M1 60 CG1 ILE 7 -5.918 13.947 ATM 61 CD1 ILE 7 -7.008 14.764 ATCM 62 C ILE 7 -4.979 10.199 ATM 63 0 ILE 7 -5.686 9.576 ATM 64 N SER 8 -4.469 9.648 ATCM 65 H SER 8 -4.039 10.240 ATCUM 66 CA SER 8 -4.629 8.226 ATCM 67 CB SER 8 -6.079 7.930 1 ATCM 68 OG SER 8 -6.236 6.581 E A'TCM 69 HG SER 8 -7.179 6.384 5 ATM 70 C SER 8 -3.685 7.798 5 ATlM 71 0 SER 8 -3.607 8.454 5 ATM 72 N PRO 9 -2.830 6.798 5 ATCM 73 CD PRO 9 -2.665 6.076 5 ATCM 74 CA PRO 9 -1.706 6.548 5 ATCM 75 CB PRO 9 -0.709 5.793 5 ATM 76 CG PRO 9 -1.572 5.093 5 55.071 56.308 56.389 57.674 58.586 57.778 54.848 54.745 54.805 55.050 54.508 54.575 53.836 54.158 53.972 55.415 56.629 54.772 53.824 55.423 54.799 55.244 55.170 54.527 55.249 56.034 54.151 I 53.499 53.842 1 53.450 1 53.064 1 3.022 0 52.707 1 1.664 1 .2.965 1 4.238 1 2.055 1 2.932 1 3.920 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1

L.

L.

L.

L.

.1 1 1.00 5.19 1.00 8.81 1.00 13.30 1.00 15.76 1.00 22.97 1.00 22.20 1.00 3.46 1.00 4.04 1.00 2.00 0.00 0.00 1.00 5.26 1.00 3.67 1.00 13.50 ).00 0.00 00 2.01 00 9.57 00 15.36 .00 8.03 .00 0.00 .00 7.62 .00 5.96 .00 2.71 .00 2.00 .00 2.01 .00 11.96 .00 17.57 .00 12.78 00 0.00 00 12.24 00 6.63 00 12.33 00 0.00 00 19.11 00 17.14 00 23.27 00 22.82 00 25.68 00 25.08 00 26.18

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FEBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

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FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

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-33a a a

ATOM

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ATOM

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77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 96 97 98 99 100 101 102 103 104 I 105 I 106 107 108 H 109 HH 110 IS 111 H 112 HI 113 C 114 0 115 N c

N

H

C

C

0

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a

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CH

c 0

N

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11 12 121 22

PRO

PRO

GLY

SGLY

A GLY

GLY

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ASP

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A ASP

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1 ASP 2 ASP

ASP

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GLY

GLY

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ARG

ARG

ARG

ARG

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ARG

ARG

ARG

ARG

ARG

ARG

ARG

ARG

ARG

ARG

ARG

ARG

THR

9 9 10 10 10 10 10 11 11 11 11 11 11 11 11 11 12 12 12 12 12 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 14 -3.034 -1.272 -0.602 -1.373 -0.241 0.479 -0.018 -0.664 0.992 0.767 0.713 1.591 -0.204 2.438 3.364 2.637 1.858 3.958 4.976 6.183 4.488 3.572 5.357 5.756 4.624 5.130 4.963 5.508 4.154 4.023 4.540 3.414 3.490 3.595 2.873 4.720 5.414 3.392 5.01 5.98 6.69 5.16 4.15 3.911 3.62( 3.84( 2.585 1.862 2.804 3.686 2.635 3.073 2.273 4.372 4.932 4.948 4.585 4.621 4.222 3.918 4.030 2.552 1.555 0.296 0.361 1.005 -0.435 0.266 0.450 0.864 1.415 1.557 2.001 4.537 4.995 4 50.737 8 49.728 6 49.796 8 48.531 4 48.412 6 49.386 6 47.208 46.504 47.006 45.675 44.493 44.377 43.659 47.085 47.190 46.898 46.696 47.081 46.015 46.262 44.833 44.840 43.667 43.526 43.724 44.418 45.870 46.370 C 46.567 1 47.877 1 48.341 0 48.399 0 45.961 1 44.977 0 46.485 0 42.369 1 41.459 1 1.00 1.00 0.00 30.17 28.78 0.00 1.

1.1 1.I 1.C 10.

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0( 0.01 1.0( 1.0( 1.0C 1.00 0.00 L.00 1.00 1.00 1.00 1.00 0.00 L.00 L.00 .00 .00 .00 .00 .00 .00 .00 00 32.81 00 34.72 00 37.49 00 30.71 )0 0.00 )0 28.23 )0 23.26 )0 21.83 )0 13.66 0 23.38 0 29.86 0 31.65 0 31.53 0 0.00 0 34.79 0 37.89 0 38.20 40.35 0.00 S43.98 i 48.12 56.08 64.50 70.55 0.00 73.54 74.82 0.00 0.00 75.14 0.00 0.00 40.88 41.05 -2.056 5.766 50.778 1.00 28.63

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

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FKBP

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FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

4.531 42.328 1.00 36.51 a a

ATCM

ATCM

AICM

ATM

ATCM

ATCM

ATCM

ATOM

ATM

ATCM

ATCM

ATCM

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A'1T14 A'ItM 15 ATCM

A'CM

ATCM

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AfCM

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A'IM

A7TM

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ATM

A'1U4

AT(M

ATCM

ATM

ATCM

ATM

ATCM

ATM

116 H THR 117 CA HR 118 CB THR 119 OG1 THR 120 1 IHR 121 CG2 THR 122 C IHR 123 0 THR 124 N PHE 125 H PHE 126 CA PHE 127 CB PHE 128 OG PHE 129 CD1 PHE 130 CD2 PHE 131 CE1 PHE 132 CE2 PHE 133 CZ PHE 134 C PHE 135 0 PHE 136 N PRO 137 CD PRO 138 CA PRO 139 CB PRO 140 CG PRO 141 C PRO 142 0 PRO 143 N LYS 144 H LYS 145 CA LYS 146 CB LYS 147 CG LYS 148 CD LYS 149 CE LYS 150 NZ LYS 151 HZ1 LYS 152 HZ2 LYS 153 HZ3 LYS 154 C LYS 14 14 14 14 14 14 14 14 15 15 15 15 15 15 15 15 15 15 15 15 16 16 16 16 16 16 16 17 17 17 17 17 17 17 17 17 17 17 17 2.944 2.654 1.296 1.477 0.659 0.722 2.416 1.373 3.430 4.257 3.354 4.725 5.202 5.046 5.732 5.400 6.089 5.919 2.902 3.176 2.232 2.068 1.814 0.852 0.905 2.998 3.580 3.408 3.044 4.463 4.856 5.973 5.425 6.545 6.050 5.395 5.550 6.857 4.031 3.906 5.085 4.362 2.945 2.484 4.651 6.589 7.023 7.364 6.922 8.822 9.405 9.018 9.885 7.756 9.499 7.363 8.237 9.358 8.739 10.532 11.493 11.122 12.243 12.310 11.672 12.683 10.958 10. 054 11.441 10.356 9.427 8.075 7.050 5.706 5.316 5.803 5.061 S42.915 S41.199 41.010 41.172 40.952 39.621 41.356 41.846 41.000 40.707 40.970 41.330 42.701 43.775 42.936 45.062 44.218 45.283 39.596 38.557 39.571 40.671 38.296 38.710 40.215 37.512 37.895 36.467 36.366 35.572 34.563 35.030 35.497 35.721 36.174 35.466 37.081 36.292 0.0 1.0 1.0 l.0' 0.0 1.01 1.0C 1.0 1.0 0.OC l.OC l.OC 1.0oC 1.0oC 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 0.00 0.00 0 0.00 0 31.82 0 34.22 0 31.38 0 0.00 0 29.70 0 28.19 25.30 27.12 S0.00 S30.73 S30.56 S31.81 I 31.26 S31.84 i 28.40 31.05 31.16 34.59 32.29 35.21 32.43 36.14 33.90 34.16 38.59 40.62 44.97 0.00 49.95 53.22 61.47 69.15 73.13 72.77 0.00 0.00 0.00

EKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FRBP

FKBP

EKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

12.703 34.823 1.00 50.23 6t 6 6 6* 66 *6 6* 6 6**6 ATOM 155 0 LYS 17 ATOM 156 N ARG 18 ATOM 157 H ARG 18 ATOM 158 CA ARG 18 ATIM 159 CB ARG 18 ATM 160 COG ARG 18 ATCM 161 CD ARG 18 ATOM 162 NE ARG 18 ATOM 163 HE ARG 18 ATM 164 CZ ARG 18 ATIM 165 NHFl ARG 18 ATCM 166 HH11 ARG 18 ATM 167 HH12 ARG 18 ATCM 168 NH2 ARG 18 15 ATCM 169 HH21 ARG 18 ATM 170 HH22 ARG 18 ATIM 171 C ARG 18 ATCM 172 0 ARG 18 ATM 173 N GLY 19 ATOM 174 H GLY 19 ATOM 175 CA GLY 19 ATCM 176 C GLY 19 ATOM 177 0 GLY 19 ATOM 178 N GLN 20 ATCM 179 H GLN 20 AT'M 180 CA GLN 20 ATCM 181 CB GLN 20 ATOM 182 G GUEN 20 ATOM 183 CD GUN 20 ATICM 184 OE1 GLN 20 ATOM 185 NE2 GLN 20 ATCM 186 HE21 GUN 20 ATCM 187 HE22 GLN 20 AT'M 188 C GUN 20 ATCM 189 0 GUN 20 ATCM 190 N THR 21 ATOM 191 H THR 21 AT'M 192 CA THR 21 ATM 193 CB THR 21 2.8E 4.93 5.78 4.66 5.96 5.75 7.03 8.001 8.691 7.99! 8.954 9.674 8.923 7.000 6.256 6.994 3.965 4.440 2.775 2.437 2.037 0.878 0.242 0.603 1.278 -0.571 -0.290 0.907 0.945 1.852 -0.064 -0.781 -0.025 -1.784 -1.641 -2.957 -2.993 -4.185 -5.398 12 12.81 8 13.67 2 13.55 6 14.90 8 15.67: 5 17.03- 0 17.57: 5 18.00f 3 17.37! 5 19.203 1 19.52E 18.876 20.425 I 20.052 19.798 20.950 14.637 13.832 15.209 15.781 15.058 14.072 13.821 13.509 13.579 12.655 11.586 10.723 9.516 9.355 8.672 8.854 7.895 13.458 14.558 12.836 11.964 13.406 13.137 3 34.389 1.00 51.36 2 34.718 1.00 48.43 3 35.190 0.00 0.00 8 33.986 1.00 46.13 1 33.732 1.00 47.22 1 33.092 1.00 53.52 2 32.467 1.00 60.93 3 33.466 1.00 68.56 33.748 0.00 0.00 34.054 1.00 71.82 34.910 1.00 73.41 35.143 0.00 0.00 35.358 0.00 0.00 33.826 1.00 74.07 33.207 0.00 0.00 34.267 0.00 0.00 32.652 1.00 44.43 31.844 1.00 44.85 32.491 1.00 41.63 33.210 0.00 0.00 31.246 1.00 36.64 31.281 1.00 33.71 30.256 1.00 31.30 32.454 1.00 31.51 33.162 0.00 0.00 32.647 1.00 27.89 33.702 1.00 27.47 33.416 1.00 29.05 34.305 1.00 28.73 35.112 1.00 29.95 34.191 1.00 29.76 33.542 0.00 0.00 34.776 0.00 0.00 33.096 1.00 26.36 33.652 1.00 23.69 32.994 1.00 23.74 32.525 0.00 0.00 33.551 1.00 19.78 32.648 1.00 18.09

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

**66 6 6*66 *6 6 66 66 -3.6- *4

S.

S

ATCM

ATOM

ARM

ATOM

AM

ATOM

ATCM

AWI

AICM

ATM

ATCM

ATOM

A'IMt

ATCM

15 ATCM

A'IUI

ATCM4 ATCM ATM

ATOM

ATCM

ATCM

ATCM

ATOM

ATCM

ATCIM

ATOM

ATM

ATCMX

ATOM

ATOM

ATM

ATCM

ATCM

ATM

ATCM

ATCM

ATOM

194 OG1 THR 195 I1 THR 196 CG2 IHR 197 C THR 198 0 THR 199 N CYS 200 H CYS 201 CA CYS 202 CB CYS 203 SG CYS 204 C CYS 205 0 CYS 206 N VAL 207 H VAL 208 CA VAL 209 CB VAL 210 OG1 VAL 211 OG2 VAL 212 C VAL 213 0 VAL 214 N VAL 215 H VAL 216 CA VAL 217 CB VAL 218 031 VAL 219 OG2 VAL 220 C VAL 221 0 VAL 222 N HIS 223 H HIS 224 CA HIS 225 CB HIS 226 OG HIS 227 CD2 HIS 228 ND1 HIS 229 HD1 HIS 230 CE1 HIS 231 NE2 HIS 232 HE2 HIS 21 21 21 21 21 22 22 22 22 22 22 22 23 23 23 23 23 23 23 23 24 24 24 24 24 24 24 24 25 25 25 25 25 25 25 25 25 25 25 -5.10: -4.66" -6.62- -4.50; -4.89! -4.39C -4.044 -4.794 -4.056 -2.300 -6.301 -6.840 -6.991 -6.547 -8.371 -9.180 -10.658 -8.972 -8.353 -7.678 -8.946 -9.395 -8.896 -7.806 -6.481 -8.238 -10.237 -11.078 -10.481 -9.837 -11.588 -12.462 -13.789 -14.625 -14.420 -13.990 -15.591 -15.738 -16.532 3 13.576 7 12.831 1 13.882 12.869 11.707 13.744 14.636 13.421 14.322 14.464 13.589 14.676 12.485 11.617 12.542 11.314 11.483 11.121 12.579 11.765 13.622 14.274 13.840 14.883 14.535 16.276 14.309 14.804 14.041 13.454 14.671 13.611 13.412 12.348 14.398 4 15.194 4 13.959 4 12.715 4 31.319 30.862 33.159 34.945 35.112 35.939 35.726 37.302 38.281 37.959 37.492 37.284 37.760 37.634 38.232 37.743 38.043 36.264 39.770 40.416 40.342 39.762 41.782 42.170 41.524 41.784 42.333 41.583 43.617 44.074 44.346 3 45.015 3 44.351 44.335 1 43.625 1 13.216 0 13.204 1 [3.619 1 1.00 0.00 1.00 1.0 1.0' 1.01 0.0( 1.0 1.0( 1.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 ).00 1.00 1.00 00 L.00 L.00 .00 .00 .00 0 19.51 0 21.36 0 15.33 0 0.00 0 7.92 0 4.88 9.58 7.02 1 8.66 4.33 0.00 6.31 3.87 2.00 5.84 11.82 17.38 10.13 0.00 5.89 3.59 2.00 2.66 7.13 8.15 8.15 0.00 5.84 2.00 2.00 2.01 6.75 0.00 2.00 2.00 25.65 0.00 15.30

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

12.146 43.449 0.00 0.00 -37a as a a a a. a.

ATGM

ATCM

ATCM

ATOM

ATCM

ATOM

ATCM

ATCM

ATCM

ATOM

ATOM ATM

ATCM

ATOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATCM

ATM

ATCM

ATCM

ATCM

ATOM

ATCM

ATOM

ATCM

ATCM

233 C HIS 234 0 HIS 235 N TYR 236 H TYR 237 CA TYR 238 CB TYR 239 OG TYR 240 CD1 TYR 241 CE1 TYR 242 CD2 TYR 243 CE2 TYR 244 CZ TYR 245 OH TYR 246 HH TYR 247 C TYR 248 0 TYR 249 N THR 250 H THR 251 CA TOHR 252 CB THR 253 031 THR 254 B31 THR 255 032 THR 256 C THR 257 0 THR 258 N GLY 259 H GLY 260 CA GLY 261 C GLY 262 0 GLY 263 N MET 264 H MET 265 CA MET 266 CB MET 267 CG MET 268 SD MET 269 CE MET 270 C MET 271 0 MET 25 25 26 26 26 26 26 26 26 26 26 26 26 26 26 26 27 27 27 27 27 27 27 27 27 28 28 28 28 28 29 29 29 29 29 29 29 29 29 -10.08! -11.45( -12.07: -10.95( -9.95C -10.57C -11.017 -11.725 -10.831 -11.536 -11.982 -12.704 -12.792 -12.057 -13.162 -11.778 -11.030 -12.469 -13.138 -13.987 -13.409 -13.957 -11.436 -10.365 -11.664 -12.274 -10.813 -11.438 -12.646 -10.619 -9.683 -11.091 -11.512 -10.445 -11.065 -12.824 -10.033 -8.847 5 15.23' 5 16.86' L 17.15! 17.84( 18.827 19.83S 21.08C 21.939 19.497 20.342 21.551 22.348 21.935 18.638 18.746 19.056 18.611 20.164 19.737 18.606 17.851 20.891 21.213 20.891 22.419 22.498 23.538 24.437 24.729 24.887 24.601 25.812 25.047 24.128 22.500 22.854 26.845 26.630 3 46.125 7 45.414 5 44.712 46.389 45.770 I 44.824 i 45.279 44.434 43.495 42.651 43.122 42.274 41.411 47.045 46.515 48.276 48.735 48.924 50.219 49.972 49.785 50.779 49.273 49.784 48.779 48.038 49.128 50.175 50.131 51.117 51.122 52.138 53.404 53.999 54.510 54.721 52.477 52.242 3 1.00 8.08 1.00 2.00 0.00 0.00 1.00 2.00 1.00 3.39 1.00 8.68 1.00 7.15 1.00 11.31 1.00 11.88 1.00 1.00 1.00 0.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 0.00 1.00 8.71 9.36 9.02 0.00 2.60 2.96 8.98 0.00 3.70 3.82 5.37 0.00 2.73 2.00 2.00 5.64 0.00 8.04 8.15 9.73 4.38 0.00 6.14 -11.013 15.611 45.409 1.00 5.86

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FEBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

1.00 11.72 1.00 14.88 1.00 7.90 1.00 5.60 1.00 6.50 1.00 5.89 -38- ATM 272 N LEU 30 -10.477 28.013 52.923 1.00 11.28 FKBP ATOM 273 H LEU 30 -11.444 28.168 52.902 0.00 0.00 FKBP ATCM 274 CA LEU 30 -9.561 29.028 53.443 1.00 14.74 FKBP ATM 275 CB LEU 30 -10.281 30.379 53.572 1.00 12.99 FKBP ATOM 276 OG LEU 30 -10.887 30.967 52.292 1.00 10.36 FKBP ATOM 277 CD1 LEU 30 -12.064 31.842 52.668 1.00 12.99 FKBP ATOM 278 CD2 LEU 30 -9.848 31.761 51.510 1.00 3.34 FKBP ATCM 279 C LEU 30 -9.042 28.573 54.805 1.00 14.12 FKBP ATM 280 0 LEU 30 -9.664 27.732 55.453 1.00 16.16 FKBP ATIM 281 N GLU 31 -7.944 29.169 55.262 1.00 14.66 FKBP ATOM 282 H GLU 31 -7.506 29.828 54.682 0.00 0.00 FKBP ATCM 283 CA GLU 31 -7.266 28.722 56.483 1.00 17.28 FKBP AT3 CM 284 CB GLU 31 -6.294 29.799 56.962 1.00 14.61 FKBP ATM 285 CG GLU 31 -5.818 29.586 58.382 1.00 22.25 FKBP 15 ATCM 286 CD GLU 31 -4.510 30.284 58.698 1.00 26.77 FKBP t* ATM 287 OE1 GLU 31 -4.245 31.362 58.107 1.00 21.74 FKBP ATCM 288 OE2 GLU 31 -3.774 29.762 59.576 1.00 23.08

FKBP

ATIM 289 C GLU 31 -8.187 28.313 57.642 1.00 18.96 FKBP ATCM 290 0 GLU 31 -8.008 27.258 58.262 1.00 18.93 FKBP 20

K

A'T 291 N ASP 32 -9.238 29.090 57.855 1.00 17.34 FKBP ATCM 292 H ASP 32 -9.405 29.814 57.223 0.00 0.00 FKBP ATCM 293 CA ASP 32 -10.116 28.866 58.996 1.00 19.84 FKBP ATCM 294 CB ASP 32 -10.894 30.142 59.308 1.00 27.98 FKBP ATM: M 295 OG ASP 32 -11.601 30.704 58.090 1.00 34.72 FKBP ATOM 296 OD1 ASP 32 -12.727 30.254 57.801 1.00 32.49 FKBP ATCM 297 OD2 ASP 32 -11.023 31.588 57.415 1.00 43.34 FKBP *oo: ATCM 298 C ASP 32 -11.096 27.713 58.816 1.00 18.08

FKBP

ATCM 299 0 ASP 32 -11.986 27.541 59.638 1.00 17.85 FKBP ATOM 300 N GLY 33 -10.994 26.998 57.697 1.00 18.90 FKBP ATCM 301 H GLY 33 -10.204 27.111 57.137 0.00 0.00 FKBP ATM 302 CA GLY 33 -11.909 25.896 57.417 1.00 14.65 FKBP ATOM 303 C GLY 33 -13.146 26.270 56.616 1.00 10.95 FKBP ATCM 304 0 GLY 33 -14.020 25.437 56.370 1.00 11.28 FKBP AT'M 305 N LYS 34 -13.235 27.536 56.230 1.00 5.53 FKBP ATM 306 H LYS 34 -12.565 28.159 56.564 0.00 0.00 FKBP ATCM 307 CA LYS 34 -14.320 27.999 55.379 1.00 7.65 FKBP ATM 308 CB LYS 34 -14.270 29.521 55.255 1.00 15.91 FKBP ATCM 309 OG LYS 34 -15.468 30.131 54.554 1.00 23.47 FKBP ATOM 310 CD LYS 34 -15.360 31.646 54.513 1.00 34.71 FKBP PCT/US96/16953

A'ITM

ATM

ATOM

ATCM

ATM

A'ItJ

ATOM

ATlCM

ATCM

ATM

ATM

ATOM

ATCM

ATCM

ATM

ATM

A'ItX,

ATM

MATM

ATCMI

ATCM

ATCM

ATM

ATCM

ATCMI

ATCM

ATCM

A'IM

ATCM

ATCM

ATOM

ATCM

ATM

IU14 AIUA~f 311 CE LYS 312 NZ LYS 313 HZ1 LYS 314 HZ2 LYS 315 HZ3 LYS 316 C LYS 317 0 LYS 318 N LYS 319 H LYS 320 CA LYS 321 CB LYS 322 OG LYS 323. CD LYS 324 CE LYS 325 NZ LYS 326 HZ1 LYS 327 HZ2 LYS 328 HZ3 LYS 329 C LYS 330 0 LYS 331 N PHE 332 H PHE 333 CA PHE 334 CB PHE 335 CG PHE 336 CD1 PHE 337 CD2 PHE 338 CE1 PHE 339 CE2 PHE 340 CZ PHE 341 C PHE 342 0 PHE 343 N ASP 344 H ASP 345 CA ASP 346 CB ASP 347 CG ASP 348 ODI ASP 349 OD2 ASP 34 34 34 34 34 34 34 35 35 35 35 35 35 35 35 35 35 35 35 35 36 36 36 36 36 36 36 36 36 36 36 36 37 37 37 37 37 37 37 -15.21 13.80! -13.47! -13.19( -13.745 -14.222 -13.29C -15.06- -15.554 -15.178 -16.269 -16.379 -17.142 -18.637 -19.352 -19.180 -19.004 -20.373 -15.520 -16.387 -14.796 -13.981 -15.167 -14.077 -12.728 -11.660 -12.470 -10.350 -11.167 -10.110 -15.553 -16.499 -14.972 -14.365 -15.201 -14.340 -14.583 -15.679 -13.665 3 32.24 5 32.63! 5 33.32- 5 31.79: 33.05! 27.36! 27.65- 26.371 26.012 25.719 24.657 23.854 22.573 22.803 21.501 20.892 21.025 21.681 26.736 27.596 26.690 26.149 27.504 28.541 27.959 28.108 27.442 27.758 27.092 27.250 26.696 27.050 25.507 25.202 24.672 25.220 24.518 23.968 24.565 5 55.918 1.00 38.38 5 56.227 1.00 41.83 1 55.520 0.00 0.00 2 56.185 0.00 0.00 5 57.176 0.00 0.00 53.991 1.00 7.56 53.232 1.00 3.26 53.757 1.00 8.73 54.530 0.00 0.00 52.459 1.00 8.15 .52.511 1.00 2.40 51.249 1.00 7.41 51.484 1.00 11.33 51.464 1.00 15.67 51.304 1.00 20.77 52.129 0.00 0.00 50.450 0.00 0.00 51.212 0.00 0.00 51.378 1.00 13.32 51.587 1.00 16.59 50.257 1.00 12.19 50.278 0.00 0.00 49.098 1.00 8.93 48.753 1.00 4.86 48.415 1.00 3.36 49.295 1.00 4.33 47.151 1.00 7.57 48.916 1.00 5.11 46.766 1.00 5.95 47.648 1.00 2.00 47.861 1.00 11.24 47.152 1.00 9.15 47.738 1.00 11.21 48.445 0.00 0.00 46.568 1.00 8.81 45.416 1.00 12.70 44.091 1.00 11.57 43.855 1.00 7.88 43.254 1.00 15.66

FKBP

EKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

A.

A

A

ATCM

ATCM

ATOM

ATOM

ATOM

ATOM

ATCM

ATIM

ATOM

ATM

ATCM

ATOM

15 ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATCM

ATCOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATOM

ATOM

ATOM

ATCM

ATCM

ATCM

ATOM

ATCM

350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 C ASP 0 ASP N SER H SER CA SER CB SER OG SER H3 SER C SER 0 SER N SER H SER CA SER CB SER OG SER HG SER C SER 0 SER N ARG H ARG CA ARG CB ARG CG ARG CD ARG NE ARG HE ARG CZ ARG NEH ARG HHl ARG HH12 ARG NH2 ARG HH21 ARG HH22 ARG C ARG -14.874 -13.905 -15.751 -16.607 -15.461 -15.954 -15.979 -15.613 -16.108 -17.313 -15.339 -14.397 -15.840 -14.682 -13.861 -14.195 -16.762 -17.547 -16.624 -16.027 -17.441 -16.800 -15.385 -14.978 -13.546 -12.924 -13.031 -11.727 -11.112 -11.374 -13.812 -14.794 -13.417 -18.883 -19.798 -19.085 -18.307 -20.435 -20.375 23.199 22.904 22.291 22.613 20.850 20.223 18.804 18.490 20.110 20.210 19.252 19.223 18.414 17.758 16.976 17.054 17.317 16.751 16.994 17.536 15.972 15.538 15.003 14.243 13.940 14.683 12.714 12.527 13.308 11.597 11.673 11.785 10.752 16.433 15.612 17.746 18.340 18.315 19.784 46.864 47.545 46.450 46.095 46.493 47.800 47.722 48.571 45.349 45.168 44.684 44.967 43.584 42.825 43.683 44.589 44.088 43.324 45.376 45.944 46.025 47.345 47.220 48.484 48.561 48.660 48.497 48.631 48.782 48.585 48.262 48.128 48.214 46.270 46.350 46.370 46.438 46.454 46.879 1.00 2.00 1.00 2.01 1.00 2.52 0.00 0.00 1.00 2.33 1.00 12.19 1.00 9.54 0.00 0.00 1.00 2.00 1.00 2.31 1.00 2.00 0.00 0.00 1.00 3.72 1.00 3.50 1.00 3.28 0.00 0.00 1.00 9.63 1.00 6.74 1.00 13.48 0.00 0.00 1.00 12.15 1.00 4.43 1.00 2.00 1.00 3.29 1.00 4.66 0.00 0.00 1.00 2.00 1.00 2.00 0.00 0.00 0.00 0.00 1.00 2.00 0.'00 0.00 0.00 0.00 1.00 17.11 1.00 17.06 1.00 20.79 0.00 0.00 1.00 26.68 1.00 26.55

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

ARG

ASP

ASP

387 CA ASP 388 CB ASP -41a

AIM

AGUI

ATCM

ATCM

ATUA

ATEM

ATCM

ATCM

ATCM

A2CM

ATCM

A~IUCI

AM

ATOM

A'IU3 A EUM ATaKI

ATC

AUGH

ATO

A'ITM

ATOM4 MMK1

ATCM

ATOM

AI4

ATCM

AMM

ATO

5 ARM A'04l

ATCM

A'ITM

A7TO

ATOM

ATM AIM

ATOR

3 3 3 3 3 3 3 39 39 39 39 40 40 40 40 40 40 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 89 CG ASP 90 OD1 ASP 91 OD2 ASP 92 C ASP 93 O ASP 94 N AMG )5 H ARG 6 CA ARG 7 CB ARG 8 CG ARG 9 CD ARG 0 NE ARG 1 HE ARG 2 CZ ARG 3 NH1 ARG I HH11 ARG 5 3H12 ARG SNH2 ARG SHH21 ARG HH22 ARG C AR O ARG N ASN H ASN CA ASN CB ASN CG ASN OD1 ASN ND2 ASN HD21 ASN HD22 ASN C ASN 0 ASN N LYS H LYS CA LYS CB LYS CG LYS CD LYS 41 41 41 41 41 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 43 43 43 43 43 43 43 43 43 43 43 44 44 44 44 44 44 -19.641 -19.251 -19.426 -21.187 -22.416 -20.447 -19.519 -21.006 -20.168 -19.815 -18.697 -17.703 -17.911 -16.491 -15.684 -16.002 -14.773 -16.070 -16.655 -15.156 -21.051 -21.679 -20.302 3 -19.786 1 -20.290 1 -21.724 1 -21.808 1 -20.789 1 -23.025 1 -23.786 1 -23.041 1 -19.628 1 -20.087 1: -18.475 14 -18.152 1E -17.874 14 -18.554 15 -18.478 15 -18.796 17 19.9 19.0 21.1( 18.2C 18.08 18.30 18.59 18.12 18.86 20.30 20.84 19.814 18.86S 20.058 19.045 18.108 19.213 21.306 22.080 21.465 L6.642 L6.252 15.832 6.217 4.392 3.852 2.455 1.802 1.987 2.557 1.094 1.078 3.228 1.696 5.288 .757 .879 .755 93 48.1 )1 48.8 7 48.5 6 45.1; 5 45.1C 7 44.01 5 44.12 4 42.67 5 41.62 2 41.97 0 41.08 1 40.76 40.92 40.27: 39.978 40.125 39.600 40.089 40.328 39.719 42.320 41.338 43.064 43.793 42.840 42.869 43.431 43.662 43.662 43.466 44.043 41.498 40.740 41.275 41.984 39.947 39.148 37.638 95 1.00 34.97 52 1.00 38.14 59 1.00 36.52 4 1.00 30.48 6 1.00 31.53 8 1.00 31.99 0 0.00 0.00 6 1.00 26.25 5 1.00 22.17 6 1.00 26.16 9 1.00 29.95 9 1.00 40.62 S0.00 0.00 1.00 44.80 1.00 43.55 0.00 0.00 0.00 0.00 1.00 47.04 0.00 0.00 0.00 0.00 1.00 25.62 1.00 29.04 1.00 20.94 0.00 0.00 1.00 21.52 1.00 23.52 1.00 28.90 1.00 28.67 1.00 33.33 0.00 0.00 0.00 0.00 1.00 20.93 1.00 21.51 1.00 20.83 0.00 0.00 1.00 19.75 1.00 24.43 1.00 23.61

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FRBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

KBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

.084 36.965 1.00 29.64 -42- ATM 428 CE LYS 44 -20.212 17.565 ATCM 429 NZ LYS 44 -20.543 18.848 ATCM 430 HZ1 LYS 44 -20.497 18.697 ATCM 431 HZ2 LYS 44 -19.853 19.580 ATM 432 HZ3 LYS 44 -21.496 19.168 ATM 433 C LYS 44 -16.361 15.014 ATCM 434 0 LYS 44 -15.928 16.029 ATCM 435 N PRO 45 -15.545 14.014 ATM 436 CD PRO 45 -15.909 12.612 ATOM 437 CA PRO 45 -14.093 14.182 AM 438 CB PRO 45 -13.539 12.779 ATCM 439 CG PRO 45 -14.679 11.871 ATOM 440 C PRO 45 -13.496 15.228 ATICM 441 0 PRO 45 -13.942 15.399 ATCM 442 N PHE 46 -12.501 15.942 ATM 443 H PHE 46 -12.151 15.695 ATM 444 CA PEE 46 -11.825 16.989 ATM 445 CB PHE 46 -11.346 18.068 ATCM 446 3G PHE 46 -10.549 19.165 ATM 447 CD1 PHE 46 -9.192 19.284 ATM 448 CD2 PHE 46 -11.180 20.149 ATUM 449 CE1 PHE 46 -8.472 20.369 ATCM 450 CE2 PHE 46 -10.475 21.243 ATCM 451 CZ PHE 46 -9.117 21.357 ATM 452 C PHE 46 -10.644 16.371 ATCM 453 0 PHE 46 -9.984 15.479 ATOM 454 N LYS 47 -10.421 16.782 ATM 455 H LYS 47 -11.004 17.458 ATCM4 456 CA LYS 47 -9.293 16.255 ATM 457 CB LYS 47 -9.770 15.421 ATOM 458 CG LYS 47 -10.510 14.147 ATM 459 CD LYS 47 -11.587 13.853 ATCM 460 CE LYS 47 -11.326 12.543 ATM 461 NZ LYS 47 -11.608 11.397 ATCIM 462 HZ1 LYS 47 -12.594 11.462 ATM 463 HZ2 LYS 47 -10.981 11.442 ATC4 464 HZ3 LYS 47 -11.471 10.498 ATCM 465 C LYS 47 -8.435 17.389 ATM 466 0 LYS 47 -8.943 18.449 37.282 36.583 35.555 36.854 36.846 40.049 40.596 39.695 39.438 39.830 39.557 39.886 38.887 37.753 39.389 40.268 38.637 39.615 38.980 39.246 38.222 38.779 37.749 38.030 37.898 38.421 36.655 36.253 35.893 34.700 35.058 34.032 33.312 34.216 34.542 35.042 33.712 35.395 35.061 1.00 34.29 1.00 38.07 0.00 0.00 0.00 0.00 0.00 0.00 1.00 17.91 1.00 21.43 1.00 16.30 1.00 17.34 1.00 17.48 1.00 14.90 1.00 19.40 1.00 15.55 1.00 17.90 1.00 11.92 0.00 0.00 1.00 10.26 1.00 7.26 1.00 2.00 1.00 2.00 1.00 2.00 1.00 2.30 1.00 2.00 1.00 5.96 1.00 10.45 1.00 16.71 1.00 9.72 0.00 0.00 1.00 4.83 1.00 5.22 1.00 8.65 1.00 11.93 1.00 10.86 1.00 15.06

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

0.00 0.00 0.00 1.00 1.00 0.00 0.00 0.00 2.00 2.00 -43-

ATCM

ATOM

ATM

ATOM

ATCM

ATOM

ATM

ATCM

ATCM

ATOM

A'IM

MATC ATCM

ATIM

ATCIM

A'ItJA

ATCM

ATCM

ATCM

AM

ATCM

ACM

ATC1

ATCM

ATCM

ATCM

ATCI

ATM

ATCM

ATCM

ATCM

ATM

ATCM

ATOM

ATCM

467 N PHE 468 H PHE 469 CA PHE 470 CB PHE 471 cG PE 472 CD1 PHE 473 CD2 PHE 474 CE1 PHE 475 CE2 PHE 476 CZ PHE 477 C PHE 478 0 PE 479 .N MET 480 H MET 481 CA MET 482 CB MET 483 CG MET 484 SD MET 485 CE MET 486 C MET 487 0 MET 488 N LEJ 489 H LEU 490 CA LEJ 491 CB LEU 492 OG LEU 493 CD1 LEU 494 CD2 LEU 495 C LEU 496 0 LEU 497 N GLY 498 H GLY 499 -CA GLY 500 C GLY 501 0 GLY 502 N LYS 503 H LYS 504 CA LYS 505 CB LYS 48 48 48 48 48 48 48 48 48 48 48 48 49 49 49 49 49 49 49 49 49 50 50 50 50 50 50 50 50 50 51 51 51 51 51 52 52 52 52 -7.125 17.205 35.472 1.00 2.00 -6.799 16.438 35.994 0.00 0.00 -6.191 18.157 34.896 1.00 6.26 -5.964 19.323 35.875 1.00 2.45 -4.948 19.036 36.942 1.00 4.20 -5.254 18.188 38.005 1.00 2.00 -3.650 19.548 36.837 1.00 2.00 -4.282 17.837 38.936 1.00 2.00 -2.664 19.200 37.769 1.00 2.59 -2.983 18.340 38.817 1.00 2.53 -4.866 17.469 34.538 1.00 10.81 -4.480 16.476 35.159 1.00 16.65 -4.181 17.984 33.526 1.00 13.39 -4.543 18.774 33.084 0.00 0.00 -2.892 17.437 33.113 1.00 16.66 -2.690 17.663 31.614 1.00 22.76 -1.538 16.885 31.016 1.00 32.61 -0.985 17.585 29.454 1.00 46.48 -0.812 16.105 28.435 1.00 45.16 -1.768 18.109 33.898 1.00 16.05 -1.749 19.332 34.046 1.00 17.38 -0.852 17.314 34.433 1.00 16.03 -0.925 16.348 34.258 0.00 0.00 0.166 17.848 35.336 1.00 16.25 0.587 16.777 36.350 1.00 16.08 1.737 17.151 37.290 1.00 15.77 1.189 17.731 38.587 1.00 17.22 2.591 15.923 37.561 1.00 17.09 1.398 18.380 34.606 1.00 18.27 2.130 17.629 33.962 1.00 17.62 1.659 19.671 34.773 1.00 24.68 1.071 20.196 35.347 0.00 0.00 2.832 20.281 34.163 1.00 28.29 2.511 21.451 33.246 1.00 30.06 3.312 22.367 33.092 1.00 31.10 1.283 21.482 32.739 1.00 31.85 0.651 20.805 33.051 0.00 0.00 0.883 22.452 31.724 1.00 30.54 -0.281 21.887 30.899 1.00 33.91

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

-44- ATM 506 OG LYS 52 -0.110 20.427 30.479 9

ATOM

ATM

ATM

ATM

ATOM

ATCM

ATOM

ATCIM

ATM

ATM

ATOM

ATOM

ATOM

ATCM

ATM

ATM

ATCM

ATM

ATCM

ATOM

ATCM

ATOM

ATCM

ATM

ATM

ATM

ATOM

ATCM

ATOM

ATM

ATOM

ATM

ATM

ATM

ATM

AM

AICM

AT(M

507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 CD LYS CE LYS NZ LYS HZ1 LYS HZ2 LYS HZ3 LYS C LYS 0 LYS N GUN H GUN CA GN CB GLN CG .GLN CD GN OE1 GUN NE2 GUN 523 HE21 GEN 524 HE22 GiN 525 526 527 528 529 530 531 532 C GUN O GiN N GLU H GLU CA GLU CB GLU CG GU CD GU OEI GLU OE2 GLU C GUJ 0 GLU N VAL H VAL CA VAL CB VAL CGI VAL CG2 VAL C VAL 0 VAL 1.015 1.708 2.954 3.632 2.732 3.361 0.475 -0.349 1.025 1.847 0.572 1.219 2.599 3.585 3.854 4.096 3.837 4.723 -0.950 -1.456 -1.672 -1.188 -3.126 -3.666 -4.296 -4.414 -3.543 -5.339 -3.741 -4.873 -3.035 -2.142 -3.513 -3.774 -4.995 -2.573 -2.500 -1.369 20.263 18.913 18.849 19.546 19.066 17.895 23.795 24.498 24.130 23.671 25.282 26.571 26.848 25.737 25.432 25.098 25.352 24.391 25.457 26.570 24.344 23.505 24.378 23.022 23.020 21.628 21.242 20.896 24.762 25.238 24.444 24.084 24.731 23.446 22.759 22.538 25.559 25.737 29.458 29.584 28.767 29.134 27.773 28.831 32.323 31.741 33.490 33.747 34.279 33.768 34.333 34.025 32.865 35.067 35.970 34.821 34.313 34.380 34.338 34.304 34.306 33.878 32.516 31.960 31.157 32.368 35.642 35.696 36.722 36.580 38.071 38.849 38.309 38.761 38.849 38.408 1.00 38.74 1.00 44.12 1.00 44.68 1.00 46.84 0.00 0.00 0.00 0.00 0.00 0.00 1.00 27.06 1.00 30.79 1.00 21.58 0.00 0.00 1.00 18.83 1.00 25.35 1.00 34.50 1.00 42.12 1.00 46.61 1.00 46.53 0.00 0.00 0.00 0.00 1.00 15.57 1.00 17.17 1.00 12.00 0.00 0.00 1.00 6.49 1.00 6.66 1.00 4.63 1.00 11.57 1.00 18.19 1.00 10.83 1.00 5.69 1.00 4.44 1.00 4.70 0.00 0.00 1.00 6.95 1.00 3.43 1.00 9.22 1.00 2.21 1.00 9.75 1.00 9.34

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FEKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

1

ATOM

A7MM ATOa

ATM

AI1

AIM~

ATMO

ATM

ATItM

ATCM

ATM

ATCM

ATOM

M

ATOM

ATM

ATOM

20 At

ATOM

ATM

ATOM

ATOK

ATM

AOMM

ATOM.

AMM

A'I~yl

ATOM

A'IU,4

ATOM

A'I,'I

ATOM

ATOM

ATCM

ATOM

ATM

ATOM

545 N ILE 546 H ILE 547 CA 1 1

E

548 CB ILE 549 CG2 ILE 550 CG1 ILE 551 CD ILE 552 C ILE 553 O ILE 554 N ARG 555 H ARG 556 CA ARG 557 CB ARG 558 c ARG 559 CD ARG '560 NE ARG 561 HE ARG 562 cz ARG 563 NH1 ARG 564 HH11 ARG 565 HL2 ARG 566 NH2M ARG 567 H21 ARG 568 HH22 ARG 569 C ARG 570 O ARG 571 N GLY 572 H GLY 573 CA GLY 574 C GLY 575 O GLY 576 N TRP 577 H TRP 578 CA TRP 579 CB TRP 580 CG TRP 581 CD2 TRP 582 CE2 TRP 583 CE3 TRP 56 56 56 56 56 56 56 56 56 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 58 58 58 58 58 59 59 59 59 59 59 59 59 -2.887 -3.799 -1.964 -2.674 -3.665 -3.377 -4.003 -0.734 -0.759 0.353 0.284 1.648 2.707 4.115 5.090 6.447 6.567 7.535 8.728 8.794 9.551 7.430 6.534 8.258 1.700 2 2.321 2 1.084 2 0.719 2 0.973 2 0.326 2 0.633 2 -0.567 2 -0.838 2 -1.177 2 -2.399 2 -3.672 2 -4.707 2] -5.725 21 -4.874 20 25.8 26.7 27.36 28.44 26.26 26.75 25.96 24.72 26.65 27.62 26.01 27.09 26.573 27.708 27.196 26.228 27.957 27.390 26.398 27.954 29.277 29.712 29.836 5.006 3.946 5.349 6.253 4.402 3.080 2.043 3.124 4.004 L.927 2.294 2.138 .189 .386 .193 14 40.322 35 40.869 5 42.123 9 41.701 3 42.920 6 44.206 2 41.286 9 41.270 1 41.615 7 41.637 3 41.850 L 42.058 42.013 42.068 42.189 42.278 42.208 42.332 42.443 42.380 42.124 42.038 42.149 43.014 42.901 1 44.142 1 44.227 C 45.240 1 44.849 1 45.438 1 43.856 1 43.525 0 43.269 1 42.443 1 43.172 1 42.889 1 43.843 1 0.

1.0 1.

1.0 1.0 1.C 1.0 1.0 1.0 0.0 1.0 1.0( 1.0( 1.0( 1. OC 1.0 1.00 1.00 0.00 0.00 L. 00 1.00 .00 0.00 L.00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 00 0.00 00 10.94 00 9.38 )0 9.44 0 4.02 0 2.00 0 12.55 0 15.13 0 10.58 0 0.00 0 12.39 0 13.28 0 16.07 0 18.63 29.56 0.00 29.74 S34.84 0.00 0.00 24.22 0.00 0.00 15.27 16.77 13.48 0.00 12.25 9.23 8.04 6.52 0.00 2.00 2.00 2.87 4.49 5.98 26.026 40.031 1.00 12.04

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FEBP

FKBP

FKBP

FKBP

FKBP

FEKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

r r r 4 L.921 1.00 2.00 -46- ATCM 584 CD1 TRP 59 -4.093 22.857 44.252 1.00 2.00 FKBP ATOM 585 NE1 TRP 59 -5.327 22.413 44.659 1.00 4.48 FKBP ATCM 586 HE1 TRP 59 -5.830 22.768 45.422 0.00 0.00 FKBP ATM 587 CZ2 TRP 59 -6.897 20.615 43.859 1.00 7.28 FKBP ATOM 588 CZ3 TRP 59 -6.043 19.433 41.939 1.00 4.10 FKBP ATCM 589 CH2 TRP 59 -7.033 19.648 42.900 1.00 2.01 FKBP ATCM 590 C TRP 59 -0.215 21.196 42.365 1.00 3.20 FKBP ATOM 591 0 TRP 59 -0.186 19.969 42.345 1.00 9.79 FKBP ATM 592 N GLU 60 0.507 21.955 41.550 1.00 3.19 FKBP ATIM 593 H GLU 60 0.323 22.919 41.539 0.00 0.00 FKBP ATCM 594 CA GLU 60 1.484 21.388 40.636 1.00 5.73 FKBP ATCM 595 CB GLU 60 2.142 22.502 39.819 1.00 10.18 FKBP ATCM 596 OG GLU 60 2.585 22.086 38.415 1.00 13.55 FKBP ATOM 597 CD GLU 60 1.463 22.147 37.398 1.00 16.71. FKBP 15 ATCM 598 OE1 GLU 60 1.649 22.793 36.348 1.00 22.45 FKBP ATCM 599 OE2 GLU 60 0.393 21.551 37.640 1.00 19.83 FKBP ATCM 600 C GLU 60 2.538 20.587 41.395 1.00 8.89 FKBP ATOM 601 0 GLU 60 2.703 19.395 41.150 1.00 14.67 FKBP ATIM 602 N GLU 61 3.116 21.189 42.428 1.00 11.93 FKBP 20 ATCM 603 H GUI 61 2.859 22.117 42.606 0.00 0.00 FKBP ATOM 604 CA GLU 61 4.123 20.510 43.249 1.00 15.22 FKBP ATCM 605 CB GLU 61 5.053 21.533 43.916 1.00 18.18 FKBP ATCM 606 OG GLU 61 5.177 22.868 43.171 1.00 28.20 FKBP ATCM 607 CD GLU 61 6.615 23.314 42.926 1.00 31.43 FKBP 25 ATOCM 608 OE1 GU 61 7.478 23.101 43.807 1.00 35.07 FKBP ATCM 609 OE2 GJU 61 6.865 23.933 41.867 1.00 34.62 FKBP ATOM 610 C GLU 61 3.519 19.581 44.315 1.00 14.96 FKBP ATCM 611 0 GLU 61 4.101 18.558 44.663 1.00 21.59 FKBP ATCM 612 N GLY 62 2.355 19.938 44.840 1.00 16.29 FKBP ATCM 613 H GLY 62 1.970 20.809 44.617 0.00 .0.00 FKBP ATOM 614 CA GLY 62 1.687 19.077 45.801 1.00 12.82 FKBP ATCM 615 C GLY 62 1.281 17.734 45.219 1.00 12.55 FKBP ATCM 616 0 GLY 62 1.782 16.697 45.639 1.00 12.58 FKBP ATOM 617 N VAL 63 0.438 17.764 44.190 1.00 12.60 FKBP ATCM 618 H VAL 63 0.172 18.639 43.830 0.00 0.00 FKBP ATM4 619 CA VAL 63 -0.092 16.550 43.570 1.00 12.62 FKBP ATOM 620 CB VAL 63 -1.164 16.899 42.511 1.00 7.73 FKBP ATCM 621 CG1 VAL 63 -1.788 15.628 41.954 1.00 7.25 FKBP ATOM 622 CG2 VAL 63 -2.234 17.780 43.122 1.00 3.26 FKBP -47a a a 1

A

I

A

A

A

A

A

A

A'

A'

A'!

A1

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

ATM 623 C ATM 624 0 ATOM 625 N ATOM 626 H ATCM 627 CA ATOM 628 CB ATCM 629 C ATCM 630 0 ATOM 631 N ATM 632 H ATiM 633 CA ATCM 634 CB kTM 635 .G3 TCOM 636 CD LTCM 637 OE1 LTOM 638 NE2 LTIM 639 HE21 TOM 640 HE22 TOM 641 C TOM 642 0 TOM 643 N ICM 644 H CTM 645 CA ICM 646 CB [OM 647 CG [CM 648 SD [CM 649 CE 1 OM 650 C OM 651 0 0M 652 N 0M 653 H OM 654 CA CM 655 CB CM 656 OG S CM 657 HG S OM 658 C S IM 659 0 S 4M 660 N V M 661 H V

VAL

VAL

ALA

ALA

ALA

ALA

ALA

ALA

GLN

GLN

GEN

GLN

GLN

GUN

GLN

GLN

GLN

GLN

GLN

GLMT

MET

MET

MET

MET

MET

MET

3ER SER

SER

ER

ER

ER

ER

ER

AL

AL

63 63 64 64 64 64 64 64 65 65 65 65 65 65 65 65 65 65 65 65 66 66 66 66 66 66 66 66 66 67 67 67 67 67 67 67 67 68 68 0.99i 0.92' 2.041 2.009! 3.19E 4.203 3.856 4.548 3.657 3.161 4.202 4.359 5.473 5.524 5.543 5.516 5.428 5.596 3.325 3.694 2.094 1.872 1.119 0.286 -0.487 -2.084 -3.186 1.186 1.705 0.832 0.710 0.727 1.649 1.250 1.986 -0.721 -1.556 -1.055 -0.361 7 14.446 3 16.305 17.279 15.570 16.542 14.687 13.726 15.026 15.844 14.233 15.097 16.118 16.996 16.500 18.307 18.638 18.845 13.037 12.226 13.034 13.655 12.044 12.651 13.766 14.610 13.301 10.788 10.831 z 9.643 4 9.638 4 8.409 4 7.317 4 6.897 4 7.045 4 7.926 4 8.364 4 7.115 4 6.855 4 42.958 42.416 42.315 41.905 41.338 42.976 42.656 44.245 44.449 45.353 46.606 46.542 47.782 48.910 47.580 46.667 48.387 45.706 46.553 45.210 44.491 45.646 45.616 46.628 46.495 1 46.911 1 14.774 1 43.660 1 15.346 1 16.319 0 14.565 1 5.134 1 6.427 1 7.038 0 4.518 1 5.309 1 3.523 1 2.883 0.

1.

1.

0.I .1.

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0( 1.00 0.0( 0.0C 0.00 1.0 1.00C l.00 D.00 1.00 1.00 1.00 1.00 L.00 L.00 L.00 ).00 .00 .00 .00 .00 .00 .00 .00 .00 .00 00 18.69 00 15.67 00 0.00 00 14.59 00 13.86 )0 16.87 )0 19.52 )0 16.81 10 0.00 '0 14.57 0 15.78 0 27.03 0 35.69 0 39.86 0 36.82 0 0.00 0 0.00 0 11.92 12.99 8.83 I 0.00 i 9.40 5.56 3.07 12.38 12.15 13.38 16.22 13.44 0.00 11.42 7.60 7.91 0.00 12.45 14.85 12.38 0.00 15.674 42.921 1.00 15.97

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

EKBP

FKBP

FKBP

FKBP

EKBP

FKBP

FKBP

EKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

-48-

C

ATCM

ATOM

ATIM

ATOM

ATCM

ATOM

ATCM

ATCM

ATCM

ATCIM

ATOM

ATCM

ATOM

ATOM

ATM

ATCM

ATOM

AICM

ATOM

ATM

ATOM

ATCM

ATOM

AITCM

ATOM

ATCM

ATCM

ATCM

ATCMI

ATOM

ATOM

ATCM

662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700

CA

CB

OG1 OG2

C

0

N

H

CA

C

0

N

H

CA

CB

OG

CD

OE1 NE2 HE21 HE22

C

O

N

H

CA

CB

OG

CD

NE

HE

CZ

NH1 HH11 HH12 NH2 HH21 HH22

C

VAL

VAL

VAL

VAL

VAL

VAL

GLY

GLY

GLY

GLY

GLY

GIN

GIN

GLN

GIN

GIN

GUN

GIN

GEN

GEN

GIN

GIN

ARG

ARG

ARG

ARG

ARG

ARG

A.RG

ARG

ARG

ARG

ARG

ARG

ARG

ARG

68 68 68 68 68 68 69 69 69 69 69 70 70 70 70 70 70 70 70 70 70 70 70 71 71 71 71 71 71 71 71 71 71 71 71 71 71 71 71 -2.647 -4.130 -2.010 -3.080 -2.603 -4.190 -4.587 -4.872 -4.755 -5.649 -3.694 -3.135 -3.357 -1.927 -1.483 -0.066 0.673 0.310 -0.298 1.237 -4.299 -4.749 -4.711 -4.639 -5.486 -6.753 -7.697 -9.066 -9.812 -9.309 -11.134 -11.708 -11.149 -12.702 -11.888 -11.460 -12.879 -4.650 5.854 5.630 6.489 6.069 5.033 6.630 7.362 6.114 7.061 7.132 7.859 7.917 8.660 9.161 10.064 10.555 10.028 11.586 11.997 11.850 9.830 10.400 10.082 9.362 11.274 10.873 12.010 11.504 12.542 13.289 12.564 13.525 14.237 13.542 11.640 10.906 11.654 12.208 42.067 41.800 40.874 44.532 44.999 44.992 44.469 46.162 47.344 48.185 47.354 46.548 48.515 48.395 49.524 49.331 48.505 50.067 50.702 49.896 48.671 47.691 49.904 50.543 50.246 50.997 51.228 51.639 52.347 52.735 52.475 53.183 53.609 53.282 51.890 51.361 52.011 51.114 1.00 5.10 1.00 5.86 1.00 1.00 1.00 1.00 0.00 2.65 9.51 13.72 7.92 0.00 1.00 8.54 1.00 7.63 1.00 12.92 1.00 3.17 0.00 0.00 1.00 2.00 1.00 2.57 1.00 10.26 1.00 10.61 1.00 18.69 1.00 11.45 0.00 0.00 0.00 0.00 1.00 2.00 1.00 3.88 1.00 5.36 0.00 0.00 1.00 5.53 1.00 2.00 1.00 2.00 1.00 3.25 1.00 8.85 0.00 0.00 1.00 18.29 1.00 25.79 0.00 0.00 0.00 0.00 1.00 23.05 0.00 0.00 0.00 0.00 1.00 3.03 -2.457 6.756 43.314 1.00 10.06

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

C.

-49- ATOM 701 O ARG 71 -4.006 11.764 52.060 1.00 4.39 FKBP

ATM

ATCM

ATCM

ATCM

ATOM

ATCM

ATM

ATCM

ATCM

ATCM

ATOM

ATM

ATM

ATICM

ATIM

ATM

ATM

ATCMI

ATM

ATCM

ATOM

ATCM

ATCM

ATCM4

ATOM

ATOM

ATCM

ATOM

ATCM

ATM

ATM

ATOM

ATOM

ATCM

ATM

702 N ALA 703 H ALA 704 CA ALA 705 CB ALA 706 C ALA 707 0 ALA 708 N LYS 709 H LYS 710 CA LYS 711 CB LYS 712 OG LYS 713 CD LYS 714 CE LYS 715 NZ LYS 716 HZ1 LYS 717 HZ2 LYS 718 HZ3 LYS 719 C LYS 720 0 LYS 721 N LEJ 722 H LEU 723 CA LEU 724 CB LEU 725 CG LEUI 726 CD1 LEU 727 CD2 LEU 728 C LEU 729 0 LEU 730 N THR 731 H T'HR 732 CA THR 733 CB THR 734 OG1 THR 735 HG1 THR 736 CG2 THR 737 C THR 738 0 THR 739 N ILE 72 -4.628 13.489 72 -5.218 13.805 72 -3.725 14.428 72 -2.456 14.557 72 -4.326 15.803 72 -5.376 16.145 73 -3.766 16.490 73 -3.101 16.042 73 -4.121 17.861 73 -4.387 18.018 73 -4.104 19.408 73 -4.807 19.628 73 -4.136 20.729 73 -5.033 21.240 73 -5.238 20.469 73 -5.920 21.583 73 -4.569 22.019 73 -2.943 18.713 73 -1.794 18.396 74 -3.212 19.628 74 -4.064 19.565 74 -2.218 20.582 74 -2.303 20.706 74 -1.440 19.791 74 -1.789 18.330 74 -1.663 20.157 4 74 -2.403 21.962 1 74 -3.449 22.600 5 75 -1.385 22.431 5 75 -0.717 21.784 5 75 -1.383 23.796 5 75 -0.905 23.830 5 75 -1.957 23.327 5 75 -2.720 23.901 5 75 -0.556 25.238 5 75 -0.513 24.654 5 75 0.683 24.416 5 76 -1.180 25.508 5: 50.7' 50.05 51.42 50.63 51.65 51.11 52.63: 53.19; 52.91' .54.41 54.95 56.287 57.086 58.148 58.817 57.728 58.657 52.488 52.814 51.566 51.121 51.082 49.560 18.695 18.947 47.241 51.695 1.515 2.405 2.717 2.913 4.397 5.227 5.117 4.861 2.000 1.846 1.234 74 1.00 2.46 4 0.00 0.00 ;5 1.00 2.00 6 1.00 2.00 4 1.00 4.21 9 1.00 10.57 2 1.00 8.68 9 0.00 0.00 7 1.00 4.13 0 1.00 6.40 6 1.00 13.82 1.00 15.85 1.00 18.32 1.00 22.33 0.00 0.00 0.00 0.00 0.00 0.00 1.00 4.72 1.00 6.20 1.00 6.47' 0.00 0.00 1.00 8.06 1.00 12.85 1.00 11.86 1.00 11.50 1.00 12.57 1.00 8.90 1.00 14.56 1.00 7.32 0.00 0.00 1.00 6.76 1.00 6.87 1.00 2.01 0.00 0.00 1.00 3.73 1.00 6.27 1.00 5.48 1.00 10.43

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

ATM 740 H ILE 76 -2.141 25.633 51.388 0.00 0.00 FKBP ATCM 741 CA ILE 76 -0.542 26.284 50.167 1.00 11.16 FKBP ATCM 742 CB ILE 76 -1.326 26.090 48.830 1.00 6.31 FKBP ATCM 743 OG2 ILE 76 -0.653 26.827 47.719 1.00 9.44 FKBP ATM 744 OG1 ILE 76 -1.388 24.601 48.459 1.00 5.62 FKBP ATOM 745 CD1 ILE 76 -2.630 24.205 47.691 1.00 2.00 FKBP ATCM4 746 C ILE 76 -0.454 27.788 50.522 1.00 12.21 FKBP ATOM 747 0 ILE 76 -1.476 28.460 50.752 1.00 13.89 FKBP ATCM 748 N SER 77 0.768 28.287 50.692 1.00 10.50 FKBP AM 749 H SER 77 1.535 27.692 50.566 0.00 0.00 FKBP ATCM 750 CA SER 77 0.947 29.700 51.009 1.00 11.73 FKBP ATCM 751 CB SER 77 2.354 29.978 51.571 1.00 11.33 FKBP ATIM 752 OG SER 77 3.405 29.669 50.667 1.00 18.57 FKBP AM 753 3 SER 77 4.140 30.103 51.109 0.00 0.00 FKBP 15 ATCM 754 C SER 77 0.681 30.566 49.790 1.00 12.45 FKBP ATCM 755 0 SER 77 0.922 30.149 48.662 1.00 15.48 FKBP ATCM 756 N PRO 78 0.151 31.778 49.998 1.00 14.32 FKBP ATM 757 CD PRO 78 0.192 32.544 51.251 1.00 18.10 FKBP ATCM 758 CA PRO 78 -0.362 32.607 48.906 1.00 14.95 FKBP 20 ATCM 759 CB PRO 78 -0.594 33.957 49.573 1.00 15.74 FKBP ATCM 760 0G PRO 78 0.309 33.944 50.759 1.00 15.85 FKBP ATCM 761 C PRO 78 0.574 32.728 47.710 1.00 15.21 FKBP ATM 762 0 PRO 78 0.109 32.790 46.576 1.00 20.63 FKBP ATM 763 N ASP 79 1.882 32.698 47.956 1.00 13.60 FKBP ATCM 764 H ASP 79 2.162 32.697 48.889 0.00 0.00 FKBP ATCM 765 CA ASP 79 2.877 32.679 46.874 1.00 19.42 FKBP ATCM 766 CB ASP 79 4.305 32.510 47.424 1.00 28.97 FKBP ATCM 767 CG ASP 79 4.599 33.401 48.629 1.00 37.43 FKBP A'CM 768 OD1 ASP 79 5.657 33.195 49.270 1.00 39.71 FKBP ATCM 769 OD2 ASP 79 3.792 34.306 48.939 1.00 45.91 FKBP ATOM 770 C ASP 79 2.616 31.548 45.877 1.00 17.87 FKBP ATM4 771 0 ASP 79 2.547 31.777 44.676 1.00 20.31 FKBP ATCM 772 N TYR 80 2.442 30.335 46.392 1.00 15.45 FKBP ATM 773 H TYR 80 2.347 30.254 47.356 0.00 0.00 FKBP ATCM 774 CA TYR 80 2.142 29.178 45.557 1.00 12.31 FKBP ATCM 775 CB TYR 80 2.611 27.897 46.234 1.00 10.17 FKBP ATCM 776 CG TYR 80 4.082 27.626 46.070 1.00 9.13 FKBP ATCM 777 CD1 TYR 80 5.022 28.600 46.373 1.00 5.08 FKBP ATCM 778 CE1 TYR 80 6.373 28.303 46.419 1.00 6.16 FKBP I. 9 *9 9 a *9*a 9a .9

ATOM

ATOM

ATCM

ATIM

ATM

ATOM

ATOM

ATM

ATM

ATOM

ATCM

ATOM

ATCM

ATOM

ATCM

ATOM

ATCM

ATOM

ATCM

ATOM

ATOM

25 ATOM

ATOM

ATCM

ATOM

ATCM

ATCM

ATOM

ATCM

ATOM

ATM

ATOM

ATM

ATCM

779 CD2 TYR 780 CE2 TYR 781 CZ TYR 782 OH TYR 783 HH TYR 784 C TYR 785 0 TYR 786 N ALA 787 H ALA 788 CA ALA 789 CB ALA 790 C ALA 791 0 ALA 792 N TYR .793 H TYR 794 CA TYR 795 CB TYR 796 0G TYR 797 CD1 TYR 798 CE1 TYR 799 CD2 TYR 800 CE2 TYR 801 CZ TYR 802 OH TYR 803 HH TYR 804 C TYR 805 0 TYR 806 N GLY 807 H GLY 808 CA GLY 809 C GLY 810 0 GLY 811 N ALA 812 H ALA 813 CA ALA 814 CB ALA 815 C ALA 816 0 ALA 817 N THR 80 80 80 80 80 80 80 81 81 81 81 81 81 82 82 82 82 82 82 82 82 82 82 82 82 82 82 83 83 83 83 83 84 84 84 84 84 84 85 4.536 5.889 6.801 8.124 8.729 0.657 0.194 -0.104 0.347 -1.536 -2.362 -1.973 -1.507 -2.886 -3.142 -3.462 -4.982 -5.676 -6.283 -6.918 -5.724 -6.357 -6.946 -7.546 -7.818 -2.869 -3.388 -1.763 -1.475 -0.972 -1.681 -2.708 -1.099 3 -0.306 3 -1.639 3 -0.640 4 -2.965 3 -3.823 4 -3.131 3 26.34 26.03 27.02 26.68: 27.40) 29.03: 27.93 30.115 31.010 30.071 29.899 31.342 31.630 32.106 32.049 33.239 33.249 32.084 31.091 30.013 31.975 30.904 29.930 28.871 28.255 34.591 35.646 34.539 33.662 35.719 36.878 36.728 38.055 38.078 39.270 10.394 .9.637 0.230 9.247 7 45.781 7 45.827 1 46.159 3 46.343 8 46.126 3 45.227 6 44.907 45.344 45.423 45,028 46.312 44.290 43.192 44.874 45.838 44.147 44.324 43.658 44.415 43.804 42.262 41.648 42.425 41.800 42.478 44.552 44.183 45.288 45.571 C 45.566 1 46.233 1 46.910 1 46.055 1 45.480 0 46.628 1 46.455 1 45.982 1 46.618 1 44.726 1 1.1 1.0 1.0 1.( 0.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0( 1.00 1.00 1.0( 1.00 1.00 1.00 1.00 1.00 1.00 1.00 3.00 L.00 L.00 L.00 .00 .00 .00 .00 .00 .00 .00 .00 00 12.62 00 15.72 00 13.97 )0 19.55 )0 0.00 )0 9.68 10 9.28 10 9.06 '0 0.00 0 8.94 0 10.95 0 11.59 0 14.63 0 13.59 0 0.00 0 15.87 0 15.49.

19.64 18.02 4 16.50 1 19.36 i 12.44 12.60 12.06 0.00 15.70 15.54 17.13 0.00 15.64 20.32 23.74 19.06 0.00 15.70 19.93 13.85 14.46 17.88

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

-52- 1 .fTU.r 01 0 TT f' Tr' tu.L oj.o n "iR. O) -Z.4/U J .bb9 44.303 0.00 0.00 FKBP ATCM 819 CA THR 85 -4.308 39.623 43.934 1.00 24.03 FKBP ATCM 820 CB THR 85 -4.036 39.482 42.419 1.00 21.29 FKBP ATOM 821 031 THR 85 -3.482 38.185 42.150 1.00 28.80 FKBP AICM 822 HG1 THR 85 -4.132 37.483 42.316 0.00 0.00 FKBP ATCM 823 CG2 IHR 85 -3.054 40.541 41.956 1.00 16.23 FKBP ATCM 824 C 'HR 85 -5.537 38.787 44.254 1.00 24.35 FKBP

A

T

CM 825 0 THR 85 -6.660 39.189 43.954 1.00 27.70 FKBP ATCM 826 N GLY 86 -5.304 37.579 44.761 1.00 25.09 FKBP ATCM 827 H GLY 86 -4.382 37.292 44.914 0.00 0.00 FKBP ATCM 828 CA GLY 86 -6.388 36.655 45.020 1.00 19.79 FKBP ATCM 829 C GLY 86 -7.151 36.310 43.759 1.00 21.57 FKBP ATM 830 0 GLY 86 -6.589 36.200 42.659 1.00 18.32 FKBP ATCM 831 N HIS 87 -8.454 36.149 43.930 1.00 21.72 FEBP 15 AICM 832 H HIS 87 -8.780 36.318 44.827 0.00 0.00 FKBP ATIM 833 CA HIS 87 -9.355 35.858 42.828 1.00 24.25 FKBP

A

TCM 834 CB HIS 87 -9.432 34.350 42.568 1.00 25.61 FKBP ATOM 835 CG HIS 87 -10.134 33.994 41.292 1.00 29.60 FKBP ATCM 836 CD2 HIS 87 -11.360 33.466 41.064 1.00 27.65 FKBP AIM 837 ND1 HIS 87 -9.564 34.185 40.050 1.00 31.39 FKBP AUTCM 838 HD1 HIS 87 -8.690 34.592 39.843 0.00 0.00 FKBP AITM 839 CE1 HIS 87 -10.405 33.783 39.115 1.00 32.76 FKBP M 840 NE2 HIS 87 -11.503 33.347 39.703 1.00 30.12 FKBP SATCM 841 HE2 HIS 87 -12.329 33.167 39.202 0.00 0.00 FKBP 25 ATOM 842 C HIS 87 -10.727 36.387 43.212 1.00 22.13 FKBP ATCM 843 0 HIS 87 -11.356 35.891 44.152 1.00 27.18 FKBP ATCM 844 N PRO 88 -11.105 37.531 42.639 1.00 19.63 FKBP ATCM 845 CD PRO 88 -10.357 38.290 41.620 1.00 20.36 FKBP ATO4 846 CA PRO 88 -11.989 38.403 43.410 1.00 18.79 FKBP ATCM 847 CB PRO 88 -11.946 39.707 42.626 1.00 18.51 FKBP ATCM 848 CG PRO 88 -10.550 39.713 42.059 1.00 16.30 FKBP ATCM 849 C PRO 88 -13.399 37.848 43.580 1.00 18.22 FKBP ATCM 850 0 PRO 88 -13.974 37.286 42.650 1.00 21.77 FKBP ATCM 851 N GLY 89 -13.851 37.819 44.828 1.00 15.16 FKBP ATC4 852 H GLY 89 -13.303 38.201 45.539 0.00 0.00 FKBP AT'M 853 CA GLY 89 -15.160 37.271 45.120 1.00 12.28 FKBP ATM 854 C GLY 89 -15.116 35.891 45.749 1.00 13.88 FKBP ATCM 855 0 GLY 89 -16.142 35.385 46.211 1.00 13.05 FKBP ATOM 856 N ILE 90 -13.932 35.289 45.812 1.00 12.11 FKBP -53p p *c p p p

C

p p 2 2 15 1

A

20 A

A

A

A

A

A'

A

A

A'I

AT

AT

AT']

Al

AT

AT

A

AT

AT

AT

AT

AICM 857 H ILE 90 -13.164 ATCM 858 CA ILE 90 -13.831 ATM 859 CB ILE 90 -13.950 ATCM 860 CG2 ILE 90 -13.063 ATCM 861 031 ILE 90 -13.590 ATIM 862 CD1 ILE 90 -14.036 ATOM 863 C ILE 90 -12.577 ATCM 864 0 ILE 90 -12.663 AlCM 865 N ILE 91 -11.416 AICM 866 H ILE 91 -11.413 AT0M4 867 CA ILE 91 -10.150 ATOM 868 CB ILE 91 -9.091 AT1M 869 CG2 ILE 91 -7.873 ATaM 870 O31 ILE 91 -9.681 ATCM 871 CD1 ILE 91 -10.163 T1v 872 C ILE 91 -9.584 LTUM 873 0 ILE 91 -9.285 MTCM 874 N PRO 92 -9.520 LTCM 875 CD PRO 92 -9.964 TCM 876 CA PRO 92 -9.007 TCM 877 CB PRO 92 -9.421 TM 878 CG PRO 92 -9.477 TOM 879 C PRO 92 -7.492 TIM 880 0 PRO 92 -6.815 10M 881 N PRO 93 -6.966 TOM 882 CD PRO 93 -7.700 ICM 883 CA PRO 93 -5.518 3 [TO 884 CB PRO 93 -5.380 4 I[M 885 G0 PRO 93 -6.629 4 [CM 886 C PRO 93 -4.743 3 .CM 887 0 PRO 93 -5.160 3 CM 888 N HIS 94 -3.609 3 CM 889 H HIS 94 -3.476 3 0M 890 CA HIS 94 -2.701 3 IM 891 CB HIS 94 -2.366 3 CM 892 CG HIS 94 -1.762 3.

CM 893 CD2 HIS 94 -2.313 4 CM 894 ND1 HIS 94 -0.455 3! IM 895 HD1 HIS 94 0.241 3f 35.74 33.92 32.87 .33.25 31.47 30.36 33.67 33.13, 34.01 34.38( 33.91! 33.08! 32.881 31.762 30.821 35.324 36.025 35.797 35.110 37.143 37.381 36.019 37.264 36.290 38.493 39.762 38.704 t0.217 10.717 '7.999 7.971 7.424 7.286 6.830 7.855 9.103 0.308 9.165 8.484 12 45.410 8 46.328 '5 45.177 2 44.007 8 45.688 1 44.764 0 47.150 4 48.247 3 46.600 0 45.696 47.328 46.559 47.428 46.041 47.084 47.520 46.539 48.781 50.011 49.062 50.514 51.107 48.855 48.516 48.923 48.785 48.833 48.941 48.308 49.933 1 51.090 1 49.563 1 48.598 0 50.538 1 51.608 1 51.061 1 50.781 1 50.621 1 50.761 0 0.00 1.00 1.00 1.00 1.1 1.1 1.0 1.0 1.0 O.0 1.0 1.0 1.0 1.0 1.0 1.0( 1.0( 1.0O 1.01 1.00 1.00 1. 00 1.00 1.00 1.00 1.00 1.00 1.00 L.00 '.00 .00 .00 .00 .00 .00 .00 .00 00 28.28 00 34.25 00 14.47 )0 15.69 )0 12.99 )0 0.00 >0 9.92 )0 6.38 '0 2.00 0 4.55 0 3.68 0 15.34 0 13.98 0 17.29 0 14.17 0 12.40 10.67 11.96 1 14.30 1 17.48 15.65 18.15 16.50 17.10 22.16 16.97 20.11 15.46 0.00 14.40 12.10 15.95 16.10 16.58 0.00 0.00 17.75 23.54 24.28

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

EKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

pp..

pp p p pp -54-

CO

C.

C C C

ATOM

ATCM

ATOM

ATOMM

ATOM

ATCM

ATM

ATOM

ATOM

ATIM

ATM

ATCM

ATCM

ATCM

15 ATUM

ATCM

ATCM

ATOM

ATCM

ATM

ATM

ATOM

ATCM

25 ATM

ATOM

ATCM

ATOM

ATOM

ATOM

ATOME

ATCM

ATOM

ATCM

ATCM

ATOM

ATOM

896 CE1 HIS 897 NE2 HIS 898 HE2 HIS 899 C HIS 900 0 HIS 901 N ALA 902 H ALA 903 CA ALA 904 CB ALA 905 C ALA 906 0 ALA 907 N THR 908 H THR 909 CA THR 910 CB THR 911 OG1 THR 912 G31 THR 913 CG2 THR 914 C T'HR 915 0 THR 916 N LEU 917 H LEU 918 CA LEU 919 CB LEU 920 OG LEU 921 CD1 LEU 922 CD2 LEU 923 C LEU 924 0 LEU 925 N VAL 926 H VAL 927 CA VAL 928 CB VAL 929 CG1 VAL 930 CG2 VAL 931 C VAL 932 0 VAL 933 N PHE 934 H PHE 94 94 94 94 94 95 95 95 95 95 95 96 96 96 96 96 96 96 96 96 97 97 97 97 97 97 97 97 97 98 98 98 98 98 98 98 98 99 99 -1.342 41.063 -1.470 41.979 -3.176 35.531 -2.380 .34.843 -4.403 35.112 -4.911 35.568 -4.982 33.954 -6.365 33.676 -4.132 32.683 -3.691 32.260 -3.801 32.165 -3.847 32.758 -3.319 30.797 -2.740 30.568 -1.655 31.480 -1.236 31.644 -2.240 29.139 -4.501 29.852 -5.569 30.025 -4.349 28.937 -3.495 28.902 -5.406 27.976 -5.672 27.930 -5.948 29.193 -5.831 28.841 -7.326 29.758 -5.083 26.557 -3.926 26.123 -6.121 25.814 -7.012 26.221 -5.968 24.407 -6.461 24.079 -6.144 22.638 -5.824 25.011 -6.801 23.602 -8.012 23.836 -6.166 22.622 -5.202 22.540 50.171 49.833 51.202 51.836 50.915 50.215 51.576 51.026 51.516 50.456 52.691 53.468 52.831 54.254 54.472 53.620 54.430 52.600 53.212 51.642 51.157 51.332 49.826 49.011 47.534 49.318 51.814 51.815 52.167 52.183 52.476 53.900 54.230 54.917 1.00 21.63 0.00 0.00 1.00 13.30 1.00 16.61 1.00 6.56 0.00 0.00 1.00 7.81 1.00 2.72 1.00 10.01 1.00 10.42 1.00 12.98 0.00 0.00 1.00 12.92 1.00 9.93 1.00 11.98 0.00 0.00 1.00 3.68 1.00 14.35 1.00 14.86 -0.230 40.351 50.086 1.00 20.16

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

FKBP

e

C

Ce..

CC C Ce

CC

1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 8.43 0.00 3.80 3.61 6.56 2.62 6.52 5.71 7.74 2.33 0.00 3.09 4.96 2.00 2.00 7.78 8.13 7.58 0.00 51.491- 1.00 51.346 1.00 50.853 1.00 50.970 0.00 00 0 S* *0 0 *0 0 o

ATCM

ATCM

ATM

ATOM

ATM

ATOM

ATCM

ATOM

ATCM

ATCM

ATOM

ATOM

ATM

ATOM

15 ATOM

ATICM

ATM

ATM

20 AICM

ATCM

ATCM

ATOM

ATCM

ATCM

ATM

ATCM

ATOM

ATOM

ATOM

ATOM

ATCM

ATM

ATCM

ATOM

ATOM

ATM

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TRP

TRP

:S 2024 :S 2024 U 2025 U 2025 U 2025 U 2025 L 2025 J 2025 U 2025 U 2025 J 2025 J 2025 P 2026 2026 2026 2026 2026 2026 2026 2026 2026 2027 2027 2027 2027 2027 2027 2027 2027 2027 2027 2027 2027 2027 2027 2027 2027 2028 -13.728 23.558 17.158 1.00 19.84 -13.541 24.012 18.280 1.00 22.62 -12.963 23.906 16.127 1.00 20.21 -13.279 23.712 15.223 0.00 0.00 -11.732 24.686 16.318 1.00 20.43 -10.969 24.846 14.994 1.00 27.02 -10.961 23.614 14.089 1.00 41.60 -10.550 23.937 12.652 1.00 47.27 -9.330 23.903 12.369 1.00 54.42 -11.440 24.219 11.810 1.00 37.45 -12.037 26.074 16.875 1.00 17.30 -11.268 26.641 17.651 1.00 15.80 -13.159 26.625 16.444 1.00 15.93 -13.715 26.119 15.820 0.00 0.00 -13.552 27.971 16.816 1.00 18.01 -14.806 28.354 16.021 1.00 21.46 -15.619 29.490 16.603 1.00 28.72 -16.931 30.032 15.505 1.00 34.40 -15.938 30.642 14.095 1.00 36.70 -13.805 28.060 18.325 1.00 18.72 -13.257 28.927 19.012 1.00 18.88 -14.553 27.092 18.845 1.00 18.28 -14.929 26.414 18.243 0.00 0.00 -14.890 27.047 20.263 1.00 16.52 -16.087 26.129 20.481 1.00 14.68 -17.381 26.861 20.453 1.00 16.26 -17.870 27.760 21.450 1.00 16.49 -19.120 28.239 21.003 1.00 15.26 -17.373 28.214 22.681 1.00 18.70 -18.322 26.831 19.466 1.00 16.17 -19.370 27.656 19.789 1.00 13.89 -20.150 27.816 19.215 0.00 0.00 -19.886 29.142 21.745 1.00 17.88 -18.133 29.114 23.421 1.00 17.25 -19.376 29.565 22.950 1.00 21.47 -13.736 26.609 21.159 1.00 15.61 -13.561 27.129 22.254 1.00 18.72 -12.906 25.702 20.665 1.00 11.04 -13.152 25.290 19.807 0.00 0.00

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

HIS 2028 ATOM 1208 CA HIS 2028 -11.735 25.275 21.412 1.00 10.15 FRAP

ATCM

ATOM

ATCM

ATOM

ATCM

ATCIM

ATOM

ATCM

ATCM

ATCM

ATOM

ATOM

ATCM

ATOM

ATOM

ATCM

ATCM

ATOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATOM

ATCM

ATCM

ATCOM

ATOM

ATOM

ATOM

ATCM

1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 CB HIS OG HIS CD2 HIS ND1 HIS HD1 HIS CE1 HIS NE2 HIS HE2 HIS C HIS O HIS N GLU H GLU CA GLU CB GLU CG GLU CD GLU OE1 GLU OE2 GLU C GLU O GLU N GLY H GLY CA GLY C GLY 0 GLY N LEU H LEU CA LEU CB LEU CG LEU CD1 LEUJ CD2 LEU C LEU O LEU N GLU H GLU CA GLU CB GLU 2028 2028 2028 2028 2028 2028 2028 2028 2028 2028 2029 2029 2029 2029 2029 2029 2029 2029 2029 2029 2030 2030 2030 2030 2030 2031 2031 2031 2031 2031 2031 2031 2031 2031 2032 2032 2032 2032 -10.920 -9.821 -9.786 -8.575 -8.284 -7.814 -8.527 -8.221 -10.827 -10.401 -10.360 -10.688 -9.433 -8.601 -9.401 -8.554 -8.624 -7.828 -10.133 -9.533 -11.433 -11.843 -12.214 -12.307 -11.837 -12.767 -13.130 -12.805 -13.382 -14.869 -15.347 -15.656 -11.441 -11.337 -10.386 -10.522 -9.068 -8.028 24.282 23.642 22.484 24.215 25.084 23.433 22.377 21.579 26.424 26.519 27.167 27.017 28.257 28.592 28.822 28.678 29.570 27.664 29.508 30.277 29.634 29.093 30.696 30.538 31.390 29.368 28.749 29.012 27.612 27.475 26.079 28.530 29.088 29.538 28.657 28.216 28.756 27.986 20.604 21.389 22.091 21.529 21.180 22.276 22.629 23.119 21.805 22.941 20.817 19.900 21.093 19.843 18.565 17.307 16.429 17.191 21.642 22.392 21.380 20.670 21.997 23.504 24.257 23.932 23.264 25.341 25.511 25.192 25.568 25.936 26.024 27.168 25.348 24.483 25.957 1.00 9.23 1.00 10.39 1.00 8.51 1.00 13.26 0.00 0.00 1.00 15.69 1.00 18.29 0.00 0.00 1.00 10.27 1.00 10.19 1.00 19.72 0.00 0.00 1.00 27.56 1.00 34.06 1.00 44.39 1.00 50.63 1.00 54.55 1.00 51.32 1.00 27.45 1.00 29.68 1.00 25.66 0.00 0.00 1.00 21.35 1.00 16.02 1.00 17.01 1.00 11.25 0.00 0.00 1.00 6.54 1.00 2.00 1.00 2.25 1.00 2.00 1.00 2.00 1.00 10.09 1.00 16.95 1.00 8.34 0.00 0.00 1.00 12.37

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP 25.146 -1.00 16.26

ATOM

ATOM

ATCM

ATOM

ATCM

ATCM

ATCM

ATCM

ATOM

ATOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

A

T

M

ATOM

ATOM

ATaMx

ATCM

AICM

ATOM

ATCMI

ATCM

ATCM

ATCM

ATCM

AT

ATICM

ATCM

ATOM

ATOM

ATOM

ATOI

A TOI A'IU'c

AIM

A IO1 124' 1241 124 125( 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 127.6 1277 1278 1279 1280 1281 1282 1283 1284 1285 7 OG GLU 8 CD GLU SOE1 GLU 0 OE2 GLU SC GLU O 0 GLU N GLU SH GLU CA GLU CB GU OG GLU CD GLU OE1 GLU OE2 GLU C GUJ C GLU O GLUJ N ALA H ALA CA ALA CB ALA C ALA O ALA N SER H SER CA SER CB SER OG SER HG SER C SER 0 SER N ARG H ARG CA ARG 2 CB ARG 2 CG ARG 2 CD ARG 2 NE ARG 2 HE ARG 2 CZ ARG 2 2032 2032 2032 2032 2032 2032 2033 2033 2033 2033 2033 2033 2033 2033 2033 2033 2034 2034 2034 2034 2034 2034 2035 2035 2035 2035 2035 2035 2035 2035 2036 2036 2036 !036 !036 036 036 ;036 036 -5.79: -4.61' -6.24: -8.62! -8.26- -8.83- -9.243 -8.462 -8.631 -7.834 -8.155 -7.793 -8.759 -9.308 -8.808 -10.600 -10.945 -11.509 -12.920 -11.101 -10.907 -10.811 -10.871 -10.482 -10.357 -9.012 -8.700 -9.201 -9.171 -8.195 -8.314 -6.934 -5.959 -4.695 -4.229 -3.637 -2.897 -4.055 2 26.772 7 27.092 L 25.611 9 30.210 30.588 31.053 30.710 32.473 33.140 34.437 35.152 36.346 34.530 33.226 34.068 32.933 32.334 33.572 33.101 33.257 34.157 31.988 31.330 31.543 30.016 29.595 29.696 32.193 32.734 32.265 31.862 32.909 32.792 33.631 34.185 35.515 35.626 2 36.595 2 25.235 1.00 30.03 24.948 1.00 31.98 25.078 1.00 32.01 26.154 1.00 12.81 27.261 1.00 21.81 25.147 1.00 11.47 24.323 0.00 0.00 25.225 1.00 12.69 23.854 1.00 19.44 23.650 1.00 30.82 22.319 1.00 42.12 22.186 1.00 44.44 21.408 1.00 39.63 26.254 1.00 10.31 26.994 1.00 6.92 26.275 1.00 6.18 25.587 0.00 0.00 27.205 1.00 2.76 26.943 1.00 2.50 28.641 1.00 6.07 29.453 1.00 11.33 28.903 1.00 8.47 28.175 0.00 0.00 30.250 1.00 4.56 30.294 1.00 2.00 30.200 1.00 7.26 29.288 0.00 0.00 30.749 1.00 5.40 31.846 1.00 11.51 29.886 1.00 3.96 28.998 0.00 0.00 30.233 1.00 6.68 29.065 1.00 7.24 29.210 1.00 17.54 27.860 1.00 17.93 27.997 1.00 18.57 i8.628 0.00 0.00 !7.344 1.00 20.32 -6.692 27.831 25.861 1.00 23.62

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

-64-

ATCM

ATM

ATCM

A'TM

ATCM

ATCM

ATCM

A'U1A

ATCM

ATOUM

ATOM

ATCM

ATCM

ATCM

A'IuiM

ATOM

ATOM

ATfM

ATOM

ATOM

ATCM

AqIM 25 ATCOM

ATCM

ATCM

ATM

ATCM

ATOM

ATCM

ATOM

ATCM

ATCM

ATOM

ATOM

ATCM

1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 C ARG 2036 O ARG 2036 N LEU 2037 H LEU 2037 CA LEU 2037 CB LEU 2037 CG LEU 2037 CD1 LEU 2037 CD2 LEU 2037 C LEU 2037 0 LEU 2037 N TYR 2038 H TYR 2038 CA TYR 2038 CB TYR 2038 CG TYR 2038 CD1 TYR 2038 CE1 TYR 2038 CD2 TYR 2038 CE2 TYR 2038 CZ TYR 2038 CH TYR 2038 HH TYR 2038 C TYR 2038 0 TYR 2038 N PHE 2039 H PHE 2039 CA PHE 2039 CB PHE 2039 CG PHE 2039 CD1 PHE 2039 CD2 PHE 2039 CE1 PHE 2039 1286 NH1 ARG 2036 1287 HH11 ARG 2036 1288 HH12 ARG 2036 1289 NH2 ARG 2036 1290 HH21 ARG 2036 1291 HH22 ARG 2036 -3.456 -2.689 -3.766 -5.080 -5.564 -5.391 -7.110 -6.463 -8.041 -8.541 -8.309 -9.163 -8.302 -9.130 -7.624 -9.004 -8.626 -10.020 -10.327 -10.693 -12.006 -12.761 -12.942 -13.555 -13.230 -13.850 -14.006 -14.596 -15.321 -9.811 -9.408 -9.481 -9.764 -8.717 -8.665 -9.988 -10.540 -10.745 -11.828 37.762 37.827 38.572 36.518 35.653 37.341 34.382 34.872 35.057 34.590 36.466 37.034 37.375 37.388 38.713 36.692 37.583 35.886 35.266 35.930 35.138 35.090 36.239 36.181 33.880 33.810 34.962 34.893 34.267 35.403 36.164 34.113 33.595 33.455 31.950 31.281 31.147 30.902 30.656 27.540 1.00 24.32 28.180 27.045 26.505 26.375 26.030 30.624 31.548 29.964 29.261 30.214 29.084 27.873 26.613 28.110 31.543 32.295 31.832 31.130 33.132 33.071 34.375 35.143 36.391 34.884 36.131 36.880 38.123 38.078 34.277 35.158 34.235 33.452 35.299 35.054 35.235 36.510 34.131 36.680 0.00 0.00 0.00 0.00 1.00 20.76 0.00 0.00 0.00 0.00 1.00 9.31 1.00 12.91 1.00 10.78 0.00 0.00 1.00 8.83 1.00 9.75 1.00 8.95 1.00 11.32 1.00 7.83 1.00 12.66 1.00 17.85 1.00 11.90 0.00 0.00 1.00 11.68 1.00 9.29 1.00 12.17 1.00 10.58 1.00 17.63 1.00 17.46 1.00 17.47 1.00 18.99 1.00 22.39 0.00 0.00 1.00 13.86 1.00 17.65 1.00 13.85 0.00 0.00 1.00 10.83 1.00 2.58 1.00 6.64 1.00 4.84 1.00 2.79 1.00 5.26

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

ATCM 1325 CE2 PHE 2039 -12.039 30.408 4 4.

ATOM

ATCM

ATOM

ATCM

ATOM

ATM

ATM

ATCM

AT]M

ATCIM

ATCM

ATIM

ATM

ATCM

ATCM

ATCM

ATCM

ATOM

ATOM

ATCM

ATOM

ATCM

ATOM

25 ATM

ATOM

ATOM

ATIM

ATM

ATM

ATOM

ATCM

ATCM

ATCM

ATCM

ATOM

ATCM

ATCM

ATCM

1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 SCZ PHE C PHE O PHE N GLY H GLY CA GLY C GLY O GLY N GLU H GLU CA GUJ CB GLU CG GLU CD GLU OE1 GLU OE2 GLU C GLU O GLU N ARG H ARG CA ARG CB ARG CG ARG CD ARG NE ARG HE ARG CZ ARG NH1 ARG 2039 2039 2039 2040 2040 2040 2040 2040 2041 2041 2041 2041 2041 2041 2041 2041 2041 2041 2042 2042 2042 2042 2042 2042 2042 2042 2042 2042 2042 2042 2042 2042 i042 !042 i042 :043 043 043 -12.58 -7.30, -6.86: -6.61! -7.06( -5.221 -4.954 -3.957 -5.815 -6.555 -5.590 -5.476 -5.030 -3.792 -3.772 -2.844 -6.689 -6.754 -7.626 -7.540 -8.785 -8.389 -8.704 -7.650 -6.318 -6.148 -5.273 -4.097 -4.011 -3.309 -5.398 -6.289 -4.609 -9.485 -10.031 -9.560 -9.152 -10.219 1 30.287 6 33.980 1 34.248 9 34.155 0 34.013 L 34.544 36.026 36.384 36.881 36.502 38.328 38.867 37.856 38.302 39.459 37.489 39.108 40.330 38.392 37.419 39.011 39.691 38.869 37.815 38.396 39.074 38.026 38.606 39.312 38.359 37.089 4 36.673 4 36.857 4 40.015 3 41.009 3 39.689 3 38.845 3 40.545 3 34.292 1.00 2.18 35.563 1.00 4.94 35.460 1.00 14.37 36.579 1.00 15.23 34.336 1.00 17.70 33.471 0.00 0.00 34.369 1.00 19.07 34.561 1.00 19.43 35.180 1.00 24.65 34.012 1.00 17.18 33.494 0.00 0.00 34.019 1.00 16.74 32.589 1.00 21.26 31.544 1.00 34.57 30.785 1.00 39.88 30.303 1.00 41.61 30.664 1.00 43.16 34.733 1.00 16.00 34.629 1.00 16.19 35.340 1.00 16.54 35.364 0.00 0.00 35.974 1.00 17.30 37.283 1.00 21.74 38.515 1.00 29.43 38.736 1.00 31.60 38.627 1.00 34.93 37.940 0.00 0.00 39.358 1.00 41.93 39.146 1.00 43.89 38.444 0.00 0.00 39.710 0.00 0.00 10.296 1.00 42.95 10.485 0.00 0.00 10.865 0.00 0.00 35.074 1.00 15.46 5.550 1.00 17.81 3.789 1.00 13.57 3.525 0.00 0.00 2.805 1.00 12.63

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

4144 444444 1354 HH11 ARG 1355 HH12 ARG 1356 NH2 ARG 1357 HH21 ARG 1358 HH22 ARG 1359 C ARG 1360 0 ARG 1361 N ASN 1362 H ASN 1363 CA ASN 2 2 2 2 2 2 -66- ATCM4 ATCm

ATOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATOM

ATCM

ATCM

ATCM

ATM

ATCM

ATIM

ATCM

ATCM

ATCM

ATIM

ATCM

ATM

ATOM

ATM

ATCM

25 ATCM

ATCM

ATCM

ATCM

ATOM

AICM

ATM

ATCM

ATCM

ATOM

ATCM

AICM

ATM

ATM

ATM

A'0M 1364 CB ASN 1365 G03 ASN 1366 OD1 ASN 1367 ND2 ASN 1368 HD21 ASN 1369 HD22 ASN 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 C ASN 0 ASN N VAL H VAL CA VAL CB VAL 031 VAL CG2 VAL C VAL 0 VAL N LYS H LYS CA LYS CB LYS CG LYS CD LYS CE LYS NZ LYS HZ1 LYS HZ2 LYS HZ3 LYS C LYS 0 LYS N GLY H GLY CA GLY C GLY 0 GLY N MET H MET CA MET CB MET CG MET f 2043 2043 2043 2043 2043 2043 2043 2043 2044 2044 2044 2044 2044 2044 2044 2044 2045 2045 2045 2045 2045 2045 2045 2045 2045 2045 2045 2045 2045 2046 2046 2046 2046 2046 2047 2047 2047 2047 2047 -9.673 -10.778 -8.725 -7.861 -8.951 -11.589 -11.704 -12.622 -12.407 -13.996 -14.942 -16.254 -14.280 -14.599 -15.607 -14.013 -13.326 -14.387 -13.791 -13.868 -12.848 -13. 013 -12.203 -11.194 -12.477 -12.387 -13.912 -14.697 -12.640 -12.091 -12.063 -12.716 -13.079 -12.944 -12.639 -13.555 -13.571 -14.762 -9.322 40.702 31.567 1.00 9.40 41.92E 42.457 42.382 41.929 43.171 39.985 39.254 40.329 40.817 39.930 40.079 39.343 39.541 40.724 40.328 41.873 42.230 42.614 44.027 44.787 45.913 46.763 48.015 47.773 48.555 48.579 41.880 41.616 41.493 41.759 40.767 39.427 39.138 38.632 38.911 37.327 36.520 36.725 S30.734 1.00 13.89 30.805 1.00 13.79 29.941 1.00 19.98 29.933 0.00 0.00 29.415 0.00 0.00 32.399 1.00 11.08 31.410 1.00 15.73 33.164 1.00 7.83 33.986 0.00 0.00 32.841 1.00 8.89 34.049 1.00 4.93 33.783 1.00 2.00 35.300 1.00 6.55 31.680 1.00 12.31 31.111 1.00 16.97 31.366 1.00 15.26 31.961 0.00 0.00 30.158 1.00 18.66 30.205 1.00 20.39 28.894 1.00 27.87 28.846 1.00 36.04 27.592 1.00 39.79 27.646 1.00 42.34 27.696 0.00 0.00 28.491 0.00 0.00 26.791 0.00 0.00 28.890 1.00 15.74 27.982 1.00 15.10 28.885 1.00 13.71 29.647 0.00 0.00 27.768 1.00 11.16 27.486 1.00 11.68 26.350 1.00 12.25 28.522 1.00 14.02 29.412 0.00 0.00 28.317 1.00 12.90 29.625 1.00 9.26 30.521 1.00 6.02

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

-67-

S

S S

ATOM

ATCM

ATCM

AICMU

ATOM

ATCM

ATCM

ATCM

ATCOM

ATOM

ATM

ATCM

ATCM

ACM

ATCM

ATCM

ATCM

ATOM

ATOM

ATCM

25 A'IrM

ATOM

ATOM

ATCM

ATM

ATIM

ATOM

ATCM

ATOM

ATOM

ATOM

ATM

ATCM

ATOM

1403 1404 1405 1406 SD MET CE MET C MET O MET 2047 2047 2047 2047 2 2

J

3 a

I

1 1 1 1 1 1 1.

1.

1.

1407 N PHE 2048 1408 H PHE 2048 1409 CA PHE 2048 1410 CB PHE 2048 1411 0G PHE 2048 1412 CD1 PHE 2048 1413 CD2 PHE 2048 1414 CE1 PHE 2048 1415 CE2 PE 2048 1416 CZ PHE 2048 1417 -C PHE 2048 1418 0 PEE 2048 1419 N GLU 2049 1420 H GLU 2049 1421 CA GUJ 2049 L422 CB GLU 2049 L423 CG GLU 2049 L424 CD GLU 2049 L425 OE1 GLU 2049 L426 OE2 GLU 2049 L427 C GLU 2049 .428 0 GLU 2049 .429 N VAL 2050 .430 H VAL 2050 .431 CA VAL 2050 .432 CB VAL 2050 433 CGI1 VAL 2050 434 CG2 VAL 2050 435 C VAL 2050 436 0 VAL 2050 437 N LEU 2051 438 H LEU 2051 439 CA LEU 2051 440 CB LEU 2051 441 OG LEU 2051 -16.86! -14.954 -15.27E -15.71C -15.41C -16.992 -17.754 -18.356 -18.849 -18.506 -19.481 -19.137 -19.623 -16.785 -17.540 -15.754 -15.274 -15.368 -14.144 -14.432 -13.244 -13.006 -12.598 -15.072 -15.771 -14.120 -13.667 -13.800 -12.318 -11.942 -12.039 -14.693 -14.244 -15.981 -16.263 -16.971 -17.122 -17.216 5 35.461 1 37.413 36.624 38.465 39.078 38.707 39.849 39.479 38.201 40.442 37.901 40.138 38.875 39.054 38.619 39.843 40.244 40.161 41.090 42.512 43.464 44.240 43.492 38.890 38.579 38.096 38.388 36.807 36.446 36.639 35.006 35.680 34.799 35.775 36.655 34.764 34.686 33.329 31.714 1.00 4.80 27.691 1.00 14.99 26.816 1.00 20.34 28.001 1.00 13.61 28.703 0.00 0.00 27.324 1.00 12.00 28.012 1.00 15.37 29.357 1.00 19.64 29.600 1.00 20.36 30.352 1.00 17.04 30.806 1.00 12.14 31.552 1.00 7.86 31.774 1.00 2.66 25.839 1.00 11.47 24.968 1.00 9.57 25.558 1.00 10.97 26.315 0.00 0.00 24.189 1.00 12.08 24.187 1.00 18.49 24.700 1.00 28.61 24.566 1.00 32.92 25.521 1.00 34.23 23.489 1.00 32.94 23.387 1.00 10.88 22.427 1.00 12.08 23.862 1.00 10.17 24.675 0.00 0.00 23.247 1.00 10.01 23.457 1.00 6.62 24.901 1.00 11.08 22.995 1.00 11.04 23.781 1.00 14.92 24.529 1.00 20.63 23.454 1.00 12.19 23.111 0.00 0.00 23.816 1.00 9.54 25.336 1.00 8.37 26.046 1.00 8.86 -15.175 35.189 31.335 1.00 6.46

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

SLSS

*SSS**

S. S S S

S.

-68- ATCM 1442 CD1 LEU 2051 -16.110 32.395 25.592 1.00 5.79 FRAP a.

a

ATOM

ATCM

ATCM

ATOM

ATOM

ATCM

ATM

ATCM

ATIM

ATM4

ATOM

ATOM

ATCM

ATM

ATC4

ATOM

ATCM

A

ATCM

ATCM

ATCM

ATM

25 ATCM

ATM

ATCM

ATM

ATCIM

ATCM

ATCM

ATCM

ATOM

ATIM

ATCM

ATCM

It!4i IlIl A L 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 CD2 LEUJ C LEIJ 0 LEU N GLU H GLU CA GLU CB GLU CG GUJ CD GLU OE1 GLU OE2 GLU C GLU 0 GLU N PRO CD PRO CA PRO CB PRO OG PRO C PRO 0 PRO N LEU H LEU CA LEJ CB LEU OG LEU CD1 LEU CD2 LEU C LEU 0 LEU N HIS H HIS CA HIS CB HIS CG HIS CD2 HIS ND1 HIS HD1 HIS 2051 2051 2051 2052 2052 2052 2052 2052 2052 2052 2052 2052 2052 2053 2053 2053 2053 2053 2053 2053 2054 2054 2054 2054 2054 2054 2054 2054 2054 2055 2055 2055 2055 2055 2055 2055 2055 2055 -17.118 33.550 -18.310 35.117 -19.052 34.237 -18.562 36.413 -17.932 37.078 -19.837 36.897 -19.980 38.399 -21.396 38.835 -21.530 40.343 -22.567 40.884 -20.605 40.987 -20.059 36.587 -21.045 35.948 -19.085 36.922 -18.104 38.004 -18.978 36.374 -17.537 36.674 -17.265 37.981 -19.301 34.882 -20.157 34.520 -18.588 34.021 -17.894 34.386 -18.813 32.574 -17.897 31.859 -16.431 32.303 -15.603 31.503 -15.873 32.146 -20.267 32.247 -20.928 31.510 -20.805 32.908 -20.241 33.532 -22.205 32.716 -22.533 33.366 -22.237 32.495 -21.136 32.399 -23.118 31.542 -24.025 31.364 -22.569 30.891 27.538 23.188 22.752 23.042 23.408 22.525 22.792 23.103 23.220 22.772 23.766 21.044 20.693 20.175 20.386 18.814 18.444 19.139 18.639 17.837 19.362 19.944 19.304 20.296 20.307 21.299 18.921 19.621 18.895 20.632 21.142 20.965 22.310 23.491 24.270 23.952 23.581 24.960 1.00 2.00 1.00 10.79 1.00 14.03 1.00 11.63 0.00 0.00 1.00 13.53 1.00 18.53 1.00 29.17 1.00 34.41 1.00 39.61 1.00 36.83 1.00 9.88 1.00 11.10 1.00 9.83 1.00 7.70 1.00 9.97 1.00 12.18 1.00 11.41 1.00 11.69 1.00 15.54 1.00 12.26 0.00 0.00 1.00 7.01 1.00 2.00 1.00 2.00 1.00 2.00 1.00 12.00 1.00 6.82 1.00 7.84 1.00 4.28 0.00 0.00 1.00 5.58 1.00 5.95 1.00 2.00 1.00 2.00 1.00 2.00 0.00 0.00 1.00 2.00

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

ATC 1480 CE1 HIS

ATM

ATCM

ATCM

ATM

ATCM

ATCM

ATCM

ATCM

ATOM

ATM

ATCM

ATCM4

ATCM

ATM

ATCM

ATOM

ATCM

ATCM

ATCM

ATCM

ATC4

ATM

ATM

ATCM

ATCM

ATCM

ATCM

ATM

ATOM

ATCM

ATCM

AIM

ATCM

ATCM

ATCM

ATCM

ATM

ATM

1481 NE2 HIS 2055 1482 HE2 HIS 2055 1483 C HIS 2055 1484 0 HIS 2055 1485 N ALA 2056 1486 H ALA 2056 1487 CA ALA 2056 1488 CB AIA 2056 1489 C ALA 2056 1490 0 ALA 2056 1491 N MET 2057 1492 H MET 2057 1493 CA MET 2057 1494 CB MET 2057 1495 CG03 MET 2057 1496 SD MET 2057 1497 CE MET 2057 1498 C MET 2057 1499 0 MET 2057 1500 N MET 2058 1501 H MET 2058 1502 CA MET 2058 1503 CB MET 2058 1504 CG MET 2058 1505 SD MET 2058 1506 CE MET 2058 1507 C MET 2058 1508 0 MET 2058 1509 N GU 2059 1510 H GLU 2059 1511 CA GLU 2059 1512 CB GU 2059 1513 CG GLU 2059 1514 CD GLU 2059 1515 OE1 GLU 2059 1516 OE2 GLU 2059 1517 C GLU 2059 1518 0 GLU 2059 -21.362 31.384 25.166 1.00 3.10 -20.608 30.877 25.532 0.00 0.00 -23.118 33.276 19.884 1.00 8.31 -24.215 32.765 19.667 1.00 14.91 -22.644 34.290 19.170 1.00 10.33 -21.767 34.651 19.397 0.00 0.00 -23.442 34.935 18.130 1.00 10.51 -22.729 36.161 17.619 1.00 9.92 -23.731 33.985 16.974 1.00 14.24 -24.885 33.829 16.556 1.00 17.21 -22.680 33.340 16.476 1.00 11.79 -21.792 33.596 16.814 0.00 0.00 -22.810 32.294 15.469 1.00 15.13 -21.452 31.642 15.231 1.00 17.94 -20.692 32.266 14.087 1.00 27.92 -18.979 31.767 14.037 1.00 39.79 -18.164 33.353 14.482 1.00 41.99 -23.842 31.222 15.834 1.00 17.76 -24.808 31.000 15.100 1.00 16.63 -23.679 30.615 17.005 1.00 20.22 -22.898 30.870 17.543 0.00 0.00 -24.617 29.603 17.489 1.00 21.71 -24.359 29.323 18.969 1.00 20.36 -22.991 28.760 19.256 1.00 15.47 -22.714 27.281 18.302 1.00 20.16 -23.353 26.049 19.380 1.00 12.03 -26.074 30.032 17.295 1.00 25.10 -26.865 29.330 16.659 1.00 28.18 -26.375 31.246 17.742 1.00 25.58 -25.654 31.794 18.125 0.00 0.00 -27.725 31.798 17.694 1.00 26.53 -27.759 33.099 18.504 1.00 26.67 -29.007 33.941 18.330 1.00 28.36 -28.701 35.344 17.828 1.00 34.40 -27.515 35.648 17.560 1.00 37.80 -29.653 36.146 17.699 1.00 36.02 -28.224 32.039 16.261 1.00 24.75 -29.425 32.148 16.022 1.00 24.66 -27.303 32.057 15.307 1.00 23.58

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

ATCM 1519 N ARG 2060

ATCM

ATCM

ATCM

ATCM

A'IUfi4

ATCM

ATM

ATCM

I0AIU,4

ATM

ATCM

ATCM

ATCM

ATCM

ATOM

ATCM

ATIM

ATCM

ATM

ATCM

AM

ATCMI

ATM

ATCM

ATCMI

ATIM

ATOM

ATOM

ATOM

ATOM

ATOM

ATCM

ATM

ATCM

AM

ATM

ATM

1520 H ARG 1521 CA ARG 1522 CB ARG 1523 OG ARG 1524 CD ARG 1525 NE ARG 1526 HE ARG 1527 CZ ARG 1528 NHI ARG 1529 HH1 ARG 1530 HH12 ARG 1531 NH2 ARG 1532 HH21 ARG 1533 HH22 ARG 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 C ARG 0 ARG N GLY H GLY CA GLY C GLY O GLY N PRO CD PRO CA PRO CB PRO CG PRO C PRO 0 PRO N GIN H GIN CA GIN CB GUN CG GN CD GIN OE1 GIN NE2 GIN 2060 2060 2060 2060 2060 2060 2060 2060 2060 2060 2060 2060 2060 2060 2060 2060 2061 2061 2061 2061 2061 2062 2062 2062 2062 2062 2062 2062 2063 2063 2063 2063 2063 2063 2063 2063 2063 2063 2063 -26.365 -27.660 -26.547 -26.338 -27.275 -28.381 -28.189 -29.635 -30.590 -30.376 -31.526 -29.933 -29.220 -30.874 -27.992 -28.925 -27.246 -26.500 -27.597 -26.442 -25.500 -26.516 -27.590 -25.740 -26.204 -27.072 -26.051 -27.208 -25.048 -24.240 -25.258 -24.384 -25.131 -24.186 -23.139 -24.556 -25.396 -23.899 -24.930 31.985 32.296 33.091 34.497 35.527 35.927 36.558 35.492 35.982 36.677 35.646 34.533 34.125 34.210 31.021 31.013 29.945 30.030 28.662 27.751 28.198 26.448 25.836 25.433 24.110 24.467 25.463 25.349 25.729 26.065 25.668 26.700 27.922 29.035 28.776 30.280 30.488 30.917 24.278 15.562 0.00 0.00 13.914 1.00 27.89 13.224 1.00 31.68 13.808 1.00 33.63 13.173 1.00 36.15 14.046 1.00 35.55 14.770 0.00 0.00 13.924 1.00 37.00 14.704 1.00 38.84 15.389 0.00 0.00 14.601 0.00 0.00 13.057 1.00 33.57 12.486 0.00 0.00 12.967 0.00 0.00 13.117 1.00 26.90 12.317 1.00 26.30 13.351 1.00 27.44 13.976 0.00 0.00 12.758 1.00 23.84 12.361 1.00 25.08 11.690 1.00 29.79 12.695 1.00 21.10 13.489 1.00 18.97 11.976 1.00 19.45 12.585 1.00 14.25 13.734 1.00 14.98 10.487 1.00 21.95 10.085 1.00 26.38 9.670 1.00 21.33 10.056 0.00 0.00 8.224 1.00 22.88 7.510 1.00 25.75 7.002 1.00 30.23 6.545 1.00 37.47 5.945 1.00 42.91 6.822 1.00 34.40 7.270 0.00 0.00 6.484 0.00 0.00 7.701 1.00 19.53

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

1556 HE21 GIN 1557 HE22 GIN 1558 C GIN -71p.

P P

C

ATOM

ATCM

ATM

ATCM

ATCM

ATOM

ATCM

ATOM

ATOM

ATOM

ATOM

ATCM

ATCM

15 ATCM ATCM4

ATCM

ATOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATOM

ATOM

ATIM ATM

ATCM

ATOM

ATM

ATOM

ATCM

ATOCM

ATCM

ATOM

ATOM

ATOM

155 1561 156: 156 156: 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 9 0 GIN 0 N THR 1 H THR 2 CA THR 3CB THR SOG1 THR SH31 THR OG2 THR C THR 0 THR N LEU H LEU CA LEU CB LEU G LEUJ CD1 LEU CD2 LEU C LEU O LEU N LYS H LYS CA LYS CB LYS OG LYS CD LYS CE LYS NZ LYS HZI LYS HZ2 LYS HZ3 LYS C LYS 0 LYS N GLU H GLU 2 CA GLU 2 CB GLU 2 CG GLU 2 CD GLU 2 OE1 GLU 2 2063 2064 2064 2064 2064 2064 2064 2064 2064 2064 2065 2065 2065 2065 2065 2065 2065 2065 2065 2066 2066 2066 2066 2066 2066 2066 2066 2066 2066 2066 2066 2066 2067 !067 !067 067 :067 ;067 067 -25.78 -23.68 -23.11 -23.22 -21.68.

-21.21 -21.14! -21.128 -23.743 -24.272 -23.483 -23.146 -23.813 -23.667 -24.909 -26.158 -24.658 -22.940 -23.445 -21.649 -21.297 -20.707 -19.297 -18.442 -17.028 -16.122 -16.549 -16.491 -17.527 -15.912 -21.072 -20.704 -21.548 -21.556 -21.998 -22.143 -20.877 -21.032 -22.161 1 23.56 5 23.88 4 24.47' 0 22.59: 3 22.55: 3 22.46! 5 21.52S 3 23.812 1 21.471 21.725 20.231 20.079 19.063 17.808 16.954 17.819 16.129 18.949 18.670 19.264 19.271 19.308 19.636 18.438 18.870 17.672 16.861 17.449 16.533 16.043 20.317 20.148 21.479 21.672 22.508 23.842 24.292 25.619 26.174 8 7.181 1.00 22.10 0 7.897 1.00 16.77 7 8.406 0.00 0.00 3 7.423 1.00 17.61 L 7.414 1.00 18.02 5 8.763 1.00 16.37 8.956 0.00 0.00 6.763 1.00 19.18 8.322 1.00 17.50 9.402 1.00 19.82 7.922 1.00 17.20 7.018 0.00 0.00 8.731 1.00 13.79 7.879 1.00 17.73 7.614 1.00 18.83 7.466 1.00 19.10 6.365 1.00 14.71 9.988 1.00 13.22 11.070 1.00 12.57 9.848 1.00 9.29 8.935 0.00 0.00 10.976 1.00 8.13 10.475 1.00 2.00 10.157 1.00 2.00 9.846 1.00 2.00 9.553 1.00 9.62 8.378 1.00 5.28 7.520 0.00 0.00 8.514 0.00 0.00 8.283 0.00 0.00 12.070 1.00 11.53 13.226 1.00 16.33 11.646 1.00 14.92 10.692 0.00 0.00 12.569 1.00 15.78 11.835 1.00 22.50 11.105 1.00 25.09 10.365 1.00 25.97 10.309 1.00 16.81

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

C

C

C

C C

C.

*5 *0

ATCM

ATCM

ATCM

ATCM

ATCM

ATCOM

ATM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATOM

ATOM

ATOM

ATCM

ATCM

ATOM

25 A'IM

ATOM

ATOM

ATM

ATCM

ATOM

ATO'M

ATC4

ATCM

ATOM

ATM

ATCM

ATCM

ATCM

ATM

1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 OE2 GLU 2067 C GLU 2067 0 GLU 2067 N THR 2068 H THR 2068 CA THR 2068 CB THR 2068 0G1 THR 2068 H1 THR 2068 CG2 THR 2068 C THR 2068 O THR 2068 N SER 2069 H SER 2069 CA SER 2069 CB SER 2069 OG SER 2069 HG SER 2069 C SER 2069 0 SER 2069 N PHE 2070 H PHE 2070 CA PHE 2070 CB PHE 2070 OG PHE 2070 CD1 PHE 2070 CD2 PHE 2070 CE1 PHE 2070 CE2 PHE 2070 CZ PHE 2070 C PHE 2070 0 PHE 2070 N ASN 2071 H ASN 2071 CA ASN 2071 CB ASN 2071 CG ASN 2071 ODI ASN 2071 ND2 ASN 2071 -20.002 -23.336 -23.693 -24.096 -23.847 -25.345 -26.140 -26.656 -25.961 -27.317 -25.120 -25.625 -24.303 -23.872 -24.066 -23.234 -21.951 -21.427 -23.404 -23.865 -22.371 -21.960 -21.786 -20.732 -20.154 -18.861 -20.857 -18.272 -20.283 -18.985 -22.856 -22.752 -23.836 -23.831 -24.876 -25.689 -26.604 -27.805 -26.035 26.108 22.108 22.562 21.300 21.150 20.731 20.025 21.013 21.423 19.239 19.751 19.971 18.724 18.626 17.701 16.555 16.993 16.200 18.243 17.962 19.070 19.083 19.831 20.811 21.726 21.521 22.848 22.419 23.748 23.534 20.601 20.790 21.135 21.076 21.880 22.675 23.677 23.463 24.766 13.173 14.260 12.435 11.501 12.940 11.809 10.912 10.376 12.370 14.100 15.204 13.879 13.000 14.898 14.315 13.917 13.756 16.180 17.295 16.018 15.126 17.132 16.607 17.656 18.130 18.092 19.016 18.980 19.441 17.888 19.082 17.182 16.202 17.851 16.841 17.501 17.602 17.987 1.00 19.42 1.00 22.50 1.00 19.36 0.00 0.00 1.00 18.73 1.00 14.88 1.00 16.48 0.00 0.00 1.00 13.69 1.00 20.11 1.00 24.18 1.00 15.42 0.00 0.00 1.00 11.92 1.00 3.97 1.00 2.00 0.00 0.00 1.00 14.81 1.00 17.69 1.00 12.68 9.844 1.00 26.65 FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

S*4

S

S

SS 4.

*5 0.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 0.00 6.20 5.44 2.00 2.00 2.00 2.00 2.00 2.00 2.60 7.27 2.01 0.00 2.00 7.02 8.30 1.00 11.66 1.00 12.66 ATUM 1637 HD21 ASN 2071 -25.081 24.904 17.878 0.00 0.00 *.eS

S

S.io *5 *5*S 4 ooo.

6 S. SO

O*O*

S

S*

ATM

ATM

ATCM

AITCM

ATM

ATM

A'It1]M

ATM

ATM

A'IM

ATCM

ATCM

A'ItM

ATCM

ATCM

15 ACM4

A'IUYI

A'IU3M

ATJA

AM

AM

ATOM

20 ATCM

ATOM

ATCM

AIM

AM

25 ATC'M

ATM

ATM

ATM

ATCM

AM

AM

AM

AM

ATM

ATM

ATCM

AM

ATCM

A'IM

ATOM

AT'I

A'IcX]M 35 A'IU AT IM

A'IX'M

1641 N GUS 1642 H GiN 1643 CA GUN 1644 CB GN 1645 CG GLN 1646 CD GLN 1647 OE1 GLN 1648 NE2 GN 1649 HE21 GLN 1650.HE22 GLN 1651 C GLN 1652 0 GLN 1653 N ALA 1654 H ALA 1655 CA ALA 1656 CB ALA 1657 C ALA 1658 0 ALA 1659 N TYR 1660 H TYR 1661 CA TYR 1662 CB TYR 1663 OG TYR 1664 CD1 TYR 1665 CE1 TYR 1666 CD2 TYR 1667 CE2 TYR 1668 CZ TYR 1669 OH TYR 1670 HH TYR 1671 C TYR 1672 0 TYR 1673 N GLY 1674 H GLY 1675 CA GLY S2072 2072 2072 2072 2072 2072 2072 2072 2072 2072 2072 2072 2073 2073 2073 2073 2073 2073 2074 2074 2074 2074 2074 2074 2074 2074 2074 2074 2074 2074 2074 2074 2075 2075 2075 1638 HD22 ASN 2071 1639 C ASN 2071 1640 0 ASN 2071 -26.66.

-25.78, -26.251 -25.99 -25.64' -26.803 -27.063 -28.01C -27.941 -27.006 -28.940 -29.659 -28.875 -26.101 -26.693 -24.795 -24.426 -23.940 -22.583 -23.756 -24.013 -23.228 -23.091 -22.791 -21.330 -20.444 -19.990 -19.160 -20.045 -19.205 -18.771 -17.960 -17.868 -23.618 -23.291 -24.714 -24.997 -25.478 25.370 18.41 4 20.959 3 21.328 3 19.747 19.564 L 18.741 17.554 1 17.884 16.881 16.088 16.908 17.557 16.258 18.262 18.224 18.054 18.142 17.625 17.223 18.666 18.383 19.834 19.988 20.842 21.206 20.034 19.839 18.772 19.124 18.050 17.886 16.836 16.773 22.128 23.074 22.153 21.355 23.380 18.641 19.71: 18.14: 17.247 18.845 17.934 16.798 15.665 15.570 14.798 14.919 14.072 20.103 21.178 19.978 19.081 21.077 20.518 22.196 23.369 21.832 20.874 22.796 22.547 22.216 20.918 20.591 23.197 22.882 21.569 21.215 20.266 22.804 23.509 22.063 21.565 21.946 9 0.00 0.00 6 1.00 4.16 L 1.00 10.87 3 1.00 8.02 1 0.00 0.00 S1.00 8.00 S1.00 2.00 S1.00 6.79 S1.00 8.96 1.00 4.92 1.00 6.99 0.00 0.00 0.00 0.00 1.00 12.51 1.00 19.60 1.00 14.16 0.00 0.00 1.00 14.24 1.00 15.34 1.00 15.13 1.00 18.26 1.00 12.69 0.00 0.00 1.00 8.11 1.00 3.13 1.00 8.31 1.00 10.92 1.00 12.41 1.00 11.16 1.00 12.75 1.00 12.54 1.00 21.64 0.00 0.00 1.00 8.66 1.00 9.77 1.00 10.37 0.00 0.00 1.00 12.34

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

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-74-

OGS.

0S* S* S 0 o 000*

SS

S

0o S wOOo

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATOM

ATOM

ATCM

ATCM

ATOM

ATOM

15 ATCM

ATCM

ATM

ATCM

ATOM

ATOM

ATCM

ATM

ATCM

ATCM

25 ATCM

ATOM

ATCM

ATCM

ATM

ATCM

ATM

ATOM

ATCM

ATOM

ATOM

ATrM

ATM

ATCM

ATOM

1676 C GLY 1677 0 GLY 1678 N ARG 1679 H ARG 1680 CA ARG 1681 CB ARG 1682 OG ARG 1683 CD ARG 1684 NE ARG 1685 HE ARG 1686 CZ ARG 1687 NH. ARG 1688 Hl1 ARG 1689 HH12 ARG 1690 NH2 ARG 1691 HH21 ARG 1692 HH22 ARG

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S. Se *5 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711 1712 1713 1714 C ARG O ARG N ASP H ASP CA ASP CB ASP CG ASP OD1 ASP OD2 ASP C ASP O ASP N LEU H LEU CA LEU CB LEU OG LEU CD1 LEUJ CD2 LEUJ C LEU O LEU N MET H MET 2075 2075 2076 2076 2076 2076 2076 2076 2076 2076 2076 2076 2076 2076 2076 2076 2076 2076 2076 2077 2077 2077 2077 2077 2077 2077 2077 2077 2078 2078 2078 2078 2078 2078 2078 2078 2078 2079 2079 -26.13C -26.010 -26.770 -26.782 -27.476 -28.162 -28.703 -29.913 -31.135 -31.060 -32.351 -33.396 -33.274 -34.308 -32.532 -31.750 -33.446 -26.574 -26.861 -25.490 -25.346 -24.526 -23.332 -23.615 -24.726 -22.724 -24.035 -24.126 -23.544 -23.477 -23.064 -22.495 -21.161 -20.928 -20.010 -24.146 -23.847 -25.401 -25.579 23.79E 24.946 22.843 21.950 23.089 21.794 21.826 20.929 21.578 22.233 21.341 22.014 22.698 21.839 20.415 19.895 20.249 23.640 24.680 22.936 22.086 23.394 22.448 21.196 21.096 20.317 24.809 25.669 25.058 24.330 26.386 26.364 25.653 25.574 26.387 27.466 28.626 27.091 26.181 23.246 1.00 17.07 23.660 1.00 24.52 23.921 1.00 19.49 23.516 0.00 0.00 25.187 1.00 16.21 25.651 1.00 17.61 27.072 1.00 25.98 27.228 1.00 33.40 26.754 1.00 44.19 26.029 0.00 0.00 27.241 1.00 50.69 26.769 1.00 53.46 26.051 0.00 0.00 27.144 0.00 0.00 28.180 1.00 51.70 28.525 0.00 0.00 28.551 0.00 0.00 26.305 1.00 11.79 26.885 1.00 11.52 26.604 1.00 8.15 26.144 0.00 0.00 27.594 1.00 6.48 27.637 1.00 5.61 28.425 1.00 10.00 28.999 1.00 9.97 28.479 1.00 12.06 27.331 1.00 8.55 28.201 1.00 13.05 26.123 1.00 6.49 25.469 0.00 0.00 25.752 1.00 4.74 24.333 1.00 3.18 24.084 1.00 2.91 22.593 1.00 2.37 24.764 1.00 2.00 25.862 1.00 4.72 26.118 1.00 2.64 25.651 1.00 7.76 25.326 0.00 0.00

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

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FRAP

FRAP

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FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

ATCM4

ATCM

ATOM

AT'M

ATCM

ATCOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATCM

ATOM

ATCM

ATCM

ATCM

ATM

ATCM

ATCM

ATCM

ATCM

ATOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

AT'CM

ATCM

ATCM

ATOM

ATOM

ATOM

1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727

MET

MET

GU

GLU

GLU

GLU

GLU

GLU

1728-, OE1 GU 1729: OE2 GIIJ 1730 C GLU 1731 0 GLU 1732 N ALA 1733 H ALA 1734 CA ALA 1735 CB ALA 1736 C ALA 1737 0 ALA 1738 N GEN 1739 H GLN 1740 CA GLN 1741 CB GIN 1742 OG GLN 1743 CD GEN 1744 OE1 GLN 1745 NE2 GLN 1746 HE21 GLN 1747 HE2 GIN 1748 C GLN 1749 0 GLN 1750 N GLU 1751 H GLU 1752 CA GLU 1753 CB GLU CA MET CB MET OG MET SD MET CE MET 2079 2079 2079 2079 2079 2079 2079 2080 2080 2080 2080 2080 2080 2080 2080 2080 2080 2081 2081 2081 2081 2081 2081 2082 2082 2082 2082 2082 2082 2082 2082 2082 2082 2082 2082 2083 2083 2083 2083 -26.50' -27.802 -28.99S -29.718 -30.358 -26.686 -26.714 -26.769 -26.733 -26.928 -27.006 -27.581 -27.199 -26.827 -27.253 -25.773 -25.995 -24.555 -24.449 -23.375 -22.163 -23.591 -23.284 -24.253 -24.566 -24.557 -25.085 -25.879 -26.176 -25.360 -27.299 -27.890 -27.467 -25.558 -25.442 -26.551 -26.603 -27.523 -28.680 7 28.02, 1 27.434 28.367 28.677 27.004 28.344 29.505 27.308 26.408 27.477 26.111 26.144 24.914 25.080 23.783 28.284 29.230 27.981 27.180 28.743 28.263 30.233 31.057 30.560 29.833 31.948 32.032 33.280 33.408 33.930 32.846 32.386 32.967 32.584 33.759 31.819 30.892 32.342 31.362 25.850 1.00 14.65 1 25.295 1.00 18.67 25.363 1.00 25.96 23.724 1.00 40.57 23.294 1.00 36.64 27.330 1.00 17.59 27.716 1.00 21.68 28.158 1.00 18.54 27.770 0.00 0.00 29.599 1.00 18.17 30.286 1.00 24.46 31.708 1.00 33.04 32.530 1.00 37.28 33.714 1.00 39.48 31.991 1.00 40.40 30.191 1.00 16.44 30.940 1.00 17.68 29.756 1.00 15.30 29.211 0.00 0.00 30.149 1.00 12.75 29.373 1.00 8.47 29.912 1.00 14.17 30.767 1.00 17.02 28.809 1.00 16.91 28.233 0.00 0.00 28.477 1.00 18.00 27.048 1.00 22.74 26.739 1.00 26.79 25.268 1.00 31.68 24.509 1.00 29.64 24.838 1.00 31.52 25.460 0.00 0.00 23.886 0.00 0.00 29.439 1.00 17.54 29.768 1.00 19.50 29.875 1.00 18.34 29.552 0.00 0.00 30.826 1.00 19.36 31.021 1.00 26.08

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

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FRAP

FRAP

FRAP

FRAP

FRAP

-76-

ATCM

ATM

ATOM

ATOM

ATCM4

ATCM

ATOM

ATCM

ATOM

ATCM

ATOM

ATCM

ATCM

ATCM

AMTM

ATCM

ATOM

ATCM

ATCM

ATOM

ATOM

ATOM

ATCM

ATCM

ATOM

ATCM

ATOM

ATOM

ATOMM

ATM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATOM

A IU 4 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 1769 1770 1771 1772 1773 1774 1775 1776 1777 1778 1779 1780 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 1791 1792 SCG GLU CD GLU OE1 GUJ OE2 GLU C GLU 0 GLU N TRP H TRP CA TRP CB TRP CG TRP CD2 TRP CE2 TRP CE3 TRP CDI TRP NE1 TRP HE1 TRP CZ2 TRP CZ3 TRP CH2 TRP C TRP 0 TRP N CYS H CYS CA CYS CB CYS SG CYS C CYS O CYS N ARG H ARG CA ARG CB ARG CG ARG CD ARG NE ARG HE ARG CZ ARG NH1 ARG 2083 2083 2083 2083 2083 2083 2084 2084 2084 2084 2084 2084 2084 2084 2084 2084 2084 2084 2084 2084 2084 2084 2085 2085 2085 2085 2085 2085 2085 2086 2086 2086 2086 2086 2086 2086 2086 2086 2086 -29.802 -30.388 -30.392 -30.878 -26.863 -27.102 -25.915 -25.769 -25.139 -24.190 -24.879 -25.606 -26.292 -25.765 -25.110 -25.972 -26.397 -27.129 -26.597 -27.272 -24.348 -24.240 -23.760 -23.725 -23.062 -22.329 -20.748 -24.030 -23.718 -25.214 -25.382 -26.250 -27.476 -27.279 -28.160 -29.300 -29.553 -30.003 -31.021 31.897 33.226 34.207 33.280 32.651 33.701 31.817 30.992 32.069 30.914 29.734 29.664 28.433 30.533 28.564 27.781 26.972 28.050 30.156 28.924 33.355 34.076 33.625 32.895 34.894 34.868 34.024 36.070 37.138 35.864 35.014 36.878 36.405 36.429 35.398 35.986 36.906 35.357 35.971 31.915 1.00 40.13 31.428 1.00 46.90 32.209 1.00 48.07 30.279 1.00 52.86 32.166 1.00 13.37 32.747 1.00 17.15 32.563 1.00 6.62 32.047 0.00 0.00 33.761 1.00 3.33 34.037 1.00 5.07 34.575 1.00 6.00 35.801 1.00 10.76 35.807 1.00 14.65 36.887 1.00 9.81 33.924 1.00 10.88 34.646 1.00 17.13 34.309 0.00 0.00 36.853 1.00 14.61 37.923 1.00 11.54 37.899 1.00 16.36 33.677 1.00 4.82 34.665 1.00 10.80 32.514 1.00 7.15 31.856 0.00 0.00 32.274 1.00 7.94 30.935 1.00 2.21 30.993 1.00 14.42 32.284 1.00 11.28 32.813 1.00 13.68 31.718 1.00 10.58 31.259 0.00 0.00 31.749 1.00 11.82 30.970 1.00 16.71 29.458 1.00 22.27 28.768 1.00 36.61 28.060 1.00 45.02 28.280 0.00 0.00 27.118 1.00 49.39 26.523 1.00 48.26

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

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FRAP

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FRAP

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FRAP

FRAP

FRAP

FRAP

-77- ATM 1793 HH1 ARG 2086 -31.246 36.916 26.762 0.00 0.00

AM

ATCIM

ATM

ATCM

ATCM

ATCM

ATCM

ATOM

ATOM

ATCM

ATCM

ATM

ATOM

ATCM

ATM

ATCM

AM

ATCM

ATCM

ATM

ATM

ATOM

ATCM4

ATM

ATM

ATCM

ATCM

ATCM

ATCIM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATCM

ATM

1794 HH12 ARG 1795 NH2 ARG 1796 HH21 ARG 1797 HH22 ARG 1798 C ARG 1799 0 ARG 1800 N LYS 1801 H LYS 1802 CA LYS 1803 CB LYS 1804 OG LYS 1805 CD LYS 1806 CE LYS 1807- NZ LYS 1808 1809 1810 1811 1812 1813 1814 1815 1816 1817 1818 1819 1820 1821 1822 1823 1824 1825 1826 1827 1828 1829 1830 1831 HZ1 LYS HZ2 LYS HZ3 LYS C LYS O LYS N TYR H TYR CA TYR CB TYR COG TYR CD1 TYR CE1 TYR CD2 TYR CE2 TYR CZ TYR OH TYR HH TYR C TYR 0 TYR N MET H MET CA MET CB MET COG MET 2086 2086 2086 2086 2086 2086 2087 2087 2087 2087 2087 2087 2087 2087 2087 2087 2087 2087 2087 2088 2088 2088 2088 2088 2088 2088 2088 2088 2088 2088 2088 2088 2088 2089 2089 2089 2089 2089 -31.53E -29.673 -28.913 -30.218 -26.618 -26.536 -26.792 -26.697 -27.104 -27.217 -28.510 -28.412 -29.760 -29.640 -29.184 -29.051 -30.581 -26.038 -26.356 -24.771 -24.578 -23.693 -22.327 -21.194 -20.780 -19.817 -20.603 -19.631 -19.248 -18.308 -17.982 -23.872 -23.750 -24.238 -24.371 -24.442 -24.813 -23.637 S35.499 34.120 33.645 33.649 37.180 38.325 36.120 35.240 36.240 34.858 34.139 32.700 31.998 30.517 30.284 30.158 30.076 37.041 37.859 36.803 36.057 37.592 37.135 38.013 37.953 38.822 38.967 39.835 39.758 40.621 41.148 39.079 39.921 39.383 38.652 40.757 40.789 40.488 25.809 0.00 0.00 26.747 1.00 49.75 27.190 0.00 0.00 26.053 0.00 0.00 33.193 1.00 11.93 33.629 1.00 14.05 33.976 1.00 14.39 33.583 0.00 0.00 35.401 1.00 11.99 36.040 1.00 12.74 35.778 1.00 13.98 36.270 1.00 17.19 36.220 1.00 26.67 36.341 1.00 33.46 37.245 0.00 0.00 35.561 0.00 0.00 36.301 0.00 0.00 36.144 1.00 9.73 37.000 1.00 12.76 35.821 1.00 7.02 35.209 0.00 0.00 36.407 1.00 12.48 35.892 1.00 9.00 36.386 1.00 11.53 37.712 1.00 13.53 38.205 1.00 13.24 35.553 1.00 9.73 36.032 1.00 8.20 37.364 1.00 14.19 37.881 1.00 21.06 37.148 0.00 0.00 36.109 1.00 15.40 37.000 1.00 21.76 34.870 1.00 14.77 34.223 0.00 0.00 34.446 1.00 13.39 32.962 100 11.91 32.049 1.00 11.63

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

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FRAP

FRAP

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-78- ATCM 1832 SD MET 2089 AT4

ATOM

ATCM

ATIM

A R~

ATM

ATOM

ATM

ATM4

ATCM

ATCM

ATM

ATCM

ATM

ATCM

ATCM

ATM

ATCM

ATCM

ATCM

ATM

ATCM

A'1U4

ATOM

25 ATIM

ATCM

ATOM

ATOM

ATCM

ATM

ATCM

ATCM

ATCM

ATM

A'CM

ATCM

ATOM

ATM

ATCM

1833 CE MET 2089 1834 C MET 2089 1835 0 MET 2089 1836 N LYS 2090 1837 H LYS 2090 1838 CA LYS 2090 1839 CB LYS 2090 1840 OG LYS 2090 1841 CD LYS 2090 1842 CE LYS 2090 1843 NZ LYS 2090 1844 HZ1 LYS 2090 1845 HZ2 LYS 2090 1846 HZ3 LYS 2090 1847 C LYS 2090 1848 0 LYS 2090 1849 N SER 2091 1850 H SER 2091 1851 CA SER 2091 1852 CB SER 2091 1853 OG SER 2091 1854 HG SER 2091 1855 C SER 2091 1856 0 SER 2091 1857 N GLY 2092 1858 H GLY 2092 1859 CA GLY 2092 1860 C GLY 2092 1861 0 GLY 2092 1862 N ASN 2093 1863 H ASN 2093 1864 CA ASN 2093 1865 CB ASN 2093 1866 CG ASN 2093 1867 OD1 ASN 2093 1868 ND2 ASN 2093 1869 HD21 ASN 2093 1870 HD22 ASN 2093 -24.124 -22.62C -25.50C -25.392 -26.475 -26.475 -27.591 -28.886 -29.218 -30.240 -30.140 -30.477 -31.451 -29.826 -30.396 -27.371 -28.022 -26.466 -25.955 -26.302 -26.662 -25.722 -26.010 -24.917 -24.761 -23.903 -24.107 -22.536 -22.009 -20.913 -22.701 -23.481 -22.465 -23.572 -23.884 -25.031 -22.879 -21.955 -23.187 I 41.475 42.669 40.728 39.771 41.322 40.552 39.436 39.892 39.078 39.893 40.248 40.700 39.308 41.420 42.230 40.597 40.047 40.464 39.051 38.108 37.832 40.794 41.071 40.637 40.356 40.883 39.837 39.974 38.704 38.626 37.664 36.626 35.977 35.976 35.381 35.362 35.027 35.272 35.511 35.775 35.559 36.506 36.209 37.207 38.254 39.545 40.756 40.672 40.829 41.612 38.023 38.695 38.554 37.923 40.008 40.465 39.982 39.096 40.537 41.724 39.691 38.784 40.117 41.083 41.622 41.127 40.514 42.114 42.021 43.353 43.798 43.975 43.700 44.822 40.080 30.36! 39.331 29.75S S1.00 13.84 S1.00 2.00 1.00 11.34 S1.00 16.85 S1.00 13.58 0.00 0.00 1.00 17.01 1.00 17.48 1.00 30.54 1.00 39.03 1.00 40.52 1.00 38.43 0.00 0.00 0.00 0.00 0.00 0.00 1.00 18.18 1.00 16.27 1.00 18.19 0.00 0.00 1.00 16.08 1.00 15.61 1.00 18.00 0.00 0.00 1.00 14.61 1.00 16.95 1.00 10.93 0.00 0.00 1.00 12.47 1.00 13.42 1.00 11.96 1.00 14.42 0.00 0.00 1.00 15.72 1.00 15.84 1.00 16.87 1.00 23.23 1.00 9.23 0.00 0.00 0.00 0.00

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

-79- ATCM 1871 C ASN 2093

ATCM

ATCM

ATM

ATCM

ATCM

ATOM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATCM

ATM

ATCM

ATOM

ATCM

ATM

ATCM

ATM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATM

ATCM

ATM

ATCM

ATM

ATCM

ATCM

ATCM

ATCM

ATOM

ATCM

1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909 S0 ASN N VAL H VAL CA VAL CB VAL CG1 VAL CG2 VAL C VAL 0 VAL N LYS H LYS CA LYS CB LYS CG LYS CD LYS CE LYS NZ LYS HZ1 LYS HZ2 LYS HZ3 LYS C LYS 0 LYS N ASP H ASP CA ASP CB ASP CG ASP OD1 ASP OD2 ASP C ASP 0 ASP N LEU H LEU CA LEU CB LEU CG LEU CD1 LEU CD2 LEU 2093 2094 2094 2094 2094 2094 2094 2094 2094 2095 2095 2095 2095 2095 2095 2095 2095 2095 2095 2095 2095 2095 2096 2096 2096 2096 2096 2096 2096 2096 2096 2097 2097 2097 2097 2097 2097 2097 -21.11: -20.59S -20.652 -21.10E -19.386 -18.134 -17.885 -16.911 -19.390 -18.534 -20.415 -20.859 -20.615 -21.166 -20.193 -18.736 -17.771 -17.512 -17.131 -18.395 -16.816 -21.515 -21;.621 -22.168 -22.269 -22.850 -23.799 -24.973 -25.630 -25.238 -21.837 -21.903 -20.816 -20.814 -19.723 -18.701 -18.252 -19.451 -17.543 2 36.959 S36.466 1 36.711 S37.113 S36.003 S36.636 36.035 36.422 34.508 33.779 34.016 34.593 32.570 32.125 31.221 31.682 30.625 29.527 29.930 29.025 28.873 32.087 30.893 33.029 33.893 32.737 33.868 33.956 32.925 35.055 32.538 31.563 33.386 34.129 33.244 34.357 35.073 35.345 36.371 42.015 1.00 20.92 43.015 1.00 28.74 40.797 1.00 18.46 40.045 0.00 0.00 40.528 1.00 19.15 41.223 1.00 17.65 42.612 1.00 19.24 40.333 1.00 22.37 40.807 1.00 17.55 40.311 1.00 20.43 41.485 1.00 16.99 42.164 0.00 0.00 41.511 1.00 19.09 42.869 1.00 24.46 43.633 1.00 33.72 43.507 1.00 32.25 44.033 1.00 37.61 43.054 1.00 34.92 42.177 0.00 0.00 42.842 0.00 0.00 43.458 0.00 0.00 40.378 1.00 16.91 40.110 1.00 15.63 39.710 1.00 14.55 40.141 0.00 0.00 38.459 1.00 11.12 38.099 1.00 12.16 39.042 1.00 14.76 39.259 1.00 18.49 39.567 1.00 24.14 37.339 1.00 10.59 36.590 1.00 13.81 37.326 1.00 7.24 37.956 0.00 0.00 36.383 1.00 7.03 36.591 1.00 2.85 35.317 1.00 7.43 34.428 1.00 2.68 35.661 1.00 6.68

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

ATCM

ATOM

ATCM

AT4

ATCM

ATOM

AT

ATCM

ATCM

ATCIM

ATOM

ATM

ATCM

ATCM

ATCM

ATOM

ATCM

ATOM

ATOM

ATM

ATOM

ATCM

ATCM

ATM

25 ATOM

ATOM

ATOM

ATOM

ATOM

ATCM

AITN

ATOM

ATCM

ATCM

ATOMU

ATM

ATCM

ATOM

ATCM

1910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921 1922 1923 1924 1925 1926 1927 C LEU 0 LEU N THR H THR CA THR CB THR OG1 THR I-1 THR CG2 THR C THR O THR N GIN H GEN CA GIN CB GLN OG GEN CD GLN OE1 GIN 2097 2097 2098 2098 2098 2098 2098 2098 2098 2098 2098 2099 2099 2099 2099 2099 2099 2099 1928 NE2 GIN 2099 1929 HE21 GiN 2099 1930 HE22 GIN 2099 1931 C GLN 2099 1932 0 GIN 2099 1933 N ALA 2100 1934 H ALA 2100 1935 CA ALA 2100 1936 CB ALA 2100 1937 C ALA 2100 1938 0 ALA 2100 1939 N TRP 2101 1940 H TRP 2101 1941 CA TRP 2101 1942 CB TRP 2101 1943 OG TRP 2101 1944 CD2 TRP 2101 1945 CE2 TRP 2101 1946 CE3 TRP 2101 1947 CD1 TRP 2101 1948 NE1 TRP 2101 -19.056 -18.854 -18.847 -19.017 -18.266 -17.866 -18.952 -19.663 -16.624 -19.187 -18.733 -20.486 -20.807 -21.443 -22.843 -23.423 -23.315 -22.604 -23.989 -24.521 -23.848 -21.478 -21.842 -21.146 -21.074 -21.016 -20.953 -19.760 -19.823 -18.659 -18.717 -17.367 -16.263 -15.704 -15.003 -14.676 -14.625 -15.775 -15.158 31.873 31.190 31.410 31.985 30.082 29.853 30.231 29.576 30.666 28.940 27.967 29.070 29.834 28.076 28.371 27.264 25.887 25.017 25.697 26.407 24.808 28.072 27.085 29.211 30.018 29.323 30.796 28.586 27.736 28.801 29.421 28.222 29.010 30.029 29.798 31.057 28.646 31.378 32.008 36.504 35.499 37.735 38.512 37.954 39.429 40.288 40.325 39.781 37.521 36.924 37.772 38.297 37.293 37.838 38.720 38.084 38.580 36.959 36.558 36.587 35.768 35.147 35.178 35.723 33.738 33.348 33.277 32.394 33.988 34.743 33.627 34.300 33.420 32.198 31.662 31.500 33.581 32.525 1.00 12.75 1.00 17.71 1.00 13.89 0.00 0.00 1.00 14.50 1.00 18.86 1.00 27.76 0.00 0.00 1.00 14.88 1.00 14.65 1.00 20.42 1.00 13.41 0.00 0.00 1.00 10.97 1.00 19.13 1.00 26.63 1.00 33.37 1.00 35.83 1.00 38.47 0.00 0.00 0.00 0.00 1.00 9.33 1.00 13.05

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

1.00 0.00 1.00 1.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 9.52 0.00 3.77 2.00 2.86 2.63 2.00 0.00 3.21 3.18 4.37 5.80 7.80 5.41 5.99 1.00 13.05 -81- ATCM 1949 HE1 TRP 2101 ATCM 1950 CZ2 TRP 2101 ATCM 1951 CZ3 TRP 2101 -15.113 32.979 32.395 0.00 0.00 -13.993 31.197 30.456 1.00 6.76 -13.951 28.786 30.301 1.00 3.13 4.44 4 *4

ATOM

ATCM

ATCM

ATOM

ATCM

ATOM

ATCM

ATCM

ATOM

ATCM

ATM

ATOM

ATOM

ATOM

ATOM

ATCM

A'Ic

ATOM

ATCM

ATCM

25 ATOM

ATOM

ATOM

ATOM

ATOM

ATCM

ATOM

ATM

ATCM

ATOM

ATOM

ATOM

ATM4

ATOM

195: 195: 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 2 C-2 TRP 3 C TRP 1 0 TRP 5 N ASP H ASP CA ASP CB ASP CG ASP OD1 ASP OD2 ASP C ASP 0 ASP N LEU H LEU CA LEU CB LEU OG LEU CD1 LEU CD2 LEUJ C LEU 0 LEUX N TYR H TYR CA TYR CB TYR CG TYR CD1 TYR CE1 TYR CD2 TYR CE2 TYR CZ TYR OH TYR HH TYR C TYR 2 0 TYR 2 N TYR 2 2101 2101 2101 2102 2102 2102 2102 2102 2102 2102 2102 2102 2103 2103 2103 2103 2103 2103 2103 2103 2103 2104 2104 2104 2104 2104 2104 2104 2104 2104 2104 2104 2104 !104 !104 !105 -13.64- -17.20 -16.27 -18.091 -18.573 -18.235 -19.277 -19.127 -20.084 -18.048 -18.688 -18.144 -19.646 -19.988 -20.230 -21.589 -22.694 -23.659 -23.417 -19.314 -19.177 -18.594 -18.814 -17.605 -16.987 -17.865 -18.003 -18.772 -18.535 -19.316 -19.419 -20.122 -20.693 -16.506 -16.102 -16.054 4 4 30.052 6 26.736 1 26.065 L 26.240 26.864 24.815 24.564 23.207 22.779 22.585 24.180 23.158 24.828 25.662 24.302 24.951 24.551 25.697 23.318 24.486 23.580 25.602 26.319 25.821 27.215 28.342 29.508 30.564 28.256 29.308 30.462 31.548 31.333 24.771 24.216 24.475 3 29.791 33.960 33.501 34.807 35.388 35.005 36.099 36.759 37.436 36.637 33.686 33.248 33.029 33.421 31.794 31.537 32.512 32.675 32.012 30.577 29.756 30.530 31.162 29.482 29.602' 29.108 29.852 29.400 27.888 27.423 28.187 27.727 J 27.011 C 29.555 1 28.536 1 30.771 1 1.00 6.31 1.00 8.69 1.00 10.67 1.00 8.35 0.00 0.00 1.00 9.05 1.00 13.12 1.00 16.43 1.00 23.14 1.00 18.55 1.00 10.27 1.00 11.33 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 1.00 1.00 1.00 1.00 1.00 1.00 L.00 1.00 8.01 0.00 7.80 2.00 2.00 2.04 2.00 7.45 5.72 6.39 0.00 7.03 7.14 4.21 7.88 5.53 2.00 3.76 8.86

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

1.00 12.11 ).00 0.00 00 9.25 L.00 12.91 00 12.79 -82- ATM 1988 H TYR 2105 -16.371 25.002 31.536 0.00 0.00

ATM

ATM

ATM

ATC4

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM

ATCM4

ATCM

ATCM

ATM

ATCM

ATOM

ATM

ATM

ATM

ATCM

ATCM

ATCM

ATCM

ATM

ATM

ATCM

ATM

ATCM

ATCM

ATCM

ATM

ATOM

ATCM

ATM

ATM

ATCM

198S 199C 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 CB TYR 3G TYR CD1 TYR CE1 TYR CD2 TYR CE2 TYR CZ TYR OH TYR HH TYR C TYR 0 TYR N HIS H HIS CA HIS CB HIS CG HIS CD2 HIS ND1 HIS HD1 HIS CE1 HIS NE2 HIS HE2 HIS C HIS 0 HIS N VAL H VAL CA VAL CB VAL CGI VAL CG2 VAL C VAL 0 VAL N PHE H PHE CA PHE CB PHE 2105 2105 2105 2105 2105 2105 2105 2105 2105 2105 2105 2106 2106 2106 2106 2106 2106 2106 2106 2106 2106 2106 2106 2106 2107 2107 2107 2107 2107 2107 2107 2107 2108 2108 2108 2108 CA TYR 2105 -15.030 23.452 -14.680 23.376 -13.496 22.488 -12.288 22.693 -11.184 21.892 -13.579 21.446 -12.472 20.629 -11.276 20.866 -10.155 20.113 -9.397 20.447 -15.479 22.070 -14.702 21.307 -16.746 21.759 -17.288 22.402 -17.298 20.488 -18.705 20.326 -19.294 18.971 -20.529 18.588 -18.578 17.808 -17.628 17.736 -19.346 16.767 -20.535 17.214 -21.295 16.644 -17.315 20.332 -16.928 19.284 -17.768 21.355 -18.077 22.171 -17.797 21.281 -18.640 22.425 -20.082 22.296 -18.116 23.780 -16.384 21.294 -16.047 20.456 -15.518 22.127 -15.849 22.771 -14.109 22.164 -13.371 23.223 -11.923 23.366 30.996 32.481 32.765 32.111 32.360 33.684 33.946 33.279 33.542 33.059 30.515 29.942 30.737 31.250 30.314 30.881 30.664 30.259 30.865 31.114 30.595 30.226 29.972 28.787 28.273 28.062 28.519 26.599 25.963 26.372 26.371 26.009 25.172 26.576 27.234 26.187 27.007 26.651 1.00 10.70 S1.00 7.06 S1.00 4.50 1.00 8.17 1.00 12.33 1.00 6.11 1.00 12.32 1.00 15.17 1.00 21.26 0.00 0.00 1.00 10.02 1.00 11.77 1.00 9.87 0.00 0.00 1.00 11.64 1.00 15.15 1.00 24.44 1.00 25.83 1.00 28.70 0.00 0.00 1.00 28.91 1.00 31.03 0.00 0.00 1.00 14.72 1.00 17.34 1.00 13.33 0.00 0.00 1.00 10.31 1.00 9.70 1.00 11.91 1.00 15.79 1.00 10.92 1.00 11.27 1.00 9.62 0.00 0.00 1.00 8.05 1.00 4.20 1.00 2.00

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

ATCMI 2026 CG PHE 2108 -83- ATOM 2027 CD1 PHE 2108

ATOM

ATOM

ATOM

ATCM

ATOM

ATOM

ATCM

ATOM

ATCM

ATOM

ATM

ATOM

ATOM

ATOM

ATCM

ATCM

ATOM ATOM

ATM

ATOM

ATOM

ATOM

ATCM

ATOM

ATCM

ATOM

ATOM

ATCM

ATCM

ATOM

ATCOM

ATCM

ATCM

ATOM

ATCM

2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041: CD2 PHE CE2 PHE CZ PHE C PHE 0 PHE N ARG H ARG CA ARG CB ARG CG ARG CD ARG NE ARG HE ARG 2042 CZ ARG 2043 NH1 ARG 2044 HH11 ARG 2045 HH12 ARG 2046 NH2 ARG 2047 HH21 ARG 2048 HH22 ARG 2108 2108 2108 2108 2108 2108 2109 2109 2109 2109 2109 2109 2109 2109 2109 2109 2109 2109 2109 2109 2109 2109 2109 2110 2110 2110 2110 2110 2110 2110 2110 2110 2110 2110 2110 2110 2110 2110 -2

-J

-1 -1 -11 -i -a -a -1 -1 -11.519 24.292 -10.961 22.606 -10.170 24.461 -9.613 22.760 -9.214 23.692 -13.423 20.810 -12.685 20.368 -13.609 20.198 -14.125 20.671 -13.001 18.905 -13.358 18.476 -12.193 18.477 -11.939 17.082 -13.169 16.442 13.738 16.951 13.573 15.218 14.732 14.754 15.288 15.321 15.033 13.832 12.812 14.444 11.931 14.776 13.130 13.529 13.454 17.829 12.682 16.939 14.710 17.911 15.280 18.632 15.260 16.952 16.795 16.947 L7.418 16.320 L7.423 14.786 L6.091 14.194 .5.389 14.432 .5.762 13.332 .4.534 12.815 .3.866 13.068 .4.282 12.171 .6.633 13.017 .7.547 13.422 .6.368 12.373 25.702 27.295 25.396 27.000 26.045 26.364 25.493 27.528 28.212 27.832 29.256 30.239 30.819 31.297 31.910 30.956 31.413 32.021 31.173 30.188 29.851 29.944 26.849 26.509 26.412 26.748 25.455 25.499 26.743 26.714 26.900 26.260 27.865 27.899 27.201 28.621 28.820 28.830 29.538 1.00 4.15 1.00 3.42 1.00 8.79 1.00 9.05 1.00 12.56 1.00 9.13 1.00 10.33 1.00 11.74 0.00 0.00 1.00 12.27 1.00 18.36 1.00 32.13 1.00 43.37 1.00 53.59 0.00 0.00 1.00 54.76 1.00 54.90 0.00 0.00 0.00 0.00 1.00 53.94 0.00 0.00 0.00 0.00 1.00 11.58 1.00 11.33 1.00 10.43 0.00 0.00 1.00 10.64 1.00 12.47 1.00 19.35 1.00 31.28 1.00 41.95 0.00 0.00 1.00 41.41 1.00 36.39 0.00 0.00 0.00 0.00 1.00 36.79 0.00 0.00 0.00 0.00

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

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FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

2049 2050 2051 2052 2053 2054 2055 2056 2057 2058 2059 2060 C ARG O ARG N ARG H ARG CA ARG CB ARG OG ARG CD ARG NE ARG HE ARG CZ ARG NH1 ARG 2061 HH11 ARG 2062 HH12 ARG 2063 NH2 ARG 2064 HH21 ARG 2065 HH22 ARG -84-

C.

ATM

ATOM

ATCM

ATM

ATOM

ATCM

ATOM

ATM

ATM

ATCM

ATOM

ATCM

ATCM

15 AT3M

ATCM

ATM

ATM

ATOM

ATOM

ATOM

ATCM

ATCM

ATCM

25 ATM

ATCM

ATCM

ATCM

ATOM

ATM

ATOM

ATOM

ATOM

ATCM

ATOM

ATOM

ATCM

ATCM

ATM

AlUM! AlU'M 2066 C ARG 2067 0 ARG 2068 N ILE 2069 H ILE 2070 CA ILE 2071 CB ILE 2072 CG2 ILE 2073 OG1 ILE 2074 CD1 ILE 2075 C ILE 2076 0 ILE 2077 N SER 2078 H SER 2079 CA SER 2080 CB SER 2081 OG SER 2082 HG SER 2083 C SER 2084 0 SER 2085 OT SER 2086 OH2 WMTR 2087 HI 1MfTR 2088 H2 WAMR 2089 OH2 WATR 2090 HI WATR 2091 H2 WATR 2092 OH2 WATR 2093 HI WATR 2094 H2 WAITR 2095 OH2 WATR 2096 HI WATR 2097 H2 WATR 2098 OH2 WATR 2099 HI WATR 2100 H2 WATR 2101 OH2 WATR 2102 HI WATR 2103 H2 WATR 2104 0H2 WATR 2110 2110 2111 2111 2111 2111 2111 2111 2111 2111 2111 2112 2112 2112 2112 2112 2112 2112 2112 2112 301 301 301 302 302 302 303 303 303 304 304 304 305 305 305 306 306 306 307 -14.818 -14.494 -14.598 -14.033 -14.644 -16.148 -14.044 -14.821 -12.510 -11.963 -11.840 -12.312 -10.410 -9.590 -9.589 -9.617 -10.155 -10.552 -9.613 -13.963 -14.436 -13.909 -0.900 -1.021 -1.478 -6.938 -6.199 -6.527 -10.919 -10.331 -10.602 -21.400 -21.139 -22.356 0.813 0.278 0.156 -30.428 16.280 18.447 19.149 18.769 20.117 20.108 21.333 22.615 18.791 19.586 17.887 17.230 17.634 18.179 19.601 19.750 16.126 15.361 15.712 32.282 33.059 31.701 21.657 21.041 21.246 34.185 34.542 33.918 15.222 15.994 14.763 35.769 35.329 35.778 27.087 27.451 26.516 31.660 1 23.209 23.670 24.342 22.314 21.784 21.982 22.500 22.301 22.163 21.395 22.870 23.418 22.673 23.852 23.899 24.846 22.525 23.432 21.474 39.005 39.326 39.771 34.783 35.510 34.123 40.131 39.638 40.941 48.819 48.864 48.037 26.707 27.513 26.710 37.460 36.742 37.895 28.013

I

1.00 15.19 1.00 14.35 0.00 0.00 1.00 18.50 1.00 14.09 1.00 19.97 1.00 13.58 1.00 2.00 1.00 22.21 1.00 27.42 1.00 27.11 0.00 0.00 1.00 32.50 1.00 31.61 1.00 28.34 0.00 0.00 1.00 35.63 1.00 36.42 1.00 41.38 1.00 20.07 0.00 20.00 0.00 20.00 1.00 23.80 0.00 20.00 0.00 20.00 1.00 41.17 0.00 20.00 0.00 20.00 1.00 28.06 0.00 20.00 0.00 20.00 1.00 26.77 0.00 20.00 0.00 20.00 1.00 15.38 0.00 20.00 0.00 20.00 1.00 46.41 -14.810 17.200 24.014 1.00 12.51

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

FRAP

WATR

WATR

WATR

WATR

VRTR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WAITR

WATR

WATR

WATR

a. a.

a a a

ATOM

ATCM

ATCM

ATOM

ATOM

ATOM

ATOM

ATCM

ATOM

ATCM

ATCM

ATOM

15 ATCM4

ATOM

ATOM

ATCM

ATCM

ATCM

ATOM

ATOM

ATCM

ATM

ATOM

ATOM

ATCM

ATCM

ATCM

ATOM

ATOMv

ATCM

ATCM

ATOM

ATCM'

ATM

ATOM

ATCM

2105 2106 2107 2108 2109 2110 2111 2112 2113 2114 2115 2116 2117 2118 2119 2120 2121 2122 2123 2124 2125 2126 2127 2128 2129 2130 2131 2132 2133 2134 2135 2136 2137 2138 2139 2140 2141 2142 HI WATR 307 H2

HI

H2

OH

HI

H2 OH2

HI

H2 OH2

HI

H2 0H2

HI

H2 0H2

HI

H2 0H2

HI

H2 CH2

HI

H2 CH2

HI

H2 02

HI

H2 0H2

HI

H2 0H2 H1 H2

WATR

WATR

WATR

WATR

2 WATR

WATR

WATR

2 WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

WATR

307 308 308 308 309 309 309 310 310 310 311 311 311 312 312 312 313 313 313 314 314 314 315 315 315 316 316 316 317 317 317 318 318 318 319 319 319 -30.248 31.722 -4.519 32.837 -4.435 32.964 -4.287 31.920 -18.089 22.614 -17.511 23.005 -18.955 22.733 -22.152 21.619 -22.437 22.341 -22.872 21.464 -6.459 3.543 -6.280 2.752 -5.832 4.191 -5.993 11.471 -6.909 11.725 -5.782 11.031 -0.619 20.784 -0.854 20.074 -1.113 21.551 -5.598 26.321 -6.497 26.108 -5.118 25.491 -3.023 33.604 -2.394 34.283 -3.855 33.984 -25.006 29.561 -24.532 29.047 -25.677 28.934 -23.638 29.893 -23.016 29.169 -24.395 29.529 -7.744 6.880 -7.080 6.901 -7.480 6.116 -2.748 2.703 -3.202 3.462 -3.353 2.352 -19.295 42.654 28.946 47.558 48.515 47.465 12.803 12.138 12.394 36.180 36.738 35.569 52.877 52.368 52.543 28.804 28.881 29.653 55.049 55.637 55.388 58.876 58.602 58.861 37.769 37.516 37.469 22.950 23.605 22.652 10.609 10.621 11.101 50.272 49.564 50.785 46.777 46.395 47.432 40.303 0.00 1.00 0.00 0.00 1.00 0.00 20.00 15.92 20.00 20.00 25.97 20.00 0.0 1.0C 0.0 0.0( 1.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 0.00 0 20.00 0 41.59 0 20.00 0 20.00 32.94 20.00 20.00 18.59 20.00 20.00 19.50 20.00 20.00 36.20 20.00 20.00 26.43 20.00 20.00 41.75 20.00 20.00 16.55 20.00 20.00 20.83 20.00 20.00 -30.299 30.737 27.805 0.00 20.00

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1.00 31.05 0.00 0.00 1.00 20.00 20.00 39.42 ATOM 2143 OH2 WATR 320 -86-

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2144 2145 2146 2147 2148 2149 2150 2151 2152 2153 2154 Fa B2 H2

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320 320 321 321 321 322 322 322 323 323 323 -19.042 41.825 39.876 0.00 20.00 -18.638 0.583 -0.191 1.272 -16.781 -17.172 -15.838 -19.829 -19.808 -1-9.224 43.269 32.369 32.008 31.719 17.874 18.545 18.064 12.916 13 .873 12.538 39.991 55.901 55.428 55.776 51.246 50.688 51.228 46.549 46. 697 47.193 0.00 20.00 1.00 39.29 0.00 20.00 0.00 20.00 1.00 33.48 0.00 20.00 0.00 20.00 1.00 26.46 0.00 20.00 0.00 20.00

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mm

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Note: FKBP sequence is SEQ ID NO: 1 FRAP sequence is SEQ ID NO: 2 87 EDITORIAL NOTE APPLICATION NUMBER 66669/00 The following Sequence Listing pages 88 to 90 are part of the description. The claims pages follow on pages 91 to 97.

SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: CORNELL RESEARCH FOUNDATION, INC.

(ii) TITLE OF INVENTION: CRYSTALLINE FRAP COMPLEX (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: ARIAD Pharmaceuticals, Inc.

STREET: 26 Landsdowne Street CITY: Cambridge 15 STATE: MA COUNTRY: USA ZIP: 02139-4234 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: HEREWITH

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/005,808 FILING DATE: 23-OCT-1995 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/006,069 FILING DATE: 24-OCT-1995 -88- (viii) ATTORNEY/AGENT INFORMATION: NAME: BERSTEIN, David L.

REGISTRATION NUMBER: 31,235 REFERENCE/DOCKET NUMBER: ARIAD 350A-PCT (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 617-494-0400 TELEFAX: 617-494-0208 *r

S

*5t*

S

SS

S

INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid 15 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Sn

S

S. S 55 Gly Val Gin Val 1 Lys Arg Gly Gin 20 Gly Lys Lys Phe Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Thr Cys Val Asp Ser Ser Val His 25 Arg Asp Tyr Thr Gly Met Leu Glu Asp Phe Lys Phe Arg Asn Lys Met Leu Gly Lys Gin Glu Val Ile Arg Gly Trp Glu Gly Val Ala Gin Met Ser Val Gly Gin Thr Gly Arg Ala Lys Leu Thr 75 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala His Pro Gly lie 90 Ile Pro Pro His Ala Thr -89- Leu Val Phe Asp Val Glu Leu Leu Lys Leu Giu 100 105 0 0 .0.

*00:S 0 0 0000 *0 *0.

0..0 4, ::66.

go go INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 100 amino acids TYPE: amino acid STRA1NDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Giu Leu Ile Arg Val Ala Ile Leu Trp His Giu 1 5 10 Leu Giu Giu Ala Ser Arg Leu Tyr Phe Gly Giu 20 25 Met Phe Glu Val Leu.Glu Pro Leu His Ala.Met 35 40 Gin Thr Leu Lys Glu Thr Ser Phe Asn Gin Ala Met Trp His Giu Gly Arg Asn Val Lys Gly Met Giu Arg Gly Pro Tyr Gly Arg Asp Leu Lys Ser Gly Asn Val 50 Met Glu Ala Gin Lys Asp Leu Thr 55 Cys Arg Lys Tyr Met 75 Glu Trp 70 Asp Leu Tyr Tyr His Val Phe Arg Arg Gin Ala Trp Ile Ser Lys Gin 100 90

Claims (15)

1. A crystalline composition including a complex formed by a first protein containing an FRB domain, a second protein containing an FKBP domain and a ligand capable of forming a ternary complex with the first and second proteins, wherein the complex is characterized by the coordinates of Appendix I, or by coordinates having a root mean square deviation therefrom, with respect to conserved backbone atoms of the listed amino acids, of not more than 1.5 A.

2. A machine-readable data storage medium, including a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three- dimensional representation of a molecule or molecular complex comprising a protein containing an FRB domain, wherein the machine readable data includes data corresponding to the coordinates for the FRB domain set forth in Appendix I, or coordinates having a root mean square deviation therefrom, with respect to conserved protein backbone atoms, of not more than 1.5 A.

3. A machine-readable data storage medium comprising a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine-readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the coordinates corresponding to the second set of machine-readable data, wherein: said first set of data comprises a Fourier transform of at least a portion of the coordinates of the FRB domain set forth in Appendix I and said second set of data comprises an X-ray diffraction pattern of a molecule or molecular complex. 9**

4. A method for displaying a three dimensional representation of a composition of claim 1 which includes: PAOPERTEJhW\Amwdd spmis\2337969.222.doct1808/G3 -92- providing a machine capable of reading data stored on a machine- readable storage medium of claims 2 or 3, programmed with instructions for using said data to display a graphical three-dimensional representation of a protein or protein:ligand complex or portion thereof defined by said data, and loaded with a machine-readable storage medium of claims 2 or 3; and, permitting the machine to read said data and display the three- dimensional representation. A method for determining the three-dimensional structure of a protein containing an FRB domain, or a complex of such protein with a ligand therefor, which includes obtaining x-ray diffraction data for crystals of the protein or complex, providing three-dimensional structural coordinates for a composition of claim 1 and determining the three-dimensional structure of the protein or complex S-by analyzing the x-ray diffraction data with reference to the previous structural coordinates using molecular replacement techniques.

6. A method for determining the three-dimensional structure of a protein containing an FRB domain or co-complex of said protein with a ligand therefor, which method includes: providing structural coordinates for a composition of claim 1 and determining the three-dimensional structure of the FRB domain- containing protein or complex by homology modeling with reference to the previous structural coordinates. S7. A method for selecting a compound capable of binding to an FRB domain which includes: P:OPER\Ej\Amndel specis\2337969.222.doc-18/08/03 -93- providing coordinates of Appendix I, or coordinates having a root mean square deviation therefrom, with respect to conserved backbone atoms of the listed amino acids, of not more than 1.5 A characterizing points associated with that three-dimensional structure with respect to the favorability of interactions with one or more selected functional groups; providing a database of one or more candidate compounds; and identifying from the database those compounds having structures which best fit the points of favorable interaction with the three dimensional structure.

8. A method of claim 7 which further includes testing a compound so identified for its ability to: bind to FRAP, with or without FKBP12, inhibit the binding ofrapamycin or FKBP12:rapamycin to FRAP, and/or trigger a biological function mediated by rapamycin.

9. A method for assessing interactions with a three dimensional representation of a molecular model including a FRB domain including providing a computer system for producing a three-dimensional representation of a molecule or molecular complex, wherein said computer system includes: a machine-readable data storage medium including a data storage material encoded with machine-readable data, wherein said data includes the structure coordinates of an FRB domain; a working memory for storing instruction for processing said machine- readable data; a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine- readable data into said three-dimensional representation; and P:\OPER\EjhAmmd~d spcis\2337969.222.doc1/08O3 -94- a display coupled to said central-processing unit for displaying said three- dimensional representation; and wherein said method further includes executing instructions on a computer for: generating a three dimensional representation of a protein including an FRB domain from structural coordinates of said protein, such that the computer loads into memory thereof computer-readable data including structural coordinates of a molecule of molecular complex including an FRB domain molecular model; generating molecular models of one or more test compounds; calculating, from said molecular models, one or more possible molecular complexes which could be formed by association of said protein with said one or more test compound; and generating output data indicative of the degree of interaction and/or the location and/or the orientation of such interaction, if any. The computer system of claim 9 in which the coordinates for the FRB domain are set forth in Figure 4, or coordinates having a root mean square deviation therefrom, with respect to conserved protein backbone atoms, of not more than

11. A method for assessing interactions with a three dimensional representation of a molecular model including a FRB domain including providing a computer system for determining at least a portion of the structure coordinates corresponding to an X-ray diffraction pattern of a molecule or molecular complex, wherein said computer includes: a machine-readable data storage medium including a data storage material encoded with machine-readable data, wherein said data includes at least a **"portion of the structure coordinates of the FRB domain set forth in Figure 4 or coordinates having a root mean square deviation therefrom, with respect to conserved protein backbone atoms, of not more than o• a machine-readable data storage medium including a data storage material encoded with machine-readable data, wherein said data comprises an X-ray diffraction pattern of said molecule or molecular complex; P:\OPER\Ejb\A.mded specis2337969.222.doc-/0/03 a working memory for storing instructions for processing said machine- readable data of(a) and a central-processing unit coupled to said working memory and to said machine-readable storage medium of and for performing a Fourier transform of the machine-readable data of and for processing said machine-readable data of into structure coordinates based on the Fourier transform of the machine-readable data; and a display coupled to said central-processing unit for displaying said structure coordinates of said molecule or molecular complex; and wherein said method further includes executing instructions on a computer for: generating a three dimensional representation of a protein including an FRB domain from structural coordinates of said protein, such that the computer loads into memory thereof computer-readable data including structural coordinates of a molecule of molecular complex including an FRB domain molecular model; generating molecular models of one or more test compounds; calculating, from said molecular models, one or more possible molecular complexes which could be formed by association of said protein with said one or more test compound; and •go• generating output data indicative of the degree of interaction and/or the location and/or the orientation of such interaction, if any.

12. A method for displaying a graphical three-dimensional representation of a molecular complex including an FRB domain and at least one other compound, which method includes executing instructions on a computer for generating a three-dimensional representation of a protein including an FRB domain from structural coordinates of said protein, such that the computer loads into memory thereof computer-readable data including structural coordinates of a molecule or molecular complex including an FRB domain, generating molecular models of one or more test compounds, P:\OPER\Ej\Am dcd spcis\2337969.222.dc-18/08/03 -96- calculating, from said molecular models, one or more possible molecular complexes which could be formed by association of said protein with said one or more test compound, and displaying a graphical three-dimensional representation of the molecular complex including the FRB domain.

13. A method of claim 12, wherein said test compound is a rapamycin mimetic in which one or more structural segments of rapamycin is replaced.

14. A method of claim 13, wherein the three-dimensional representation of said FRB domain is based on coordinates of Figure 4, or based on coordinates having a root mean square deviation therefrom with respect to conserved protein backbone atoms of not more than 1.5 A. A method for assessing interactions with a three dimensional representation of a molecular model of a polypeptide including an FRB domain including: generating a molecular model of an FRB domain from structural coordinates set forth in Figure 4, or coordinates having a root mean square *deviation therefrom, with respect to conserved protein backbone atoms, of not more than 1.5A, and (ii) a molecular model of a test compound; calculating the ability of the FRB molecular model to associate with the test compound molecular model; and generating output data indicative of the degree of interaction and/or the location and/or the orientation of such interaction, if any.

16. The method of any of claims 9, 11, 12 orl 5 wherein the test compound is a protein. 9*e*

17. The method of any of claims 9, 11, 12 or 15 wherein the test compound is a peptide. 3-O3;10:32 ;Davies Collison Cave b Ud J*?u if a/ 3 97

18. The method of any of claims 9, 11, 12 or 15, wherein the test compound is a small organic molecale.

19. The method of any of claims 9, 11 or 15 wherein the test compound is a rapamycin mimetic in which one or more struictural segments of rapaxnycin is replaced. A crystalline composition according to Claim 1 or a machine-readable storage data medium according to Claims 2 or 3 or a mnethod accordinig to any one of Claims 4 to 9 or 11 to 19, or a computer system according to Claim 10 substantially as hereinbefore defined with reference to the Figures and/or Examples. Dated this 15"' day of September 2003 CORNELL RESEARCH FOUNDATION, INC. by its Patent Attorneys DAVIES COLLISON CAVE a a a. a 15/09 '03 MON 10:26 [TX/RX NO 97301