# Patent

## Title
Soluble, stable form of HDM2, crystalline forms thereof and methods of use thereof

## Abstract

The present invention discloses modified Hdm2 proteins that are soluble. In addition, the present invention discloses nucleic acids that encode the modified Hdm2 proteins of the present invention. The invention also provides crystals of modified Hdm2 proteins that are suitable for X-ray crystallization analysis. The present invention also discloses methods of using the modified Hdm2 proteins and crystals thereof to identify, select and/or design compounds that may be used as anticancer agents. The present invention further discloses compounds that bind to modified Hdm2 proteins in protein-ligand complexes.

## Background
This application claims the benefit of U.S. Provisional Application 60/461,787, filed Apr. 10, 2003, and 60/547,265, filed Feb. 24, 2004. These applications are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates to a soluble and stable form of human Double Minute 2 protein, Hdm2. The present invention further pertains to nucleic acids encoding these proteins. The present invention also relates to a process of obtaining specific samples of Hdm2 that are amenable to forming homogeneous crystals for X-ray crystallization analysis and the crystals formed thereby. The present invention also pertains to methods of using the X-ray diffractable crystals in structure-based drug design to identify compounds that can modulate the activity of the protein.
BACKGROUND OF THE INVENTION
The most commonly inactivated tumor suppressor gene in human cancer encodes the p53 protein, a transcription factor that is intimately involved in maintaining the integrity of the genome in a cell [Hall and Peters, Adv. Cancer Res., 68:67-108 (1996); Hainaut et al., Nucleic Acid Res., 25:151-157 (1997); Sherr, Cancer Res., 60:3689-95 (2000)]. In response to oncogenic stress signals, the cell triggers the p53 transcription factor to initiate either apoptosis or cell cycle arrest. Apoptosis facilitates the elimination of damaged cells from the organism, while cell cycle arrest enables damaged cells to repair genetic damage [reviewed in Ko et al., Genes & Devel. 10:1054-1072 (1996); Levine, Cell 88:323-331 (1997)]. The loss of the safeguard functions of p53 predisposes damaged cells to progress to a cancerous state. Inactivating p53 in mice consistently leads to an unusually high rate of tumors [Donehower et al., Nature, 356:215-221 (1992)].
The p53 transcription factor promotes the expression of a number of cell cycle regulatory genes, including the gene encoding the Mouse Double Minute (Mdm2) protein [see, Chene, Nature Reviews Cancer 3:102-109 (2003)]. The Mdm2 protein (designated Hdm2 in humans and Mdm2 in mice) acts to down-regulate p53 activity in an auto-regulatory manner [Wu et al, Genes Dev., 7:1126-1132 (1993); Bairak et al., EMBO J, 12:461-468 (1993)]. In the absence of oncogenic stress signals, i.e., under normal cellular conditions, the Mdm2 protein serves to maintain p53 activity at low levels [Wu et al, Genes Dev., 7:1126-1132 (1993); Barak et al., EMBO J, 12:461-468 (1993)].
Interestingly, whereas Mdm2 negative (Mdm2−/−) mice are not viable [Jones et al, Nature, 378:206-208 (1995); Montes de Oca Luna et al., Nature, 378:203-206 (1995)], additional inactivation of the p53 gene rescues Mdm2−/− mice [Jones et al, Nature, 378:206-208 (1995); Montes de Oca Luna et al., Nature, 378:203-206 (1995)]. These results indicate that the misregulation of the p53 transcription factor in the Mdm2 negative mice is the root cause of the observed lethality of the Mdm2−/− genotype, and that the regulation of p53 function relies on an appropriate balance between the two components of this p53-Mdm2 auto-regulatory system. Indeed, this balance appears to be essential for cell survival.
There are at least three ways that Mdm2 acts to downregulate p53 activity. First, Mdm2 can bind to the N-terminal transcriptional activation domain of p53 to block expression of p53-responsive genes [Kussie et al., Science, 274:948-953 (1996); Oliner et al., Nature, 362:857-860 (1993); Momand et al, Cell, 69:1237-1245 (1992)]. Second, Mdm2 shuttles p53 from the nucleus to the cytoplasm to facilitate the proteolytic degradation of p53 [Roth et al, EMBO J, 17:554-564 (1998); Freedman et al., Mol Cell Biol, 18:7288-7293 (1998); Tao and Levine, Proc. Natl. Acad. Sci. 96:3077-3080 (1999)]. Finally, Mdm2 possesses an intrinsic E3 ligase activity for conjugating ubiquitin to p53 within the ubiquitin-dependent 26S proteosome pathway [Honda et al., FEBS Lett, 420:25-27 (1997); Yasuda, Oncogene 19:1473-1476 (2000)]. Thus, Mdm2 impedes the ability of the p53 transcription factor to promote the expression of its target genes through binding p53 in the nucleus.
Attenuating the p53-Mdm2 auto-regulatory system can have a critical effect on cell homeostasis. Consistently, a correlation between the overexpression of Mdm2 and tumor formation has been reported [Chene, Nature 3:102-109 (2003)]. Since Mdm2 acts as a post-translational regulatory effector of the p53 transcription factor, compounds that hinder the ability of Hdm2/Mdm2, to interact with p53 would be anticipated to cause an immediate increase in p53 activity, and thereby rapidly promote either cell cycle arrest or apoptosis in damaged cells. Not surprisingly then, there is currently a substantial effort being made to identify new anticancer agents that hinder the ability of Hdm2 to interact with p53 [Chene, Nature 3:102-109 (2003)]. However, to date, no suitable anticancer agent has been found.
Structure-based drug design is one way to optimize the success of identifying useful antagonists of Hdm2, but use of this powerful methodology requires the three-dimensional structure of the target protein. So far, little information has been provided regarding the three-dimensional structure of Hdm2. Indeed, the only structures of Mdm2 currently available are those of Hdm2 and Xenopus Mdm2 (XMdm2), complexed with a p53 peptide, but neither crystalline form is suitable for structure-based drug design [Kussie, et al. Science, 274(5289): 948-953 (1996)]. Moreover, most of the protein-protein contacts in these crystal lattices are formed through the interaction of the exposed residues in the bound p53 peptide (D21, K24, and L25), making the p53 peptide difficult to displace, which makes it inaccessible to potential inhibitors.
In direct contrast, a successful structure-based drug design program focusing on Hdm2 requires a form of the Hdm2 protein that is amenable to crystallization in the absence of any particular binding partner. Further, the crystal form of the p53-binding pocket of the Hdm2 protein should be accessible to potential inhibitors used for testing binding or for co-structural determination. However, up until now, the solubility and stability of the free Hdm2 protein has been significantly less than that of the Hdm2-p53 peptide complex.
Thus, there is a need to obtain nucleic acids that encode an Hdm2 protein that is soluble and stable at high protein concentrations even when the protein is free of p53 or fragments thereof. In addition, there is a need to design purification procedures that lead to the preparation of an isolated active Hdm2 protein that is soluble and stable when independent of p53 or fragments thereof. In addition, there is a need to obtain reproducible crystals of Hdm2 that are of sufficient quality for X-ray crystallization analyses and structural determinations. There is also a need to provide methods for identifying inhibitors of Hdm2 through structure-based drug design and for combining potential inhibitors with the crystals of Hdm2 and analyzing their binding. Further, there is a need to obtain Hdm2 protein samples that, when combined with potential inhibitors, are amenable to forming homogenous crystals.
SUMMARY OF THE INVENTION
The present invention provides modified Hdm2 proteins that are amenable to crystallization and are soluble in E. coli extracts. The present invention further discloses a set of amino acid substitutions of the Hdm2 protein that improve its solubility and/or stability without compromising its ability to bind p53. It is a further object of the present invention to provide a modified Hdm2 protein having an amino acid substitution at one or more of the seven sites defined in Table 1. In one embodiment, the modified Hdm2 protein comprises the amino acid of SEQ ID NO: 4, wherein one or more of the seven specific amino acid residues denoted as X1-X7 of SEQ ID NO: 4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2) or said amino acid sequence comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4. In other embodiments, the modified Hdm2 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 6, 8 10 and 12; or said amino acid sequence comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4.
The present invention further provides isolated and/or recombinant nucleic acids that encode the modified Hdm2 proteins of the present invention, as well as specific peptide fragments and fusion proteins thereof. In one embodiment, the nucleic acid encodes a modified Hdm2 protein comprising the amino acid of SEQ ID NO: 4, wherein one or more of the seven specific amino acid residues denoted as X1-X7 of SEQ ID NO: 4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2) or said amino acid sequence comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4. In other embodiments, the nucleic acid encodes a modified Hdm2 protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 6, 8 10 and 12; or said amino acid sequence comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4. In certain embodiments, the nucleic acid may comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS: 5, 7, 9 and 11. The present invention further provides expression vectors that can comprise any of the nucleic acids of the present invention and a transcriptional control sequence. Preferably the nucleic acids of the present invention are operatively linked to a transcriptional control sequence in expression vectors. Host cells comprising the expression vectors are also part of the present invention. In one particular embodiment, the host cell is an E coli, cell.
In addition, the present invention provides methods for producing the above-mentioned modified Hdm2 proteins. One such embodiment comprises culturing a host cell of the present invention that expresses a nucleic acid encoding a modified Hdm2 protein of the present invention, thereby producing the modified Hdm2 protein. Methods for purifying and/or obtaining the resulting recombinant modified Hdm2 proteins are also included in the present invention, as are the purified recombinant modified Hdm2 proteins.
The present invention further provides compounds that bind to Hdm2. In one such embodiment the compound is an acetylated tripeptide. In a particular embodiment of this type the compound is Ac-6ClWAC3cE. In another embodiment the compound is Ac-6BrWAC3cE.
The present invention further provides protein-ligand complexes between the modified Hdm2 proteins of the present invention and their ligands. Preferred ligands in the complex are Ac-6ClWAC3cE and Ac-6BrWAC3cE.
Crystals comprising a modified Hdm2 protein, and/or one of the protein-ligand complexes of the present invention, also are part of the present invention. Preferably, such crystals effectively diffract X-rays for the determination of the atomic coordinates of the protein and/or of the protein-ligand complex to a resolution of greater than 5.0 Å (e.g., at least 3.0 Å, at least 2.5 Å, at least 2.0 Å, or at least 1.5 Å).
The invention provides a crystal comprising a polypeptide selected from (a) a modified Hdm2 protein, characterized by structural coordinates comprising a root mean square deviation of conserved residue backbone atoms of less than about 1.5 Å (e.g., less than about 1.0 Å, less than about 0.5 Å, or less than about 0.1 Å) when superimposed on backbone atoms described by structural coordinates of Table 3 or (b) a modified Hdm2 protein characterized by structural coordinates comprising a root mean square deviation of conserved residue backbone atoms of less than about 1.5 Å (e.g., less than about 1.0 Å, less than about 0.5 Å, or less than about 0.1 Å) when superimposed on backbone atoms described by structural coordinates of Table 4. In certain embodiments, the crystal may be complexed with a compound that binds to modified Hdm2 (e.g., Ac-6ClWAC3cE or Ac-6BrWAC3cE)
The invention also provides the three-dimensional structure of the modified Hdm2 protein. In one embodiment, the three-dimensional structure is characterized by structural coordinates comprising a root mean square deviation of conserved residue backbone atoms of less than about 1.5 Å (e.g., less than about 1.0 Å, less than about 0.5 Å, or less than about 0.1 Å) when superimposed on backbone atoms described by structural coordinates of Table 3 or 4. The present invention further provides methods of using this three-dimensional structural information in drug discovery and/or to solve corresponding structures of Hdm2 homologues, other crystalline forms of Hdm2 mutants, and co-complexes of Hdm2 and its ligands.
In another aspect of the present invention, methods are provided for obtaining a crystal comprising a modified Hdm2 protein. In one embodiment, the crystal is obtained by vapor diffusion.
In another aspect, the present invention provides a crystalline form of Hdm2 protein that is amenable to ligand soaking experiments, which enables X-ray crystallographic structural determinations to be performed on multiple Hdm2-ligand complexes in rapid succession.
The present invention further provides methods of obtaining a crystal comprising a protein-ligand complex between a ligand and a polypeptide comprising a modified Hdm2 protein. The present invention further provides methods for exchanging ligands within a crystal.
In yet another aspect, the present invention provides a method for designing, selecting and/or optimizing a compound and then evaluating it for use as an inhibitor of Hdm2. One such embodiment comprises obtaining a set of atomic coordinates that define the three-dimensional structure of the modified Hdm2 protein from a crystal of the present invention. In a related embodiment, a set of atomic coordinates that define the three-dimensional structure of the protein-ligand binding complex from a crystal of the present invention is obtained. In either case, a potential agent is then designed, selected or optimized by performing structure-based drug design with the atomic coordinates obtained. Preferably, the design or selection is performed in conjunction with computer modeling.
In another aspect, the invention provides a method for evaluating the ability of a potential inhibitor to associate with Hdm2 comprising employing computational means to perform a fitting operation between the potential inhibitor and the structure coordinates of Hdm2 and quantitating the association between the potential inhibitor and Hdm2.
A compound that is predicted to inhibit Hdm2 can be synthesized, if necessary, and subsequently contacted with Hdm2 or an active fragment thereof. The activity of the compound is then determined by an assay that measures one or more of Hdm2's activities, as described above. A compound that is predicted to inhibit Hdm2 is identified as an inhibitor of Hdm2 when there is a decrease in the activity of Hdm2 in the presence of the agent relative to in its absence.
In a related aspect of the present invention, a computer is provided that comprises a three-dimensional representation of a modified Hdm2 protein in computer memory. One such computer comprises a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, comprising the atomic coordinates of Table 3 or 4. In another embodiment, the computer comprises a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, comprising the atomic coordinates of Table 3 or 4. In yet another embodiment, the computer comprises a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, comprising the atomic coordinates of Table 3 or 4. Preferably, the computer further comprises a working memory for storing instructions for processing the machine-readable data, a central-processing unit coupled to the working memory and to the machine-readable data storage medium for processing the machine readable data into a three-dimensional representation of the modified Hdm2 protein and/or Hdm2 protein-ligand complex. More preferably, the computer includes a display coupled to the central-processing unit for displaying the three-dimensional representation.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides stable modified Hdm2 proteins produced by introducing an amino acid substitution into one or more of a unique set of amino acid residues of Hdm2. The modified Hdm2 proteins of the present invention have an improved solubility and form novel crystals that heretofore were unattainable with the wild-type Hdm2 protein. The present invention further provides methods for generating and purifying these modified Hdm2 proteins. The modified Hdm2 proteins of the present invention may be used for the structural determination of Hdm2 by X-ray crystallography and/or NMR.
Crystals of the modified Hdm2 protein and their resulting structures can be used to design high affinity inhibitors of Hdm2 that may be used in the treatment of cancer. Such drugs may be particularly useful to treat soft-tissue tumors, osteosarcomas and oesophageal carcinomas.
Structure-based drug design is the most efficient method for such drug development. In one common paradigm, a three dimensional structure is determined for a protein, e.g., the modified Hdm2, and/or a corresponding protein-ligand complex. Potential antagonists (e.g., inhibitors and/or potential drugs) of the protein are then identified and/or designed with the aid of computer modeling [Bugg et al., Scientific American, December: 92-98 (1993); West et al., TIPS, 16:67-74 (1995); Dunbrack et al., Folding & Design, 2:27-42 (1997)]. The drug candidates are then selected and tested. The most promising drug candidates are identified and then combined with the protein in a crystalline protein-ligand complex. The three-dimensional structure of the protein-ligand complex is then determined, and new potential antagonists of the protein are identified and/or designed with the aid of computer modeling. This process can then be continued in successive iterations until a lead drug candidate is identified.
Heretofore, the ability to perform structure based drug design with Hdm2 was severely hampered due to the lack of a crystalline form of the Hdm2 that is conducive for such studies. The expression and purification of a modified Hdm2 protein that when placed in a protein-ligand complex can form a monodisperse preparation, as disclosed herein, is therefore critical for the initiation of a structure based drug design program.
In addition, the present invention provides two specific ligands for Hdm2, the acetylated tripeptides, Ac-6ClWAC3cE and Ac-6BrWAC3cE. Their precise chemical structures are provided below. These tripeptides can be used to bind the modified Hdm2 proteins of the present invention to form a protein-ligand complex that is then crystallized. Such X-ray diffractable crystals can be used for structure based drug design to identify anti-tumor drugs.
Patches of hydrophobic amino acid residues on the surface of the Hdm2 protein were initially determined to be a critical factor leading to the relative insolubility of the wild-type Hdm2 protein. Interrupting these hydrophobic patches by replacing selected surface hydrophobic amino acid residues with hydrophilic amino acid alternatives were found to increase the solubility of the ensuing modified Hdm2 protein, as well as create new crystal contact sites.
The present invention therefore provides several approaches for identifying appropriate hydrophobic amino acid residues to be replaced by more hydrophilic alternatives. One such approach entails employing the Clustalw program to align the amino acid sequences of Hdm2 and Hdm4 analogs from a number of species to identify natural amino acid variations. In one particular alignment, protocol Hdm2 or Hdm4 sequences from the following species were used: Brachydanio rerio (Zebrafish), Canis familiaris (Dog), Equus caballus (Horse), Homo sapiens (Human), Mesocricetus auratus (Golden Hamster), Mus musculus (Mouse), Xenopus laevis (African Clawed Frog) and Gallus gallus (Chicken). Seven Hdm2 surface amino acid residues in the hydrophobic patches of Hdm2 were identified and the preferred alternative amino acids at those sites noted, see Table 1 below. In a related approach, the seven amino acid substitutions were selected to specifically increase Hdm2 solubility, see modified Hdm2 (HK5) having the amino acid sequence of SEQ ID NO: 12.
In still another approach, the amino acid sequences of Hdm2 and XMdm2 were compared to identify the positions of surface, solvent exposed, hydrophobic amino acid residues of the human protein that were occupied by more hydrophilic residues in the corresponding Xenopus laevis amino acid sequence. From this protein surface analysis, four potential amino acid substitutions were chosen: Human: Xenopus laevis: F55Y, Y76H, Y104S, V109S.
In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1996) (herein “Ausubel et al., 1996”).
As used herein the following terms shall have the definitions set out below:
As used herein the term “polypeptide” is used interchangeably with the term “protein” and is further meant to encompass peptides. Therefore, as used herein, a polypeptide is a polymer of two or more amino acids joined together by peptide linkages. Preferably, a polypeptide is a polymer comprising twenty or more amino acid residues joined together by peptide linkages, whereas a peptide comprises five to twenty amino acid residues joined together by peptide linkages.
As used herein a polypeptide “consisting essentially of” or that “consists essentially of” a specified amino acid sequence is a polypeptide that
(i) retains an important characteristic of the polypeptide comprising that amino acid sequence, e.g., the ability to bind p53 and/or act as a ligase for conjugating ubiquitin to p53, and
(ii) further comprises the identical amino acid sequence, except it consists of plus or minus 10% (or a lower percentage), and preferably plus or minus 5% (or a lower percentage) of the amino acid residues. In a particular embodiment, additional amino acid residues included as part of the polypeptide are part of a linked Tag, such as a C-terminal His6 Tag.
The term “generic Mdm2” is used herein to refer to the double minute 2 polypeptide from any species. When used by itself, Mdm2 refers to mouse Mdm2. When used with another species name appended to it, it refers to the Mdm2 ortholog from the named species, e.g., Xenopus Mdm2 refers to the Mdm2 ortholog from Xenopus. “Mdm2” also encompass modified forms thereof.
As used herein the terms “human Mdm2” and “Hdm2” are used interchangeably to denote the human ortholog of Mdm2. “Hdm2” also encompass modified forms thereof. Hdm2 has the GenBank accession number of M92424. Mouse Mdm2 has the GenBank accession number of X58876 [see also, published U.S. patent application 2002/0045192, published Apr. 18, 2002, the contents of which are hereby incorporated by reference in their entirety.] The amino acid sequences provided herein correspond to amino acid residues 17-125 of the full-length Hdm2 protein and retain their numerical designation from that full-length sequence. Therefore, the first amino acid residue of the wild-type Hdm2(17-125) sequence, SEQ ID NO: 2, corresponds to amino acid 17 of the full-length Hdm2 protein.
As used herein a “modified Hdm2” is identical to the wild-type Hdm2(17-125) except it has at least one amino acid substitution, i.e., it has one or more amino acid substitutions. Furthermore, a modified Hmd2 of the present invention comprises an amino acid substitution at one or more of the seven positions listed in Table 1 and denoted in SEQ ID NO: 4. Preferably, that amino acid substitution is one that is specifically defined in Table 1 (and denoted in SEQ ID NO: 4). It is also preferable that a modified Hdm2 protein of the present invention comprises 109 amino acid residues and that the positions of these 109 residues correspond to amino acid residues 17-125 of the full-length wild-type Hdm2 protein.
As used herein, a “conservative amino acid substitution” is the substitution of a functionally equivalent amino acid for an amino acid within the sequence of Hdm2. The conservative amino acid substitution can be at any position in the Hdm2 sequence except for the seven positions identified in Table 1, for which the alternatives are specifically defined. In general, a functionally equivalent amino acid is one having a similar polarity and/or molecular properties for the amino acid within the sequence. Specifically, an amino acid substitute may be selected from other members of the class to which the amino acid belongs. The class of nonpolar amino acids includes alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The class of polar neutral amino acids includes glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The class of positively charged (basic) amino acids includes arginine, and lysine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
Particularly preferred conserved amino acid exchanges are:
(a) Lys for Arg or vice versa such that a positive charge may be maintained;
(b) Glu for Asp or vice versa such that a negative charge may be maintained;
(c) Ser for Thr or vice versa such that a free —OH can be maintained;
(d) Gln for Asn or vice versa such that a free NH2 can be maintained; and
(e) Ile for Leu or for Val or vice versa as roughly equivalent hydrophobic amino acids.
As used herein the term “specific peptide fragment” is a peptide that comprises at least six amino acid residues, and preferably at least twelve amino acid residues of a modified Hdm2 protein that differs from the corresponding fragment of the wild-type Hdm2 by at least one amino acid residue. Furthermore, the different amino acid residue (or different residues) is located at a position that corresponds to one (or more) of the seven variable sites denoted in SEQ ID NO: 4 and listed Table 1 below.
As used herein the term “chimeric” protein is meant to include fusion proteins. “Chimeric” proteins of the present invention comprise at least a portion of a non-Hdm2 protein or peptide joined via a peptide bond to at least a portion of an Hdm2 protein, preferably a modified Hdm2. Chimeric proteins can have additional structural, regulatory, and/or catalytic properties. As used herein a chimeric protein can contain multiple additions to at least a portion of a modified Hdm2 protein, e.g., it can comprise both a His6Tag and a signal sequence. In a particular embodiment the chimeric protein functions as a means of detecting and/or isolating the polypeptide or fragment thereof after a recombinant nucleic acid encoding the modified Hdm2 protein or fragment thereof is expressed. Non-Hdm2 amino acid sequences are preferably either amino- or carboxy-terminal to the modified Hdm2 sequence.
As used herein, DNA and protein sequence percent identity can be determined using C, MacVector 6.0.1, Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program using the default parameters.
As used herein a “nucleic acid” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. When referring to a nucleic acid that is double stranded both the “sense” strand and the complementary “antisense” strand are intended to be included. Thus a nucleic acid that is hybridizable to SEQ ID NO: 1, for example, can be either hybridizable to the “sense” strand of SEQ ID NO: 1, which is particularly listed in the SEQUENCE LISTING, or to the “antisense” strand which can be readily determined from that SEQUENCE LISTING.
A DNA “coding sequence” is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.
A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which can then be trans-RNA spliced and translated into the protein encoded by the coding sequence.
A nucleic acid sequence is “operatively linked” to an expression control sequence when the expression control sequence controls or regulates the transcription and translation of that nucleic acid sequence. The term operatively linked includes having an appropriate start signal.
A “heterologous nucleotide sequence” as used herein is a nucleotide sequence that is added by recombinant methods to a nucleotide sequence encoding a modified Hdm2 of the present invention or encoding a fragment thereof, to form a nucleic acid that is not naturally formed in nature. Such nucleic acids can encode chimeric proteins. In addition, as used herein, a heterologous nucleotide sequence need not be a single contiguous nucleotide sequence, but can include multiple non-contiguous nucleotide sequences that have been combined with a nucleotide sequence encoding a modified Hdm2 protein of the present invention, or a portion thereof. A heterologous nucleotide sequence can comprise non-coding sequences including restriction sites, regulatory sites, promoters and the like. In still another embodiment the heterologous nucleotide can function as a means of detecting a nucleotide sequence of the present invention. The present invention provides heterologous nucleotide sequences that, when combined with nucleotide sequences encoding a modified Hdm2 protein or a fragment thereof, are necessary and sufficient to encode all of the chimeric proteins of the present invention.
The phrase “binding to” in regard to a ligand binding to a polypeptide is used herein to include any or all such specific interactions that lead to a protein-ligand binding complex. This can include processes such as covalent, ionic (electrostatic and/or charged), hydrophobic and hydrogen bonding, but does not include non-specific associations such solvent preferences.
As used herein a “ligand” of a Mdm2 protein, e.g., a modified Hdm2 protein, is a compound that binds to the polypeptide in a protein-ligand binding complex. In a specific embodiment of the present invention the ligand inhibits the ability of the Mdm2 protein to bind p53 when the ligand is bound to the Mdm2 protein in a protein-ligand binding complex. In another embodiment, the ligand inhibits the ability of Mdm2 to act as an E3 ligase for conjugating ubiquitin to p53 when the ligand is bound to the Mdm2 protein in a protein-ligand binding complex. Such ligands may also be termed an “inhibitor”.
As used herein, a “protein-ligand binding complex” or polypeptide-compound complex” is a specific association between a polypeptide and the compound that binds to it. In a preferred embodiment of the present invention, the ligand or compound is an inhibitor of the polypeptide. In a particular embodiment of this type, the binding of the inhibitor to the polypeptide occurs at the active site of the polypeptide.
As used herein “incubating a ligand with a crystal” is used interchangeably with “soaking a crystal with a ligand”. Incubating a ligand with a crystal is the contacting of a ligand with a crystal of a polypeptide under the appropriate conditions and for a sufficient time period (e.g., hours to several days) for the ligand to bind to the crystalline polypeptide and form a crystalline protein-ligand complex. Such incubating can further include contacting an excess of a substitute ligand with a crystal of a protein-ligand complex under the appropriate conditions and for a sufficient time period (e.g., hours to several days) for the substitute ligand to replace the initial ligand and form the new crystalline protein-ligand complex.
As used herein the terms “displacing”, “replacing”, and “exchanging” are used interchangeably in regard to the substitution of one ligand in a protein-ligand complex for another.
As used herein an “excess of a substitute ligand” is an amount of that ligand that is sufficient to replace 80% or more, and preferably 90% or more, of the initial ligand in a protein-ligand complex. In a particular embodiment of this type, the concentration of the substitute ligand is about ten-fold higher than the concentration of the protein-ligand complex. In a preferred embodiment, the concentration of the substitute ligand is about one hundred-fold higher than the concentration of the protein-ligand complex.
As used herein the term “X-ray diffractable crystal” is a crystal of a compound, e.g., a protein that yields a discernable diffraction pattern when subjected to 0.5 to 2.5 Å incident X-ray radiation.
As used herein an “X-ray quality crystal” is an X-ray diffractable crystal that can yield meaningful structural data of its crystalline composition when subjected to X-ray crystallographic analysis.
As used herein, and unless otherwise specified, the terms “agent”, “potential drug”, “compound”, or “test compound” are used interchangeably, and refer to chemicals that have or potentially have a use as a modulator of the activity of Mdm2. Preferably the modulator is an inhibitor of the binding complex formed between p53 and Mdm2. Preferably such agents include drugs for the treatment or prevention of a disease and/or condition involving the p53 transcription factor, e.g., cancer. Therefore, such agents may be used, as described herein, in drug assays and drug screens and the like.
As used herein a “small organic molecule” is an organic compound [or organic compound complexed with an inorganic compound (e.g., metal)] that has a molecular weight of less than 3 Kd.
As used herein the terms “approximately” and “about” are used to signify that a value is within twenty percent of the indicated value i.e., an amino acid sequence containing “approximately” 110 amino acid residues can contain between 88 and 132 amino acid residues.
As used herein the phrases “structure based rational drug design”, “structure based drug design” and “structure assisted drug design” are used interchangeably. These phrases are meant to convey a particular method of identifying and/or designing a ligand (preferably an inhibitor) for a specific target protein that includes the use of the three-dimensional structure of that protein and/or its corresponding protein-ligand complex.
Nucleic Acids Encoding Mdm2 Proteins
The nucleic acids can further comprise heterologous nucleotide sequences. In one embodiment, the nucleic acid encodes a modified Hdm2 protein comprising the amino acid of SEQ ID NO: 4, wherein one or more of the seven specific amino acid residues denoted as X1-X7 of SEQ ID NO: 4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2). In a particular embodiment of this type the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 3, wherein at least one nucleotide of one of the codons encoding one or more of the seven specific amino acid residues denoted as X1-X7 of SEQ ID NO: 4 differs from the nucleic acid sequence of wild-type Hdm2(17-125) (SEQ ID NO: 1) and wherein said codon encodes a different amino acid from that of wild-type Hdm2(17-125) (SEQ ID NO: 2).
In one embodiment, the nucleic acid encodes a modified Hdm2 protein comprising the amino acid sequence of SEQ ID NO: 6. In a particular embodiment of this type, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 5. In another embodiment, the nucleic acid encodes a modified Hdm2 protein comprising the amino acid sequence of SEQ ID NO: 8. In a particular embodiment of this type, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 7. In still another embodiment, the nucleic acid encodes a modified Hdm2 protein comprising the amino acid sequence of SEQ ID NO: 10. In a particular embodiment of this type, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 9. In yet another embodiment, the nucleic acid encodes a modified Hdm2 protein comprising the amino acid sequence of SEQ ID NO: 12. In a particular embodiment of this type, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 11. Nucleic acids that consist of the nucleotide sequences that encode the modified Hdm2 proteins of the present invention or that consist of nucleotide sequences that encode specific peptide fragments of those proteins are also provided. The present invention also includes those nucleic acids that encode a modified Hdm2 comprising one or more conservative amino acid substitutions. In certain embodiments, there may be 1-11 conservative amino acid substitutions, preferably 1, 2 or 3 conservative amino acid substitutions. In a related embodiment, the present invention provides nucleic acids that further comprise a heterologous nucleotide sequence.
Obtaining and/or constructing a cDNA that encodes a Mdm2 protein, including Hdm2 proteins and the modified Hdm2 proteins of the present invention, facilitates the production of the large quantities of protein required to perform standard enzyme assays and/or X-ray crystallographic analysis.
The present invention provides specific nucleic acid constructs that allow for the expression and isolation of large quantities of stable and active modified Hmd2 proteins. These nucleic acids can further contain heterologous nucleotide sequences. To express a recombinant protein of the present invention in a host cell, an expression vector can be constructed comprising the corresponding cDNA. The present invention therefore, provides expression vectors containing nucleic acids encoding the modified Hmd2 proteins of the present invention. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode the same or substantially the same amino acid sequence as a nucleic acid encoding a modified Hmd2 protein of the present invention may be used in the practice of the present invention. These include, but are not limited to, allelic genes, homologous genes from other species, which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change. Host cells comprising the expression vectors of the present invention are also provided. One particular host cell, an E. coli cell, is specifically exemplified below.
General methods for the cloning of cDNAs and expression of their corresponding recombinant proteins have been described [see Sambrook and Russell, Molecular Cloning, A Laboratory Manual, 3 edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor L.I. (2000)]. The particular methodology used herein is described in Example 1 below. Preferably, all of the nucleic acid constructs of the present invention are sequence confirmed.
Any technique for mutagenesis known in the art can be used to convert the native (wild-type) Hdm2 to a modified Hdm2 of the present invention, including but not limited to, in vitro site-directed mutagenesis [Hutchinson et al., J. Biol. Chem., 253:6551 (1978); Zoller and Smith, DNA, 3:479-488 (1984); Oliphant et al., Gene, 44:177 (1986); Hutchinson et al., Proc. Natl. Acad. Sci. U.S.A., 83:710 (1986)]. The use of TAB@ linkers (Pharmacia), etc. and PCR techniques also can be employed for site directed mutagenesis [see Higuchi, “Using PCR to Engineer DNA”, in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70 (1989)].
Preferably mutagenesis (i.e., modification) of an Hdm2 transcript is performed in a two step process [Wang and Malcolm, BioTechniques 26:680-682 (1999)]. In Examples 2 and 3 below, two extension reactions were performed in separate tubes in the first stage: (i) one containing the forward primer, and (ii) the other containing the reverse primer. After two cycles, the two reactions are mixed and the standard QuickChange mutagenesis procedure was carried out for an additional 18 cycles. Following amplification, the parental strand was digested with 1 Unit of Dpn1 for 2 hours and an aliquot was transformed into DH5-alpha cells [GeneWiz, New York, N.Y.]. The pET32 Xa/LIC-hdm2 (17-125) vector was used as a template.
When the modified Hdm2 has three amino acid substitutions, as described in the Example 1 below, the GENETAILOR Site-directed Mutagenesis system (Invitrogen, Carlsbad, Calif., USA) was used with the pET32 Xa/LIC-hdm2 (17-125) as the template. In this case the GENETAILOR mutagenesis was performed according to the manufacture's instruction manual, with the lone exception being the use of the pfu turbo DNA polymerase.
The Modified Hdm2 Proteins
In one embodiment, the modified Hdm2 polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 4, wherein one or more of the seven specific amino acid residues denoted as X1-X7 of SEQ ID NO: 4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2). In another embodiment, the modified Hdm2 polypeptide comprises one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4. In certain embodiments, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions in the sequence at sites other than those indicated in Table 1. In preferred embodiments, there may be 1, 2 or 3 conservative amino acid substitutions at sites other than those indicated in Table 1.
In particular embodiments, the amino acid residues at all seven of the variable positions in the amino acid sequence of SEQ ID NO: 4 are different than that of SEQ ID NO: 2. In a specific embodiment of this type, the modified Hdm2 protein is the Hdm2(HK5) comprising the amino acid sequence of SEQ ID NO: 12 or said modified Hdm2 protein comprising the amino acid sequence of SEQ ID NO: 12 comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4 as described above. In other embodiments, the modified Hdm2 protein comprises the amino acid sequence SEQ ID NO: 4 wherein the amino acid residues at six, five, four or three of the seven variable positions (X1-X7) in the amino acid sequence of SEQ ID NO: 4 are different than that of SEQ ID NO: 2. In certain embodiments, the modified Hdm2 protein comprises one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4.
In yet another embodiment, the amino acid residues at two of the seven variable positions in the amino acid sequence of SEQ ID NO: 4 are different than that of SEQ ID NO: 2. In a particular embodiment of this type the modified Hdm2 protein is Hdm2(F55Y/Y76H) protein comprising the amino acid sequence of SEQ ID NO: 10 or said modified Hdm2 protein comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4. In another embodiment, only one of the amino acid residues at the seven variable positions in the amino acid sequence of SEQ ID NO: 4 is different than that of SEQ ID NO: 2. In a particular embodiment of this type, the modified Hdm2 protein is the Hdm2(F55Y) protein comprising the amino acid sequence of SEQ ID NO: 8 or said modified Hdm2 protein comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4. In another particular embodiment, the modified Hdm2 protein is Hdm2(Y76H) protein comprising the amino acid sequence of SEQ ID NO: 6 or said modified Hdm2 protein comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4.
The present invention further provides a modified Hdm2 protein consisting of, or consisting essentially of, SEQ ID NOs: 4, wherein one or more of the seven specific amino acid residues denoted as X1-X7 of SEQ ID NO: 4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2). In certain embodiments, said modified Hdm2 protein comprises one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4. A modified Hdm2 protein is further provided that consists of, or consists essentially of, SEQ ID NOs: 6, 8, 10 or 12, or said modified Hdm2 protein comprising one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4.
Fusion proteins that comprise the modified Hdm2 proteins of the present invention are also provided, as well as specific peptide fragments of those proteins.
In one such embodiment, the modified Hdm2 protein comprises the amino acid sequence of SEQ ID NO: 4. In a preferred embodiment, the modified Hdm2 protein comprises the amino acid sequence of SEQ ID NO: 6. In one embodiment the compound is Ac-6BrWAC3cE. In a preferred embodiment, the compound is AC-6ClWAC3cE.
The amino acid sequences of the wild-type (WT) Hdm2 and the following modified Hdm2 proteins corresponding to amino acid residues 17-125 of the full-length wild-type Hdm2 are provided below:










WT Hdm2(17-125): SEQ ID NO: 2




     SQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRL



 



YDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGT



 



SVSEN



 



Modified Hdm2 (X1-X7): SEQ ID NO: 4



     SQIPASEQETX1VRPKPX2LLKLLKSVGAQKDTYTMKEVLX3YLGQYIMTK



 



RLYDEKQQHIVX4CSNDX5LGDLFGVX6SFSVKEHRKIYTMIX7RNLVVVNQQES



 



SDSGTSVSEN



 



Modified Hdm2 (Y76H): SEQ ID NO: 6



     SQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRL



 



YDEKQQHIVHCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSG



 



TSVSEN



 



Modified Hdm2 (F55Y): SEQ ID NO: 8



     SQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLYYLGQYIMTKRL



 



YDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGT



 



SVSEN



 



Modified Hdm2 (F55Y/Y76H): SEQ ID NO: 10



     SQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLYYLGQYIMTKRL



 



YDEKQQHIVHCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSG



 



TSVSEN



 



Modified Hdm2 (HK5): SEQ ID NO: 12



     SQIPASEQETKVRPKPKLLKLLKSVGAQKDTYTMKEVLHYLGQYIMTKR



 



LYDEKQQHIVKCSNDKLGDLFGVKSFSVKEHRKIYTMIYRNLVVVNQQESSDS



 



GTSVSEN






Modified Hdm2 (HK5) as indicated above has six amino acid substitutions (L27K, L33K, F55H, Y76K, L81 K, and P89K) selected to specifically increase Hdm2 solubility; only two are derived from natural variants.
Column 1 of Table 1 denotes the seven variable amino acid positions in the sequence of SEQ ID NO: 4, and presents the numbering of the amino acid positions in relation to both the full-length wild-type Hdm2 and the corresponding numbering of the wild-type Hdm2(17-125) having the amino acid sequence of SEQ ID NO: 2 (in parentheses). The seven defined positions in SEQ ID NO: 4 in which the amino acid residue can specifically vary are denoted as X1-X7 (column 2). The amino acid residues in the full-length wild-type Hmd2 occupying those seven positions are listed in column 3. All of the natural variants identified by the Clustalw alignment of the amino acid sequences of Hdm2 and Hdm4 analogs using Brachydanio rerio (Zebrafish), Canis familiaris (Dog), Equus caballus (Horse), Homo sapiens (Human), Mesocricetus auratus (Golden Hamster), Mus musculus (Mouse), Xenopus laevis (African Clawed Frog) and Gallus gallus (Chicken) are provided in column 4. All acceptable amino acids are listed in Column 5, including the amino acid residues for the seven respective positions of the wild-type Hmd2, which are in bold.







TABLE 1







Amino Acid Substitutions for SEQ ID NO: 4











A.A.
A.A.

Species



Position
Name 
Hdm2
Variants
Acceptable Variants





 27 (11)
X1
L27
Q
L, K, R, Q, E, D, S


 33 (17)
X2
L33
Q
L, K, R, Q, E, D, S


 55 (39)
X3
F55
L, Y, H
F, H, Y, K, R, Q, E, D, S


 76 (60)
X4
Y76
H
Y, H, K, R, Q, E, D, S


 81 (65)
X5
L81
A, P, C
L, K, R, Q, E, D, S, P, A


 89 (73)
X6
P89
K, V, Q, T
P, K, R, Q, E, D, S


104 (88)
X7
Y104
I, N, R, S
Y, K, R, Q, E, D, S, N









In addition, the modified Hdm2 proteins of the present invention may include conservative amino acid substitutions relative to the wild type sequence of Hdm2 (other than those at the seven positions identified in Table 1, for which the alternatives are specifically defined). In general, there are no more than 11 conservative amino acid substitutions (e.g., no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 conservative amino acid substitutions). In a preferred embodiment, there are no more than 3 conservative amino acid substitutions (0, 1, 2 or 3).
All of the modified Hdm2 proteins of the present invention also can be part of a chimeric protein. In a specific embodiment, a chimeric modified Hdm2 protein is expressed in a prokaryotic cell. Such a chimeric protein can be a fusion protein used to isolate a modified Hdm2 protein of the present invention, through the use of an affinity column that is specific for the protein fused to the modified Hdm2 protein. Examples of such fusion proteins include: a glutathione-S-transferase (GST) fusion protein, a maltose-binding protein (MBP) fusion protein, a FLAG-tagged fusion protein, or a poly-histidine-tagged fusion protein. Specific linker sequences such as a Ser-Gly linker can also be part of such a fusion protein. A chimeric modified Hdm2 protein of the present invention also can be expressed in a eukaryotic cell.
Expression of a chimeric, modified Hdm2 protein, or fragment thereof, as a fusion protein can facilitate stable expression, and/or allow for purification based on the properties of the fusion partner. Thus the purification of the recombinant polypeptides of the present invention can be simplified through the use of fusion proteins having affinity Tags. For example, GST binds glutathione conjugated to a solid support matrix, MBP binds to a maltose matrix, and poly-histidine chelates to a Ni-chelation support matrix [see Hochuli et al., Biotechnology 6:1321-1325 (1998)]. The fusion protein can be eluted from the specific matrix with appropriate buffers, or by treating with a protease that is specific for a cleavage site that has been genetically engineered in between the modified Hdm2 protein and its fusion partner. Alternatively, a modified Hdm2 protein can be combined with a marker protein such as green fluorescent protein [Waldo et al., Nature Biotech. 17:691-695 (1999); U.S. Pat. No. 5,625,048 and WO 97/26333].
Alternatively or in addition, other column chromatography steps (e.g., gel filtration, ion exchange, affinity chromatography etc.) can be used to purify the recombinant modified Hdm2 proteins of the present invention. In many cases, such column chromatography steps employ high performance liquid chromatography or analogous methods in place of the more classical gravity-based procedures. The specific details for the preferred purification procedure of the modified Hdm2 proteins of the present invention are provided in Example 1 below.
In addition, the modified Hdm2 proteins of the present invention and proteins thereof including, specific peptide fragment thereof can be chemically synthesized [see e.g., Synthetic Peptides: A User's Guide, W.H.Freeman & Co., New York, N.Y., pp. 382, Grant, ed. (1992)].
Crystallization of Protein
The modified Hdm2 proteins result in novel crystal forms not obtained with the wild-type protein. In one embodiment, the modified Hdm2 protein may be crystallized in the presence of one of a variety of different compounds (e.g., an small molecule inhibitor or a peptide from p53). Further, a compound complexed to modified Hdm2 may be exchanged in the crystal by a second compound (e.g., a potential inhibitor) by soaking the crystal in a solution containing the second compound. These crystals and the resulting structures can be used to obtain a detailed view of various inhibitors that bind to Hdm2, providing a basis for further design of potent inhibitors of Hdm2 to be used as anticancer agents.
Crystallization may be accomplished by using any of the known methods in the art (Giegé, et al., (1994) Acta Crystallogr. D50: 339-350; McPherson, (1990) Eur. J. Biochem. 189: 1-23). Such techniques include microbatch, hanging drop, seeding and dialysis. Preferably, hanging-drop vapor diffusion (McPherson, (1976) J. Biol. Chem. 251: 6300-6303) or microbatch methods (Chayen (1997) Structure 5: 1269-1274) are used. In each of these methods, it is important to promote continued crystal growth after nucleation by maintaining a supersaturated solution. In the microbatch method, polypeptide is mixed with precipitants to achieve supersaturation, the vessel is sealed and set aside until crystals appear. In the dialysis method, polypeptide is retained in a sealed dialysis membrane that is placed into a solution containing precipitant. Equilibration across the membrane increases the precipitant concentration thereby causing the polypeptide to reach supersaturation levels. It is desirable to use a modified Hdm2 protein preparation having a concentration of at least about 1 mg/mL and preferably about 5 mg/mL to about 60 mg/mL, more preferably about 20 mg/mL to about 50 mg/mL, even more preferably about 30 mg/mL to about 40 mg/mL. Crystallization may be achieved in precipitant solutions containing polyethylene glycol 1000-20,000 (PEG; average molecular weight ranging from about 1000 to about 20,000 Da), preferably about 2000 to about 6000 Da, more preferably about 5000 Da, with concentrations ranging from about 10% to about 50% (w/v). It may also be desirable to include a protein stabilizing agent. If glycerol is chosen as the protein stabilizing agent, it is preferably provided at a concentration ranging from about 0.5% to about 20%. A suitable salt, such as magnesium chloride, potassium chloride, sodium chloride, lithium chloride or sodium citrate may also be desirable in the precipitant solution, preferably in a concentration ranging from about 1 mM to about 2000 mM. The precipitant is preferably buffered to a pH of from about 6.5 to about 9.5, preferably about 7.5-8.5. Specific buffers useful in the precipitant solution may vary and are well-known in the art (Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York). Examples of useful buffers include, but are not limited to, Hepes, Tris, MES and acetate. Crystals routinely grow at a wide range of temperatures. It is, however, preferred that crystals form at temperatures between about 2° C. and about 26° C.
The crystals of the present invention have a wide range of uses. For example, high quality crystals are suitable for X-ray or neutron diffraction analysis to determine the three dimensional structure of Hdm2 and in particular to assist in the identification of the protein's effector sites. Knowledge of these sites and solvent accessible residues allow structure-based design and construction of agonists and antagonists for Hdm2.
In addition, crystallization itself can be used as a purification method. In some instances, a polypeptide or protein crystallizes from a heterogeneous mixture into crystals. Isolation of such crystals by filtration and/or centrifugation, followed by redissolving the polypeptide affords a purified solution suitable for use in growing high-quality crystals that are preferred for diffraction analysis.
Once a crystal of the present invention is grown, X-ray diffraction data can be collected. One method for determining structure with X-ray diffraction data includes use of synchrotron radiation, under standard cryogenic condition; however, alternative methods may also be used. For example, crystals can be characterized by using X-rays produced by a conventional source, such as a sealed tube or a rotating anode. Methods of characterization include, but are not limited to, precession photography, oscillation photography and diffractometer data collection.
The crystallizable compositions provided by this invention may be amenable to X-ray crystallography for providing the three-dimensional structure of a Hdm2 polypeptide. The present invention includes crystals which effectively diffract X-rays for the determination of the atomic coordinates of Hdm2 to a resolution of greater than about 5.0 Ångströms (e.g., about 4.5 Å, about 4.0 Å, about 3 Å, about 2.5 Å, about 2 Å, about 1 Å, about 0.5 Å, about 0.1 Å), preferably greater than about 4.0 Ångströms (e.g., about 3 Å, about 2.5 Å, about 2 Å, about 1 Å, about 0.5 Å, about 0.1 Å), more preferably greater than about 2.8 Ångströms (e.g., about 2.5 Å, about 2 Å, about 1 Å, about 0.5 Å, about 0.1 Å) and most preferably greater than about 2.0 Ångströms (e.g., about 1.5 Å, about 1.0 Å, about 0.5 Å, about 0.1 Å).
The present invention includes Hdm2 crystals whose three-dimensional structure is described by the structure coordinates set forth in Table 3 or 4. The scope of the present invention also includes crystals which possess structural coordinates which are similar to those set forth in Table 3 or 4; preferably, the crystals or the soluble polypeptides which are used to form the crystals exhibit Hdm2 catalytic activity and/or p53 binding (see above). In some embodiments, the crystal comprises a polypeptide that comprises the amino acid sequence of SEQ ID NO: 4, wherein one or more of the seven specific amino acid residues denoted as X1-X7 of SEQ ID NO: 4 differs from that of wild-type Hdm2(17-125) (SEQ ID NO: 2). In other embodiments, the crystal comprises a polypeptide that comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 8 and 12. In another embodiment, the modified Hdm2 polypeptide comprises one or more conservative amino acid substitutions at sites other than that of X1-X7 of SEQ ID NO: 4. Structural similarity between crystals is discussed in detail below.
The term “structure coordinates” refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a beam of X-rays by the atoms (scattering centers) of a molecule. The diffraction data are used to calculate electron density maps and to establish the positions of the individual atoms of the molecule.
Those of skill in the art will understand that a set of structure coordinates for a protein or a protein complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates will have little effect on overall shape.
The present invention includes crystals exhibiting structure coordinates which are similar to those set forth in Table 3 or 4 but for crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, additions, subtractions, rotations or translations to sets of the structure coordinates or any combinations of the above.
Alternatively, modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal may also account for variations in structure coordinates. If such variations are within an acceptable standard error as compared to the coordinates of Table 3 or 4, the resulting three-dimensional shape is considered to be the same and, accordingly, the modified crystal is considered to be within the scope of the present invention.
Various computational analyses may be necessary to determine whether a crystal is sufficiently similar to the crystals whose structural coordinates are set forth in Table 3 or 4 as to be considered the same. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, Calif.) version 4.1, and as described in the accompanying User's Guide.
The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. In general, the procedure used in Molecular Similarity to compare structures is divided into four steps: 1) input the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation; and 4) analyze the results.
Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). Since atom equivalency within QUANTA is defined by user input, for the purpose of this invention we will define equivalent atoms as protein backbone atoms (N, CA, C and O) for all conserved residues between the two structures being compared.
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 Ångströms, is reported by QUANTA.
The term “root mean square deviation” (RMSD) is a commonly known term in the art which, in general, means the square root of the arithmetic mean of the squares of the deviations from the mean distance of corresponding atoms. It is a way to express the deviation or variation from a trend or object.
For the purpose of this invention, any set of structure coordinates of a molecule that has a RMSD of conserved residue backbone atoms (N, CA, C, O) of less than about 2.0 Å when superimposed—using backbone atoms—on the relevant structure coordinates of Table 3 or 4 are considered identical and are within the scope of the present invention. Preferably the crystal is a modified Hdm2 protein as defined above. Preferably, the root mean square deviation is less than about 1.5 Å, more preferably less than 1.0 Å, even more preferably, the root mean square deviation is less than about 0.5 Å and most preferably, the root mean square deviation is less than about 0.1 Å.
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 one embodiment, the modified Hdm2 protein comprises the amino acid sequence of SEQ ID NO: 6. In a particular embodiment of this type, the crystal has the space group of P212121, having unit cell dimensions of: a=41.1, b=66.1, c=96.1 Angstroms. In a particular embodiment of this type, the modified Hdm2 protein comprises the amino acid sequence of SEQ ID NO: 10. In a particular embodiment of this type, the crystal has the space group of P212121, having unit cell dimensions of: a=38.0, b=45.3, c=64.0 Angstroms. In a related embodiment, the crystal comprises a protein-ligand binding complex with the modified Hdm2 protein.
In accordance with the present invention, the structure coordinates of the Hdm2 polypeptide and portions thereof may be stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and X-ray crystallographic analysis of a protein crystal (e.g., for producing a three-dimensional representation of Hdm2). Accordingly, one aspect of this invention provides a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table 3 or 4. The machine-readable data storage medium may also include any set of structure coordinates of a molecule that has a root mean square deviation of conserved residue backbone atoms (N, CA, C, O) of less than about 1.5 Å, preferably, less than about 1.0 Å, more preferably less than about 0.5 Å and even more preferably less than about 0.1 Å when superimposed—using backbone atoms—on the relevant structure coordinates of Table 3 or 4.
A computer system, useful in reading the machine readable data storage medium, includes a computer comprising a central processing unit (“CPU”) and a memory storage device and is also within the scope of the present invention. In general, the computer system may be any computer with an operating system such as MS-DOS, PC-DOS, Windows, OS/2, Unix, Unix variant or MaCOS. Particularly preferred computer systems are the Silicon Graphics Octane workstation or Compaq AlphaServer DS20. Other hardware systems and software packages will be known to those skilled in the art.
Input hardware coupled to the computer system by input line, may be implemented in a variety of ways. Machine-readable data of this invention may be input via the use of a modem or modems connected by a telephone line or a dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. A keyboard may also be used as an input device.
Output hardware, coupled to the computer system by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a display terminal (e.g., a cathode ray tube (CRT)) for displaying a graphical representation of the three dimensional structure of Hdm2 or a portion thereof using a program such as INSIGHT (Molecular Simulations Inc., San Diego, Calif.) or 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. In preferred embodiments, the computer possesses a display which is displaying a three dimensional representation of Hdm2 or a fragment or homologue thereof.
In operation, the central processing unit (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. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the computer system are included as appropriate throughout the following description of the data storage medium.
A magnetic data storage medium can be encoded with a machine-readable data by a computer system as described above. Storage medium may be, for example, a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation can be altered magnetically. The magnetic domains of the coating of medium may be 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 as described herein. Storage medium may also have an opening for receiving the spindle of a disk drive or other data storage device. Alternatively, an optically-readable data storage medium can be encoded with such machine-readable data, or a set of instructions. Medium can be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable.
In general, in the case of CD-ROM, as is well known, disk coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of the pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the coating.
In general, in the case of a magneto-optical disk, as is well known, disk coating has no pits, 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. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.
Structure Based Drug Design
The present invention permits the use of structure-based drug design techniques to design, select, and synthesize chemical entities, including inhibitory compounds that are capable of binding to a Hdm2 polypeptide. Also, de novo and iterative drug design methods can be used to develop drugs from the structure of the Hdm2 crystals of this invention.
One particularly useful drug design technique enabled by this invention is structure-based drug design. Structure-based drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of protein/compound complexes.
Those skilled in the art will appreciate that association of natural ligands or substrates with the binding pockets of their corresponding receptors, enzymes or specific binding proteins is the basis of many biological mechanisms of action. The term “binding pocket”, as used herein, may refer to any region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity or compound. Similarly, drugs may exert their biological effects through association with the binding pockets of receptors and/or specific binding proteins. Such association may occur with all or any part of the binding pockets. An understanding of such associations will help lead to the design of drugs having more favorable associations with the target protein, and thus, improved biological effects. Therefore, this information is valuable in designing potential protein inhibitors, such as inhibitors of Hdm2.
In iterative structure-based drug design, crystals of a series of protein/compound complexes are obtained and then the three-dimensional structure of each complex is solved. Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of a new polypeptide, solving the three-dimensional structure of the polypeptide, and comparing the associations between the new protein and previously solved protein. By observing how changes in the compound affected the protein/compound associations, these associations may be optimized.
In some cases, iterative structure-based drug design is carried out by forming successive protein-compound complexes and then crystallizing each new complex. Alternatively, a pre-formed protein crystal is soaked in the presence of an inhibitor or other binding compound, thereby forming a protein/compound complex and obviating the need to crystallize each individual protein/compound complex. Advantageously, the modified Hdm2 crystals provided by this invention may be soaked in the presence of compounds, such as Hdm2 inhibitors, substrates or other ligands to provide novel Hdm2/compound crystal complexes. In one embodiment, the complexes may be produced and screened using high throughput methods to quickly identify or design potential inhibitors of Hdm2.
The structure coordinates set forth in Table 3 or 4 can also be used to aid in obtaining structural information about another crystallized molecule or molecular complex. This may be achieved by any of a number of well-known techniques, including molecular replacement.
The structure coordinates set forth in Table 3 or 4 can also be used for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to Hdm2. In particular, structural information about another crystallized molecule or molecular complex may be obtained by well-known techniques, including molecular replacement.
Therefore, another aspect of this invention provides a method of utilizing molecular replacement to obtain structural information about a crystallized molecule or molecular complex, whose structure is unknown, comprising the steps of generating an X-ray diffraction pattern from said crystallized molecule or molecular complex and applying crystallographic phases derived from at least a portion of the structure coordinates set forth in Table 3 or 4 to the X-ray diffraction pattern to generate a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown.
Once the structure coordinates of a protein crystal have been determined, they are useful in solving the structures of other crystals. In addition, the structure of Hdm2 homologues may be determined from the structural coordinates of the present invention. For example, polypeptides may be crystallized and their structures elucidated by, for example, difference Fourier techniques and molecular replacement.
By using molecular replacement, all or part of the structure coordinates of the Hdm2 polypeptide provided by this invention (and set forth in Table 3 or 4) can be used to determine the previously unknown structure of a crystallized molecule or molecular complex more quickly and efficiently than attempting to determine such information ab initio.
Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that cannot be measured experimentally. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process. However, when the crystal structure of a protein containing a homologous portion has been solved, the phases from the known structure may provide a satisfactory estimate of the phases for the unknown structure.
Thus, this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of the modified Hdm2 crystal according to Table 3 or 4 within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed X-ray diffraction pattern amplitudes to generate an election density map of the structure whose coordinates are unknown. This, in turn, can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallized molecule or molecular complex (Lattman, “Use of the Rotation and Translation Functions”, in Meth. Enzymol., 115: 55-77 (1985); Rossman, ed., “The Molecular Replacement Method”, Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York (1972)).
Phase information from the structure coordinates of the present invention may be used to elucidate the structure of other crystals. For example, the structure of Hdm2 in complex with other atoms or molecules may be elucidated. Such complexes include, for example, those containing atoms soaked into or cocrystallized within the crystal lattice. Other structures which can be elucidated using the phase information of the present invention include for example other proteases or homologues or mutants thereof having sufficient three-dimensional structure similarity to Hdm2 complex as to be solved using molecular replacement. Also, these protein molecules in a complex with a small molecule substrate(s), inhibitor(s), transition state analog(s), product(s) or analog(s) of any of these may also be solved using the phase information of the present invention. Other complexes whose structure can be elucidated from the phase information of the present invention include a modified Hdm2 complexed with an inhibitor other than those presented herein. Complexes containing a combination of the above molecules may also be solved using the phase information of the present invention.
The structure of any portion of any crystallized molecule or molecular complex that is sufficiently homologous to any portion of the modified Hdm2 protein can be solved by this method. The difference Fourier method simply calculates an electron density map using phases calculated from the structure coordinates and observed diffraction amplitudes from a crystal of an unknown structure. This method is often used to solve structures of protein/ligand complexes where the ligand is small and does not affect the crystal form significantly.
In a preferred embodiment, the method of molecular replacement is utilized to obtain structural information about a molecule wherein the molecule comprises a Hdm2 polypeptide complex. The structure coordinates of modified Hdm2 provided by this invention are particularly useful in solving the structure of other crystal forms of Hdm2 polypeptide complexes. This approach enables the determination of the optimal sites for interaction between chemical entities, including interaction of candidate inhibitors with Hdm2.
Modified Hdm2 crystals may be studied using well-known X-ray diffraction techniques and may be refined versus X-ray data to 3 Å resolution or better to an Rfree value of about 0.40 or less using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g., Blundell & Johnson, supra; Meth. Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985)). This information may be used to optimize known Hdm2 inhibitors and to design new Hdm2 inhibitors.
Once a three-dimensional structure of a crystal comprising a modified Hdm2 protein in a protein-ligand complex is determined, the potential inhibitor of Hdm2 can be examined through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK [Dunbrack et al., Folding & Design, 2:27-42 (1997)]. This procedure can include computer fitting of potential inhibitors to the modified Hdm2 protein to ascertain how well the shape and the chemical structure of the potential modulator will interact with the modified Hdm2 protein [Bugg et al., Scientific American, December:92-98 (1993); West et al., TIBS, 16:67-74 (1995)]. Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of the modified Hdm2 protein with an inhibitor.
Generally the tighter the fit, the lower the steric hindrances, and the greater the attractive forces, the more potent the inhibitor, since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug the more likely that the drug will not interact as well with other proteins. This will minimize potential side-effects due to unwanted interactions with other proteins.
Compounds that may be used initially have been discussed by Chene [Nature 3:102-109 (2003)]. In addition, the present invention discloses the acetylated tripeptides, (a) Ac-6ClWAC3cE and (b) Ac-6BrWAC3cE, as shown respectively below, which can individually bind a modified Hmd2 protein in protein-ligand complex and form an X-ray diffractable crystal.




These ligands then can be systematically modified by computer modeling programs until one or more promising potential analogs are identified. Such analysis has been shown to be effective in the development of HIV protease inhibitors [Lam et al., Science 263:380-384 (1994); Wlodawer et al., Ann. Rev. Biochem. 62:543-585 (1993); Appelt, Perspectives in Drug Discovery and Design 1:23-48 (1993); Erickson, Perspectives in Drug Discovery and Design 1:109-128 (1993)]. Alternatively, a potential inhibitor initially can be obtained by screening a random peptide library or a chemical library. In the former case, a random peptide library can be produced by recombinant bacteriophage, for example, [Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)]. This approach may be particularly useful in this case since the natural binding partner for Mdm2 is the p53 protein. In any case, a peptide selected in this manner could then be systematically modified by computer modeling programs, as described above.
If a potential inhibitor is a small organic compound, it can be selected from a library of chemicals, as are commercially available. Alternatively, the small organic compound may be synthesized de novo. Once obtained, the potential inhibitor can be further tested in a standard binding and/or functional assay with Hdm2, a modified Hdm2 protein or active fragments thereof.
For example, a binding assay can be performed following the attachment of the Hdm2 protein to a solid support. Methods for placing Hdm2 protein on the solid support are well known in the art and include such things as linking biotin to the Hdm2 protein and linking avidin to the solid support. The solid support can be washed to remove unbound protein. A solution of a labeled potential inhibitor can be contacted with the solid support. The solid support is washed again to remove the potential inhibitor not bound to the support. The amount of labeled potential inhibitor remaining with the solid support, and thereby bound to the Hdm2 protein can be determined. Alternatively, or in addition, the dissociation constant between the labeled potential inhibitor and the Hdm2 protein, for example, can be determined. Suitable labels for either the Hdm2 protein or the potential inhibitor include, radioactive labels (e.g., 14C, 1H,) and fluorescent labels such as fluorescein isothiocyanate (FITC).
In another embodiment, a Biacore machine can be used to determine the binding constant of the Hdm2 protein with a potential inhibitor [O'Shannessy et al. Anal. Biochem. 212:457-468 (1993); Schuster et al., Nature 365:343-347 (1993)].
In addition, an inhibitor can be identified using an ELISA-based competition assay [Bottger et al., Oncogene 13:2141-2147 (1996); Bottger et al., J. Miol. Biol. 269:744-756 (1997)]. For example, the p53 protein or Hdm2-binding fragment thereof can be biotinylated and immobilized on a streptavidin-coated ELISA plate. Hdm2 is then incubated alone (in a control) or in the presence of a potential inhibitor. The Hdm2 solutions are then individually contacted with the immobilized p53 protein or Hdm2-binding fragment thereof. The binding of the Hdm2 is then determined, e.g., with a detectable anti-Hdm2 antibody. When the amount of Hdm2 detected is lower in the sample incubated with the potential inhibitor relative to the control, the potential inhibitor is identified as an inhibitor.
When a promising inhibitor is identified, a crystal comprising a protein-ligand complex of the inhibitor and a modified Hdm2 protein can be prepared. The three-dimensional structure of the resulting crystalline protein-ligand complex can then be determined by molecular replacement analysis, for example.
Molecular replacement involves the use of a known three-dimensional structure as a search model to determine the structure of a closely related molecule or protein-ligand complex in a different crystalline form. The measured X-ray diffraction properties of the new crystal are compared with the search model structure to compute the position and orientation of the protein in the new crystal. Computer programs that can be used include: X-PLOR [Brunger et al., Acta Crystallogr. A 46:585-593 (1990); Brunger et al., Acta Crystallogr. D Biol Crystallogr., 54:905-921 (1998)], CNS, (Crystallography and NMR System, a next level of XPLOR), and AMORE [Navaza, Acta Crystallographics ASO, 157-163 (1994)]. Once the position and orientation are known, an electron density map can be calculated using the search model to provide X-ray phases. Thereafter, the electron density is inspected for structural differences and the search model is modified to conform to the new structure. Using this approach, it is possible to solve the three-dimensional structures of crystals of any protein-ligand complex of the modified Hdm2 protein.
For all of the drug screening assays described herein, further refinements to the structure of the drug will generally be necessary and can be made by the successive iterations of any and/or all of the steps provided by the particular drug screening assay and/or in combination with other such drug screening assays.
A candidate drug selected by performing structure based drug design can then be assayed in situ and/or in vivo. For example, a candidate drug can be evaluated for cellular activity by incubating the candidate drug in cell cultures, e.g. using HCT-116 cells or OSA-CL cells, and then measuring its effect on cellular proliferation and expression levels of proteins that are transcriptionally regulated by p53 such as p21waf1 and Hdm2 [Chene et al., J. Mol. Biol. 299:245-256 (2000)]. A candidate drug is identified as a drug if in the presence of the drug relative to in its absence, the amount of cellular proliferation decreases and/or the amount of a protein that is transcriptionally regulated by p53 increases.
Indeed, methods of testing such potential candidate drugs in animal models are well known in the art. The potential drugs can be administered by a variety of ways including topically, orally, subcutaneously, or intraperitoneally depending on the proposed use. Generally, at least two groups of animals are used in the assay, with at least one group being a control group that is administered the administration vehicle without the potential drug.







TABLE 2







TABLE OF SEQUENCES











SEQ ID NO:
Type
Description















1
N.A.
WT Hdm2 (17-125)



2
A.A.
WT Hdm2 (17-125)



3
N.A.
modified Hdm2 (X1-X7)



4
A.A.
modified Hdm2 (X1-X7)



5
N.A.
modified Hdm2 (Y76H)



6
A.A.
modified Hdm2 (Y76H)



7
N.A.
modified Hdm2 (F55Y)



8
A.A.
modified Hdm2 (F55Y)



9
N.A.
modified Hdm2 (F55Y/Y76H)



10
A.A.
modified Hdm2 (F55Y/Y76H)



11
N.A.
modified Hdm2 (HK5)



12
A.A.
modified Hdm2 (HK5)



13
N.A.
Hdm2 (F55Y) Primer



14
N.A.
RChdm2 (F55Y) Primer



15
N.A.
Hdm2 (Y76H) Primer



16
N.A.
RChdm2 (Y76H)



17
N.A.
Y104S-GTAILOR-F Primer



18
N.A.
Y104-GTAILOR-R Primer










The present invention may be better understood by reference to the following non-limiting examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way be construed as limiting the broad scope of the invention.
EXAMPLES
Example 1
Preparation of Modified Hdm2
Preparation of Hdm2 Constructs
Hdm2 was either modified with a single amino acid change, or with a double amino acid change using the QuickChange kit (Stratagene, La Jolla, Calif., USA) and the pET32-Xa/LIC-hdm2 (17-125) vector as a template. The pET32-Xa/LIC parental vector was obtained from Novagen (San Diego, Calif.). Hdm2(F55Y), Hdm2(Y76H) and Hdm2(F55Y/Y76H) were constructed in this manner.
A modified Hdm2 having three amino acid substitutions, Hdm2(F55Y/Y76H/Y104S) was generated using GENETAILOR Site-directed Mutagenesis system (Invitrogen, Carlsbad, Calif., USA) with the vector indicated above as the template. The following primers were used to generate the above-identified modified Hdm2 proteins:










(1)
Hdm2(F55Y) Primer: SEQ ID NO:13
5′ CTATGAAAGAGGTTCTTTATTATCTTGGCCAGTATATTATGAC 3′



 


(2)
RChdm2(F55Y) Primer: SEQ ID NO:14
5′ GTCATAATATACTGGCCAAGATAATAAAGAACCTCTTTCATAG 3′


 


(3)
Hdm2(Y76H) Primer: SEQ ID NO:15
5′ GAGAAGCAACAACATATTGTACATTGTTCAAATGATCTTCTAGG 3′


 


(4)
RChdm2(Y76H) Primer: SEQ ID NO:16
5′ CCTAGAAGATCATTTGAACAATGTACAATATGTTGTTGCTTCTC 3′


 


(5)
Y104S-GTAILOR-F Primer: SEQ ID NO:17
5′ CAGGAACTTGGTAGTAGTCAATCAGCAGG 3′


 


(6)
Y104-GTAILOR-R Primer: SEQ ID NO:18
5′ GACTACTACCAAGTTCCTGGAGATCATGGT 3′






QuickChange mutagenesis was performed in two steps as previously described [Wang et al., BioTechniques 26:680-682(1999)]. In the first stage two extension reactions were performed in separate tubes; one containing the forward primer and the other containing the reverse primer. After two cycles, the two reactions were mixed and the standard QuickChange mutagenesis procedure was carried out for an additional 18 cycles. Following amplification, the parental strand was digested with 1U of Dpn1 for 2 hours and an aliquot was transformed into DH5-α cells. The GENETAILOR mutagenesis was performed according to the manufacturer's instructions except that pfu turbo DNA polymerase was used instead of the polymerase recommended by manufacture. All constructs were confirmed by sequencing (GeneWiz, New York, N.Y.).
Expression of Modified Hdm2
A colony from freshly transformed cells was grown at 37° C. to an optical density (OD) of 2.0 in 10 ml TERRIFIC broth (Mediatech, Inc.) containing 100 μg/ml carbenicilin and 1% glucose. This 10 ml culture was then used to inoculate a 1 liter culture having the same medium composition. The 1 liter culture was grown at 37° C. to an OD of 2.0, stored at 4° C. overnight, and then used to inoculate a 10 liter tank containing TERRIFIC broth and 100 μg/ml carbenicilin. The 10 liter culture was grown at 37° C. to an OD of 1.5-2.0 before lowering the temperature to 16° C. The 10 liter culture was then induced with 1 mM IPTG, and the cells were harvested 18 hours post-induction.
Purification of Modified Hdm-2 Proteins
The purification protocol as exemplified herein for Hdm2(F55Y/Y76H) is applicable for all of the modified Hdm2 proteins of the present invention.
IPTG-induced cells containing Hdm2(F55Y/Y76H) were harvested from the 10 liter fermentation, as described above. The cells were suspended in 500 ml of 50 mM Tris-Cl Buffer, pH 8.0rt (room temperature), 0.3 M NaCl, 10% (v/v) glycerol, 5 mM β-mercaptoethanol, 25 mM imidazole, 18,000 Units/liter endonuclease (ultrapure benzonase; SIGMA), and 6 ml/liter of CALBIOCHEM Protease Inhibitor Cocktail III. (All processing was performed at 4° C.) To homogenize the resulting cell suspension, it was passed through a large OMNI Mixer probe for 45 seconds, three times. The cell suspension was kept on ice for 2 minutes between each 45 second passage. The cells were then broken by three passages of the homogenized cell suspension through a Microfluidizer. The extract was recovered by centrifugation at 205,000×g for 80 minutes at 4° C.
The resulting 645 ml extract was mixed end over end for 50 minutes with 28 ml of QIAGEN Ni-NTA SUPERFLOW resin, which had been equilibrated with the equilibration buffer [50 mM Tris-Cl, pH 8.0rt, 0.3 M NaCl, 5 mM β-mercaptoethanol and 25 mM imidazole]. The supernatant was decanted, and the resin was then washed with 600 ml of the equilibration buffer. The resin was poured into a 2.6×5.3 cm column, washed with an additional 200 ml of equilibration buffer at 3.6 ml/min, and finally eluted with 50 mM Tris-Cl, pH 8.0rt, 0.1 M NaCl, 250 mM imidazole, 5 mM β-mercaptoethanol and 20% glycerol.
A 0.64 ml volume of 0.5 M CaCl2 was added to the eluted fusion protein pool (195 mg of protein in 63 ml of elution buffer). The pooled protein was then diluted to 1 mg/ml with 50 mM Tris-Cl, pH 8.0rt, 0.1 M NaCl, 10% glycerol, 5 mM CaCl2 and 5 mM β-mercaptoethanol. A 1.95 ml volume of 2000 Units/ml Factor Xa protease (NOVAGEN) was added, and the pooled protein was dialyzed overnight versus 3.87 liters of the same buffer.
A 4.33 ml volume of 1 M imidazole, pH 8.0, was added to the 200 ml of cleaved, pooled fusion protein, to bring the imidazole concentration to 24 mM. The pooled protein was then applied at a rate of 3.6 m/min to a 50 ml (2.6×9.4 cm) column of QIAGEN Ni-NTA SUPERFLOW resin that had been equilibrated with equilibration buffer. The column was then washed with the equilibration buffer.
The 230 ml flow-through was dialyzed versus three changes (6 liters, 5 liters and 5 liters) of Buffer A [25 mM Hepes-KOH, pH 7.5, 0.15 M KCl, 1 mM Na2-EDTA, 0.03% sodium azide and 5 mM dithiothreitol]. All manipulations of the modified Hdm2 were in Buffer A from this point on. The dialyzed pooled protein was concentrated to 8.5 ml with an AMICON YM10 membrane, centrifuged at 205,000×g for 15 minutes, and then applied to a 2.6×60 cm column of PHARMACIA SUPERDEX-75 at a flow rate of 0.8 ml/min. The resulting eluant was collected in 3.2 ml fractions.
Fractions 66-75 contained the purified, modified Hdm2 monomer. These fractions were pooled and a protein concentration of 3.6 mg/ml was determined using ε276=10,150 M−1 cm−1 in 20 mM sodium phosphate, pH 6.5, with 6 M guanidine hydrochloride (ExPASy-ProtParam Tool). This determination correlated with that determined by the Bradford dye binding assay (BIORAD), using bovine serum albumin as the protein standard.
EXAMPLE 2
Preparation and Crystallization of Hdm2 F55Y/Y76H-Tripeptide Complex
The modified Hdm2 (F55Y/Y76H) protein was prepared by means of QuickChange mutagenesis as disclosed in Example 1 using the appropriate primers. A small scale expression study was carried out to evaluate the solubility and expression level of this construct. The expression level of the soluble Hdm2(F55Y/Y76H) protein is 2 to 3 fold higher than wild type. 110 mgs of the Hdm2(F55Y/Y76H) was purified from a 10 L culture through the four-step purification protocol described above.
A 13 ml aliquot of the 3.6 mg/ml pool was concentrated to a 1.25 mM concentration (in 3 ml), on an AMICON 5000 mwco Ultrafree membrane. A 3 ml aliquot of 8.5 mM Ac-6ClWAC3cE (MW=535) in Buffer A was added to the concentrated aliquot. The complex was incubated at room temperature for 10 minutes, and then concentrated as above, to a final volume of 1.3 ml.
The HDM2(F55Y/Y76H) protein-tripeptide complex was crystallized using a hanging-drop vapor diffusion method. The protein (0.5 μl; 34 mg/ml) in 25 mM Hepes-potassium hydroxide, pH7.5, 0.15 M potassium chloride, 1 mM EDTA, 0.03% sodium azide and 5 mM DTT buffer was mixed with an equal volume of precipitant solution [1.4 M tri sodium citrate, 0.1 M sodium Hepes, pH 7.5] placed on the underside of a siliconized glass coverslip and sealed in close proximity to 1 ml of the precipitant solution. Crystallization plates were incubated at 22° C.; rectangular rod crystals (0.02×0.2 mm) grew over 2-30 days.
Prior to data collection, crystals were washed with the reservoir solution of the crystallization setup and transferred into the same solution with 20% glycerol added. The crystals were then flash-cooled in a nitrogen stream at 95 K. X-ray diffraction was collected using a Rigaku generator equipped with a Raxis 4++ detector. Data were integrated and scaled using the HKL package.
Data Collection Statistics:
















Resolution
50.0-1.70 Å



No. of collected reflections
135908



No. of unique reflections (F >= 0)
 12484



R-sym
 6.7%



Percent of theoretical (I/s >= 1)
98.7%



Unit Cell
a = 37.999 Å, b = 45.333 Å,




c = 63.999 Å,




α = β = γ = 90°



Space Group
P212121



Asymmetric unit
1 molecule










The crystal structure was solved using molecular replacement using the search models 1YCQ and 1YCR from the PDB. Refinement was done using the program CNX.
Theoretical Number of Reflections 9121
















Resolution Limits
50.0-1.90 Å



Number of unobserved reflections
 106 (1.2%)



Number of reflections in working set
8592 (98.8%)



Number of reflections in test set
 423 (4.6%)



Number of protein residues
87



Number of solvent atoms
44



R-factor
 0.223



R-free
 0.243



RMSD bond length
    0.0083 Å



RMSD bond angles
1.46°










The structural coordinates for the above-described Hdm2 crystal are set forth below in Table 3, which is in Protein Data Bank (PDB) file format. The numbered columns refer to the following:











Col. #
Reference
















1
Atomic coordinate records for standard groups


2
Atom serial number


3
Atom name


4
Residue name


5
Residue sequence number


6
Orthogonal coordinates for X in Angstroms


7
Orthogonal coordinates for Y in Angstroms


8
Orthogonal coordinates for Z in Angstroms


9
Occupancy


10
Temperature factor


11
Element symbol


























TABLE 3





1
2
3
4
5
6
7
8
9
10
11

























ATOM
1
CB
GLU
25
8.599
29.031
2.725
1.00
52.10
C


ATOM
2
CG
GLU
25
7.336
29.192
3.556
1.00
55.02
C


ATOM
3
CD
GLU
25
6.648
27.844
3.697
1.00
57.01
C


ATOM
4
OE1
GLU
25
5.517
27.697
3.186
1.00
58.12
O


ATOM
5
OE2
GLU
25
7.235
26.932
4.322
1.00
57.62
O


ATOM
6
C
GLU
25
10.683
30.035
1.753
1.00
47.48
C


ATOM
7
O
GLU
25
10.654
29.682
0.574
1.00
48.97
O


ATOM
8
N
GLU
25
8.564
31.353
1.814
1.00
50.30
N


ATOM
9
CA
GLU
25
9.389
30.323
2.511
1.00
49.61
C


ATOM
10
N
THR
26
11.816
30.195
2.431
1.00
43.07
N


ATOM
11
CA
THR
26
13.119
29.941
1.824
1.00
37.71
C


ATOM
12
CB
THR
26
14.267
30.244
2.807
1.00
38.14
C


ATOM
13
OG1
THR
26
14.227
31.628
3.174
1.00
38.51
O


ATOM
14
CG2
THR
26
15.613
29.942
2.163
1.00
37.88
C


ATOM
15
C
THR
26
13.216
28.482
1.385
1.00
34.76
C


ATOM
16
O
THR
26
12.823
27.579
2.120
1.00
34.28
O


ATOM
17
N
LEU
27
13.730
28.261
0.179
1.00
29.30
N


ATOM
18
CA
LEU
27
13.872
26.915
−0.365
1.00
28.75
C


ATOM
19
CB
LEU
27
13.479
26.866
−1.842
1.00
28.72
C


ATOM
20
CG
LEU
27
12.043
27.255
−2.190
1.00
29.98
C


ATOM
21
CD1
LEU
27
11.823
27.208
−3.696
1.00
30.85
C


ATOM
22
CD2
LEU
27
11.037
26.358
−1.482
1.00
30.07
C


ATOM
23
C
LEU
27
15.240
26.271
−0.161
1.00
26.20
C


ATOM
24
O
LEU
27
16.270
26.922
−0.316
1.00
26.92
O


ATOM
25
N
VAL
28
15.234
24.989
0.196
1.00
24.29
N


ATOM
26
CA
VAL
28
16.465
24.230
0.402
1.00
22.04
C


ATOM
27
CB
VAL
28
16.566
23.684
1.845
1.00
21.66
C


ATOM
28
CG1
VAL
28
16.716
24.839
2.822
1.00
20.57
C


ATOM
29
CG2
VAL
28
15.337
22.849
2.180
1.00
18.74
C


ATOM
30
C
VAL
28
16.549
23.066
−0.585
1.00
22.51
C


ATOM
31
O
VAL
28
15.526
22.500
−0.979
1.00
20.60
O


ATOM
32
N
ARG
29
17.770
22.733
−0.996
1.00
21.42
N


ATOM
33
CA
ARG
29
18.010
21.641
−1.937
1.00
23.64
C


ATOM
34
CB
ARG
29
18.869
22.116
−3.107
1.00
27.62
C


ATOM
35
CG
ARG
29
19.162
21.053
−4.151
1.00
33.83
C


ATOM
36
CD
ARG
29
20.043
21.611
−5.267
1.00
38.61
C


ATOM
37
NE
ARG
29
19.435
22.740
−5.965
1.00
42.13
N


ATOM
38
CZ
ARG
29
18.360
22.653
−6.742
1.00
45.54
C


ATOM
39
NH1
ARG
29
17.763
21.483
−6.929
1.00
47.02
N


ATOM
40
NH2
ARG
29
17.882
23.740
−7.337
1.00
46.93
N


ATOM
41
C
ARG
29
18.696
20.470
−1.227
1.00
20.05
C


ATOM
42
O
ARG
29
19.851
20.573
−0.819
1.00
18.84
O


ATOM
43
N
PRO
30
17.988
19.343
−1.078
1.00
19.58
N


ATOM
44
CD
PRO
30
16.511
19.312
−1.031
1.00
21.57
C


ATOM
45
CA
PRO
30
18.498
18.137
−0.425
1.00
20.02
C


ATOM
46
CB
PRO
30
17.278
17.234
−0.444
1.00
22.34
C


ATOM
47
CG
PRO
30
16.234
18.184
0.016
1.00
21.93
C


ATOM
48
C
PRO
30
19.678
17.493
−1.160
1.00
19.19
C


ATOM
49
O
PRO
30
19.624
17.323
−2.379
1.00
18.14
O


ATOM
50
N
LYS
31
20.737
17.144
−0.433
1.00
17.57
N


ATOM
51
CA
LYS
31
21.870
16.450
−1.044
1.00
17.54
C


ATOM
52
CB
LYS
31
23.083
16.416
−0.110
1.00
17.17
C


ATOM
53
CG
LYS
31
23.588
17.814
0.242
1.00
21.31
C


ATOM
54
CD
LYS
31
24.792
17.784
1.175
1.00
24.34
C


ATOM
55
CE
LYS
31
25.250
19.198
1.499
1.00
27.70
C


ATOM
56
NZ
LYS
31
26.424
19.216
2.411
1.00
30.99
N


ATOM
57
C
LYS
31
21.367
15.049
−1.436
1.00
18.76
C


ATOM
58
O
LYS
31
20.316
14.612
−0.961
1.00
17.24
O


ATOM
59
N
PRO
32
22.093
14.338
−2.319
1.00
18.30
N


ATOM
60
CD
PRO
32
23.329
14.791
−2.991
1.00
21.43
C


ATOM
61
CA
PRO
32
21.764
13.000
−2.820
1.00
19.62
C


ATOM
62
CB
PRO
32
23.111
12.503
−3.293
1.00
22.05
C


ATOM
63
CG
PRO
32
23.536
13.680
−4.080
1.00
21.40
C


ATOM
64
C
PRO
32
21.004
11.990
−1.947
1.00
19.34
C


ATOM
65
O
PRO
32
19.883
11.591
−2.299
1.00
17.19
O


ATOM
66
N
LEU
33
21.594
11.559
−0.833
1.00
18.45
N


ATOM
67
CA
LEU
33
20.911
10.581
0.017
1.00
18.88
C


ATOM
68
CB
LEU
33
21.788
10.020
1.142
1.00
21.73
C


ATOM
69
CG
LEU
33
23.050
9.209
0.871
1.00
28.04
C


ATOM
70
CD1
LEU
33
24.067
9.989
0.052
1.00
29.17
C


ATOM
71
CD2
LEU
33
23.663
8.738
2.191
1.00
29.05
C


ATOM
72
C
LEU
33
19.597
11.062
0.602
1.00
17.92
C


ATOM
73
O
LEU
33
18.603
10.340
0.565
1.00
16.48
O


ATOM
74
N
LEU
34
19.588
12.277
1.140
1.00
17.19
N


ATOM
75
CA
LEU
34
18.365
12.817
1.719
1.00
15.57
C


ATOM
76
CB
LEU
34
18.632
14.142
2.431
1.00
17.51
C


ATOM
77
CG
LEU
34
17.436
14.863
3.052
1.00
18.19
C


ATOM
78
CD1
LEU
34
16.656
13.966
4.007
1.00
16.62
C


ATOM
79
CD2
LEU
34
17.885
16.147
3.738
1.00
16.73
C


ATOM
80
C
LEU
34
17.316
12.995
0.638
1.00
17.16
C


ATOM
81
O
LEU
34
16.128
12.769
0.870
1.00
16.85
O


ATOM
82
N
LEU
35
17.755
13.385
−0.553
1.00
15.69
N


ATOM
83
CA
LEU
35
16.814
13.578
−1.644
1.00
18.62
C


ATOM
84
CB
LEU
35
17.508
14.148
−2.885
1.00
20.48
C


ATOM
85
CG
LEU
35
16.605
14.406
−4.097
1.00
23.23
C


ATOM
86
CD1
LEU
35
15.470
15.356
−3.733
1.00
23.45
C


ATOM
87
CD2
LEU
35
17.389
14.944
−5.293
1.00
23.24
C


ATOM
88
C
LEU
35
16.160
12.238
−1.966
1.00
19.55
C


ATOM
89
O
LEU
35
14.951
12.155
−2.195
1.00
17.74
O


ATOM
90
N
LYS
36
16.964
11.181
−1.955
1.00
18.27
N


ATOM
91
CA
LYS
36
16.453
9.859
−2.271
1.00
22.37
C


ATOM
92
CB
LYS
36
17.610
8.864
−2.413
1.00
24.10
C


ATOM
93
CG
LYS
36
17.212
7.444
−2.785
1.00
30.65
C


ATOM
94
CD
LYS
36
18.461
6.576
−2.920
1.00
32.62
C


ATOM
95
CE
LYS
36
18.128
5.145
−3.307
1.00
36.89
C


ATOM
96
NZ
LYS
36
19.362
4.312
−3.442
1.00
37.42
N


ATOM
97
C
LYS
36
15.413
9.413
−1.246
1.00
22.40
C


ATOM
98
O
LYS
36
14.435
8.751
−1.597
1.00
22.93
O


ATOM
99
N
LEU
37
15.605
9.802
0.013
1.00
22.25
N


ATOM
100
CA
LEU
37
14.659
9.432
1.065
1.00
21.53
C


ATOM
101
CB
LEU
37
15.231
9.702
2.453
1.00
24.79
C


ATOM
102
CG
LEU
37
16.511
8.980
2.865
1.00
27.14
C


ATOM
103
CD1
LEU
37
16.996
9.454
4.226
1.00
29.28
C


ATOM
104
CD2
LEU
37
16.317
7.474
2.855
1.00
29.24
C


ATOM
105
C
LEU
37
13.355
10.197
0.894
1.00
22.46
C


ATOM
106
O
LEU
37
12.269
9.636
1.053
1.00
19.80
O


ATOM
107
N
LEU
38
13.470
11.479
0.557
1.00
21.33
N


ATOM
108
CA
LEU
38
12.296
12.322
0.369
1.00
21.81
C


ATOM
109
CB
LEU
38
12.693
13.794
0.252
1.00
22.60
C


ATOM
110
CG
LEU
38
13.394
14.398
1.467
1.00
23.43
C


ATOM
111
CD1
LEU
38
13.905
15.806
1.185
1.00
24.29
C


ATOM
112
CD2
LEU
38
12.491
14.362
2.685
1.00
24.94
C


ATOM
113
C
LEU
38
11.483
11.884
−0.836
1.00
24.00
C


ATOM
114
O
LEU
38
10.254
11.986
−0.830
1.00
25.09
O


ATOM
115
N
LYS
39
12.160
11.381
−1.865
1.00
24.12
N


ATOM
116
CA
LYS
39
11.442
10.928
−3.045
1.00
28.66
C


ATOM
117
CB
LYS
39
12.339
10.855
−4.282
1.00
29.59
C


ATOM
118
CG
LYS
39
12.939
12.179
−4.728
1.00
30.58
C


ATOM
119
CD
LYS
39
13.798
11.969
−5.968
1.00
34.21
C


ATOM
120
CE
LYS
39
14.414
13.266
−6.459
1.00
35.99
C


ATOM
121
NZ
LYS
39
13.376
14.268
−6.818
1.00
37.93
N


ATOM
122
C
LYS
39
10.766
9.588
−2.803
1.00
29.02
C


ATOM
123
O
LYS
39
9.784
9.256
−3.468
1.00
30.57
O


ATOM
124
N
SER
40
11.270
8.823
−1.839
1.00
27.39
N


ATOM
125
CA
SER
40
10.671
7.526
−1.557
1.00
27.21
C


ATOM
126
CB
SER
40
11.619
6.625
−0.768
1.00
29.11
C


ATOM
127
OG
SER
40
11.934
7.200
0.484
1.00
31.61
O


ATOM
128
C
SER
40
9.371
7.739
−0.791
1.00
26.87
C


ATOM
129
O
SER
40
8.522
6.850
−0.731
1.00
24.88
O


ATOM
130
N
VAL
41
9.217
8.931
−0.222
1.00
23.16
N


ATOM
131
CA
VAL
41
8.020
9.265
0.534
1.00
24.52
C


ATOM
132
CB
VAL
41
8.321
10.392
1.553
1.00
25.80
C


ATOM
133
CG1
VAL
41
7.053
10.879
2.197
1.00
27.99
C


ATOM
134
CG2
VAL
41
9.271
9.873
2.620
1.00
25.81
C


ATOM
135
C
VAL
41
6.933
9.718
−0.436
1.00
24.07
C


ATOM
136
O
VAL
41
5.750
9.780
−0.083
1.00
22.91
O


ATOM
137
N
GLY
42
7.343
10.018
−1.665
1.00
23.67
N


ATOM
138
CA
GLY
42
6.394
10.446
−2.678
1.00
23.84
C


ATOM
139
C
GLY
42
6.685
11.764
−3.373
1.00
22.51
C


ATOM
140
O
GLY
42
6.165
12.010
−4.461
1.00
21.84
O


ATOM
141
N
ALA
43
7.508
12.616
−2.767
1.00
22.69
N


ATOM
142
CA
ALA
43
7.817
13.916
−3.361
1.00
23.42
C


ATOM
143
CB
ALA
43
8.331
14.868
−2.287
1.00
26.17
C


ATOM
144
C
ALA
43
8.830
13.811
−4.497
1.00
23.99
C


ATOM
145
O
ALA
43
9.996
13.496
−4.270
1.00
23.65
O


ATOM
146
N
GLN
44
8.369
14.091
−5.715
1.00
23.66
N


ATOM
147
CA
GLN
44
9.194
14.028
−6.925
1.00
26.00
C


ATOM
148
CB
GLN
44
8.416
13.405
−8.091
1.00
27.49
C


ATOM
149
CG
GLN
44
7.957
11.970
−7.853
1.00
29.74
C


ATOM
150
CD
GLN
44
9.160
11.066
−7.633
1.00
33.73
C


ATOM
151
OE1
GLN
44
10.295
11.436
−7.932
1.00
36.21
O


ATOM
152
NE2
GLN
44
8.915
9.878
−7.092
1.00
35.39
N


ATOM
153
C
GLN
44
9.814
15.363
−7.344
1.00
28.84
C


ATOM
154
O
GLN
44
9.895
15.669
−8.532
1.00
33.20
O


ATOM
155
N
LYS
45
10.239
16.162
−6.373
1.00
26.37
N


ATOM
156
CA
LYS
45
10.849
17.453
−6.676
1.00
26.53
C


ATOM
157
CB
LYS
45
9.937
18.606
−6.267
1.00
26.39
C


ATOM
158
CG
LYS
45
9.609
18.638
−4.794
1.00
26.84
C


ATOM
159
CD
LYS
45
8.700
19.806
−4.469
1.00
28.76
C


ATOM
160
CE
LYS
45
8.370
19.843
−2.986
1.00
27.65
C


ATOM
161
NZ
LYS
45
7.477
20.988
−2.669
1.00
30.55
N


ATOM
162
C
LYS
45
12.229
17.574
−6.037
1.00
25.49
C


ATOM
163
O
LYS
45
12.575
16.786
−5.161
1.00
24.24
O


ATOM
164
N
ASP
46
13.020
18.548
−6.480
1.00
26.71
N


ATOM
165
CA
ASP
46
14.360
18.726
−5.925
1.00
28.66
C


ATOM
166
CB
ASP
46
15.403
18.827
−7.040
1.00
35.21
C


ATOM
167
CG
ASP
46
15.144
19.991
−7.980
1.00
40.54
C


ATOM
168
OD1
ASP
46
14.152
20.724
−7.771
1.00
42.44
O


ATOM
169
OD2
ASP
46
15.936
20.172
−8.932
1.00
45.73
O


ATOM
170
C
ASP
46
14.534
19.865
−4.913
1.00
25.87
C


ATOM
171
O
ASP
46
15.587
19.975
−4.286
1.00
24.85
O


ATOM
172
N
THR
47
13.513
20.703
−4.747
1.00
23.77
N


ATOM
173
CA
THR
47
13.586
21.811
−3.788
1.00
23.33
C


ATOM
174
CB
THR
47
13.618
23.193
−4.476
1.00
25.02
C


ATOM
175
OG1
THR
47
12.413
23.394
−5.219
1.00
27.77
O


ATOM
176
CG2
THR
47
14.814
23.291
−5.398
1.00
26.67
C


ATOM
177
C
THR
47
12.398
21.764
−2.843
1.00
21.70
C


ATOM
178
O
THR
47
11.266
21.522
−3.263
1.00
21.88
O


ATOM
179
N
TYR
48
12.670
21.995
−1.563
1.00
19.26
N


ATOM
180
CA
TYR
48
11.649
21.927
−0.524
1.00
19.39
C


ATOM
181
CB
TYR
48
11.819
20.647
0.286
1.00
20.20
C


ATOM
182
CG
TYR
48
11.751
19.375
−0.517
1.00
20.69
C


ATOM
183
CD1
TYR
48
10.643
18.539
−0.433
1.00
20.32
C


ATOM
184
CE1
TYR
48
10.577
17.366
−1.170
1.00
23.35
C


ATOM
185
CD2
TYR
48
12.799
19.007
−1.362
1.00
20.03
C


ATOM
186
CE2
TYR
48
12.744
17.836
−2.107
1.00
22.54
C


ATOM
187
CZ
TYR
48
11.631
17.021
−2.007
1.00
21.66
C


ATOM
188
OH
TYR
48
11.564
15.867
−2.747
1.00
24.12
O


ATOM
189
C
TYR
48
11.761
23.093
0.444
1.00
18.48
C


ATOM
190
O
TYR
48
12.732
23.838
0.420
1.00
18.96
O


ATOM
191
N
THR
49
10.747
23.246
1.286
1.00
18.95
N


ATOM
192
CA
THR
49
10.792
24.247
2.344
1.00
19.64
C


ATOM
193
CB
THR
49
9.398
24.779
2.729
1.00
20.36
C


ATOM
194
OG1
THR
49
8.595
23.704
3.229
1.00
18.68
O


ATOM
195
CG2
THR
49
8.720
25.421
1.531
1.00
21.70
C


ATOM
196
C
THR
49
11.332
23.403
3.503
1.00
19.26
C


ATOM
197
O
THR
49
11.187
22.177
3.491
1.00
16.72
O


ATOM
198
N
MET
50
11.971
24.033
4.482
1.00
19.49
N


ATOM
199
CA
MET
50
12.496
23.287
5.621
1.00
19.92
C


ATOM
200
CB
MET
50
13.164
24.227
6.623
1.00
19.27
C


ATOM
201
CG
MET
50
13.752
23.549
7.857
1.00
21.93
C


ATOM
202
SD
MET
50
15.038
22.346
7.482
1.00
22.54
S


ATOM
203
CE
MET
50
16.405
23.430
7.220
1.00
21.56
C


ATOM
204
C
MET
50
11.366
22.505
6.292
1.00
20.51
C


ATOM
205
O
MET
50
11.556
21.358
6.717
1.00
19.62
O


ATOM
206
N
LYS
51
10.184
23.113
6.370
1.00
20.47
N


ATOM
207
CA
LYS
51
9.055
22.446
7.008
1.00
21.99
C


ATOM
208
CB
LYS
51
7.860
23.388
7.165
1.00
26.64
C


ATOM
209
CG
LYS
51
6.659
22.735
7.834
1.00
33.29
C


ATOM
210
CD
LYS
51
5.483
23.695
7.983
1.00
38.26
C


ATOM
211
CE
LYS
51
4.301
23.005
8.660
1.00
40.81
C


ATOM
212
NZ
LYS
51
3.128
23.912
8.828
1.00
42.40
N


ATOM
213
C
LYS
51
8.650
21.172
6.273
1.00
19.90
C


ATOM
214
O
LYS
51
8.205
20.210
6.898
1.00
19.38
O


ATOM
215
N
GLU
52
8.817
21.149
4.953
1.00
18.87
N


ATOM
216
CA
GLU
52
8.464
19.944
4.205
1.00
17.93
C


ATOM
217
CB
GLU
52
8.382
20.189
2.699
1.00
18.18
C


ATOM
218
CG
GLU
52
7.344
21.203
2.247
1.00
23.24
C


ATOM
219
CD
GLU
52
7.396
21.313
0.732
1.00
25.60
C


ATOM
220
OE1
GLU
52
8.396
21.846
0.213
1.00
23.72
O


ATOM
221
OE2
GLU
52
6.438
20.874
0.057
1.00
26.89
O


ATOM
222
C
GLU
52
9.486
18.855
4.482
1.00
15.85
C


ATOM
223
O
GLU
52
9.135
17.685
4.619
1.00
16.69
O


ATOM
224
N
VAL
53
10.753
19.250
4.568
1.00
16.93
N


ATOM
225
CA
VAL
53
11.825
18.293
4.825
1.00
17.04
C


ATOM
226
CB
VAL
53
13.203
18.995
4.855
1.00
16.03
C


ATOM
227
CG1
VAL
53
14.280
18.019
5.304
1.00
16.51
C


ATOM
228
CG2
VAL
53
13.534
19.547
3.467
1.00
17.18
C


ATOM
229
C
VAL
53
11.596
17.602
6.166
1.00
15.46
C


ATOM
230
O
VAL
53
11.612
16.371
6.254
1.00
15.66
O


ATOM
231
N
LEU
54
11.368
18.401
7.203
1.00
15.11
N


ATOM
232
CA
LEU
54
11.136
17.868
8.539
1.00
18.02
C


ATOM
233
CB
LEU
54
11.038
19.004
9.564
1.00
18.06
C


ATOM
234
CG
LEU
54
12.275
19.897
9.715
1.00
19.94
C


ATOM
235
CD1
LEU
54
12.017
21.075
10.651
1.00
21.06
C


ATOM
236
CD2
LEU
54
13.484
19.088
10.172
1.00
19.37
C


ATOM
237
C
LEU
54
9.887
16.989
8.551
1.00
17.44
C


ATOM
238
O
LEU
54
9.890
15.901
9.132
1.00
17.96
O


ATOM
239
N
TYR
55
8.824
17.456
7.900
1.00
19.29
N


ATOM
240
CA
TYR
55
7.582
16.686
7.826
1.00
19.06
C


ATOM
241
CB
TYR
55
6.476
17.462
7.107
1.00
21.67
C


ATOM
242
CG
TYR
55
5.208
16.659
6.914
1.00
24.34
C


ATOM
243
CD1
TYR
55
4.331
16.414
7.970
1.00
27.75
C


ATOM
244
CE1
TYR
55
3.185
15.626
7.788
1.00
28.11
C


ATOM
245
CD2
TYR
55
4.910
16.100
5.671
1.00
26.71
C


ATOM
246
CE2
TYR
55
3.778
15.316
5.479
1.00
27.50
C


ATOM
247
CZ
TYR
55
2.920
15.082
6.537
1.00
29.01
C


ATOM
248
OH
TYR
55
1.802
14.302
6.332
1.00
31.62
O


ATOM
249
C
TYR
55
7.778
15.329
7.162
1.00
17.61
C


ATOM
250
O
TYR
55
7.450
14.295
7.742
1.00
18.75
O


ATOM
251
N
TYR
56
8.314
15.332
5.943
1.00
17.90
N


ATOM
252
CA
TYR
56
8.537
14.079
5.228
1.00
15.80
C


ATOM
253
CB
TYR
56
8.959
14.322
3.781
1.00
16.36
C


ATOM
254
CG
TYR
56
7.897
14.995
2.944
1.00
19.38
C


ATOM
255
CD1
TYR
56
6.589
14.508
2.934
1.00
18.67
C


ATOM
256
CE1
TYR
56
5.610
15.083
2.132
1.00
21.66
C


ATOM
257
CD2
TYR
56
8.201
16.083
2.125
1.00
19.49
C


ATOM
258
CE2
TYR
56
7.225
16.667
1.315
1.00
20.60
C


ATOM
259
CZ
TYR
56
5.935
16.160
1.325
1.00
22.09
C


ATOM
260
OH
TYR
56
4.967
16.724
0.524
1.00
24.00
O


ATOM
261
C
TYR
56
9.463
13.067
5.893
1.00
16.42
C


ATOM
262
O
TYR
56
9.247
11.863
5.759
1.00
16.20
O


ATOM
263
N
LEU
57
10.484
13.531
6.609
1.00
17.70
N


ATOM
264
CA
LEU
57
11.362
12.579
7.283
1.00
18.25
C


ATOM
265
CB
LEU
57
12.629
13.232
7.836
1.00
22.32
C


ATOM
266
CG
LEU
57
13.582
13.831
6.810
1.00
26.55
C


ATOM
267
CD1
LEU
57
14.757
14.543
7.473
1.00
27.16
C


ATOM
268
CD2
LEU
57
14.073
12.746
5.861
1.00
29.77
C


ATOM
269
C
LEU
57
10.588
11.891
8.385
1.00
18.46
C


ATOM
270
O
LEU
57
10.782
10.704
8.641
1.00
19.38
O


ATOM
271
N
GLY
58
9.693
12.639
9.023
1.00
16.71
N


ATOM
272
CA
GLY
58
8.883
12.060
10.077
1.00
18.09
C


ATOM
273
C
CLY
58
7.958
11.030
9.458
1.00
18.69
C


ATOM
274
O
GLY
58
7.750
9.948
10.012
1.00
17.12
O


ATOM
275
N
CLN
59
7.402
11.370
8.297
1.00
18.56
N


ATOM
276
CA
GLN
59
6.494
10.470
7.585
1.00
19.64
C


ATOM
277
CB
GLN
59
5.787
11.209
6.448
1.00
21.81
C


ATOM
278
CG
GLN
59
4.918
12.363
6.913
1.00
25.45
C


ATOM
279
CD
GLN
59
3.842
11.829
7.830
1.00
29.94
C


ATOM
280
OE1
GLN
59
3.753
12.211
9.000
1.00
32.36
O


ATOM
281
NE2
GLN
59
3.015
10.934
7.304
1.00
31.47
N


ATOM
282
C
GLN
59
7.212
9.228
7.062
1.00
18.28
C


ATOM
283
O
GLN
59
6.624
8.150
6.977
1.00
18.50
O


ATOM
284
N
TYR
60
8.488
9.391
6.725
1.00
17.72
N


ATOM
285
CA
TYR
60
9.322
8.294
6.237
1.00
19.36
C


ATOM
286
CB
TYR
60
10.699
8.816
5.829
1.00
19.10
C


ATOM
287
CG
TYR
60
11.675
7.746
5.386
1.00
21.10
C


ATOM
288
CD1
TYR
60
11.634
7.221
4.095
1.00
22.00
C


ATOM
289
CE1
TYR
60
12.526
6.224
3.691
1.00
20.83
C


ATOM
290
CD2
TYR
60
12.635
7.247
6.269
1.00
21.10
C


ATOM
291
CE2
TYR
60
13.532
6.245
5.876
1.00
20.35
C


ATOM
292
CZ
TYR
60
13.469
5.740
4.585
1.00
20.69
C


ATOM
293
OH
TYR
60
14.329
4.746
4.187
1.00
20.09
O


ATOM
294
C
TYR
60
9.474
7.246
7.335
1.00
19.51
C


ATOM
295
O
TYR
60
9.283
6.046
7.113
1.00
18.29
O


ATOM
296
N
ILE
61
9.812
7.722
8.527
1.00
17.99
N


ATOM
297
CA
ILE
61
10.012
6.851
9.675
1.00
19.08
C


ATOM
298
CB
ILE
61
10.521
7.687
10.878
1.00
20.08
C


ATOM
299
CG2
ILE
61
10.452
6.869
12.164
1.00
20.84
C


ATOM
300
CG1
ILE
61
11.945
8.179
10.586
1.00
18.04
C


ATOM
301
CD1
ILE
61
12.547
9.033
11.670
1.00
20.08
C


ATOM
302
C
ILE
61
8.709
6.155
10.049
1.00
20.09
C


ATOM
303
O
ILE
61
8.692
4.975
10.406
1.00
19.98
O


ATOM
304
N
MET
62
7.606
6.879
9.933
1.00
20.53
N


ATOM
305
CA
MET
62
6.330
6.303
10.301
1.00
25.34
C


ATOM
306
CB
MET
62
5.305
7.398
10.599
1.00
28.26
C


ATOM
307
CG
MET
62
3.943
6.874
11.011
1.00
34.36
C


ATOM
308
SD
MET
62
2.800
8.211
11.360
1.00
41.93
S


ATOM
309
CE
MET
62
2.064
8.447
9.727
1.00
41.20
C


ATOM
310
C
MET
62
5.800
5.313
9.277
1.00
25.27
C


ATOM
311
O
MET
62
5.281
4.257
9.648
1.00
24.70
O


ATOM
312
N
THR
63
5.951
5.635
7.997
1.00
25.57
N


ATOM
313
CA
THR
63
5.441
4.755
6.954
1.00
28.86
C


ATOM
314
CB
THR
63
5.393
5.479
5.599
1.00
29.41
C


ATOM
315
OG1
THR
63
4.566
6.641
5.722
1.00
33.31
O


ATOM
316
CG2
THR
63
4.809
4.574
4.526
1.00
28.44
C


ATOM
317
C
THR
63
6.247
3.473
6.810
1.00
27.27
C


ATOM
318
O
THR
63
5.711
2.449
6.383
1.00
27.48
O


ATOM
319
N
LYS
64
7.526
3.510
7.168
1.00
26.66
N


ATOM
320
CA
LYS
64
8.324
2.295
7.087
1.00
26.49
C


ATOM
321
CB
LYS
64
9.730
2.517
6.523
1.00
26.55
C


ATOM
322
CG
LYS
64
9.740
3.088
5.117
1.00
29.11
C


ATOM
323
CD
LYS
64
11.150
3.247
4.571
1.00
28.43
C


ATOM
324
CE
LYS
64
11.850
1.886
4.510
1.00
29.76
C


ATOM
325
NZ
LYS
64
13.237
1.959
3.969
1.00
29.61
N


ATOM
326
C
LYS
64
8.375
1.642
8.453
1.00
25.50
C


ATOM
327
O
LYS
64
9.026
0.617
8.639
1.00
26.13
O


ATOM
328
N
ARG
65
7.678
2.255
9.403
1.00
24.76
N


ATOM
329
CA
ARG
65
7.607
1.754
10.765
1.00
25.45
C


ATOM
330
CB
ARG
65
6.672
0.542
10.843
1.00
31.11
C


ATOM
331
CG
ARG
65
5.246
0.879
10.397
1.00
35.38
C


ATOM
332
CD
ARG
65
4.303
−0.309
10.470
1.00
41.28
C


ATOM
333
NE
ARG
65
2.954
0.053
10.034
1.00
46.30
N


ATOM
334
CZ
ARG
65
1.897
−0.754
10.104
1.00
48.44
C


ATOM
335
NH1
ARG
65
2.023
−1.978
10.598
1.00
49.25
N


ATOM
336
NH2
ARG
65
0.712
−0.338
9.674
1.00
48.82
N


ATOM
337
C
ARG
65
8.979
1.472
11.378
1.00
25.07
C


ATOM
338
O
ARG
65
9.193
0.425
11.990
1.00
23.46
O


ATOM
339
N
LEU
66
9.910
2.409
11.202
1.00
22.51
N


ATOM
340
CA
LEU
66
11.259
2.246
11.745
1.00
21.93
C


ATOM
341
CB
LEU
66
12.251
3.184
11.063
1.00
21.83
C


ATOM
342
CG
LEU
66
12.406
3.035
9.552
1.00
21.63
C


ATOM
343
CD1
LEU
66
13.391
4.062
9.015
1.00
20.95
C


ATOM
344
CD2
LEU
66
12.838
1.634
9.170
1.00
20.51
C


ATOM
345
C
LEU
66
11.322
2.436
13.259
1.00
22.70
C


ATOM
346
O
LEU
66
12.325
2.115
13.893
1.00
22.69
O


ATOM
347
N
TYR
67
10.246
2.962
13.833
1.00
23.65
N


ATOM
348
CA
TYR
67
10.174
3.180
15.273
1.00
23.29
C


ATOM
349
CB
TYR
67
9.091
4.197
15.622
1.00
24.97
C


ATOM
350
CG
TYR
67
7.703
3.781
15.182
1.00
25.97
C


ATOM
351
CD1
TYR
67
6.928
2.923
15.966
1.00
27.64
C


ATOM
352
CE1
TYR
67
5.656
2.522
15.554
1.00
27.94
C


ATOM
353
CD2
TYR
67
7.172
4.227
13.972
1.00
27.22
C


ATOM
354
CE2
TYR
67
5.905
3.830
13.549
1.00
28.73
C


ATOM
355
CZ
TYR
67
5.153
2.980
14.345
1.00
29.04
C


ATOM
356
OH
TYR
67
3.899
2.594
13.930
1.00
29.83
O


ATOM
357
C
TYR
67
9.917
1.838
15.951
1.00
24.96
C


ATOM
358
O
TYR
67
9.215
0.992
15.404
1.00
26.17
O


ATOM
359
N
ASP
68
10.500
1.629
17.124
1.00
25.95
N


ATOM
360
CA
ASP
68
10.282
0.376
17.835
1.00
28.28
C


ATOM
361
CB
ASP
68
11.205
0.225
19.038
1.00
31.87
C


ATOM
362
CG
ASP
68
10.969
−1.082
19.781
1.00
35.20
C


ATOM
363
OD1
ASP
68
10.683
−1.042
20.994
1.00
36.44
O


ATOM
364
OD2
ASP
68
11.061
−2.149
19.141
1.00
36.99
O


ATOM
365
C
ASP
68
8.827
0.260
18.262
1.00
28.80
C


ATOM
366
O
ASP
68
8.286
1.157
18.907
1.00
23.20
O


ATOM
367
N
GLU
69
8.200
−0.851
17.898
1.00
32.27
N


ATOM
368
CA
GLU
69
6.799
−1.073
18.224
1.00
36.70
C


ATOM
369
CB
GLU
69
6.336
−2.428
17.684
1.00
40.26
C


ATOM
370
CG
GLU
69
4.873
−2.762
17.936
1.00
44.82
C


ATOM
371
CD
GLU
69
3.981
−1.727
17.266
1.00
48.03
C


ATOM
372
OE1
GLU
69
3.189
−2.115
16.380
1.00
50.17
O


ATOM
373
OE2
GLU
69
4.064
−0.532
17.621
1.00
50.09
O


ATOM
374
C
GLU
69
6.490
−0.956
19.719
1.00
36.29
C


ATOM
375
O
GLU
69
5.376
−0.587
20.089
1.00
35.50
O


ATOM
376
N
LYS
70
7.471
−1.245
20.574
1.00
36.45
N


ATOM
377
CA
LYS
70
7.246
−1.173
22.018
1.00
37.74
C


ATOM
378
CB
LYS
70
7.591
−2.501
22.694
1.00
40.70
C


ATOM
379
CG
LYS
70
6.766
−3.677
22.186
1.00
43.93
C


ATOM
380
CD
LYS
70
7.137
−4.979
22.889
1.00
46.97
C


ATOM
381
CE
LYS
70
6.298
−6.143
22.369
1.00
47.54
C


ATOM
382
NZ
LYS
70
6.634
−7.434
23.042
1.00
48.98
N


ATOM
383
C
LYS
70
7.928
−0.005
22.730
1.00
37.66
C


ATOM
384
O
LYS
70
7.557
0.348
23.852
1.00
39.60
O


ATOM
385
N
GLN
71
8.918
0.595
22.080
1.00
33.78
N


ATOM
386
CA
GLN
71
9.615
1.749
22.643
1.00
31.65
C


ATOM
387
CB
GLN
71
11.073
1.425
22.967
1.00
33.12
C


ATOM
388
CG
GLN
71
11.259
0.266
23.920
1.00
35.60
C


ATOM
389
CD
GLN
71
12.739
0.054
24.162
1.00
36.23
C


ATOM
390
OE1
GLN
71
13.361
−0.811
23.548
1.00
35.70
O


ATOM
391
NE2
GLN
71
13.305
0.826
25.081
1.00
35.71
N


ATOM
392
C
GLN
71
9.530
2.706
21.466
1.00
29.31
C


ATOM
393
O
GLN
71
10.488
2.864
20.713
1.00
23.70
O


ATOM
394
N
GLN
72
8.378
3.349
21.322
1.00
27.87
N


ATOM
395
CA
GLN
72
8.142
4.223
20.185
1.00
29.09
C


ATOM
396
CB
GLN
72
6.650
4.530
20.041
1.00
32.14
C


ATOM
397
CG
GLN
72
5.800
3.283
19.830
1.00
35.48
C


ATOM
398
CD
GLN
72
4.345
3.680
19.685
1.00
37.97
C


ATOM
399
OE1
GLN
72
3.689
3.324
18.707
1.00
40.02
O


ATOM
400
NE2
GLN
72
3.836
4.431
20.654
1.00
39.88
N


ATOM
401
C
GLN
72
8.975
5.480
20.002
1.00
27.04
C


ATOM
402
O
GLN
72
8.862
6.134
18.968
1.00
28.04
O


ATOM
403
N
HIS
73
9.814
5.823
20.973
1.00
25.52
N


ATOM
404
CA
HIS
73
10.638
7.017
20.817
1.00
26.50
C


ATOM
405
CB
HIS
73
10.876
7.728
22.155
1.00
29.45
C


ATOM
406
CG
HIS
73
11.649
6.914
23.147
1.00
33.08
C


ATOM
407
CD2
HIS
73
11.240
6.122
24.167
1.00
34.44
C


ATOM
408
ND1
HIS
73
13.025
6.847
23.136
1.00
34.94
N


ATOM
409
CE1
HIS
73
13.432
6.048
24.109
1.00
35.16
C


ATOM
410
NE2
HIS
73
12.369
5.596
24.748
1.00
34.82
N


ATOM
411
C
HIS
73
11.968
6.628
20.170
1.00
25.41
C


ATOM
412
O
HIS
73
12.763
7.494
19.797
1.00
24.29
O


ATOM
413
N
ILE
74
12.200
5.322
20.031
1.00
22.09
N


ATOM
414
CA
ILE
74
13.439
4.827
19.433
1.00
19.61
C


ATOM
415
CB
ILE
74
13.967
3.575
20.159
1.00
19.95
C


ATOM
416
CG2
ILE
74
15.274
3.118
19.517
1.00
20.34
C


ATOM
417
CG1
ILE
74
14.185
3.888
21.640
1.00
15.63
C


ATOM
418
CD1
ILE
74
14.696
2.712
22.442
1.00
20.76
C


ATOM
419
C
ILE
74
13.275
4.472
17.966
1.00
20.12
C


ATOM
420
O
ILE
74
12.440
3.642
17.593
1.00
19.78
O


ATOM
421
N
VAL
75
14.083
5.107
17.131
1.00
18.20
N


ATOM
422
CA
VAL
75
14.034
4.856
15.703
1.00
17.89
C


ATOM
423
CB
VAL
75
14.193
6.171
14.901
1.00
17.74
C


ATOM
424
CG1
VAL
75
14.278
5.862
13.413
1.00
20.35
C


ATOM
425
CG2
VAL
75
13.025
7.106
15.185
1.00
19.50
C


ATOM
426
C
VAL
75
15.152
3.909
15.297
1.00
18.16
C


ATOM
427
O
VAL
75
16.326
4.218
15.493
1.00
18.79
O


ATOM
428
N
HIS
76
14.788
2.754
14.745
1.00
18.90
N


ATOM
429
CA
HIS
76
15.775
1.785
14.271
1.00
20.21
C


ATOM
430
CB
HIS
76
15.334
0.332
14.487
1.00
19.35
C


ATOM
431
CG
HIS
76
15.229
−0.064
15.924
1.00
18.87
C


ATOM
432
CD2
HIS
76
14.203
0.011
16.804
1.00
20.15
C


ATOM
433
ND1
HIS
76
16.292
−0.602
16.618
1.00
17.09
N


ATOM
434
CE1
HIS
76
15.924
−0.841
17.864
1.00
18.81
C


ATOM
435
NE2
HIS
76
14.661
−0.478
18.003
1.00
18.94
N


ATOM
436
C
HIS
76
16.102
2.027
12.810
1.00
21.94
C


ATOM
437
O
HIS
76
15.285
1.781
11.923
1.00
21.85
O


ATOM
438
N
CYS
77
17.314
2.496
12.567
1.00
19.97
N


ATOM
439
CA
CYS
77
17.743
2.787
11.217
1.00
22.10
C


ATOM
440
CB
CYS
77
17.956
4.292
11.050
1.00
21.37
C


ATOM
441
SG
CYS
77
19.086
4.999
12.249
1.00
23.82
S


ATOM
442
C
CYS
77
18.889
1.937
10.685
1.00
22.14
C


ATOM
443
O
CYS
77
19.434
2.212
9.619
1.00
21.97
O


ATOM
444
N
SER
78
19.251
0.899
11.434
1.00
22.19
N


ATOM
445
CA
SER
78
20.304
−0.010
11.000
1.00
24.97
C


ATOM
446
CB
SER
78
20.689
−1.008
12.098
1.00
24.27
C


ATOM
447
OG
SER
78
19.588
−1.819
12.475
1.00
23.96
O


ATOM
448
C
SER
78
19.746
−0.727
9.772
1.00
27.08
C


ATOM
449
O
SER
78
18.578
−1.098
9.752
1.00
29.90
O


ATOM
450
N
ASN
79
20.562
−0.896
8.739
1.00
31.08
N


ATOM
451
CA
ASN
79
20.102
−1.579
7.530
1.00
33.84
C


ATOM
452
CB
ASN
79
19.475
−2.939
7.856
1.00
37.91
C


ATOM
453
CG
ASN
79
20.430
−3.867
8.583
1.00
40.98
C


ATOM
454
OD1
ASN
79
20.087
−4.436
9.623
1.00
44.02
O


ATOM
455
ND2
ASN
79
21.639
−4.014
8.050
1.00
41.58
N


ATOM
456
C
ASN
79
19.130
−0.741
6.688
1.00
33.03
C


ATOM
457
O
ASN
79
18.500
−1.260
5.765
1.00
31.20
O


ATOM
458
N
ASP
80
18.995
0.543
7.013
1.00
28.81
N


ATOM
459
CA
ASP
80
18.098
1.425
6.263
1.00
24.65
C


ATOM
460
CB
ASP
80
16.875
1.821
7.091
1.00
23.49
C


ATOM
461
CG
ASP
80
15.880
2.662
6.307
1.00
24.25
C


ATOM
462
OD1
ASP
80
14.807
2.115
5.975
1.00
23.20
O


ATOM
463
OD2
ASP
80
16.143
3.855
6.033
1.00
22.88
O


ATOM
464
C
ASP
80
18.890
2.645
5.811
1.00
24.06
C


ATOM
465
O
ASP
80
19.814
3.078
6.505
1.00
21.20
O


ATOM
466
N
LEU
81
18.546
3.188
4.647
1.00
21.17
N


ATOM
467
CA
LEU
81
19.257
4.354
4.131
1.00
23.85
C


ATOM
468
CB
LEU
81
18.638
4.843
2.819
1.00
27.45
C


ATOM
469
CG
LEU
81
19.280
6.057
2.138
1.00
29.98
C


ATOM
470
CD1
LEU
81
20.759
5.822
1.861
1.00
32.81
C


ATOM
471
CD2
LEU
81
18.548
6.421
0.845
1.00
32.11
C


ATOM
472
C
LEU
81
19.313
5.486
5.155
1.00
21.14
C


ATOM
473
O
LEU
81
20.249
6.285
5.156
1.00
18.90
O


ATOM
474
N
LEU
82
18.325
5.546
6.042
1.00
18.54
N


ATOM
475
CA
LEU
82
18.318
6.598
7.052
1.00
15.44
C


ATOM
476
CB
LEU
82
17.019
6.590
7.857
1.00
16.76
C


ATOM
477
CG
LEU
82
16.897
7.650
8.953
1.00
16.50
C


ATOM
478
CD1
LEU
82
17.090
9.052
8.382
1.00
17.63
C


ATOM
479
CD2
LEU
82
15.559
7.538
9.694
1.00
17.39
C


ATOM
480
C
LEU
82
19.516
6.405
7.969
1.00
15.53
C


ATOM
481
O
LEU
82
20.070
7.373
8.495
1.00
15.91
O


ATOM
482
N
GLY
83
19.916
5.145
8.139
1.00
14.77
N


ATOM
483
CA
GLY
83
21.058
4.824
8.978
1.00
16.75
C


ATOM
484
C
GLY
83
22.341
5.353
8.365
1.00
19.93
C


ATOM
485
O
GLY
83
23.236
5.824
9.075
1.00
19.30
O


ATOM
486
N
ASP
84
22.445
5.278
7.042
1.00
20.23
N


ATOM
487
CA
ASP
84
23.640
5.790
6.376
1.00
19.77
C


ATOM
488
CB
ASP
84
23.694
5.372
4.903
1.00
22.40
C


ATOM
489
CG
ASP
84
23.784
3.865
4.712
1.00
24.25
C


ATOM
490
OD1
ASP
84
24.006
3.137
5.700
1.00
24.82
O


ATOM
491
OD2
ASP
84
23.627
3.407
3.562
1.00
25.98
O


ATOM
492
C
ASP
84
23.670
7.314
6.487
1.00
20.03
C


ATOM
493
O
ASP
84
24.739
7.919
6.577
1.00
19.62
O


ATOM
494
N
LEU
85
22.488
7.925
6.511
1.00
16.11
N


ATOM
495
CA
LEU
85
22.385
9.377
6.578
1.00
17.87
C


ATOM
496
CB
LEU
85
20.972
9.827
6.194
1.00
19.94
C


ATOM
497
CG
LEU
85
20.673
11.320
6.099
1.00
24.54
C


ATOM
498
CD1
LEU
85
21.547
11.960
5.019
1.00
24.28
C


ATOM
499
CD2
LEU
85
19.205
11.575
5.770
1.00
24.55
C


ATOM
500
C
LEU
85
22.779
9.897
7.965
1.00
18.33
C


ATOM
501
O
LEU
85
23.522
10.875
8.078
1.00
18.30
O


ATOM
502
N
PHE
86
22.297
9.247
9.022
1.00
18.48
N


ATOM
503
CA
PHE
86
22.646
9.686
10.372
1.00
19.70
C


ATOM
504
CB
PHE
86
21.491
9.506
11.370
1.00
22.89
C


ATOM
505
CG
PHE
86
20.283
10.375
11.092
1.00
25.20
C


ATOM
506
CD1
PHE
86
19.282
10.504
12.051
1.00
29.11
C


ATOM
507
CD2
PHE
86
20.175
11.114
9.915
1.00
26.71
C


ATOM
508
CE1
PHE
86
18.195
11.359
11.850
1.00
26.77
C


ATOM
509
CE2
PHE
86
19.091
11.972
9.702
1.00
28.12
C


ATOM
510
CZ
PHE
86
18.100
12.095
10.673
1.00
28.90
C


ATOM
511
C
PHE
86
23.943
9.104
10.927
1.00
19.63
C


ATOM
512
O
PHE
86
24.464
9.594
11.927
1.00
21.85
O


ATOM
513
N
GLY
87
24.468
8.074
10.272
1.00
18.21
N


ATOM
514
CA
GLY
87
25.713
7.467
10.715
1.00
20.13
C


ATOM
515
C
GLY
87
25.620
6.699
12.021
1.00
22.27
C


ATOM
516
O
GLY
87
26.612
6.552
12.737
1.00
21.79
O


ATOM
517
N
VAL
88
24.425
6.210
12.332
1.00
21.59
N


ATOM
518
CA
VAL
88
24.186
5.448
13.555
1.00
22.13
C


ATOM
519
CB
VAL
88
23.716
6.351
14.724
1.00
22.68
C


ATOM
520
CG1
VAL
88
24.830
7.306
15.130
1.00
24.59
C


ATOM
521
CG2
VAL
88
22.463
7.117
14.324
1.00
23.26
C


ATOM
522
C
VAL
88
23.105
4.408
13.305
1.00
21.88
C


ATOM
523
O
VAL
88
22.271
4.576
12.417
1.00
21.47
O


ATOM
524
N
PRO
89
23.116
3.307
14.074
1.00
22.24
N


ATOM
525
CD
PRO
89
23.982
2.960
15.221
1.00
23.24
C


ATOM
526
CA
PRO
89
22.110
2.267
13.901
1.00
20.32
C


ATOM
527
CB
PRO
89
22.807
1.064
14.503
1.00
23.05
C


ATOM
528
CG
PRO
89
23.286
1.669
15.787
1.00
22.08
C


ATOM
529
C
PRO
89
20.770
2.609
14.559
1.00
20.59
C


ATOM
530
O
PRO
89
19.775
1.912
14.343
1.00
21.33
O


ATOM
531
N
SER
90
20.743
3.688
15.341
1.00
18.62
N


ATOM
532
CA
SER
90
19.520
4.097
16.035
1.00
17.25
C


ATOM
533
CB
SER
90
19.109
3.069
17.085
1.00
18.96
C


ATOM
534
OG
SER
90
20.118
2.972
18.080
1.00
16.15
O


ATOM
535
C
SER
90
19.640
5.447
16.724
1.00
17.40
C


ATOM
536
O
SER
90
20.742
5.937
16.990
1.00
17.90
O


ATOM
537
N
PHE
91
18.486
6.040
17.013
1.00
15.60
N


ATOM
538
CA
PHE
91
18.422
7.308
17.718
1.00
16.44
C


ATOM
539
CB
PHE
91
18.750
8.512
16.828
1.00
19.56
C


ATOM
540
CG
PHE
91
17.793
8.708
15.682
1.00
19.09
C


ATOM
541
CD1
PHE
91
17.923
7.972
14.512
1.00
19.65
C


ATOM
542
CD2
PHE
91
16.756
9.634
15.783
1.00
21.34
C


ATOM
543
CE1
PHE
91
17.033
8.153
13.450
1.00
20.79
C


ATOM
544
CE2
PHE
91
15.858
9.824
14.728
1.00
20.20
C


ATOM
545
CZ
PHE
91
15.999
9.083
13.561
1.00
20.92
C


ATOM
546
C
PHE
91
17.043
7.491
18.334
1.00
18.30
C


ATOM
547
O
PHE
91
16.058
6.939
17.846
1.00
17.76
O


ATOM
548
N
SER
92
16.992
8.248
19.422
1.00
18.06
N


ATOM
549
CA
SER
92
15.737
8.575
20.083
1.00
20.21
C


ATOM
550
CB
SER
92
15.913
8.741
21.587
1.00
20.33
C


ATOM
551
OG
SER
92
14.676
9.099
22.181
1.00
22.41
O


ATOM
552
C
SER
92
15.224
9.875
19.468
1.00
20.17
C


ATOM
553
O
SER
92
16.010
10.786
19.203
1.00
19.59
O


ATOM
554
N
VAL
93
13.920
9.970
19.226
1.00
20.06
N


ATOM
555
CA
VAL
93
13.381
11.198
18.647
1.00
22.08
C


ATOM
556
CB
VAL
93
11.867
11.074
18.371
1.00
21.37
C


ATOM
557
CG1
VAL
93
11.608
9.904
17.429
1.00
22.86
C


ATOM
558
CG2
VAL
93
11.114
10.891
19.681
1.00
22.21
C


ATOM
559
C
VAL
93
13.614
12.384
19.583
1.00
22.18
C


ATOM
560
O
VAL
93
13.425
13.538
19.197
1.00
20.43
O


ATOM
561
N
LYS
94
14.038
12.098
20.811
1.00
24.14
N


ATOM
562
CA
LYS
94
14.298
13.153
21.784
1.00
25.77
C


ATOM
563
CB
LYS
94
14.142
12.620
23.213
1.00
30.87
C


ATOM
564
CG
LYS
94
15.094
11.484
23.557
1.00
36.14
C


ATOM
565
CD
LYS
94
14.888
10.965
24.976
1.00
40.51
C


ATOM
566
CE
LYS
94
13.467
10.431
25.164
1.00
42.75
C


ATOM
567
NZ
LYS
94
13.224
9.907
26.539
1.00
45.31
N


ATOM
568
C
LYS
94
15.677
13.781
21.558
1.00
24.88
C


ATOM
569
O
LYS
94
15.992
14.825
22.129
1.00
24.06
O


ATOM
570
N
GLU
95
16.488
13.144
20.715
1.00
23.25
N


ATOM
571
CA
GLU
95
17.823
13.653
20.393
1.00
22.14
C


ATOM
572
CB
GLU
95
18.761
12.526
19.940
1.00
24.17
C


ATOM
573
CG
GLU
95
19.031
11.470
21.011
1.00
28.59
C


ATOM
574
CD
GLU
95
19.963
10.399
20.463
1.00
31.12
C


ATOM
575
OE1
GLU
95
21.188
10.511
20.674
1.00
33.36
O


ATOM
576
OE2
GLU
95
19.473
9.430
19.847
1.00
32.84
O


ATOM
577
C
GLU
95
17.733
14.757
19.346
1.00
20.96
C


ATOM
578
O
GLU
95
18.275
14.639
18.245
1.00
18.40
O


ATOM
579
N
HIS
96
17.049
15.839
19.702
1.00
20.19
N


ATOM
580
CA
HIS
96
16.860
16.952
18.782
1.00
20.60
C


ATOM
581
CB
HIS
96
16.067
18.089
19.431
1.00
19.83
C


ATOM
582
CG
HIS
96
14.680
17.702
19.844
1.00
21.58
C


ATOM
583
CD2
HIS
96
13.987
16.552
19.675
1.00
22.20
C


ATOM
584
ND1
HIS
96
13.827
18.572
20.490
1.00
24.09
N


ATOM
585
CE1
HIS
96
12.667
17.975
20.700
1.00
23.77
C


ATOM
586
NE2
HIS
96
12.737
16.748
20.215
1.00
24.59
N


ATOM
587
C
HIS
96
18.105
17.502
18.110
1.00
19.96
C


ATOM
588
O
HIS
96
18.112
17.693
16.897
1.00
18.54
O


ATOM
589
N
ARG
97
19.163
17.750
18.873
1.00
19.16
N


ATOM
590
CA
ARG
97
20.343
18.309
18.241
1.00
21.59
C


ATOM
591
CB
ARG
97
21.402
18.733
19.254
1.00
23.22
C


ATOM
592
CG
ARG
97
22.605
19.332
18.560
1.00
26.59
C


ATOM
593
CD
ARG
97
23.697
19.791
19.503
1.00
29.57
C


ATOM
594
NE
ARG
97
24.804
20.353
18.733
1.00
30.13
N


ATOM
595
CZ
ARG
97
25.877
20.930
19.263
1.00
31.77
C


ATOM
596
NH1
ARG
97
25.998
21.028
20.581
1.00
30.89
N


ATOM
597
NH2
ARG
97
26.825
21.417
18.470
1.00
30.56
N


ATOM
598
C
ARG
97
20.927
17.382
17.187
1.00
20.19
C


ATOM
599
O
ARG
97
21.341
17.836
16.124
1.00
18.40
O


ATOM
600
N
LYS
98
20.945
16.083
17.472
1.00
20.40
N


ATOM
601
CA
LYS
98
21.462
15.113
16.512
1.00
22.67
C


ATOM
602
CB
LYS
98
21.499
13.710
17.125
1.00
25.02
C


ATOM
603
CG
LYS
98
21.910
12.590
16.172
1.00
28.25
C


ATOM
604
CD
LYS
98
23.290
12.753
15.572
1.00
32.85
C


ATOM
605
CE
LYS
98
23.591
11.577
14.640
1.00
35.25
C


ATOM
606
NZ
LYS
98
24.936
11.653
13.997
1.00
38.28
N


ATOM
607
C
LYS
98
20.636
15.113
15.232
1.00
21.36
C


ATOM
608
O
LYS
98
21.177
15.067
14.124
1.00
20.55
O


ATOM
609
N
ILE
99
19.322
15.183
15.395
1.00
17.78
N


ATOM
610
CA
ILE
99
18.409
15.168
14.261
1.00
16.72
C


ATOM
611
CB
ILE
99
16.962
15.036
14.757
1.00
18.32
C


ATOM
612
CG2
ILE
99
15.999
15.108
13.577
1.00
18.94
C


ATOM
613
CG1
ILE
99
16.813
13.713
15.522
1.00
18.48
C


ATOM
614
CD1
ILE
99
15.432
13.460
16.100
1.00
20.38
C


ATOM
615
C
ILE
99
18.556
16.406
13.379
1.00
15.71
C


ATOM
616
O
ILE
99
18.745
16.289
12.166
1.00
16.59
O


ATOM
617
N
TYR
100
18.473
17.594
13.970
1.00
16.29
N


ATOM
618
CA
TYR
100
18.649
18.801
13.168
1.00
15.73
C


ATOM
619
CB
TYR
100
18.384
20.081
13.959
1.00
17.85
C


ATOM
620
CG
TYR
100
16.958
20.305
14.383
1.00
18.03
C


ATOM
621
CD1
TYR
100
15.989
20.628
13.433
1.00
18.59
C


ATOM
622
CE1
TYR
100
14.686
20.920
13.805
1.00
19.33
C


ATOM
623
CD2
TYR
100
16.585
20.272
15.724
1.00
19.08
C


ATOM
624
CE2
TYR
100
15.277
20.564
16.108
1.00
20.31
C


ATOM
625
CZ
TYR
100
14.337
20.889
15.142
1.00
20.85
C


ATOM
626
OH
TYR
100
13.047
21.200
15.505
1.00
20.48
O


ATOM
627
C
TYR
100
20.028
18.898
12.547
1.00
17.08
C


ATOM
628
O
TYR
100
20.177
19.362
11.417
1.00
17.10
O


ATOM
629
N
THR
101
21.038
18.455
13.282
1.00
17.01
N


ATOM
630
CA
THR
101
22.395
18.547
12.771
1.00
18.10
C


ATOM
631
CB
THR
101
23.421
18.061
13.818
1.00
19.38
C


ATOM
632
OG1
THR
101
23.409
18.950
14.944
1.00
19.40
O


ATOM
633
CG2
THR
101
24.827
18.023
13.219
1.00
21.23
C


ATOM
634
C
THR
101
22.563
17.729
11.509
1.00
17.82
C


ATOM
635
O
THR
101
23.123
18.207
10.523
1.00
18.96
O


ATOM
636
N
MET
102
22.055
16.503
11.515
1.00
14.87
N


ATOM
637
CA
MET
102
22.241
15.673
10.345
1.00
18.86
C


ATOM
638
CB
MET
102
22.051
14.200
10.687
1.00
22.58
C


ATOM
639
CG
MET
102
22.968
13.769
11.831
1.00
25.73
C


ATOM
640
SD
MET
102
24.729
14.046
11.491
1.00
29.72
S


ATOM
641
CE
MET
102
25.017
12.908
10.134
1.00
26.51
C


ATOM
642
C
MET
102
21.366
16.115
9.181
1.00
17.36
C


ATOM
643
O
MET
102
21.716
15.918
8.019
1.00
17.66
O


ATOM
644
N
ILE
103
20.242
16.746
9.488
1.00
17.10
N


ATOM
645
CA
ILE
103
19.360
17.217
8.432
1.00
18.31
C


ATOM
646
CB
ILE
103
17.971
17.598
9.004
1.00
18.43
C


ATOM
647
CG2
ILE
103
17.137
18.308
7.942
1.00
19.89
C


ATOM
648
CG1
ILE
103
17.261
16.336
9.503
1.00
21.17
C


ATOM
649
CD1
ILE
103
15.895
16.591
10.129
1.00
21.70
C


ATOM
650
C
ILE
103
19.997
18.442
7.778
1.00
18.58
C


ATOM
651
O
ILE
103
20.091
18.521
6.552
1.00
18.12
O


ATOM
652
N
TYR
104
20.461
19.384
8.598
1.00
17.58
N


ATOM
653
CA
TYR
104
21.063
20.600
8.065
1.00
19.92
C


ATOM
654
CB
TYR
104
21.408
21.597
9.171
1.00
17.24
C


ATOM
655
CG
TYR
104
20.245
22.073
10.015
1.00
19.35
C


ATOM
656
CD1
TYR
104
18.920
21.924
9.590
1.00
17.10
C


ATOM
657
CE1
TYR
104
17.861
22.409
10.365
1.00
19.27
C


ATOM
658
CD2
TYR
104
20.479
22.714
11.227
1.00
17.95
C


ATOM
659
CE2
TYR
104
19.439
23.196
12.001
1.00
19.03
C


ATOM
660
CZ
TYR
104
18.137
23.044
11.571
1.00
18.59
C


ATOM
661
OH
TYR
104
17.125
23.531
12.363
1.00
18.75
O


ATOM
662
C
TYR
104
22.308
20.332
7.224
1.00
18.77
C


ATOM
663
O
TYR
104
22.544
21.020
6.234
1.00
17.01
O


ATOM
664
N
ARG
105
23.095
19.332
7.608
1.00
19.90
N


ATOM
665
CA
ARG
105
24.303
19.019
6.850
1.00
22.21
C


ATOM
666
CB
ARG
105
25.252
18.112
7.634
1.00
23.00
C


ATOM
667
CG
ARG
105
25.786
18.721
8.916
1.00
25.04
C


ATOM
668
CD
ARG
105
26.724
17.753
9.616
1.00
27.11
C


ATOM
669
NE
ARG
105
27.269
18.306
10.853
1.00
27.31
N


ATOM
670
CZ
ARG
105
27.986
17.604
11.724
1.00
28.44
C


ATOM
671
NH1
ARG
105
28.241
16.324
11.488
1.00
27.73
N


ATOM
672
NH2
ARG
105
28.442
18.175
12.831
1.00
28.80
N


ATOM
673
C
ARG
105
24.007
18.433
5.479
1.00
22.30
C


ATOM
674
O
ARG
105
24.893
18.333
4.632
1.00
24.22
O


ATOM
675
N
ASN
106
22.752
18.054
5.267
1.00
20.53
N


ATOM
676
CA
ASN
106
22.317
17.497
3.993
1.00
21.19
C


ATOM
677
CB
ASN
106
21.721
16.097
4.143
1.00
19.77
C


ATOM
678
CG
ASN
106
22.744
15.084
4.612
1.00
22.79
C


ATOM
679
OD1
ASN
106
23.423
14.470
3.788
1.00
23.07
O


ATOM
680
ND2
ASN
106
22.843
14.879
5.924
1.00
19.66
N


ATOM
681
C
ASN
106
21.414
18.414
3.191
1.00
20.46
C


ATOM
682
O
ASN
106
20.657
17.967
2.324
1.00
19.35
O


ATOM
683
N
LEU
107
21.510
19.703
3.497
1.00
20.37
N


ATOM
684
CA
LEU
107
20.722
20.740
2.845
1.00
22.38
C


ATOM
685
CB
LEU
107
19.663
21.303
3.791
1.00
21.77
C


ATOM
686
CG
LEU
107
18.618
20.323
4.316
1.00
23.03
C


ATOM
687
CD1
LEU
107
17.706
20.996
5.334
1.00
21.74
C


ATOM
688
CD2
LEU
107
17.816
19.716
3.169
1.00
22.14
C


ATOM
689
C
LEU
107
21.586
21.872
2.306
1.00
23.14
C


ATOM
690
O
LEU
107
22.618
22.205
2.884
1.00
22.39
O


ATOM
691
N
VAL
108
21.158
22.445
1.189
1.00
23.48
N


ATOM
692
CA
VAL
108
21.846
23.569
0.566
1.00
26.51
C


ATOM
693
CB
VAL
108
22.484
23.158
−0.776
1.00
28.25
C


ATOM
694
CG1
VAL
108
23.128
24.364
−1.432
1.00
31.06
C


ATOM
695
CG2
VAL
108
23.511
22.064
−0.543
1.00
30.49
C


ATOM
696
C
VAL
108
20.795
24.647
0.304
1.00
25.74
C


ATOM
697
O
VAL
108
19.766
24.363
−0.301
1.00
24.47
O


ATOM
698
N
VAL
109
21.042
25.871
0.767
1.00
25.08
N


ATOM
699
CA
VAL
109
20.096
26.958
0.548
1.00
28.59
C


ATOM
700
CB
VAL
109
20.491
28.224
1.325
1.00
30.38
C


ATOM
701
CG1
VAL
109
19.457
29.320
1.088
1.00
30.40
C


ATOM
702
CG2
VAL
109
20.600
27.907
2.809
1.00
30.06
C


ATOM
703
C
VAL
109
20.007
27.285
−0.942
1.00
31.09
C


ATOM
704
O
VAL
109
21.017
27.538
−1.600
1.00
30.68
O


ATOM
705
N
VAL
110
18.784
27.273
−1.457
1.00
34.26
N


ATOM
706
CA
VAL
110
18.497
27.512
−2.869
1.00
40.68
C


ATOM
707
CB
VAL
110
17.092
26.951
−3.231
1.00
41.37
C


ATOM
708
CG1
VAL
110
16.776
27.222
−4.691
1.00
42.09
C


ATOM
709
CG2
VAL
110
17.046
25.456
−2.948
1.00
41.30
C


ATOM
710
C
VAL
110
18.586
28.950
−3.376
1.00
45.42
C


ATOM
711
O
VAL
110
18.320
29.904
−2.643
1.00
45.46
O


ATOM
712
N
ASN
111
18.980
29.063
−4.644
1.00
50.62
N


ATOM
713
CA
ASN
111
19.090
30.319
−5.380
1.00
54.41
C


ATOM
714
CB
ASN
111
18.454
30.130
−6.764
1.00
57.01
C


ATOM
715
CG
ASN
111
18.469
31.386
−7.600
1.00
59.53
C


ATOM
716
OD1
ASN
111
17.413
31.886
−7.991
1.00
60.59
O


ATOM
717
ND2
ASN
111
19.660
31.900
−7.891
1.00
60.11
N


ATOM
718
C
ASN
111
18.521
31.547
−4.662
1.00
54.78
C


ATOM
719
O
ASN
111
17.400
31.982
−4.938
1.00
56.01
O


ATOM
720
C1
SCH
996
−1.000
8.674
15.423
1.00
58.94


ATOM
721
C2
SCH
996
0.074
7.820
16.068
1.00
58.85


ATOM
722
O1
SCH
996
−0.100
7.261
17.153
1.00
59.47


ATOM
723
N1
SCH
996
1.202
7.745
15.337
1.00
57.09


ATOM
724
C3
SCH
996
2.471
7.578
16.042
1.00
55.53


ATOM
725
C4
SCH
996
3.102
8.922
16.429
1.00
58.30


ATOM
726
O2
SCH
996
3.020
9.892
15.673
1.00
59.95


ATOM
727
N2
SCH
996
3.724
8.908
17.631
1.00
61.62


ATOM
728
C5
SCH
996
4.020
10.154
18.329
1.00
63.29


ATOM
729
C6
SCH
996
2.706
10.796
18.790
1.00
65.39


ATOM
730
O3
SCH
996
2.516
12.011
18.716
1.00
65.59


ATOM
731
N3
SCH
996
1.822
9.901
19.269
1.00
67.92


ATOM
732
C7
SCH
996
0.554
10.407
19.793
1.00
70.11


ATOM
733
C8
SCH
996
0.346
9.699
20.930
1.00
70.97


ATOM
734
O4
SCH
996
0.223
10.153
22.077
1.00
71.51


ATOM
735
O5
SCH
996
0.325
8.321
20.830
1.00
71.56


ATOM
736
C9
SCH
996
3.406
6.688
15.192
1.00
49.97


ATOM
737
C10
SCH
996
−0.504
10.428
18.687
1.00
70.56


ATOM
738
C11
SCH
996
−0.315
11.537
17.664
1.00
71.51


ATOM
739
C12
SCH
996
−1.305
12.653
17.941
1.00
72.44


ATOM
740
O6
SCH
996
−0.991
13.651
18.515
1.00
72.59


ATOM
741
O7
SCH
996
−2.556
12.416
17.482
1.00
72.61


ATOM
742
C13
SCH
996
4.852
6.796
15.638
1.00
44.76


ATOM
743
C14
SCH
996
5.890
7.608
15.051
1.00
41.72


ATOM
744
C15
SCH
996
7.096
7.390
15.825
1.00
40.91


ATOM
745
N4
SCH
996
6.776
6.472
16.833
1.00
40.79


ATOM
746
C16
SCH
996
5.458
6.131
16.715
1.00
43.06


ATOM
747
C17
SCH
996
5.912
8.510
13.946
1.00
40.02


ATOM
748
C18
SCH
996
7.115
9.193
13.606
1.00
37.25


ATOM
749
C19
SCH
996
8.298
8.983
14.370
1.00
36.32


ATOM
750
C20
SCH
996
8.295
8.082
15.474
1.00
37.47


ATOM
751
CL1
SCH
996
9.737
9.840
13.965
1.00
29.32


ATOM
752
C21
SCH
996
4.747
11.137
17.396
1.00
62.87


ATOM
753
C22
SCH
996
4.877
9.854
19.566
1.00
62.94


ATOM
754
C23
SCH
996
6.279
9.364
19.188
1.00
62.44


ATOM
755
C24
SCH
996
6.166
10.673
17.043
1.00
62.33


ATOM
756
C25
SCH
996
7.006
10.356
18.280
1.00
62.08


ATOM
757
C1
SCH
998
7.020
14.306
19.529
1.00
32.04


ATOM
758
C2
SCH
998
8.132
14.220
18.499
1.00
30.99


ATOM
759
O1
SCH
998
9.294
13.953
18.813
1.00
31.33


ATOM
760
N1
SCH
998
7.711
14.463
17.243
1.00
28.90


ATOM
761
C3
SCH
998
8.715
14.626
16.190
1.00
28.20


ATOM
762
C4
SCH
998
9.050
16.095
15.923
1.00
28.60


ATOM
763
O2
SCH
998
8.184
16.880
15.527
1.00
28.06


ATOM
764
N2
SCH
998
10.342
16.402
16.159
1.00
26.21


ATOM
765
C5
SCH
998
10.807
17.775
16.036
1.00
28.33


ATOM
766
C6
SCH
998
9.978
18.673
16.963
1.00
33.18


ATOM
767
O3
SCH
998
9.634
19.808
16.630
1.00
35.64


ATOM
768
N3
SCH
998
9.686
18.088
18.140
1.00
37.61


ATOM
769
C7
SCH
998
8.697
18.731
19.006
1.00
42.54


ATOM
770
C8
SCH
998
9.245
18.545
20.404
1.00
42.96


ATOM
771
O4
SCH
998
9.535
19.446
21.049
1.00
44.48


ATOM
772
O5
SCH
998
9.382
17.262
20.693
1.00
43.12


ATOM
773
C9
SCH
998
8.244
13.892
14.923
1.00
27.77


ATOM
774
C10
SCH
998
7.290
18.240
18.661
1.00
44.92


ATOM
775
C11
SCH
998
6.730
18.812
17.368
1.00
48.30


ATOM
776
C12
SCH
998
5.583
19.752
17.684
1.00
49.87


ATOM
777
O6
SCH
998
5.601
20.902
17.365
1.00
51.11


ATOM
778
O7
SCH
998
4.554
19.175
18.348
1.00
50.51


ATOM
779
C13
SCH
998
9.158
14.149
13.740
1.00
25.37


ATOM
780
C14
SCH
998
10.480
13.618
13.499
1.00
25.02


ATOM
781
C15
SCH
998
10.902
14.099
12.199
1.00
25.73


ATOM
782
N4
SCH
998
9.868
14.895
11.699
1.00
25.53


ATOM
783
C16
SCH
998
8.852
14.918
12.609
1.00
25.55


ATOM
784
C17
SCH
998
11.350
12.777
14.254
1.00
25.02


ATOM
785
C18
SCH
998
12.617
12.399
13.724
1.00
25.30


ATOM
786
C19
SCH
998
13.020
12.858
12.437
1.00
26.55


ATOM
787
C20
SCH
998
12.171
13.711
11.677
1.00
24.81


ATOM
788
CL1
SCH
998
14.529
12.355
11.778
1.00
26.71


ATOM
789
C21
SCH
998
10.639
18.264
14.593
1.00
26.42


ATOM
790
C22
SCH
998
12.279
17.852
16.457
1.00
26.82


ATOM
791
C23
SCH
998
13.181
17.061
15.496
1.00
25.79


ATOM
792
C24
SCH
998
11.554
17.510
13.617
1.00
24.84


ATOM
793
C25
SCH
998
13.025
17.532
14.048
1.00
24.21


ATOM
794
C1
SCH
999
14.304
27.533
13.501
1.00
20.77


ATOM
795
C2
SCH
999
14.911
27.965
12.179
1.00
20.52


ATOM
796
O1
SCH
999
14.277
28.618
11.350
1.00
21.90


ATOM
797
N1
SCH
999
16.183
27.557
12.024
1.00
19.32


ATOM
798
C3
SCH
999
16.883
27.892
10.787
1.00
20.88


ATOM
799
C4
SCH
999
16.080
27.517
9.526
1.00
22.08


ATOM
800
O2
SCH
999
15.657
26.372
9.370
1.00
21.93


ATOM
801
N2
SCH
999
15.894
28.551
8.669
1.00
22.51


ATOM
802
C5
SCH
999
15.213
28.377
7.382
1.00
24.88


ATOM
803
C6
SCH
999
13.709
28.188
7.602
1.00
26.16


ATOM
804
O3
SCH
999
12.953
27.935
6.665
1.00
27.24


ATOM
805
N3
SCH
999
13.318
28.331
8.879
1.00
27.90


ATOM
806
C7
SCH
999
11.886
28.201
9.143
1.00
31.35


ATOM
807
C8
SCH
999
11.241
29.583
9.052
1.00
34.43


ATOM
808
O4
SCH
999
10.006
29.558
9.037
1.00
35.86


ATOM
809
O5
SCH
999
11.994
30.531
8.919
1.00
36.30


ATOM
810
C9
SCH
999
18.277
27.245
10.824
1.00
20.43


ATOM
811
C10
SCH
999
11.653
27.295
10.349
1.00
31.35


ATOM
812
C11
SCH
999
12.066
25.844
10.129
1.00
29.83


ATOM
813
C12
SCH
999
11.716
25.026
11.360
1.00
32.05


ATOM
814
O6
SCH
999
12.531
24.381
11.942
1.00
31.48


ATOM
815
O7
SCH
999
10.416
25.092
11.730
1.00
32.95


ATOM
816
C13
SCH
999
19.093
27.591
9.595
1.00
21.99


ATOM
817
C14
SCH
999
19.603
26.675
8.606
1.00
21.11


ATOM
818
C15
SCH
999
20.326
27.456
7.626
1.00
22.79


ATOM
819
N4
SCH
999
20.243
28.793
8.029
1.00
23.21


ATOM
820
C16
SCH
999
19.518
28.863
9.184
1.00
22.90


ATOM
821
C17
SCH
999
19.512
25.260
8.455
1.00
21.66


ATOM
822
C18
SCH
999
20.126
24.621
7.341
1.00
23.05


ATOM
823
C19
SCH
999
20.833
25.388
6.371
1.00
22.06


ATOM
824
C20
SCH
999
20.937
26.803
6.511
1.00
22.85


ATOM
825
CL1
SCH
999
21.550
24.594
5.014
1.00
26.59


ATOM
826
C21
SCH
999
15.747
27.140
6.639
1.00
23.66


ATOM
827
C22
SCH
999
15.409
29.639
6.533
1.00
24.90


ATOM
828
C23
SCH
999
16.867
29.820
6.129
1.00
24.75


ATOM
829
C24
SCH
999
17.176
27.321
6.103
1.00
23.22


ATOM
830
C25
SCH
999
17.379
28.625
5.329
1.00
23.88


ATOM
831
OH2
WAT
1001
3.395
9.112
−1.435
1.00
16.59
O


ATOM
839
OH2
WAT
1002
24.775
12.618
6.376
1.00
17.52
O


ATOM
832
OH2
WAT
1003
18.514
−0.561
14.622
1.00
18.25
O


ATOM
835
OH2
WAT
1004
15.044
24.301
11.181
1.00
18.80
O


ATOM
856
OH2
WAT
1005
20.867
15.283
20.436
1.00
24.20
O


ATOM
842
OH2
WAT
1006
9.724
26.049
6.031
1.00
24.72
O


ATOM
860
OH2
WAT
1007
6.074
13.931
10.197
1.00
24.81
O


ATOM
857
OH2
WAT
1008
21.757
8.195
18.608
1.00
25.80
O


ATOM
833
OH2
WAT
1009
12.107
26.746
4.500
1.00
25.85
O


ATOM
843
OH2
WAT
1010
16.620
1.807
2.942
1.00
26.33
O


ATOM
870
OH2
WAT
1011
7.466
20.307
9.569
1.00
26.55
O


ATOM
848
OH2
WAT
1012
11.574
22.642
13.779
1.00
26.80
O


ATOM
834
OH2
WAT
1013
13.759
−1.012
20.605
1.00
28.49
O


ATOM
840
OH2
WAT
1014
25.743
20.101
15.817
1.00
28.57
O


ATOM
855
OH2
WAT
1015
0.544
11.546
5.002
1.00
29.35
O


ATOM
844
OH2
WAT
1016
21.356
8.460
22.469
1.00
30.69
O


ATOM
836
OH2
WAT
1017
14.502
−0.707
6.212
1.00
31.40
O


ATOM
841
OH2
WAT
1018
11.775
14.532
17.517
1.00
31.42
O


ATOM
851
OH2
WAT
1019
22.475
1.722
7.609
1.00
31.99
O


ATOM
859
OH2
WAT
1020
5.732
18.551
−1.162
1.00
32.12
O


ATOM
872
OH2
WAT
1021
23.725
20.929
22.606
1.00
34.79
O


ATOM
862
OH2
WAT
1022
23.860
26.433
1.644
1.00
34.96
O


ATOM
863
OH2
WAT
1023
5.394
16.356
11.571
1.00
35.71
O


ATOM
861
OH2
WAT
1024
21.332
30.796
5.811
1.00
36.70
O


ATOM
847
OH2
WAT
1025
9.148
−2.863
15.900
1.00
37.64
O


ATOM
858
OH2
WAT
1026
14.166
16.525
23.729
1.00
37.84
O


ATOM
866
OH2
WAT
1027
11.334
30.280
5.618
1.00
38.35
O


ATOM
864
OH2
WAT
1028
18.007
18.195
−4.362
1.00
40.37
O


ATOM
871
OH2
WAT
1029
11.860
21.767
17.854
1.00
40.71
O


ATOM
874
OH2
WAT
1030
8.276
27.446
8.045
1.00
41.07
O


ATOM
846
OH2
WAT
1031
7.632
18.152
11.360
1.00
42.63
O


ATOM
865
OH2
WAT
1032
25.898
10.512
16.626
1.00
43.37
O


ATOM
854
OH2
WAT
1033
8.159
−1.042
14.070
1.00
44.00
O


ATOM
852
OH2
WAT
1034
28.464
14.740
9.077
1.00
46.88
O


ATOM
845
OH2
WAT
1035
2.078
6.181
4.658
1.00
47.60
O


ATOM
838
OH2
WAT
1036
26.484
15.271
4.182
1.00
47.99
O


ATOM
867
OH2
WAT
1037
23.657
28.439
−1.144
1.00
50.33
O


ATOM
837
OH2
WAT
1038
13.538
21.361
20.180
1.00
54.13
O


ATOM
869
OH2
WAT
1039
25.590
16.309
16.444
1.00
54.19
O


ATOM
850
OH2
WAT
1040
26.845
13.685
16.859
1.00
55.86
O


ATOM
873
OH2
WAT
1041
27.978
4.166
14.080
1.00
55.87
O


ATOM
868
OH2
WAT
1042
20.489
1.898
−2.662
1.00
56.16
O


ATOM
853
OH2
WAT
1043
17.178
10.875
−6.741
1.00
59.20
O


ATOM
849
OH2
WAT
1044
25.481
2.906
8.796
1.00
63.46
O


END









EXAMPLE 3
Preparation and Crystallization of Hdm2 Y76H-Tripeptide Complex
Production and Crystallization of Modified Hdm2(Y76H):
The modified Hdm2(Y76H) protein was produced using the QuickChange site-directed mutagenesis method as discussed in Example 1 except that only the primers for Y76H were used in mutagenesis. The p53 peptide analog, Ac-6ClWAC3cE, disclosed and defined above, was dissolved in the same buffer and added to the Hdm2(Y76H) protein solution.
The single mutant HDM2 (17-125) Y76H-tripeptide complex was crystallized using a hanging-drop vapor diffusion method. The protein-peptide solution (1 μl; 6-10 mg/ml) in buffer A was mixed with an equal volume of precipitant [0.1 M Tris, pH 8-9, 35% PEG 4000, and 0.0 to 0.2 M magnesium chloride], placed on the underside of a siliconized glass coverslip and sealed in close proximity to 1 ml of the precipitant solution. Vapor diffusion crystallization experiments were conducted using the hanging drop method. Specifically, crystals were grown from a droplet containing a mixture of 0.5-2.0 μl of protein and 0.5-1.0 μl of the precipitant solution. Crystallization plates were incubated at 4° C.; rectangular rod crystals (0.1×0.1×0.3 mm) grew over 2-30 days.
Prior to data collection, crystals were washed with the reservoir solution of the crystallization setup and transferred into the same solution with 10% glycerol added. The crystals were then flash-cooled in a nitrogen stream at 95 K. X-ray diffraction was collected using a Riga generator equipped with a Praxis 4++ detector. Data were integrated and scaled using the HKL package.
Data Collection Statistics:
















Resolution
50.0-.20 Å



No. of collected reflections
13234



No. of unique reflections (F >= 0)
 4341



R-sym
 6.2%



Percent of theoretical (I/s >= 1)
84.8%



Unit Cell
a = 41.1 Å, b = 42.7 Å,




c = 53.777 Å,




α = β = γ = 90°



Space Group
P212121



Asymmetric unit
1 molecule










The crystal structure was solved using molecular replacement using the search models 1YCQ and 1YCR from the PDB. Refinement was done using the program CNX.
















Theoretical number of reflections
5854



Resolution Limits
50.0-2.1 Å



Number of unobserved reflections
1529 (26.1%)



Number of reflections in working set
4325 (73.9%)



Number of reflections in test set
 197 (3.4%)



Number of protein residues
 87



Number of solvent atoms
  0



R-factor
  0.45



R-free
  0.51



RMSD bond length
    0.014 Å



RMSD bond angles
1.97°










The structural coordinates for the above-described Hdm2 crystal are set forth below in Table 4.

















TABLE 4





1
2
3
4
5
6
7
8
9
10
11

























ATOM
1
CB
GLU
25
7.387
−0.822
5.902
1.00
31.40
C


ATOM
2
CG
GLU
25
7.019
−0.298
4.509
1.00
35.01
C


ATOM
3
CD
GLU
25
7.873
−0.972
3.442
1.00
37.01
C


ATOM
4
OE1
GLU
25
7.321
−1.714
2.600
1.00
35.86
O


ATOM
5
OE2
GLU
25
9.101
−0.744
3.437
1.00
38.68
O


ATOM
6
C
GLU
25
5.081
−0.176
6.662
1.00
30.27
C


ATOM
7
O
GLU
25
4.374
−1.161
6.879
1.00
31.21
O


ATOM
8
N
GLU
25
6.922
−0.708
8.375
1.00
29.47
N


ATOM
9
CA
GLU
25
6.567
−0.166
7.021
1.00
31.52
C


ATOM
10
N
THR
26
4.610
0.938
6.116
1.00
28.18
N


ATOM
11
CA
THR
26
3.217
1.047
5.711
1.00
24.93
C


ATOM
12
CB
THR
26
2.441
2.031
6.611
1.00
24.28
C


ATOM
13
OG1
THR
26
2.654
1.706
7.990
1.00
23.56
O


ATOM
14
CG2
THR
26
0.955
1.959
6.304
1.00
22.48
C


ATOM
15
C
THR
26
3.096
1.533
4.264
1.00
24.10
C


ATOM
16
O
THR
26
3.633
2.581
3.908
1.00
22.83
O


ATOM
17
N
LEU
27
2.405
0.760
3.431
1.00
19.92
N


ATOM
18
CA
LEU
27
2.177
1.160
2.047
1.00
20.55
C


ATOM
19
CB
LEU
27
2.052
−0.044
1.107
1.00
22.12
C


ATOM
20
CG
LEU
27
3.257
−0.991
1.005
1.00
24.55
C


ATOM
21
CD1
LEU
27
2.945
−2.199
0.113
1.00
22.27
C


ATOM
22
CD2
LEU
27
4.506
−0.259
0.513
1.00
22.59
C


ATOM
23
C
LEU
27
0.899
1.995
2.057
1.00
19.40
C


ATOM
24
O
LEU
27
−0.124
1.568
2.599
1.00
18.49
O


ATOM
25
N
VAL
28
0.956
3.190
1.476
1.00
20.26
N


ATOM
26
CA
VAL
28
−0.206
4.075
1.457
1.00
21.18
C


ATOM
27
CB
VAL
28
−0.090
5.200
2.523
1.00
19.01
C


ATOM
28
CG1
VAL
28
0.094
4.599
3.897
1.00
19.71
C


ATOM
29
CG2
VAL
28
1.057
6.125
2.183
1.00
20.08
C


ATOM
30
C
VAL
28
−0.481
4.741
0.118
1.00
21.98
C


ATOM
31
O
VAL
28
0.431
4.966
−0.679
1.00
23.19
O


ATOM
32
N
ARG
29
−1.755
5.042
−0.120
1.00
23.38
N


ATOM
33
CA
ARG
29
−2.188
5.722
−1.338
1.00
25.82
C


ATOM
34
CB
ARG
29
−3.239
4.904
−2.089
1.00
27.99
C


ATOM
35
CG
ARG
29
−2.761
3.515
−2.489
1.00
30.54
C


ATOM
36
CD
ARG
29
−3.838
2.738
−3.220
1.00
33.91
C


ATOM
37
NE
ARG
29
−3.398
1.395
−3.590
1.00
37.13
N


ATOM
38
CZ
ARG
29
−2.478
1.134
−4.511
1.00
39.21
C


ATOM
39
NH1
ARG
29
−1.896
2.131
−5.168
1.00
41.39
N


ATOM
40
NH2
ARG
29
−2.135
−0.122
−4.771
1.00
39.25
N


ATOM
41
C
ARG
29
−2.743
7.082
−0.905
1.00
26.04
C


ATOM
42
O
ARG
29
−3.723
7.163
−0.162
1.00
25.87
O


ATOM
43
N
PRO
30
−2.100
8.167
−1.358
1.00
25.80
N


ATOM
44
CD
PRO
30
−0.679
8.055
−1.738
1.00
24.67
C


ATOM
45
CA
PRO
30
−2.394
9.579
−1.111
1.00
22.90
C


ATOM
46
CB
PRO
30
−1.203
10.259
−1.751
1.00
22.35
C


ATOM
47
CG
PRO
30
−0.114
9.428
−1.255
1.00
26.65
C


ATOM
48
C
PRO
30
−3.699
10.094
−1.723
1.00
22.50
C


ATOM
49
O
PRO
30
−4.062
9.701
−2.835
1.00
21.62
O


ATOM
50
N
LYS
31
−4.411
10.954
−1.001
1.00
22.08
N


ATOM
51
CA
LYS
31
−5.593
11.574
−1.590
1.00
23.03
C


ATOM
52
CB
LYS
31
−6.451
12.381
−0.620
1.00
25.31
C


ATOM
53
CG
LYS
31
−7.122
11.655
0.501
1.00
27.67
C


ATOM
54
CD
LYS
31
−7.906
12.701
1.266
1.00
29.82
C


ATOM
55
CE
LYS
31
−8.654
12.122
2.423
1.00
30.22
C


ATOM
56
NZ
LYS
31
−9.401
13.196
3.133
1.00
31.71
N


ATOM
57
C
LYS
31
−5.065
12.527
−2.648
1.00
21.77
C


ATOM
58
O
LYS
31
−3.906
12.951
−2.590
1.00
21.83
O


ATOM
59
N
PRO
32
−5.909
12.873
−3.629
1.00
20.24
N


ATOM
60
CD
PRO
32
−7.253
12.301
−3.838
1.00
20.22
C


ATOM
61
CA
PRO
32
−5.608
13.770
−4.736
1.00
19.66
C


ATOM
62
CB
PRO
32
−6.996
14.120
−5.227
1.00
21.15
C


ATOM
63
CG
PRO
32
−7.598
12.787
−5.277
1.00
21.31
C


ATOM
64
C
PRO
32
−4.799
15.018
−4.348
1.00
18.96
C


ATOM
65
O
PRO
32
−3.832
15.368
−5.022
1.00
17.76
O


ATOM
66
N
LEU
33
−5.190
15.682
−3.263
1.00
20.65
N


ATOM
67
CA
LEU
33
−4.497
16.901
−2.853
1.00
22.91
C


ATOM
68
CB
LEU
33
−5.412
17.805
−2.025
1.00
27.03
C


ATOM
69
CG
LEU
33
−6.690
18.264
−2.733
1.00
30.44
C


ATOM
70
CD1
LEU
33
−7.567
19.092
−1.796
1.00
32.45
C


ATOM
71
CD2
LEU
33
−6.385
19.036
−4.014
1.00
31.10
C


ATOM
72
C
LEU
33
−3.172
16.659
−2.141
1.00
22.13
C


ATOM
73
O
LEU
33
−2.277
17.507
−2.169
1.00
20.13
O


ATOM
74
N
LEU
34
−3.041
15.500
−1.507
1.00
22.44
N


ATOM
75
CA
LEU
34
−1.788
15.170
−0.845
1.00
22.32
C


ATOM
76
CB
LEU
34
−1.967
14.032
0.158
1.00
22.46
C


ATOM
77
CG
LEU
34
−0.686
13.562
0.851
1.00
25.03
C


ATOM
78
CD1
LEU
34
0.092
14.709
1.488
1.00
22.68
C


ATOM
79
CD2
LEU
34
−0.984
12.449
1.846
1.00
25.15
C


ATOM
80
C
LEU
34
−0.833
14.775
−1.959
1.00
20.98
C


ATOM
81
O
LEU
34
0.370
15.027
−1.892
1.00
18.72
O


ATOM
82
N
LEU
35
−1.403
14.172
−2.996
1.00
22.16
N


ATOM
83
CA
LEU
35
−0.644
13.746
−4.158
1.00
22.50
C


ATOM
84
CB
LEU
35
−1.499
12.888
−5.086
1.00
21.79
C


ATOM
85
CG
LEU
35
−0.799
12.451
−6.371
1.00
21.31
C


ATOM
86
CD1
LEU
35
0.509
11.738
−6.069
1.00
22.29
C


ATOM
87
CD2
LEU
35
−1.709
11.605
−7.249
1.00
21.81
C


ATOM
88
C
LEU
35
−0.120
14.950
−4.913
1.00
23.62
C


ATOM
89
O
LEU
35
0.965
14.909
−5.489
1.00
24.50
O


ATOM
90
N
LYS
36
−0.892
16.029
−4.887
1.00
26.45
N


ATOM
91
CA
LYS
36
−0.517
17.247
−5.581
1.00
30.02
C


ATOM
92
CB
LYS
36
−1.739
18.156
−5.759
1.00
31.51
C


ATOM
93
CG
LYS
36
−1.468
19.454
−6.494
1.00
34.42
C


ATOM
94
CD
LYS
36
−2.742
20.280
−6.633
1.00
37.59
C


ATOM
95
CE
LYS
36
−2.483
21.584
−7.381
1.00
40.02
C


ATOM
96
NZ
LYS
36
−3.723
22.409
−7.540
1.00
42.11
N


ATOM
97
C
LYS
36
0.624
17.964
−4.864
1.00
30.99
C


ATOM
98
O
LYS
36
1.321
18.782
−5.460
1.00
32.96
O


ATOM
99
N
LEU
37
0.828
17.645
−3.590
1.00
31.87
N


ATOM
100
CA
LEU
37
1.909
18.274
−2.841
1.00
33.15
C


ATOM
101
CB
LEU
37
1.504
18.569
−1.395
1.00
34.78
C


ATOM
102
CG
LEU
37
0.316
19.498
−1.144
1.00
37.22
C


ATOM
103
CD1
LEU
37
0.039
19.625
0.347
1.00
36.79
C


ATOM
104
CD2
LEU
37
0.540
20.873
−1.760
1.00
38.28
C


ATOM
105
C
LEU
37
3.153
17.395
−2.866
1.00
32.75
C


ATOM
106
O
LEU
37
4.277
17.889
−2.747
1.00
31.66
O


ATOM
107
N
LEU
38
2.945
16.093
−3.040
1.00
30.45
N


ATOM
108
CA
LEU
38
4.054
15.155
−3.080
1.00
28.31
C


ATOM
109
CB
LEU
38
3.578
13.726
−2.805
1.00
27.70
C


ATOM
110
CG
LEU
38
2.878
13.360
−1.492
1.00
27.23
C


ATOM
111
CD1
LEU
38
2.383
11.925
−1.547
1.00
27.09
C


ATOM
112
CD2
LEU
38
3.750
13.597
−0.262
1.00
24.95
C


ATOM
113
C
LEU
38
4.713
15.230
−4.451
1.00
28.49
C


ATOM
114
O
LEU
38
5.888
14.897
−4.598
1.00
29.16
O


ATOM
115
N
LYS
39
3.956
15.684
−5.449
1.00
27.07
N


ATOM
116
CA
LYS
39
4.480
15.783
−6.806
1.00
28.73
C


ATOM
117
CB
LYS
39
3.373
15.754
−7.861
1.00
33.27
C


ATOM
118
CG
LYS
39
2.504
14.521
−7.920
1.00
37.75
C


ATOM
119
CD
LYS
39
1.487
14.719
−9.035
1.00
41.51
C


ATOM
120
CE
LYS
39
0.550
13.539
−9.187
1.00
45.31
C


ATOM
121
NZ
LYS
39
−0.430
13.767
−10.292
1.00
47.13
N


ATOM
122
C
LYS
39
5.304
17.036
−7.034
1.00
25.77
C


ATOM
123
O
LYS
39
6.154
17.062
−7.916
1.00
27.98
O


ATOM
124
N
SER
40
5.064
18.071
−6.240
1.00
24.34
N


ATOM
125
CA
SER
40
5.795
19.311
−6.427
1.00
23.75
C


ATOM
126
CB
SER
40
5.033
20.501
−5.845
1.00
24.25
C


ATOM
127
OG
SER
40
4.844
20.354
−4.451
1.00
24.47
O


ATOM
128
C
SER
40
7.180
19.229
−5.825
1.00
23.75
C


ATOM
129
O
SER
40
8.029
20.066
−6.099
1.00
26.76
O


ATOM
130
N
VAL
41
7.414
18.203
−5.018
1.00
26.35
N


ATOM
131
CA
VAL
41
8.701
18.055
−4.367
1.00
25.90
C


ATOM
132
CB
VAL
41
8.550
18.299
−2.846
1.00
26.80
C


ATOM
133
CG1
VAL
41
7.800
17.139
−2.204
1.00
25.77
C


ATOM
134
CG2
VAL
41
9.905
18.491
−2.202
1.00
28.50
C


ATOM
135
C
VAL
41
9.308
16.674
−4.597
1.00
26.12
C


ATOM
136
O
VAL
41
10.480
16.450
−4.300
1.00
28.42
O


ATOM
137
N
GLY
42
8.523
15.751
−5.144
1.00
24.94
N


ATOM
138
CA
GLY
42
9.049
14.416
−5.363
1.00
22.59
C


ATOM
139
C
GLY
42
8.438
13.615
−6.495
1.00
22.27
C


ATOM
140
O
GLY
42
7.720
14.143
−7.347
1.00
19.19
O


ATOM
141
N
ALA
43
8.740
12.321
−6.490
1.00
21.23
N


ATOM
142
CA
ALA
43
8.252
11.387
−7.491
1.00
23.12
C


ATOM
143
CB
ALA
43
8.939
10.043
−7.326
1.00
22.78
C


ATOM
144
C
ALA
43
6.740
11.202
−7.448
1.00
26.26
C


ATOM
145
O
ALA
43
6.137
11.093
−6.382
1.00
26.47
O


ATOM
146
N
GLN
44
6.144
11.182
−8.632
1.00
29.51
N


ATOM
147
CA
GLN
44
4.711
10.994
−8.814
1.00
34.61
C


ATOM
148
CB
GLN
44
4.254
11.798
−10.044
1.00
38.60
C


ATOM
149
CG
GLN
44
2.772
11.752
−10.418
1.00
44.52
C


ATOM
150
CD
GLN
44
2.343
10.341
−10.743
1.00
47.57
C


ATOM
151
OE1
GLN
44
1.459
9.781
−10.093
1.00
50.77
O


ATOM
152
NE2
GLN
44
2.959
9.760
−11.768
1.00
49.38
N


ATOM
153
C
GLN
44
4.452
9.486
−8.949
1.00
33.65
C


ATOM
154
O
GLN
44
4.970
8.844
−9.857
1.00
34.09
O


ATOM
155
N
LYS
45
3.660
8.925
−8.040
1.00
33.88
N


ATOM
156
CA
LYS
45
3.353
7.493
−8.071
1.00
34.47
C


ATOM
157
CB
LYS
45
4.586
6.652
−7.744
1.00
31.48
C


ATOM
158
CG
LYS
45
5.205
6.911
−6.388
1.00
28.37
C


ATOM
159
CD
LYS
45
6.411
6.017
−6.214
1.00
24.70
C


ATOM
160
CE
LYS
45
7.084
6.228
−4.883
1.00
24.73
C


ATOM
161
NZ
LYS
45
8.267
5.329
−4.749
1.00
24.48
N


ATOM
162
C
LYS
45
2.151
7.096
−7.218
1.00
36.41
C


ATOM
163
O
LYS
45
1.924
7.670
−6.157
1.00
36.79
O


ATOM
164
N
ASP
46
1.375
6.120
−7.693
1.00
39.70
N


ATOM
165
CA
ASP
46
0.178
5.678
−6.974
1.00
41.06
C


ATOM
166
CB
ASP
46
−0.641
4.672
−7.792
1.00
42.39
C


ATOM
167
CG
ASP
46
−1.137
5.241
−9.105
1.00
43.83
C


ATOM
168
OD1
ASP
46
−0.864
4.624
−10.156
1.00
45.18
O


ATOM
169
OD2
ASP
46
−1.799
6.301
−9.086
1.00
43.47
O


ATOM
170
C
ASP
46
0.391
5.118
−5.573
1.00
40.30
C


ATOM
171
O
ASP
46
−0.539
5.103
−4.769
1.00
40.91
O


ATOM
172
N
THR
47
1.605
4.671
−5.268
1.00
38.48
N


ATOM
173
CA
THR
47
1.868
4.100
−3.953
1.00
35.27
C


ATOM
174
CB
THR
47
1.860
2.564
−4.000
1.00
34.96
C


ATOM
175
OG1
THR
47
0.592
2.115
−4.482
1.00
35.07
O


ATOM
176
CG2
THR
47
2.083
1.986
−2.614
1.00
34.48
C


ATOM
177
C
THR
47
3.165
4.561
−3.312
1.00
33.33
C


ATOM
178
O
THR
47
4.224
4.569
−3.942
1.00
34.15
O


ATOM
179
N
TYR
48
3.055
4.951
−2.048
1.00
30.30
N


ATOM
180
CA
TYR
48
4.180
5.435
−1.256
1.00
27.10
C


ATOM
181
CB
TYR
48
4.048
6.925
−0.949
1.00
24.18
C


ATOM
182
CG
TYR
48
3.995
7.837
−2.150
1.00
21.88
C


ATOM
183
CD1
TYR
48
5.152
8.418
−2.659
1.00
19.95
C


ATOM
184
CE1
TYR
48
5.096
9.298
−3.727
1.00
20.60
C


ATOM
185
CD2
TYR
48
2.782
8.153
−2.747
1.00
19.91
C


ATOM
186
CE2
TYR
48
2.716
9.027
−3.812
1.00
20.90
C


ATOM
187
CZ
TYR
48
3.872
9.598
−4.298
1.00
20.62
C


ATOM
188
OH
TYR
48
3.795
10.481
−5.347
1.00
22.83
O


ATOM
189
C
TYR
48
4.269
4.685
0.061
1.00
26.72
C


ATOM
190
O
TYR
48
3.275
4.158
0.558
1.00
27.60
O


ATOM
191
N
THR
49
5.470
4.620
0.616
1.00
27.57
N


ATOM
192
CA
THR
49
5.647
4.001
1.916
1.00
27.42
C


ATOM
193
CB
THR
49
7.100
3.558
2.171
1.00
26.03
C


ATOM
194
OG1
THR
49
7.953
4.708
2.191
1.00
24.83
O


ATOM
195
CG2
THR
49
7.562
2.595
1.092
1.00
26.14
C


ATOM
196
C
THR
49
5.317
5.167
2.836
1.00
26.91
C


ATOM
197
O
THR
49
5.606
6.313
2.506
1.00
27.92
O


ATOM
198
N
MET
50
4.692
4.891
3.969
1.00
28.56
N


ATOM
199
CA
MET
50
4.345
5.953
4.905
1.00
30.04
C


ATOM
200
CB
MET
50
3.773
5.358
6.190
1.00
28.54
C


ATOM
201
CG
MET
50
3.355
6.367
7.246
1.00
30.69
C


ATOM
202
SD
MET
50
2.058
7.489
6.688
1.00
30.76
S


ATOM
203
CE
MET
50
2.888
9.022
6.804
1.00
31.08
C


ATOM
204
C
MET
50
5.605
6.746
5.233
1.00
30.79
C


ATOM
205
O
MET
50
5.546
7.896
5.664
1.00
31.44
O


ATOM
206
N
LYS
51
6.753
6.132
4.992
1.00
31.31
N


ATOM
207
CA
LYS
51
8.008
6.764
5.343
1.00
31.72
C


ATOM
208
CB
LYS
51
8.996
5.654
5.711
1.00
34.50
C


ATOM
209
CG
LYS
51
10.325
6.042
6.283
1.00
39.52
C


ATOM
210
CD
LYS
51
11.086
4.747
6.577
1.00
43.25
C


ATOM
211
CE
LYS
51
12.449
4.991
7.201
1.00
46.68
C


ATOM
212
NZ
LYS
51
12.318
5.719
8.503
1.00
47.10
N


ATOM
213
C
LYS
51
8.503
7.745
4.265
1.00
28.34
C


ATOM
214
O
LYS
51
9.297
8.637
4.559
1.00
25.46
O


ATOM
215
N
GLU
52
8.011
7.600
3.032
1.00
24.36
N


ATOM
216
CA
GLU
52
8.388
8.523
1.956
1.00
23.10
C


ATOM
217
CB
GLU
52
8.166
7.936
0.560
1.00
23.72
C


ATOM
218
CG
GLU
52
8.895
6.653
0.213
1.00
26.58
C


ATOM
219
CD
GLU
52
8.521
6.256
−1.209
1.00
25.01
C


ATOM
220
OE1
GLU
52
7.629
5.400
−1.371
1.00
24.55
O


ATOM
221
OE2
GLU
52
9.123
6.785
−2.164
1.00
25.12
O


ATOM
222
C
GLU
52
7.468
9.738
2.088
1.00
21.67
C


ATOM
223
O
GLU
52
7.843
10.860
1.740
1.00
18.74
O


ATOM
224
N
VAL
53
6.263
9.495
2.599
1.00
18.59
N


ATOM
225
CA
VAL
53
5.268
10.545
2.763
1.00
19.55
C


ATOM
226
CB
VAL
53
3.896
9.949
3.143
1.00
19.33
C


ATOM
227
CG1
VAL
53
2.896
11.062
3.434
1.00
19.57
C


ATOM
228
CG2
VAL
53
3.394
9.067
2.016
1.00
20.00
C


ATOM
229
C
VAL
53
5.703
11.518
3.841
1.00
19.68
C


ATOM
230
O
VAL
53
5.559
12.727
3.684
1.00
19.09
O


ATOM
231
N
LEU
54
6.253
10.983
4.926
1.00
21.56
N


ATOM
232
CA
LEU
54
6.732
11.811
6.022
1.00
24.05
C


ATOM
233
CB
LEU
54
7.118
10.949
7.227
1.00
25.58
C


ATOM
234
CG
LEU
54
5.962
10.130
7.805
1.00
27.53
C


ATOM
235
CD1
LEU
54
6.406
9.184
8.917
1.00
27.26
C


ATOM
236
CD2
LEU
54
4.854
11.055
8.278
1.00
27.23
C


ATOM
237
C
LEU
54
7.917
12.628
5.530
1.00
23.85
C


ATOM
238
O
LEU
54
8.066
13.800
5.876
1.00
24.62
O


ATOM
239
N
PHE
55
8.747
12.002
4.702
1.00
22.58
N


ATOM
240
CA
PHE
55
9.908
12.674
4.145
1.00
21.31
C


ATOM
241
CB
PHE
55
10.796
11.716
3.357
1.00
20.68
C


ATOM
242
CG
PHE
55
11.981
12.387
2.725
1.00
20.30
C


ATOM
243
CD1
PHE
55
13.012
12.888
3.507
1.00
18.41
C


ATOM
244
CD2
PHE
55
12.048
12.549
1.346
1.00
19.84
C


ATOM
245
CE1
PHE
55
14.085
13.538
2.928
1.00
18.68
C


ATOM
246
CE2
PHE
55
13.120
13.201
0.759
1.00
18.51
C


ATOM
247
CZ
PHE
55
14.140
13.696
1.552
1.00
19.56
C


ATOM
248
C
PHE
55
9.512
13.850
3.269
1.00
20.62
C


ATOM
249
O
PHE
55
9.963
14.971
3.493
1.00
23.48
O


ATOM
250
N
TYR
56
8.670
13.599
2.270
1.00
19.21
N


ATOM
251
CA
TYR
56
8.243
14.671
1.382
1.00
17.94
C


ATOM
252
CB
TYR
56
7.459
14.142
0.182
1.00
17.19
C


ATOM
253
CG
TYR
56
8.259
13.265
−0.752
1.00
17.01
C


ATOM
254
CD1
TYR
56
9.447
13.726
−1.310
1.00
16.35
C


ATOM
255
CE1
TYR
56
10.130
12.987
−2.251
1.00
15.96
C


ATOM
256
CD2
TYR
56
7.781
12.024
−1.158
1.00
17.74
C


ATOM
257
CE2
TYR
56
8.457
11.273
−2.105
1.00
18.06
C


ATOM
258
CZ
TYR
56
9.627
11.763
−2.651
1.00
18.15
C


ATOM
259
OH
TYR
56
10.268
11.055
−3.637
1.00
17.16
O


ATOM
260
C
TYR
56
7.506
15.830
2.034
1.00
18.40
C


ATOM
261
O
TYR
56
7.708
16.974
1.632
1.00
20.22
O


ATOM
262
N
LEU
57
6.666
15.559
3.034
1.00
18.61
N


ATOM
263
CA
LEU
57
5.964
16.652
3.699
1.00
19.85
C


ATOM
264
CB
LEU
57
4.869
16.162
4.655
1.00
20.61
C


ATOM
265
CG
LEU
57
3.644
15.424
4.115
1.00
23.15
C


ATOM
266
CD1
LEU
57
2.757
14.911
5.243
1.00
23.67
C


ATOM
267
CD2
LEU
57
2.843
16.320
3.162
1.00
21.07
C


ATOM
268
C
LEU
57
6.942
17.560
4.425
1.00
19.93
C


ATOM
269
O
LEU
57
6.778
18.777
4.429
1.00
20.07
O


ATOM
270
N
GLY
58
7.970
16.968
5.023
1.00
18.96
N


ATOM
271
CA
GLY
58
8.959
17.764
5.724
1.00
19.61
C


ATOM
272
C
GLY
58
9.735
18.612
4.736
1.00
20.66
C


ATOM
273
O
GLY
58
9.973
19.801
4.968
1.00
19.89
O


ATOM
274
N
GLN
59
10.132
18.003
3.622
1.00
21.02
N


ATOM
275
CA
GLN
59
10.873
18.730
2.600
1.00
22.78
C


ATOM
276
CB
GLN
59
11.519
17.776
1.599
1.00
22.59
C


ATOM
277
CG
GLN
59
12.505
16.839
2.250
1.00
25.03
C


ATOM
278
CD
GLN
59
13.576
17.674
2.912
1.00
26.69
C


ATOM
279
OE1
GLN
59
13.877
17.502
4.090
1.00
28.42
O


ATOM
280
NE2
GLN
59
14.139
18.612
2.160
1.00
27.94
N


ATOM
281
C
GLN
59
9.980
19.745
1.900
1.00
21.11
C


ATOM
282
O
GLN
59
10.458
20.730
1.342
1.00
20.60
O


ATOM
283
N
TYR
60
8.675
19.504
1.943
1.00
20.96
N


ATOM
284
CA
TYR
60
7.725
20.427
1.340
1.00
21.51
C


ATOM
285
CB
TYR
60
6.350
19.786
1.207
1.00
20.18
C


ATOM
286
CG
TYR
60
5.290
20.727
0.684
1.00
19.26
C


ATOM
287
CD1
TYR
60
5.127
20.946
−0.677
1.00
18.65
C


ATOM
288
CE1
TYR
60
4.145
21.796
−1.148
1.00
18.43
C


ATOM
289
CD2
TYR
60
4.444
21.390
1.563
1.00
18.15
C


ATOM
290
CE2
TYR
60
3.465
22.238
1.106
1.00
19.04
C


ATOM
291
CZ
TYR
60
3.314
22.437
−0.251
1.00
19.66
C


ATOM
292
OH
TYR
60
2.316
23.266
−0.705
1.00
17.66
O


ATOM
293
C
TYR
60
7.631
21.661
2.221
1.00
21.23
C


ATOM
294
O
TYR
60
7.592
22.793
1.737
1.00
21.02
O


ATOM
295
N
ILE
61
7.615
21.414
3.525
1.00
20.45
N


ATOM
296
CA
ILE
61
7.514
22.463
4.518
1.00
19.88
C


ATOM
297
CB
ILE
61
7.305
21.841
5.913
1.00
16.63
C


ATOM
298
CG2
ILE
61
7.405
22.907
6.990
1.00
15.17
C


ATOM
299
CG1
ILE
61
5.961
21.112
5.956
1.00
14.67
C


ATOM
300
CD1
ILE
61
5.660
20.431
7.295
1.00
13.50
C


ATOM
301
C
ILE
61
8.789
23.293
4.518
1.00
24.11
C


ATOM
302
O
ILE
61
8.779
24.463
4.915
1.00
24.81
O


ATOM
303
N
MET
62
9.882
22.695
4.052
1.00
27.53
N


ATOM
304
CA
MET
62
11.158
23.399
4.010
1.00
31.34
C


ATOM
305
CB
MET
62
12.331
22.454
4.293
1.00
34.73
C


ATOM
306
CG
MET
62
12.295
21.791
5.665
1.00
41.21
C


ATOM
307
SD
MET
62
13.705
20.685
5.949
1.00
45.94
S


ATOM
308
CE
MET
62
15.034
21.883
6.155
1.00
46.15
C


ATOM
309
C
MET
62
11.394
24.154
2.710
1.00
30.78
C


ATOM
310
O
MET
62
11.975
25.236
2.723
1.00
31.93
O


ATOM
311
N
THR
63
10.929
23.607
1.591
1.00
30.29
N


ATOM
312
CA
THR
63
11.146
24.278
0.318
1.00
31.93
C


ATOM
313
CB
THR
63
10.913
23.331
−0.879
1.00
32.45
C


ATOM
314
OG1
THR
63
11.653
22.121
−0.691
1.00
34.57
O


ATOM
315
CG2
THR
63
11.383
23.992
−2.168
1.00
33.58
C


ATOM
316
C
THR
63
10.222
25.476
0.164
1.00
30.66
C


ATOM
317
O
THR
63
10.550
26.430
−0.535
1.00
32.12
O


ATOM
318
N
LYS
64
9.072
25.437
0.825
1.00
30.47
N


ATOM
319
CA
LYS
64
8.144
26.554
0.750
1.00
30.88
C


ATOM
320
CB
LYS
64
6.715
26.113
0.433
1.00
31.95
C


ATOM
321
CG
LYS
64
6.592
25.412
−0.903
1.00
33.47
C


ATOM
322
CD
LYS
64
5.163
25.021
−1.188
1.00
35.44
C


ATOM
323
CE
LYS
64
4.269
26.251
−1.223
1.00
35.32
C


ATOM
324
NZ
LYS
64
2.851
25.905
−1.507
1.00
36.49
N


ATOM
325
C
LYS
64
8.218
27.370
2.021
1.00
30.38
C


ATOM
326
O
LYS
64
7.504
28.356
2.190
1.00
31.61
O


ATOM
327
N
ARG
65
9.096
26.929
2.914
1.00
31.08
N


ATOM
328
CA
ARG
65
9.350
27.604
4.176
1.00
32.41
C


ATOM
329
CB
ARG
65
10.296
28.776
3.921
1.00
33.62
C


ATOM
330
CG
ARG
65
11.569
28.220
3.304
1.00
38.45
C


ATOM
331
CD
ARG
65
12.660
29.194
2.949
1.00
39.91
C


ATOM
332
NE
ARG
65
13.766
28.425
2.375
1.00
42.85
N


ATOM
333
CZ
ARG
65
14.793
28.944
1.713
1.00
43.34
C


ATOM
334
NH1
ARG
65
14.878
30.255
1.530
1.00
45.53
N


ATOM
335
NH2
ARG
65
15.729
28.146
1.221
1.00
43.64
N


ATOM
336
C
ARG
65
8.099
27.982
4.955
1.00
30.80
C


ATOM
337
O
ARG
65
7.856
29.145
5.259
1.00
28.75
O


ATOM
338
N
LEU
66
7.310
26.965
5.274
1.00
31.27
N


ATOM
339
CA
LEU
66
6.077
27.149
6.020
1.00
31.53
C


ATOM
340
CB
LEU
66
5.082
26.044
5.687
1.00
30.56
C


ATOM
341
CG
LEU
66
4.742
25.973
4.201
1.00
30.97
C


ATOM
342
CD1
LEU
66
3.800
24.809
3.914
1.00
31.85
C


ATOM
343
CD2
LEU
66
4.156
27.290
3.700
1.00
28.41
C


ATOM
344
C
LEU
66
6.391
27.171
7.507
1.00
32.00
C


ATOM
345
O
LEU
66
5.505
27.320
8.342
1.00
36.99
O


ATOM
346
N
TYR
67
7.669
27.018
7.822
1.00
32.16
N


ATOM
347
CA
TYR
67
8.147
27.010
9.196
1.00
29.34
C


ATOM
348
CB
TYR
67
9.300
26.023
9.365
1.00
30.19
C


ATOM
349
CG
TYR
67
10.505
26.362
8.517
1.00
30.32
C


ATOM
350
CD1
TYR
67
11.362
27.393
8.877
1.00
31.21
C


ATOM
351
CE1
TYR
67
12.453
27.726
8.097
1.00
31.23
C


ATOM
352
CD2
TYR
67
10.775
25.668
7.348
1.00
31.49
C


ATOM
353
CE2
TYR
67
11.867
25.995
6.559
1.00
31.76
C


ATOM
354
CZ
TYR
67
12.702
27.025
6.939
1.00
30.71
C


ATOM
355
OH
TYR
67
13.790
27.351
6.163
1.00
30.69
O


ATOM
356
C
TYR
67
8.584
28.410
9.599
1.00
26.76
C


ATOM
357
O
TYR
67
8.974
29.212
8.755
1.00
27.18
O


ATOM
358
N
ASP
68
8.505
28.710
10.887
1.00
26.00
N


ATOM
359
CA
ASP
68
8.931
30.012
11.376
1.00
24.98
C


ATOM
360
CB
ASP
68
8.536
30.213
12.832
1.00
24.44
C


ATOM
361
CG
ASP
68
8.916
31.581
13.354
1.00
25.82
C


ATOM
362
OD1
ASP
68
8.229
32.064
14.280
1.00
27.13
O


ATOM
363
OD2
ASP
68
9.876
32.189
12.829
1.00
23.05
O


ATOM
364
C
ASP
68
10.456
30.065
11.239
1.00
26.08
C


ATOM
365
O
ASP
68
11.171
29.230
11.809
1.00
20.81
O


ATOM
366
N
GLU
69
10.954
31.038
10.481
1.00
26.25
N


ATOM
367
CA
GLU
69
12.393
31.179
10.299
1.00
27.50
C


ATOM
368
CB
GLU
69
12.711
32.341
9.355
1.00
31.03
C


ATOM
369
CG
GLU
69
14.200
32.606
9.160
1.00
36.70
C


ATOM
370
CD
GLU
69
14.885
31.383
8.579
1.00
40.21
C


ATOM
371
OE1
GLU
69
14.189
30.407
8.224
1.00
42.79
O


ATOM
372
OE2
GLU
69
16.133
31.400
8.486
1.00
41.52
O


ATOM
373
C
GLU
69
13.155
31.330
11.617
1.00
25.94
C


ATOM
374
O
GLU
69
14.320
30.956
11.705
1.00
25.86
O


ATOM
375
N
LYS
70
12.493
31.856
12.645
1.00
23.46
N


ATOM
376
CA
LYS
70
13.141
32.048
13.937
1.00
23.45
C


ATOM
377
CB
LYS
70
12.882
33.449
14.490
1.00
26.70
C


ATOM
378
CG
LYS
70
13.362
34.567
13.582
1.00
28.84
C


ATOM
379
CD
LYS
70
14.858
34.477
13.329
1.00
31.67
C


ATOM
380
CE
LYS
70
15.327
35.607
12.415
1.00
33.30
C


ATOM
381
NZ
LYS
70
16.788
35.549
12.150
1.00
34.03
N


ATOM
382
C
LYS
70
12.762
30.989
14.965
1.00
22.06
C


ATOM
383
O
LYS
70
13.147
31.077
16.123
1.00
21.58
O


ATOM
384
N
GLN
71
11.995
29.993
14.541
1.00
21.15
N


ATOM
385
CA
GLN
71
11.588
28.913
15.436
1.00
20.20
C


ATOM
386
CB
GLN
71
10.497
29.383
16.407
1.00
20.24
C


ATOM
387
CG
GLN
71
10.110
28.357
17.460
1.00
20.50
C


ATOM
388
CD
GLN
71
9.029
28.928
18.363
1.00
20.46
C


ATOM
389
OE1
GLN
71
8.365
29.908
18.019
1.00
24.60
O


ATOM
390
NE2
GLN
71
8.865
28.330
19.535
1.00
18.17
N


ATOM
391
C
GLN
71
11.088
27.853
14.461
1.00
20.18
C


ATOM
392
O
GLN
71
9.880
27.643
14.280
1.00
20.12
O


ATOM
393
N
GLN
72
12.056
27.188
13.840
1.00
20.81
N


ATOM
394
CA
GLN
72
11.806
26.189
12.813
1.00
22.77
C


ATOM
395
CB
GLN
72
13.119
25.855
12.110
1.00
23.53
C


ATOM
396
CG
GLN
72
13.723
27.086
11.456
1.00
27.22
C


ATOM
397
CD
GLN
72
15.027
26.744
10.776
1.00
29.38
C


ATOM
398
OE1
GLN
72
15.497
25.604
10.824
1.00
30.31
O


ATOM
399
NE2
GLN
72
15.626
27.741
10.137
1.00
30.16
N


ATOM
400
C
GLN
72
11.028
24.940
13.195
1.00
21.80
C


ATOM
401
O
GLN
72
10.603
24.178
12.328
1.00
20.25
O


ATOM
402
N
HIS
73
10.829
24.737
14.488
1.00
20.18
N


ATOM
403
CA
HIS
73
10.076
23.585
14.949
1.00
21.21
C


ATOM
404
CB
HIS
73
10.627
23.080
16.287
1.00
22.75
C


ATOM
405
CG
HIS
73
10.662
24.117
17.366
1.00
24.88
C


ATOM
406
CD2
HIS
73
11.670
24.911
17.801
1.00
25.16
C


ATOM
407
ND1
HIS
73
9.565
24.435
18.136
1.00
26.05
N


ATOM
408
CE1
HIS
73
9.895
25.378
19.001
1.00
27.37
C


ATOM
409
NE2
HIS
73
11.167
25.684
18.818
1.00
27.33
N


ATOM
410
C
HIS
73
8.569
23.857
14.955
1.00
19.30
C


ATOM
411
O
HIS
73
7.762
23.018
15.353
1.00
15.05
O


ATOM
412
N
ILE
74
8.202
25.049
14.495
1.00
20.68
N


ATOM
413
CA
ILE
74
6.800
25.442
14.408
1.00
20.38
C


ATOM
414
CB
ILE
74
6.517
26.761
15.170
1.00
17.27
C


ATOM
415
CG2
ILE
74
5.082
27.186
14.930
1.00
16.94
C


ATOM
416
CG1
ILE
74
6.788
26.585
16.673
1.00
12.85
C


ATOM
417
CD1
ILE
74
5.940
25.522
17.350
1.00
10.25
C


ATOM
418
C
ILE
74
6.433
25.631
12.931
1.00
22.21
C


ATOM
419
O
ILE
74
7.068
26.401
12.207
1.00
19.54
O


ATOM
420
N
VAL
75
5.406
24.914
12.489
1.00
22.66
N


ATOM
421
CA
VAL
75
4.968
24.994
11.106
1.00
20.85
C


ATOM
422
CB
VAL
75
4.656
23.596
10.552
1.00
19.84
C


ATOM
423
CG1
VAL
75
4.268
23.697
9.081
1.00
17.74
C


ATOM
424
CG2
VAL
75
5.870
22.678
10.745
1.00
19.97
C


ATOM
425
C
VAL
75
3.730
25.866
10.931
1.00
22.04
C


ATOM
426
O
VAL
75
2.682
25.605
11.523
1.00
19.88
O


ATOM
427
N
HIS
76
3.860
26.907
10.117
1.00
19.99
N


ATOM
428
CA
HIS
76
2.742
27.797
9.850
1.00
20.86
C


ATOM
429
CB
HIS
76
3.140
29.249
10.074
1.00
20.76
C


ATOM
430
CG
HIS
76
3.595
29.532
11.471
1.00
18.48
C


ATOM
431
CD2
HIS
76
4.799
29.914
11.958
1.00
17.24
C


ATOM
432
ND1
HIS
76
2.764
29.401
12.561
1.00
18.78
N


ATOM
433
CE1
HIS
76
3.436
29.693
13.661
1.00
17.42
C


ATOM
434
NE2
HIS
76
4.673
30.007
13.323
1.00
17.51
N


ATOM
435
C
HIS
76
2.250
27.541
8.429
1.00
22.01
C


ATOM
436
O
HIS
76
2.906
27.886
7.448
1.00
20.06
O


ATOM
437
N
CYS
77
1.076
26.934
8.333
1.00
25.10
N


ATOM
438
CA
CYS
77
0.503
26.582
7.049
1.00
27.53
C


ATOM
439
CB
CYS
77
0.499
25.055
6.882
1.00
26.53
C


ATOM
440
SG
CYS
77
−0.321
24.144
8.202
1.00
29.08
S


ATOM
441
C
CYS
77
−0.805
27.255
6.632
1.00
28.98
C


ATOM
442
O
CYS
77
−1.442
26.851
5.660
1.00
28.62
O


ATOM
443
N
SER
78
−1.206
28.283
7.377
1.00
31.46
N


ATOM
444
CA
SER
78
−2.404
29.043
7.035
1.00
34.18
C


ATOM
445
CB
SER
78
−2.859
29.936
8.186
1.00
32.67
C


ATOM
446
OG
SER
78
−1.858
30.878
8.515
1.00
32.54
O


ATOM
447
C
SER
78
−1.935
29.883
5.851
1.00
37.37
C


ATOM
448
O
SER
78
−0.941
30.602
5.966
1.00
40.89
O


ATOM
449
N
ASN
79
−2.637
29.782
4.725
1.00
38.16
N


ATOM
450
CA
ASN
79
−2.281
30.491
3.490
1.00
38.25
C


ATOM
451
CB
ASN
79
−1.490
31.788
3.729
1.00
41.43
C


ATOM
452
CG
ASN
79
−2.269
32.814
4.535
1.00
44.32
C


ATOM
453
OD1
ASN
79
−1.804
33.293
5.568
1.00
47.01
O


ATOM
454
ND2
ASN
79
−3.464
33.155
4.062
1.00
45.45
N


ATOM
455
C
ASN
79
−1.535
29.579
2.522
1.00
36.47
C


ATOM
456
O
ASN
79
−1.050
30.026
1.481
1.00
36.50
O


ATOM
457
N
ASP
80
−1.439
28.301
2.879
1.00
33.49
N


ATOM
458
CA
ASP
80
−0.782
27.304
2.038
1.00
30.65
C


ATOM
459
CB
ASP
80
0.597
26.908
2.567
1.00
28.08
C


ATOM
460
CG
ASP
80
1.300
25.894
1.666
1.00
27.51
C


ATOM
461
OD1
ASP
80
2.347
26.248
1.088
1.00
28.18
O


ATOM
462
OD2
ASP
80
0.818
24.749
1.528
1.00
24.49
O


ATOM
463
C
ASP
80
−1.696
26.089
1.926
1.00
30.18
C


ATOM
464
O
ASP
80
−2.399
25.750
2.876
1.00
32.29
O


ATOM
465
N
LEU
81
−1.701
25.447
0.763
1.00
29.44
N


ATOM
466
CA
LEU
81
−2.547
24.279
0.548
1.00
28.49
C


ATOM
467
CB
LEU
81
−2.242
23.632
−0.806
1.00
30.75
C


ATOM
468
CG
LEU
81
−3.069
22.396
−1.179
1.00
32.89
C


ATOM
469
CD1
LEU
81
−4.564
22.720
−1.167
1.00
32.68
C


ATOM
470
CD2
LEU
81
−2.658
21.821
−2.530
1.00
33.32
C


ATOM
471
C
LEU
81
−2.428
23.240
1.667
1.00
27.48
C


ATOM
472
O
LEU
81
−3.327
22.422
1.861
1.00
26.60
O


ATOM
473
N
LEU
82
−1.328
23.284
2.411
1.00
25.83
N


ATOM
474
CA
LEU
82
−1.121
22.326
3.485
1.00
25.93
C


ATOM
475
CB
LEU
82
0.335
22.334
3.953
1.00
27.37
C


ATOM
476
CG
LEU
82
0.714
21.341
5.051
1.00
26.40
C


ATOM
477
CD1
LEU
82
0.391
19.918
4.617
1.00
28.04
C


ATOM
478
CD2
LEU
82
2.183
21.459
5.440
1.00
25.48
C


ATOM
479
C
LEU
82
−2.075
22.622
4.640
1.00
26.61
C


ATOM
480
O
LEU
82
−2.394
21.736
5.434
1.00
24.04
O


ATOM
481
N
GLY
83
−2.539
23.870
4.713
1.00
25.97
N


ATOM
482
CA
GLY
83
−3.476
24.255
5.751
1.00
23.73
C


ATOM
483
C
GLY
83
−4.816
23.613
5.437
1.00
24.97
C


ATOM
484
O
GLY
83
−5.531
23.137
6.324
1.00
23.63
O


ATOM
485
N
ASP
84
−5.155
23.592
4.154
1.00
26.10
N


ATOM
486
CA
ASP
84
−6.404
22.994
3.705
1.00
28.17
C


ATOM
487
CB
ASP
84
−6.695
23.378
2.259
1.00
29.18
C


ATOM
488
CG
ASP
84
−6.829
24.868
2.065
1.00
31.27
C


ATOM
489
OD1
ASP
84
−6.896
25.604
3.073
1.00
30.35
O


ATOM
490
OD2
ASP
84
−6.856
25.298
0.891
1.00
33.57
O


ATOM
491
C
ASP
84
−6.345
21.477
3.817
1.00
28.68
C


ATOM
492
O
ASP
84
−7.369
20.818
3.992
1.00
29.33
O


ATOM
493
N
LEU
85
−5.138
20.931
3.728
1.00
28.47
N


ATOM
494
CA
LEU
85
−4.946
19.491
3.787
1.00
28.25
C


ATOM
495
CB
LEU
85
−3.572
19.113
3.231
1.00
29.46
C


ATOM
496
CG
LEU
85
−3.224
17.628
3.151
1.00
31.51
C


ATOM
497
CD1
LEU
85
−4.226
16.913
2.258
1.00
31.47
C


ATOM
498
CD2
LEU
85
−1.809
17.393
2.634
1.00
32.07
C


ATOM
499
C
LEU
85
−5.125
19.006
5.229
1.00
27.80
C


ATOM
500
O
LEU
85
−5.923
18.105
5.500
1.00
26.59
O


ATOM
501
N
PHE
86
−4.386
19.618
6.149
1.00
25.85
N


ATOM
502
CA
PHE
86
−4.466
19.262
7.561
1.00
25.42
C


ATOM
503
CB
PHE
86
−3.153
19.569
8.288
1.00
25.61
C


ATOM
504
CG
PHE
86
−1.984
18.745
7.833
1.00
24.56
C


ATOM
505
CD1
PHE
86
−0.723
18.972
8.362
1.00
25.69
C


ATOM
506
CD2
PHE
86
−2.158
17.687
6.955
1.00
25.22
C


ATOM
507
CE1
PHE
86
0.343
18.158
8.033
1.00
24.12
C


ATOM
508
CE2
PHE
86
−1.096
16.866
6.620
1.00
24.92
C


ATOM
509
CZ
PHE
86
0.156
17.100
7.161
1.00
25.70
C


ATOM
510
C
PHE
86
−5.626
19.871
8.342
1.00
25.22
C


ATOM
511
O
PHE
86
−5.984
19.374
9.403
1.00
27.68
O


ATOM
512
N
GLY
87
−6.220
20.936
7.821
1.00
25.48
N


ATOM
513
CA
GLY
87
−7.308
21.572
8.538
1.00
24.46
C


ATOM
514
C
GLY
87
−6.846
22.301
9.793
1.00
24.27
C


ATOM
515
O
GLY
87
−7.577
22.374
10.778
1.00
25.23
O


ATOM
516
N
VAL
88
−5.628
22.835
9.763
1.00
23.15
N


ATOM
517
CA
VAL
88
−5.067
23.582
10.892
1.00
21.50
C


ATOM
518
CB
VAL
88
−4.183
22.697
11.837
1.00
22.47
C


ATOM
519
CG1
VAL
88
−5.014
21.573
12.444
1.00
23.92
C


ATOM
520
CG2
VAL
88
−2.985
22.136
11.082
1.00
21.60
C


ATOM
521
C
VAL
88
−4.200
24.704
10.341
1.00
19.98
C


ATOM
522
O
VAL
88
−3.544
24.541
9.317
1.00
19.52
O


ATOM
523
N
PRO
89
−4.196
25.871
11.001
1.00
21.92
N


ATOM
524
CD
PRO
89
−4.796
26.279
12.289
1.00
20.40
C


ATOM
525
CA
PRO
89
−3.366
26.958
10.488
1.00
21.32
C


ATOM
526
CB
PRO
89
−4.009
28.164
11.135
1.00
21.47
C


ATOM
527
CG
PRO
89
−4.109
27.664
12.548
1.00
21.05
C


ATOM
528
C
PRO
89
−1.881
26.800
10.808
1.00
21.71
C


ATOM
529
O
PRO
89
−1.029
27.350
10.114
1.00
20.80
O


ATOM
530
N
SER
90
−1.579
26.025
11.845
1.00
22.01
N


ATOM
531
CA
SER
90
−0.197
25.803
12.257
1.00
24.39
C


ATOM
532
CB
SER
90
0.475
27.107
12.704
1.00
25.95
C


ATOM
533
OG
SER
90
−0.191
27.688
13.806
1.00
30.18
O


ATOM
534
C
SER
90
−0.076
24.728
13.329
1.00
23.46
C


ATOM
535
O
SER
90
−1.048
24.407
14.010
1.00
25.46
O


ATOM
536
N
PHE
91
1.119
24.161
13.462
1.00
23.68
N


ATOM
537
CA
PHE
91
1.375
23.126
14.463
1.00
22.17
C


ATOM
538
CB
PHE
91
0.869
21.755
14.002
1.00
22.00
C


ATOM
539
CG
PHE
91
1.527
21.265
12.739
1.00
23.50
C


ATOM
540
CD1
PHE
91
1.133
21.751
11.497
1.00
23.67
C


ATOM
541
CD2
PHE
91
2.575
20.360
12.798
1.00
21.96
C


ATOM
542
CE1
PHE
91
1.775
21.342
10.339
1.00
22.42
C


ATOM
543
CE2
PHE
91
3.220
19.949
11.645
1.00
23.74
C


ATOM
544
CZ
PHE
91
2.818
20.442
10.414
1.00
22.47
C


ATOM
545
C
PHE
91
2.862
23.032
14.812
1.00
21.03
C


ATOM
546
O
PHE
91
3.700
23.714
14.225
1.00
22.64
O


ATOM
547
N
SER
92
3.162
22.177
15.781
1.00
18.50
N


ATOM
548
CA
SER
92
4.517
21.909
16.234
1.00
17.97
C


ATOM
549
CB
SER
92
4.626
21.948
17.750
1.00
17.24
C


ATOM
550
OG
SER
92
5.941
21.622
18.147
1.00
15.01
O


ATOM
551
C
SER
92
4.938
20.538
15.718
1.00
19.41
C


ATOM
552
O
SER
92
4.134
19.607
15.711
1.00
16.66
O


ATOM
553
N
VAL
93
6.186
20.417
15.273
1.00
19.68
N


ATOM
554
CA
VAL
93
6.676
19.142
14.766
1.00
20.81
C


ATOM
555
CB
VAL
93
8.052
19.290
14.079
1.00
22.21
C


ATOM
556
CG1
VAL
93
8.008
20.418
13.061
1.00
21.22
C


ATOM
557
CG2
VAL
93
9.134
19.522
15.119
1.00
20.18
C


ATOM
558
C
VAL
93
6.819
18.131
15.897
1.00
21.81
C


ATOM
559
O
VAL
93
7.136
16.969
15.662
1.00
24.55
O


ATOM
560
N
LYS
94
6.579
18.568
17.127
1.00
22.80
N


ATOM
561
CA
LYS
94
6.696
17.670
18.267
1.00
23.16
C


ATOM
562
CB
LYS
94
7.400
18.364
19.431
1.00
25.99
C


ATOM
563
CG
LYS
94
6.681
19.571
19.966
1.00
29.97
C


ATOM
564
CD
LYS
94
7.486
20.206
21.088
1.00
34.85
C


ATOM
565
CE
LYS
94
8.855
20.641
20.553
1.00
34.82
C


ATOM
566
NZ
LYS
94
9.710
21.284
21.583
1.00
37.75
N


ATOM
567
C
LYS
94
5.343
17.088
18.669
1.00
20.95
C


ATOM
568
O
LYS
94
5.253
16.252
19.570
1.00
19.08
O


ATOM
569
N
GLU
95
4.297
17.538
17.983
1.00
19.27
N


ATOM
570
CA
GLU
95
2.937
17.055
18.212
1.00
18.96
C


ATOM
571
CB
GLU
95
1.919
18.145
17.873
1.00
18.81
C


ATOM
572
CG
GLU
95
2.046
19.387
18.743
1.00
21.00
C


ATOM
573
CD
GLU
95
1.017
20.418
18.319
1.00
22.03
C


ATOM
574
OE1
GLU
95
−0.095
20.444
18.888
1.00
20.78
O


ATOM
575
OE2
GLU
95
1.338
21.228
17.429
1.00
23.38
O


ATOM
576
C
GLU
95
2.751
15.823
17.334
1.00
18.97
C


ATOM
577
O
GLU
95
2.008
15.853
16.359
1.00
17.30
O


ATOM
578
N
HIS
96
3.426
14.735
17.698
1.00
20.49
N


ATOM
579
CA
HIS
96
3.374
13.503
16.919
1.00
22.53
C


ATOM
580
CB
HIS
96
4.357
12.468
17.460
1.00
22.80
C


ATOM
581
CG
HIS
96
5.778
12.924
17.429
1.00
22.99
C


ATOM
582
CD2
HIS
96
6.331
14.078
16.987
1.00
24.26
C


ATOM
583
ND1
HIS
96
6.818
12.157
17.903
1.00
22.37
N


ATOM
584
CE1
HIS
96
7.952
12.818
17.754
1.00
25.34
C


ATOM
585
NE2
HIS
96
7.683
13.987
17.200
1.00
25.87
N


ATOM
586
C
HIS
96
2.016
12.865
16.703
1.00
23.09
C


ATOM
587
O
HIS
96
1.741
12.375
15.611
1.00
25.80
O


ATOM
588
N
ARG
97
1.153
12.871
17.712
1.00
23.57
N


ATOM
589
CA
ARG
97
−0.145
12.253
17.509
1.00
22.11
C


ATOM
590
CB
ARG
97
−0.852
11.940
18.829
1.00
22.67
C


ATOM
591
CG
ARG
97
−2.215
11.295
18.608
1.00
25.29
C


ATOM
592
CD
ARG
97
−2.946
10.947
19.895
1.00
24.16
C


ATOM
593
NE
ARG
97
−4.238
10.344
19.586
1.00
25.87
N


ATOM
594
CZ
ARG
97
−5.067
9.819
20.481
1.00
25.09
C


ATOM
595
NH1
ARG
97
−4.748
9.801
21.769
1.00
25.97
N


ATOM
596
NH2
ARG
97
−6.236
9.339
20.086
1.00
24.37
N


ATOM
597
C
ARG
97
−1.019
13.095
16.596
1.00
20.82
C


ATOM
598
O
ARG
97
−1.837
12.563
15.849
1.00
22.99
O


ATOM
599
N
LYS
98
−0.833
14.409
16.638
1.00
19.91
N


ATOM
600
CA
LYS
98
−1.614
15.294
15.787
1.00
19.73
C


ATOM
601
CB
LYS
98
−1.487
16.749
16.228
1.00
20.60
C


ATOM
602
CG
LYS
98
−2.252
17.723
15.331
1.00
23.23
C


ATOM
603
CD
LYS
98
−2.091
19.164
15.794
1.00
23.00
C


ATOM
604
CE
LYS
98
−2.611
19.344
17.205
1.00
24.33
C


ATOM
605
NZ
LYS
98
−2.463
20.740
17.674
1.00
25.97
N


ATOM
606
C
LYS
98
−1.181
15.160
14.331
1.00
18.95
C


ATOM
607
O
LYS
98
−2.013
15.142
13.423
1.00
18.06
O


ATOM
608
N
ILE
99
0.126
15.047
14.120
1.00
16.71
N


ATOM
609
CA
ILE
99
0.664
14.935
12.777
1.00
18.14
C


ATOM
610
CB
ILE
99
2.205
15.049
12.794
1.00
18.88
C


ATOM
611
CG2
ILE
99
2.764
14.887
11.382
1.00
17.52
C


ATOM
612
CG1
ILE
99
2.612
16.408
13.372
1.00
16.52
C


ATOM
613
CD1
ILE
99
4.102
16.610
13.445
1.00
18.05
C


ATOM
614
C
ILE
99
0.252
13.632
12.105
1.00
19.52
C


ATOM
615
O
ILE
99
−0.096
13.633
10.929
1.00
20.84
O


ATOM
616
N
TYR
100
0.278
12.520
12.836
1.00
21.34
N


ATOM
617
CA
TYR
100
−0.140
11.258
12.234
1.00
21.76
C


ATOM
618
CB
TYR
100
0.388
10.029
12.982
1.00
20.52
C


ATOM
619
CG
TYR
100
1.880
9.828
12.897
1.00
21.94
C


ATOM
620
CD1
TYR
100
2.456
9.359
11.724
1.00
22.57
C


ATOM
621
CE1
TYR
100
3.812
9.131
11.633
1.00
22.83
C


ATOM
622
CD2
TYR
100
2.708
10.070
13.982
1.00
22.53
C


ATOM
623
CE2
TYR
100
4.068
9.847
13.901
1.00
22.55
C


ATOM
624
CZ
TYR
100
4.613
9.374
12.724
1.00
22.83
C


ATOM
625
OH
TYR
100
5.962
9.125
12.642
1.00
24.71
O


ATOM
626
C
TYR
100
−1.637
11.164
12.051
1.00
22.16
C


ATOM
627
O
TYR
100
−2.120
10.375
11.240
1.00
20.79
O


ATOM
628
N
THR
101
−2.375
11.980
12.789
1.00
22.43
N


ATOM
629
CA
THR
101
−3.818
11.949
12.659
1.00
20.93
C


ATOM
630
CB
THR
101
−4.501
12.622
13.867
1.00
20.70
C


ATOM
631
OG1
THR
101
−4.220
11.862
15.049
1.00
21.16
O


ATOM
632
CG2
THR
101
−6.013
12.703
13.661
1.00
19.19
C


ATOM
633
C
THR
101
−4.176
12.684
11.382
1.00
20.33
C


ATOM
634
O
THR
101
−4.980
12.199
10.583
1.00
19.32
O


ATOM
635
N
MET
102
−3.551
13.843
11.186
1.00
20.81
N


ATOM
636
CA
MET
102
−3.803
14.664
10.006
1.00
21.77
C


ATOM
637
CB
MET
102
−3.128
16.031
10.119
1.00
22.96
C


ATOM
638
CG
MET
102
−3.563
16.875
11.305
1.00
25.26
C


ATOM
639
SD
MET
102
−2.691
18.457
11.303
1.00
26.02
S


ATOM
640
CE
MET
102
−1.054
17.961
11.843
1.00
25.10
C


ATOM
641
C
MET
102
−3.326
13.982
8.735
1.00
21.84
C


ATOM
642
O
MET
102
−3.959
14.101
7.689
1.00
22.57
O


ATOM
643
N
ILE
103
−2.215
13.259
8.828
1.00
20.60
N


ATOM
644
CA
ILE
103
−1.672
12.580
7.664
1.00
20.83
C


ATOM
645
CB
ILE
103
−0.220
12.115
7.937
1.00
21.14
C


ATOM
646
CG2
ILE
103
0.262
11.190
6.832
1.00
20.31
C


ATOM
647
CG1
ILE
103
0.685
13.350
8.046
1.00
20.27
C


ATOM
648
CD1
ILE
103
2.144
13.056
8.323
1.00
20.70
C


ATOM
649
C
ILE
103
−2.568
11.414
7.248
1.00
20.54
C


ATOM
650
O
ILE
103
−2.897
11.288
6.072
1.00
20.36
O


ATOM
651
N
TYR
104
−2.974
10.569
8.194
1.00
20.06
N


ATOM
652
CA
TYR
104
−3.879
9.475
7.843
1.00
18.57
C


ATOM
653
CB
TYR
104
−4.007
8.411
8.939
1.00
17.25
C


ATOM
654
CG
TYR
104
−2.810
7.512
9.145
1.00
18.10
C


ATOM
655
CD1
TYR
104
−2.836
6.214
8.649
1.00
19.08
C


ATOM
656
CE1
TYR
104
−1.795
5.339
8.849
1.00
18.97
C


ATOM
657
CD2
TYR
104
−1.688
7.920
9.851
1.00
17.72
C


ATOM
658
CE2
TYR
104
−0.621
7.043
10.059
1.00
20.75
C


ATOM
659
CZ
TYR
104
−0.691
5.746
9.551
1.00
20.88
C


ATOM
660
OH
TYR
104
0.323
4.835
9.749
1.00
19.81
O


ATOM
661
C
TYR
104
−5.259
9.907
7.370
1.00
19.95
C


ATOM
662
O
TYR
104
−5.991
9.105
6.794
1.00
21.99
O


ATOM
663
N
ARG
105
−5.626
11.162
7.602
1.00
17.59
N


ATOM
664
CA
ARG
105
−6.915
11.623
7.121
1.00
17.20
C


ATOM
665
CB
ARG
105
−7.491
12.768
7.959
1.00
16.31
C


ATOM
666
CG
ARG
105
−7.847
12.404
9.383
1.00
17.10
C


ATOM
667
CD
ARG
105
−8.413
13.609
10.125
1.00
16.21
C


ATOM
668
NE
ARG
105
−8.799
13.284
11.495
1.00
16.48
N


ATOM
669
CZ
ARG
105
−9.313
14.159
12.358
1.00
19.13
C


ATOM
670
NH1
ARG
105
−9.495
15.427
12.003
1.00
17.52
N


ATOM
671
NH2
ARG
105
−9.676
13.760
13.572
1.00
19.14
N


ATOM
672
C
ARG
105
−6.798
12.023
5.663
1.00
17.20
C


ATOM
673
O
ARG
105
−7.789
12.336
5.021
1.00
16.63
O


ATOM
674
N
ASN
106
−5.574
11.989
5.147
1.00
19.37
N


ATOM
675
CA
ASN
106
−5.302
12.366
3.766
1.00
20.38
C


ATOM
676
CB
ASN
106
−4.507
13.663
3.704
1.00
20.43
C


ATOM
677
CG
ASN
106
−5.251
14.812
4.339
1.00
20.58
C


ATOM
678
OD1
ASN
106
−6.143
15.390
3.723
1.00
23.31
O


ATOM
679
ND2
ASN
106
−4.885
15.160
5.562
1.00
17.80
N


ATOM
680
C
ASN
106
−4.659
11.281
2.919
1.00
21.95
C


ATOM
681
O
ASN
106
−4.027
11.558
1.896
1.00
20.86
O


ATOM
682
N
LEU
107
−4.823
10.042
3.361
1.00
21.77
N


ATOM
683
CA
LEU
107
−4.264
8.899
2.666
1.00
21.73
C


ATOM
684
CB
LEU
107
−2.737
8.860
2.832
1.00
20.40
C


ATOM
685
CG
LEU
107
−2.118
8.762
4.235
1.00
19.18
C


ATOM
686
CD1
LEU
107
−2.558
7.498
4.968
1.00
18.29
C


ATOM
687
CD2
LEU
107
−0.602
8.811
4.171
1.00
16.48
C


ATOM
688
C
LEU
107
−4.943
7.622
3.150
1.00
24.57
C


ATOM
689
O
LEU
107
−5.476
7.574
4.261
1.00
26.59
O


ATOM
690
N
VAL
108
−4.950
6.604
2.300
1.00
24.85
N


ATOM
691
CA
VAL
108
−5.535
5.314
2.637
1.00
23.27
C


ATOM
692
CB
VAL
108
−6.585
4.884
1.594
1.00
25.05
C


ATOM
693
CG1
VAL
108
−6.727
3.374
1.592
1.00
26.97
C


ATOM
694
CG2
VAL
108
−7.923
5.523
1.917
1.00
23.65
C


ATOM
695
C
VAL
108
−4.435
4.259
2.704
1.00
22.36
C


ATOM
696
O
VAL
108
−3.534
4.240
1.866
1.00
23.25
O


ATOM
697
N
VAL
109
−4.494
3.397
3.714
1.00
21.67
N


ATOM
698
CA
VAL
109
−3.504
2.336
3.865
1.00
20.80
C


ATOM
699
CB
VAL
109
−3.455
1.801
5.316
1.00
19.45
C


ATOM
700
CG1
VAL
109
−2.544
0.583
5.389
1.00
17.84
C


ATOM
701
CG2
VAL
109
−2.941
2.892
6.256
1.00
18.14
C


ATOM
702
C
VAL
109
−3.787
1.173
2.910
1.00
21.74
C


ATOM
703
O
VAL
109
−4.915
0.696
2.811
1.00
22.57
O


ATOM
704
N
VAL
110
−2.754
0.739
2.195
1.00
22.61
N


ATOM
705
CA
VAL
110
−2.871
−0.365
1.249
1.00
23.79
C


ATOM
706
CB
VAL
110
−1.791
−0.279
0.158
1.00
22.99
C


ATOM
707
CG1
VAL
110
−1.997
−1.377
−0.864
1.00
23.58
C


ATOM
708
CG2
VAL
110
−1.828
1.081
−0.495
1.00
24.01
C


ATOM
709
C
VAL
110
−2.722
−1.703
1.961
1.00
24.53
C


ATOM
710
O
VAL
110
−3.639
−2.521
1.944
1.00
28.90
O


ATOM
711
OH2
WAT
901
6.275
31.112
15.565
1.00
15.47
O


ATOM
712
OH2
WAT
902
0.081
30.036
10.221
1.00
17.32
O


ATOM
713
OH2
WAT
903
0.127
15.519
19.408
1.00
7.93
O


ATOM
714
OH2
WAT
904
7.030
2.992
5.951
1.00
1.12
O


ATOM
715
OH2
WAT
905
−8.260
9.003
4.793
1.00
16.56
O


ATOM
716
OH2
WAT
906
−0.287
23.410
17.398
1.00
20.45
O


ATOM
717
OH2
WAT
907
11.907
15.641
5.635
1.00
12.75
O


ATOM
718
OH2
WAT
908
6.231
3.121
−2.803
1.00
30.59
O


ATOM
719
OH2
WAT
909
14.427
27.473
−1.777
1.00
32.06
O


ATOM
720
OH2
WAT
910
1.367
29.323
5.015
1.00
30.79
O


ATOM
721
OH2
WAT
911
−3.588
−4.814
0.678
1.00
24.03
O


ATOM
722
OH2
WAT
912
3.828
6.836
−11.268
1.00
35.90
O


ATOM
723
OH2
WAT
913
8.152
2.609
8.906
1.00
34.73
O


ATOM
724
OH2
WAT
914
2.691
4.733
10.966
1.00
33.58
O


ATOM
725
OH2
WAT
916
−5.458
26.444
7.758
1.00
45.56
O


ATOM
726
OH2
WAT
917
−8.076
13.274
16.506
1.00
48.77
O


ATOM
727
OH2
WAT
918
−7.491
17.267
10.217
1.00
40.88
O


ATOM
728
OH2
WAT
919
−3.101
7.362
−4.725
1.00
53.52
O


ATOM
729
OH2
WAT
920
1.887
19.751
−8.003
1.00
32.67
O


ATOM
730
OH2
WAT
921
−8.379
15.505
−1.840
1.00
19.94
O


ATOM
731
OH2
WAT
922
12.181
19.059
−4.016
1.00
25.92
O


ATOM
732
OH2
WAT
923
−8.305
16.544
7.450
1.00
29.31
O


ATOM
733
OH2
WAT
924
−10.379
14.823
6.843
1.00
19.67
O


ATOM
734
C1
SCH
999
14.935
15.343
10.195
1.00
58.18
C


ATOM
735
C2
SCH
999
13.897
15.023
11.252
1.00
58.15
C


ATOM
736
O1
SCH
999
14.212
14.616
12.373
1.00
58.19
O


ATOM
737
N1
SCH
999
12.629
15.230
10.832
1.00
57.26
N


ATOM
738
C3
SCH
999
11.571
15.353
11.839
1.00
54.45
C


ATOM
739
C4
SCH
999
10.855
14.032
12.129
1.00
55.03
C


ATOM
740
O2
SCH
999
11.268
12.976
11.642
1.00
55.46
O


ATOM
741
N2
SCH
999
9.780
14.154
12.934
1.00
56.28
N


ATOM
742
C5
SCH
999
9.102
12.951
13.392
1.00
58.10
C


ATOM
743
C6
SCH
999
10.025
12.199
14.360
1.00
59.43
C


ATOM
744
O3
SCH
999
10.530
11.115
14.068
1.00
59.32
O


ATOM
745
N3
SCH
999
10.203
12.847
15.527
1.00
61.53
N


ATOM
746
C7
SCH
999
11.400
12.542
16.310
1.00
62.93
C


ATOM
747
C8
SCH
999
11.113
11.660
17.534
1.00
62.40
C


ATOM
748
O4
SCH
999
11.912
11.709
18.416
1.00
60.48
O


ATOM
749
O5
SCH
999
10.616
10.479
17.100
1.00
62.41
O


ATOM
750
C9
SCH
999
10.586
16.476
11.453
1.00
49.76
C


ATOM
751
C10
SCH
999
12.105
13.839
16.718
1.00
64.21
C


ATOM
752
C11
SCH
999
13.629
13.756
16.707
1.00
65.67
C


ATOM
753
C12
SCH
999
14.196
14.646
17.803
1.00
66.24
C


ATOM
754
O6
SCH
999
14.526
15.775
17.594
1.00
66.36
O


ATOM
755
O7
SCH
999
14.290
14.052
19.016
1.00
65.17
O


ATOM
756
C13
SCH
999
9.685
16.072
10.295
1.00
46.19
C


ATOM
757
C14
SCH
999
8.313
16.472
10.068
1.00
43.66
C


ATOM
758
C15
SCH
999
7.872
15.829
8.846
1.00
42.20
C


ATOM
759
N4
SCH
999
8.951
15.079
8.368
1.00
43.85
N


ATOM
760
C16
SCH
999
10.006
15.227
9.219
1.00
44.78
C


ATOM
761
C17
SCH
999
7.411
17.318
10.780
1.00
43.03
C


ATOM
762
C18
SCH
999
6.085
17.525
10.294
1.00
39.51
C


ATOM
763
C19
SCH
999
5.651
16.887
9.095
1.00
39.12
C


ATOM
764
C20
SCH
999
6.542
16.041
8.370
1.00
40.06
C


ATOM
765
CL1
SCH
999
4.034
17.133
8.522
1.00
34.69
CL


ATOM
766
C21
SCH
999
8.779
12.041
12.203
1.00
57.67
C


ATOM
767
C22
SCH
999
7.820
13.335
14.135
1.00
58.12
C


ATOM
768
C23
SCH
999
6.793
13.968
13.201
1.00
56.83
C


ATOM
769
C24
SCH
999
7.749
12.676
11.271
1.00
57.14
C


ATOM
770
C25
SCH
999
6.477
13.064
12.015
1.00
56.55
C


END









The present invention is not to be limited in scope by the specific embodiments describe 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 scope of the appended claims.
It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.
Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

## Claims

1. A purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.

2. A compound represented by a structural formula which is selected from the group consisting of

3. A crystal comprising a complex between a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10 and a compound represented by the structural formula:




wherein said crystal is in space group P212121 and comprises unit cell dimensions a=37.999 Å, b=45.333 Å, c=63.999 Å, where α=β=Υ=90°.

4. A crystal comprising a complex between a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 and a compound represented by the structural formula:




wherein said crystal is in space group P212121 and comprises unit cell dimensions a=41.1 Å, b=42.7 Å, c=53.777 Å, where α=β=Υ=90°.

5. The crystal of claim 3 wherein the complex in said crystal is characterized by structural coordinates set forth in Table 3.

6. The crystal of claim 4 wherein the complex in said crystal is characterized by structural coordinates set forth in Table 4.

7. The polypeptide of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 6.

8. The polypeptide of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 8.

9. The polypeptide of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 10.

10. The polypeptide of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 12.