# Patent

## Title
Isolated GRP94 ligand binding domain polypeptide and nucleic acid encoding same, crystalline form of same, and screening methods employing same

## Abstract

An isolated GRP94 ligand binding domain polypeptide, a three-dimensional crystal structure of the same, and methods of using the same to design modulators of Hsp90 proteins.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 10/260,104, filed Sep. 30, 2002, now U.S. Pat. No. 7,691,969 which itself is based on and claims priority to U.S. Provisional Patent Application Ser. No. 60/326,291, filed Oct. 1, 2001. The disclosure of each of these applications is herein incorporated by referenced in its entirety.


TECHNICAL FIELD
The present invention relates to compositions and methods pertaining to the modulation of molecular chaperone function by regulatory ligands. In a preferred embodiment, the present invention relates to an isolated and purified GRP94 ligand binding domain (LBD) polypeptide, a crystallized form of the GRP94 LBD in complex with a ligand, and to screening methods associated therewith.












Table of Abbreviations











8-ANS
1,8-anilinonaphthalenesulfonate


APC
antigen presenting cells


BiP
ER hsp70 homolog


bis-ANS
4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid


BMDC
bone marrow-derived dendritic cells


BN-PAGE
blue native polyacrylamide gel electrophoresis


CEA
carcinoembryonic antigen(s)


CT
computed tomographic


CTL
cytotoxic T lymphocyte(s)


DC
dendritic cells


DMEM
Dulbecco's modified Eagle's medium


DTH
delayed-type hypersensitivity


ER
endoplasmic reticulum


GALT
gut-associated lymphoid tissue


GRP94
glucose regulated protein of 94 kDa, ER paralog of



the Hsp90 family of chaperones


GST
glutathione S-transferase


HIV
human immunodeficiency virus


HPLC
high pressure liquid chromatography


hr
hour(s)


hsp(s)
heat shock protein(s)


HSP70
heat shock protein of 70 kDa


Hsp90
any member of the Hsp90 family of chaperones


HSP90
heat shock protein of 90 kDa


HSV
herpes simplex virus


IFN
interferon


Ig
immunoglobulin


IGF-1
insulin-like growth factor


IgG
immunoglobulin G


IL
interleukin


LBD
ligand binding domain


MHC
major histocompatability complex


min
minute


MLTC
mixed lymphocyte tumor cell assay


NECA
N-ethylcarboxamidoadenosine


PDI
protein disulfide isomerase


PSA
prostate-specific antigen


RSV
respiratory syncytial virus


RT
room temperature


SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel



electrophoresis


TAP
transporter associated with antigen presentation



complex


TFA
trifluoroacetic acid


TNF
tumor necrosis factor










Amino Acid Abbreviations









Single-
Three-



Letter Code
Letter Code
Name





A
Ala
Alanine


V
Val
Valine


L
Leu
Leucine


I
Ile
Isoleucine


P
Pro
Proline


F
Phe
Phenylalanine


W
Trp
Tryptophan


M
Met
Methionine


G
Gly
Glycine


S
Ser
Serine


T
Thr
Threonine


C
Cys
Cysteine


Y
Tyr
Tyrosine


N
Asn
Asparagine


Q
Gln
Glutamine


D
Asp
Aspartic Acid


E
Glu
Glutamic Acid


K
Lys
Lysine


R
Arg
Arginine


H
His
Histidine










Functionally Equivalent Codons











3-
1-



Amino Acid
Letter
Letter
Codons





Alanine
Ala
A
GCA; GCC; GCG; GCU


Cysteine
Cys
C
UGC; UGU


Aspartic Acid
Asp
D
GAC; GAU


Glumatic acid
Glu
E
GAA; GAG


Phenylalanine
Phe
F
UUC; UUU


Glycine
Gly
G
GGA; GGC; GGG; GGU


Histidine
His
H
CAC; CAU


Isoleucine
Ile
I
AUA; AUC; AUU


Lysine
Lys
K
AAA; AAG


Methionine
Met
M
AUG


Asparagine
Asn
N
AAC; AAU


Proline
Pro
P
CCA; CCC; CCG; CCU


Glutamine
Gln
Q
CAA; CAG


Threonine
Thr
T
ACA; ACC; ACG; ACU


Valine
Val
V
GUA; GUC; GUG; GUU


Tryptophan
Trp
W
UGG


Tyrosine
Tyr
Y
UAC; UAU


Leucine
Leu
L
UUA; UUG; CUA; CUC; CUG; CUU


Arginine
Arg
R
AGA; AGG; CGA; CGC; CGG; CGU


Serine
Ser
S
ACG; AGU; UCA; UCC; UCG; UCU









BACKGROUND
The pursuit of approaches for treatment and prevention of cancer and infectious diseases represents an ongoing effort in the medical community. Recent efforts to combat cancer and infectious disease have included attempts to induce or enhance immune responses in subjects suffering from a type of cancer or an infectious disease. See, e.g. Srivastava et al. (1998) Immunity 8:657-665.
Ischemia/reperfusion injury is a significant source of morbidity and mortality in a number of clinical disorders, including myocardial infarction, cerebrovascular disease, and peripheral vascular disease. In addition, ischemia/reperfusion is relevant to the function of transplanted organs and to the recovery expedience following any cardiovascular surgery. See Fan et al. (1999) J Mol Med 77:577-596. Thus, the identification of cellular protective mechanisms against ischemia-induced damage is a central goal for therapy of, for example, heart attacks, strokes, and neurodegenerative diseases, as well as for improvement of recovery following surgery or transplantation.
The Hsp90 class of molecular chaperones are among the most abundant proteins in eukaryotic cells. Hsp90 family members are phylogenetically ubiquitous whereas the endoplasmic reticulum paralog of HSP90, GRP94 (gp96, ERp99, endoplasmin), is found only in higher plants and metazoans (Nicchitta (1998) Curr Opin Immunol 10:103-109). The Hsp90 family of proteins are known to be involved in directing the proper folding and trafficking of newly synthesized proteins and in conferring protection to the cell during conditions of heat shock, oxidative stress, nutrient stress, and other physiological stress scenarios (Toft (1998) Trends Endocrinol Metab 9:238-243; Pratt (1998) Proc Soc Exp Biol Med 217:420-434). Under such stress conditions, protein folding, protein oligomeric assembly, and protein stability can be profoundly disrupted. It is the function of the Hsp90 family of proteins, in concert with other molecular chaperones, to assist in preventing and reversing stress-induced inactivation of protein structure and function.
At a molecular level, HSP90 function in protein folding is known to require the activity of a series of co-chaperones and accessory molecules, including Hsp70, p48Hip, p60Hop, p23, and FKBP52 (Prodromou et al. (1999) EMBO J 18:754-762; Johnson et al. (1996) J Steroid Biochem Mol Biol 56:31-37; Chang et al. (1997) Mol Cell Biol 17:318-325; Duina et al. (1996) Science 274:1713-1715; Chen et al. (1996) Mol Endocrinol 10:682-693; Smith et al. (1993) J Biol Chem 268:18365-18371; Dittmar et al. (1998) J Biol Chem 273:7358-7366; Kosano et al. (1998) J Biol Chem 273:3273-3279). These co-chaperones and accessory molecules participate in both concerted and sequential interactions with HSP90 and thereby serve to regulate its chaperone activity (Buchner (1999) Trends Biochem Sci 24:136-141; Pratt et al. (1996) Exs 77:79-95; Pratt (1998) Proc Soc Exp Biol Med 217:420-434; Caplan (1999) Trends Cell Biol 9:262-268).
In addition to the contribution of co-chaperone proteins to the regulation of HSP90 function, recent crystallographic studies have identified an ATP/ADP binding pocket in the N-terminal domain of yeast and human HSP90, suggesting that HSP90 activity is regulated through cyclic ATP binding and hydrolysis, as has been established for the Hsp70 family of chaperones (Kassenbrock & Kelly (1989) EMBO J 8:1461-1467; Flynn et al. (1989) Science 245:385-390; Palleros et al. (1991) Proc Natl Acad Sci USA 88:519-523; Sriram et al. (1997) Structure 5:403-14; Prodromou et al. (1997) Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910; Csermely & Kahn (1991) J Biol Chem 266:4943-4950; Csermely et al. (1993) J Biol Chem 268:1901-1907; Sullivan et al. (1997) J Biol Chem 272:8007-8012; Scheibel et al. (1997) J Biol Chem 272:18608-18613; Scheibel et al. (1998) Proc Natl Acad Sci USA 95:1495-1499; Panaretou et al. (1998) EMBO J 17:4829-4836; Caplan (1999) Trends Cell Biol 9:262-268; Grenert et al. (1999) J Biol Chem 274:17525-17533).
It has also been reported that HSP90 contains motifs bearing significant similarities to the Walker “A” and “B” sequences associated with ATP binding (Csermely & Kahn (1991) J Biol Chem 266:4943-4950; Jakob et al. (1996) J Biol Chem 271:10035-10041). Although these sequences are substantially different from the consensus sequences found among serine and tyrosine kinases, they are homologous to the ATP binding sequence seen in the Hsp70 family of proteins (Csermely & Kahn (1991) J Biol Chem 266:4943-4950). Consistent with sequence predictions, ATP binding, autophosphorylation activity, and ATPase activity have all been demonstrated for HSP90, though these findings are not without controversy (Csermely & Kahn (1991) J Biol Chem 266:4943-4950; Nadeau et al. (1993) J Biol Chem 268:1479-1487, Jakob et al. (1996) J Biol Chem 271:10035-10041; Grenert et al. (1999) J Biol Chem 274:17525-17533; Scheibel et al. (1997) J Biol Chem 272:18608-18613; Prodromou et al. (1997) Cell 90:65-75).
In part because of the very low affinity of HSP90 for ATP, a role for ATP in the regulation of HSP90 function remained under question until crystallographic resolution of the N-terminal domain of yeast and human HSP90 in association with bound adenosine nucleotides (Prodromou et al. (1997) Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910). Aided by atomic scale structural insights, amino acid residues critical for ATP binding and hydrolysis were subsequently identified and analyzed (Prodromou et al. (1997) Cell 90:65-75; Panaretou et al. (1998) EMBO J 17:4829-4836; Obermann et al. (1998) J Cell Biol 143:901-910). Thus, in the human HSP90, aspartate 93 (D128 for GRP94; D79 for yeast HSP90) provides a direct hydrogen bond interaction with the N6 group of the purine moiety of the adenosine ring and is essential for ATP binding (Prodromou et al. (1997) Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910). Glutamate 47 (E82 for GRP94; E33 for yeast HSP90) was postulated to play an important catalytic role in ATP hydrolysis, based both on its location relative to bound nucleotide and through comparison with the ATP binding domain of E. coli DNA gyrase B (Prodromou et al. (1997) Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910). In subsequent mutagenesis studies of yeast HSP90, it was observed that the D79 mutant was deficient in ATP binding and that E47 mutants were deficient in ATP hydrolysis activity (Obermann et al. (1998) J Cell Biol 143:901-910; Panaretou et al. (1998) EMBO J 17:4829-4836). As further evidence for a function of these residues in HSP90 activity, yeast containing either mutant form of HSP90 were inviable (Obermann et al. (1998) J Cell Biol 143:901-910; Panaretou et al. (1998) EMBO J 17:4829-4836).
Progress in the development of Hsp90-based therapeutic and other applications has been impeded by a lack of characterization of ligand interactions of Hsp90 proteins, including GRP94. Despite the above-described characterization of ATP interaction with HSP90, evidence in support of intrinsic ATP binding and ATPase activities with respect to GRP94 is controversial and, as with HSP90, a clear consensus regarding the molecular basis of an adenosine nucleotide-mediated regulation of GRP94-substrate interactions has yet to emerge (Jakob et al. (1996) J Biol Chem 271:10035-10041; Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156; Li and Srivastava (1993) EMBO J 12:3143-3151; Csermely et al. (1995) J Biol Chem 270:6381-6388; Csermely et al. (1998) Pharmacol Ther 79:129-168).
What is needed, then, is characterization of ligand interactions at the ligand binding pocket of a HSP90 protein, in particular GRP94 and HSP90. To this end, the present invention discloses an isolated and purified GRP94 LBD polypeptide as well as X-ray crystallographic methods that have been used to elucidate GRP94 LBD three-dimensional structure. Prior to the disclosure of the present application, high-quality GRP94 crystals as required for X-ray crystallographic experiments have been unavailable. The solved crystal structure of GRP94 provides structural details that facilitate the design of Hsp90 protein modulators. Using such methods, the active and inactive structural conformations of GRP94 and HSP90 are elucidated, and the regulative capacity of several compounds to induce the active or inactive conformation is also demonstrated. The disclosure herein also provides drug screening methods pertaining to the biological activity of Hsp90 proteins. Thus, the present invention meets a long-standing need in the art for methods and compositions that contribute to the understanding, diagnosis and treatment of disorders related to Hsp90 protein function.
SUMMARY
A substantially pure GRP94 ligand binding domain polypeptide in crystalline form is disclosed. In one embodiment, the crystalline form has unit cell has lattice constants of a=99.889 Å, b=89.614 Å, c=60.066 Å; β=90.132°; space group symmetry C2; and 2 GRP94+NECA complexes in the asymmetric unit. In another embodiment, the crystalline form has lattice constants of the unit cell has lattice constants of a=89.200 Å, b=99.180 Å, c=63.071 Å; α=β=γ=90.0°; space group symmetry C222(1); and 1 GRP94+NECA complex in the asymmetric unit.
Optionally, the GRP94 polypeptide has the amino acid sequence of SEQ ID NO:6. Preferably, the GRP94 polypeptide is in complex with a ligand, more preferably the ligand is NECA. Most preferably, the GRP94 polypeptide has a crystalline structure further characterized by the coordinates corresponding to Tables 1, 2 or 3.
A method for determining the three-dimensional structure of a crystallized GRP94 ligand binding domain polypeptide to a resolution of about 1.8 Å or better is also disclosed. The method comprises: (a) crystallizing a GRP94 ligand binding domain polypeptide; and (b) analyzing the GRP94 ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized GRP94 ligand binding domain polypeptide, whereby the three-dimensional structure of a crystallized GRP94 ligand binding domain polypeptide is determined to a resolution of about 1.8 Å or better.
A method of generating a crystallized GRP94 ligand binding domain polypeptide is also disclosed. In a preferred embodiment, the method comprises: (a) incubating a solution comprising a GRP94 ligand binding domain with a reservoir; and (b) crystallizing the GRP94 ligand binding domain polypeptide using the hanging drop method, whereby a crystallized GRP94 ligand binding domain polypeptide is generated.
Further, a method of designing a modulator of an Hsp90 protein is disclosed. The method comprises: (a) designing a potential modulator of an Hsp90 protein that will make interactions with amino acids in the ligand binding site of the Hsp90 protein based upon the atomic structure coordinates of a GRP94 ligand binding domain polypeptide; (b) synthesizing the modulator; and (c) determining whether the potential modulator modulates the activity of the Hsp90 protein, whereby a modulator of an Hsp90 protein is designed.
Additionally, a method of designing a modulator that selectively modulates the activity of a GRP94 polypeptide is disclosed. The method comprises: (a) obtaining a crystalline form of a GRP94 ligand binding domain polypeptide; determining the three-dimensional structure of the crystalline form of the GRP94 ligand binding domain polypeptide; and (c) synthesizing a modulator based on the three-dimensional structure of the crystalline form of the GRP94 ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of a GRP94 polypeptide is designed.
A method for identifying a GRP94 modulator is also disclosed. The method comprises: (a) providing atomic coordinates of a GRP94 ligand binding domain to a computerized modeling system; and (b) modeling ligands that fit spatially into the binding pocket of the GRP94 ligand binding domain to thereby identify a GRP94 modulator.
A method of identifying a modulator that selectively modulates the activity of a GRP94 polypeptide compared to other Hsp90 polypeptides is also disclosed. The method comprises: (a) providing atomic coordinates of a GRP94 ligand binding domain polypeptide to a computerized modeling system; and (b) modeling a ligand that fits into the binding pocket of a GRP94 ligand binding domain and that interacts with conformationally constrained residues of a GRP94 ligand binding domain polypeptide conserved among Hsp90 subtypes, whereby a modulator that selectively modulates the activity of a GRP94 polypeptide compared to other Hsp90 polypeptides is identified.
Further, a method of identifying an Hsp90 modulator that selectively modulates the biological activity of one Hsp90 polypeptide compared to GRP94 is disclosed. The method comprises: (a) providing an atomic structure coordinate set describing a GRP94 ligand binding domain polypeptide and at least one other atomic structure coordinate set describing an Hsp90 ligand binding domain, each ligand binding domain comprising a ligand binding site; (b) comparing the atomic structure coordinate sets to identify at least one difference between the sets; (c) designing a candidate ligand predicted to interact with the difference of step (b); (d) synthesizing the candidate ligand; and (e) testing the synthesized candidate ligand for an ability to selectively modulate an Hsp90 as compared to GRP94, whereby an Hsp90 modulator that selectively modulates the biological activity of the Hsp90 compared to GRP94 is identified.
Additionally, a method of designing a modulator of a GRP94 polypeptide is disclosed. The method comprises: (a) selecting a candidate GRP94 ligand; (b) determining which amino acid or amino acids of a GRP94 polypeptide interact with the ligand using a three-dimensional model of a crystallized protein comprising a GRP94 ligand binding domain polypeptide; (c) identifying in a biological assay for GRP94 activity a degree to which the ligand modulates the activity of the GRP94 polypeptide; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the GRP94 polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) synthesizing a chemical compound with the selected chemical modification to form a modified ligand; (f) contacting the modified ligand with the GRP94 polypeptide; (g) identifying in a biological assay for GRP94 activity a degree to which the modified ligand modulates the biological activity of the GRP94 polypeptide; and (h) comparing the biological activity of the GRP94 polypeptide in the presence of modified ligand with the biological activity of the GRP94 polypeptide in the presence of the unmodified ligand, whereby a modulator of a GRP94 polypeptide is designed.
An assay method for identifying a compound that inhibits binding of a ligand to a GRP94 polypeptide is also disclosed. The assay method comprises: (a) designing a test inhibitor compound based on the three dimensional atomic coordinates of a GRP94 ligand binding domain polypeptide; incubating a GRP94 polypeptide with a ligand in the presence of a test inhibitor compound; determining an amount of ligand that is bound to the GRP94 polypeptide, wherein decreased binding of ligand to the GRP94 protein in the presence of the test inhibitor compound relative to binding of ligand in the absence of the test inhibitor compound is indicative of inhibition; and identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed, whereby a compound that inhibits binding of a ligand to a GRP94 polypeptide is identified.
A method of screening a plurality of compounds for a modulator of a GRP94 ligand binding domain polypeptide is also provided. The method comprises: (a) providing a library of test samples; (b) contacting a GRP94 ligand binding domain polypeptide with each test sample; (c) detecting an interaction between a test sample and the GRP94 ligand binding domain polypeptide; (d) identifying a test sample that interacts with the GRP94 ligand binding domain polypeptide; and (e) isolating a test sample that interacts with the GRP94 ligand binding domain polypeptide, whereby a plurality of compounds is screened for a modulator of a GRP94 ligand binding domain polypeptide.
An isolated GRP94 LBD polypeptide is also disclosed. In one embodiment, the isolated polypeptide has the sequence of any of SEQ ID NOs:4 or 6. An isolated nucleic acid molecule encoding a GRP94 LBD polypeptide is also disclosed, as is a chimeric gene comprising the nucleic acid molecule to a heterologous promoter, a vector comprising the chimeric gene, and a host cell comprising the chimeric gene. Methods of detecting the GRP LBD polypeptide and nucleic acid encoding the same are also disclosed, as is an antibody that specifically recognizes a GRP94 LBD polypeptide.
A method for identifying a substance that modulates GRP94 LBD function is also disclosed. The method comprises: (a) isolating a GRP94 LBD polypeptide; (b) exposing the isolated GRP94 polypeptide to a plurality of substances; (c) assaying binding of a substance to the isolated GRP94 polypeptide; and (d) selecting a substance that demonstrates specific binding to the isolated GRP94 LBD polypeptide.
Accordingly, it is an object of the present invention to provide a three dimensional structure of the ligand binding domain of a GRP94. This and other objects are achieved in whole or in part by the present invention.
An object of the invention having been stated hereinabove, other objects will be evident as the description proceeds, when taken in connection with the accompanying Drawings and Laboratory Examples as best described hereinbelow.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a graph depicting Prodan binding to GRP94 independent of GRP94 structural state. Fluorescence emission wavelength scans of 0.5 μM native or heat shocked (hs) GRP94 were performed following exposure to 5 μM Prodan for 30 minutes. Values represent the maximal fluorescence relative to that occurring with an identical concentration of heat shocked GRP94. Experiments were conducted at excitation wavelengths of 360 nm (Prodan). All spectra were background corrected.
FIG. 1B is a graph depicting 8-ANS binding to GRP94, and dependence of such binding on GRP94 structural state. Fluorescence emission wavelength scans of 0.5 μM native or heat shocked (hs) GRP94 were performed following exposure to 5 μM 8-ANS for 30 minutes. Values represent the maximal fluorescence relative to that occurring with an identical concentration of heat shocked GRP94. Experiments were conducted at excitation wavelengths of 372 nm (8-ANS). All spectra were background corrected.
FIG. 1C is a graph depicting bis-ANS binding to GRP94, and dependence of such binding on GRP94 structural state. Fluorescence emission wavelength scans of 0.5 μM native or heat shocked (hs) GRP94 were performed following exposure to 5 μM bis-ANS for 20 hours. Values represent the maximal fluorescence relative to that occurring with an identical concentration of heat shocked GRP94. Experiments were conducted at excitation wavelengths of 393 nm (bis-ANS). All spectra were background corrected.
FIG. 1D is a graph depicting a time course of bis-ANS binding to GRP94. Values represent the maximal fluorescence relative to that occurring with an identical concentration of heat shocked GRP94. Experiments were conducted at excitation wavelengths of 393 nm (bis-ANS). All spectra were background corrected.
FIG. 2A is a graph depicting kinetic analysis of bis-ANS interactions with heat shocked GRP94. The concentration dependence of bis-ANS binding to heat shocked GRP94 was conducted under experimental conditions of fixed bis-ANS concentration (50 nM) and increasing GRP94 concentration, as indicated.
FIG. 2B is a Klotz plot representation of bis-ANS/GRP94 binding data. Half maximal binding occurs at 110 nM GRP94. Excitation wavelength, 393 nm. Emission wavelength, 475 nm.
FIG. 3 is a digital image of a Coomassie Blue stained gel depicting that bis-ANS and heat shock increase GRP94 proteolysis sensitivity. GRP94 (5 μg, 5 μM) was incubated with 50 μM bis-ANS for one hour at 37° C. or heat shocked for 15 minutes at 50° C. Samples were then digested with 0.1% trypsin for 30 minutes at 37° C. and analyzed on 12.5% SDS-PAGE gels. Lane 1, 5 μg of undigested GRP94; lane 2, control native GRP94 incubated with trypsin; lane 3, bis-ANS treated GRP94 digested with trypsin; lane 4, GRP94 heat shocked then digested with trypsin.
FIG. 4 is a digital image of a Coomassie Blue stained gel depicting that bis-ANS and heat shock induce GRP94 multimerization. GRP94 was heat shocked at 50° C. for 0-15 minutes or incubated with 10-fold molar excess of bis-ANS and the structural state of the protein analyzed on 5-18% native blue polyacrylamide gradient gels. The mobilities of GRP94 dimers, tetramers, hexamers, and octamers are shown. Molecular weight standards are indicated to the right of FIG. 4.
FIG. 5 is a graph depicting that circular dichroism spectra of native, heat shocked, and bis-ANS treated GRP94 are identical. Circular dichroism spectra of 1 μM GRP94 native (diamonds); heat shocked (dot and dash); and treated 2 hours with 10 μM bis-ANS (dotted) are shown. Spectra were collected as described in Examples 1-8 below.
FIG. 6A is a digital image of a Coomassie Blue stained gel depicting that radicicol blocks bis-ANS structural transitions. GRP94 (5 μM) was preincubated for one hour at 37° C. with 0-500 μM radicicol and subsequently incubated for one hour at 37° C. with 50 μM bis-ANS, trypsinized, and the trypsin digestion pattern analyzed by SDS-PAGE.
FIG. 6B is a graph depicting that radicicol blocks heat shock and bis-ANS binding. GRP94 (0.5 μM) was preincubated with 0-10 μM radicicol for one hour, heat shocked, and subsequently incubated with 1 μM bis-ANS. Bis-ANS binding was determined by spectrofluorometry with bis-ANS binding to native GRP94 in the absence of radicicol shown for comparison. Excitation 393 nm, emission 410-600 nm.
FIG. 7A is a graph depicting that bis-ANS and heat shock stimulate GRP94 chaperone activity. Citrate synthase enzyme was diluted to 0.15 μM into buffer containing no GRP94, 1 μM native GRP94, heat shocked GRP94, or GRP94 which had been preincubated for two hours with 10 μM bis-ANS, and citrate synthase aggregation at 43° C. was monitored by light scattering at 500 nm in a thermostatted spectrofluorometer.
FIG. 7B is a bar graph depicting that bis-ANS and heat shock stimulate GRP94 peptide binding activity. Native, heat shocked, or bis-ANS treated GRP94 were incubated with a 10-fold molar excess of 125I-VSV8 peptide for 30 minutes at 37° C. Free peptide was removed by spin column chromatography and bound radioactive peptide quantitated by gamma counting.
FIG. 8 is a bar graph depicting that GRP94 and Hsp90 exhibit differential ligand binding. NECA and ATP binding to GRP94 was performed in the presence of 20 nM [3H]-NECA (closed bars) or 50 μM [32P]ATP (hatched bars) for 1 hour at 4° C. Bound versus free nucleotide were separated by vacuum filtration. PEI treated glass filters (S&S #32, Schleicher and Schuell of Keene, N.H.) were used for the NECA binding assay while nitrocellulose filters (S&S BA85, Schleicher and Schuell of Keene, N.H.) were used to measure ATP binding. The data presented are averages of triplicate points and are corrected for nonspecific ligand binding.
FIG. 9A is a Scatchard plot depicting characterization of NECA binding to GRP94. GRP94 was incubated with increasing concentrations of NECA for 1 hour at 4° C. as described in Materials and Methods. Bound versus free NECA were then separated by vacuum filtration with glass filters pretreated in 0.3% PEI.
FIG. 9B is a saturation curve depicting characterization of NECA binding to GRP94. The curve is plotted with respect to GRP94 dimer concentration. The maximal binding stoichiometry is 1 molecule of NECA per molecule of GRP94 dimer.
FIG. 9C is a graph depicting stoichiometry of GRP94 binding to NECA (solid oval) and radicicol (solid rectangle). NECA and radicicol binding to GRP94 was assayed by isothermal titration calorimetry. GRP94 was present at a concentration of 5 μM. NECA titrations were performed with a 152 μM NECA stock whereas radicicol titrations were performed with a 115 μM stock. ITC data were collected as μcal/sec versus time and the area under individual injection peaks, determined with the instrument software, was plotted.
FIG. 10A is a graph depicting a competition assay for NECA by the Hsp90 family inhibitors, geldanamycin (♦) and radicicol (▪). GRP94 was incubated with 20 nM [3H]-NECA and increasing concentrations of competitors for 1 hour at 4° C. Bound NECA was separated from free by vacuum filtration with glass filters pre-treated in 0.3% PEI. All data points represent the average of triplicates points minus background (nonspecific NECA binding in the absence of protein).
FIG. 10B is a graph depicting a competition assay for NECA by ATP (♦), ADP (▪), and AMP (▴). GRP94 was incubated with 20 nM 3H-NECA and increasing concentrations of competitors for 1 hour at 4° C. Bound NECA was separated from free by vacuum filtration with glass filters pre-treated in 0.3% PEI. All data points represent the average of triplicate points minus background (nonspecific NECA binding in the absence of protein).
FIG. 10C is a graph depicting a competition assay for NECA by adenosine (▴), and cAMP (▪). GRP94 was incubated with 20 nM [3H]-NECA and increasing concentrations of competitors for 1 hour at 4° C. Bound NECA was separated from free by vacuum filtration with glass filters pre-treated in 0.3% PEI. All data points represent the average of triplicates points minus background (nonspecific NECA binding in the absence of protein).
FIG. 11 is a bar graph depicting that ligand binding specificity of GRP94 to the adenosine base. GRP94 was incubated with 20 nM [3H]-NECA and competitors, all at 50 μM final concentration for 1 hour at 4° C., and bound vs. free NECA was separated by vacuum filtration with glass filters pretreated in 0.3% PEI.
FIG. 12 is a graph depicting that binding of ATP, ADP, and AMP to GRP94 is sensitive to Mg2+ concentration. GRP94 was incubated for 1 hour at 4° C. in 50 mM Tris, 20 nM [3H]-NECA and one of the following concentrations of competitor: 3.1×10−6 M ATP, 3.1×10−5 M ADP, 6×10−4 M AMP, or 3.1×10−5 M adenosine. Reactions were performed in the presence of 10 mM Mg(OAc)2 (hatched bars) or in the presence of nominal, endogenous magnesium (closed bars). Bound vs. free NECA was separated by vacuum filtration with glass filters pretreated in 0.3% PEI.
FIG. 13A is a bar graph depicting the effects of NECA on GRP94 autophosphorylation. 25 μl reactions consisting of 1 μM GRP94 (closed bars), 0.15 mM γ-32PATP (6000 cpm/pmol), 10 mM Mg(OAc)2, and 50 mM K-Hepes, pH 7.4) were incubated for 1 hour at 37° C. One (1) unit casein kinase II (hatched bars) was incubated in the above conditions with the addition of 4 μM casein. Competitors were added to the appropriate samples with a final concentration of 180 μM NECA in 3.6% DMSO, 180 μM radicicol in 3.6% DMSO, 5 μg/ml heparin, 5 mM GTP, or 3.6% DMSO. Phosphorylated species were quantitated on a Fuji MACBAS1000TM phosphorimaging system, and the average PSL units of three independent experiments are displayed.
FIG. 13B is a bar graph depicting ATP hydrolysis in the presence and absence of GRP94. 100 μl reactions consisting of 1 μM GRP94 monomer, various concentrations of MgATP (pH 7.0), and 50 mM K-Hepes, pH 7.4, were incubated for two hours at 37° C. ATP and ADP were separated on a Hewlett Packard HPLC using a Partisil SAX column. Spontaneous ATP hydrolysis was determined in the absence of protein. Hydrolysis in the presence of GRP94 is indicated by closed bars and spontaneous hydrolysis is indicated by the hatched bars.
FIG. 14 is a graph depicting ligand-induced conformational changes of GRP94. GRP94 (50 μg/ml) was incubated in buffer A supplemented with 10 mM Mg(OAc)2 and the following concentrations of ligands for 1 hour at 37° C.: 50 μM NECA, 50 μM geldanamycin, 2.5 mM ATP, or 2.5 mM ADP. Samples were excited at a wavelength of 295 nm and the tryptophan emission spectra were recorded from 300-400 nm. All spectra were corrected by subtraction of spectra obtained in buffer alone or buffer+ligand samples.



BRIEF DESCRIPTION OF SEQUENCES IN THE SEQUENCE LISTING
SEQ ID NOs:1 and 2 are, respectively, a DNA sequence encoding a wild type full-length human GRP94 (GENBANK® Accession No. NM—003299) and the amino acid sequence (GENBANK® Accession No. NP—003290) of a human GRP94 encoded by the DNA sequence.
SEQ ID NOs:3 and 4 are, respectively, a DNA sequence encoding a wild type ligand binding domain of a human GRP94 and the amino acid sequence of a human GRP94 (residues 22-337) encoded by the DNA sequence.
SEQ ID NOs:5 and 6 are, respectively, a DNA sequence encoding a ligand binding domain of a canine GRP94 (residues 22-337) and the amino acid sequence of a canine GRP94 encoded by the DNA sequence.
SEQ ID NO:7 is peptide VSV8.
SEQ ID NO:8 is a peptide that maps to residues 271-281 of the N-terminal domain of GRP94.
SEQ ID NO:9 is a peptide that maps to amino acids 210-222 of the human Hsp90 sequence.
DETAILED DESCRIPTION
Disclosed herein is the characterization of ligand interactions of GRP94, and where applicable Hsp90, wherein ligand binding to the N-terminal nucleotide binding domain of GRP94, and in some instances, Hsp90, elicits a conformational change that converts GRP94, and in some instances, Hsp90, from an inactive to an active conformation, and wherein the chaperone and peptide binding activities of GRP94, and where applicable, Hsp90, are markedly stimulated. Also disclosed herein is the characterization of ligand interactions of GRP94, and where applicable Hsp90, wherein ligand binding to the N-terminal nucleotide binding domain of GRP94, and in some instances, Hsp90, inhibits a conformational change that converts GRP94, and in some instances, Hsp90, from an inactive to an active conformation, and wherein the activities of GRP94, and where applicable, Hsp90, are markedly inhibited. Particularly, disclosed herein is an isolated and purified GRP94 ligand binding domain (LBD) polypeptide, and a crystalline form thereof. In a preferred embodiment, the crystalline form further comprises a ligand.
Also disclosed herein are methods of screening for ligands that bind to the GRP94 LBD and inhibit protein activity and/or protein conformational activation in a manner similar and/or related to that observed with geldanamycin and radicicol. Such ligands can function as potential anti-tumor therapeutics, among other applications.
Until disclosure of the present invention presented herein, the ability to obtain crystalline forms of the ligand binding domain of GRP94 has not been realized. And until disclosure of the present invention presented herein, a detailed three-dimensional crystal structure of a GRP94 LBD polypeptide has not been solved.
In addition to providing structural information, crystalline polypeptides provide other advantages. For example, the crystallization process itself further purifies the polypeptide, and satisfies one of the classical criteria for homogeneity. In fact, crystallization frequently provides unparalleled purification quality, removing impurities that are not removed by other purification methods such as HPLC, dialysis, conventional column chromatography, and other methods. Moreover, crystalline polypeptides are sometimes stable at ambient temperatures and free of protease contamination and other degradation associated with solution storage. Crystalline polypeptides can also be useful as pharmaceutical preparations. Finally, crystallization techniques in general are largely free of problems such as denaturation associated with other stabilization methods (e.g., lyophilization). Once crystallization has been accomplished, crystallographic data provides useful structural information that can assist the design of compounds that can serve as modulators (e.g. agonists or antagonists), as described herein below. In addition, the crystal structure provides information useful to map a ligand binding domain, which can then be mimicked by a chemical entity that can serve as an antagonist or agonist.
I. definitions
While the following terms are believed to have well defined meanings in the art, the following definitions are set forth to facilitate explanation of the invention.
Following long-standing patent law convention, the terms “a” and “an” mean “one or more” when used in this application, including the claims.
As used herein the term “Hsp90 protein” is meant to refer to any of the Hsp90 class of molecular chaperones that are among the most abundant proteins in eukaryotic cells, and to biologically active fragments of such proteins. The term “HSP90 protein” refers to an individual member of this class, exemplified by canine HSP90 (GENBANK® Accession No. U01153) and mouse HSP90 (SwissProt Accession No. P08113), and to biologically active fragments thereof. Hsp90 family members are phylogenetically ubiquitous whereas the endoplasmic reticulum paralog of HSP90, GRP94 (gp96, ERp99, endoplasmin) is found only in higher plants and metazoans (Nicchitta (1998) Curr Opin Immunol 10:103-109). The Hsp90 family of proteins are involved in directing the proper folding and trafficking of newly synthesized proteins and in conferring protection to the cell during conditions of heat shock, oxidative stress, hypoxic/anoxic conditions, nutrient deprivation, other physiological stresses, and disorders or traumas that promote such stress conditions such as, for example, stroke and myocardial infarction.
As used herein, the terms “ligand binding domain (LBD) of the Hsp90 protein”, “Hsp90 LBD”, “GRP94 LBD”, and “HSP90 LBD” are used interchangeably and mean that region of an Hsp90 protein, preferably a GRP94 polypeptide or a HSP90 polypeptide, where a ligand binds. Even more preferably, the GRP94 LBD comprises amino acid residues 69-337 of mammalian GRP94 (e.g., human (SEQ ID NO: 2) or canine (GENBANK® Accession No. AAA17708)), or amino acid residues 22-337 of human GRP94 (which exhibits the same behavior and activity of the 69-337 LBD; see SEQ ID NO: 4)).
As used herein, the terms “binding pocket of the GRP94 ligand binding domain”, “GRP94 ligand binding pocket” and “GRP94 binding pocket” are used interchangeably, and refer to the large cavity within the GRP94 ligand binding domain (LBD) where a ligand can bind. This cavity can be empty, or can contain water molecules or other molecules from the solvent, or can contain ligand atoms. The binding pocket also includes regions of space near the “main” binding pocket that not occupied by atoms of GRP94 but that are near the “main” binding pocket, and that are contiguous with the “main” binding pocket.
“Antigenic molecule” as used herein refers to the peptides with which GRP94 or HSP90 endogenously associates in vivo (e.g., in infected cells or precancerous or cancerous tissue) as well as exogenous antigens/immunogens (i.e., not complexed with GRP94 or HSP90 in vivo) or antigenic/immunogenic fragments and derivatives thereof.
The term “biological activity” is meant to refer to a molecule having a biological or physiological effect in a subject. Adjuvant activity is an example of a biological activity. Activating or inducing production of other biological molecules having adjuvant activity is also a contemplated biological activity.
The term “adjuvant activity” is meant to refer to a molecule having the ability to enhance or otherwise modulate the response of a vertebrate subject's immune system to an antigen.
The term “immune system” includes all the cells, tissues, systems, structures and processes, including non-specific and specific categories, that provide a defense against antigenic molecules, including potential pathogens, in a vertebrate subject. As is well known in the art, the non-specific immune system includes phagocytic cells such as neutrophils, monocytes, tissue macrophages, Kupffer cells, alveolar macrophages, dendritic cells and microglia. The specific immune system refers to the cells and other structures that impart specific immunity within a host. Included among these cells are the lymphocytes, particularly the B cell lymphocytes and the T cell lymphocytes. These cells also include natural killer (NK) cells. Additionally, antibody-producing cells, like B lymphocytes, and the antibodies produced by the antibody-producing cells are also included within the term “immune system”.
The term “immune response” is meant to refer to any response to an antigen or antigenic determinant by the immune system of a vertebrate subject. Exemplary immune responses include humoral immune responses (e.g. production of antigen-specific antibodies) and cell-mediated immune responses (e.g. lymphocyte proliferation), as defined herein below.
The term “systemic immune response” is meant to refer to an immune response in the lymph node-, spleen-, or gut-associated lymphoid tissues wherein cells, such as B lymphocytes, of the immune system are developed. For example, a systemic immune response can comprise the production of serum IgG's. Further, systemic immune response refers to antigen-specific antibodies circulating in the blood stream and antigen-specific cells in lymphoid tissue in systemic compartments such as the spleen and lymph nodes.
The terms “humoral immunity” or “humoral immune response” are meant to refer to the form of acquired immunity in which antibody molecules are secreted in response to antigenic stimulation.
The terms “cell-mediated immunity” and “cell-mediated immune response” are meant to refer to the immunological defense provided by lymphocytes, such as that defense provided by T cell lymphocytes when they come into close proximity to their victim cells. A cell-mediated immune response also comprises lymphocyte proliferation. When “lymphocyte proliferation” is measured, the ability of lymphocytes to proliferate in response to specific antigen is measured. Lymphocyte proliferation is meant to refer to B cell, T-helper cell or CTL cell proliferation.
The term “CTL response” is meant to refer to the ability of an antigen-specific cell to lyse and kill a cell expressing the specific antigen. As described herein below, standard, art-recognized CTL assays are performed to measure CTL activity.
“Adoptive immunotherapy” as used herein refers to a therapeutic approach with particular applicability to cancer whereby immune cells with an antitumor reactivity are administered to a tumor-bearing host, with the aim that the cells mediate either directly or indirectly, the regression of an established tumor.
An “immunogenic composition” is meant to refer to a composition that can elicit an immune response. A vaccine is contemplated to fall within the meaning of the term “immunogenic composition”, in accordance with the present invention.
The term “a biological response modifier” is meant to refer to a molecule having the ability to enhance or otherwise modulate a subject's response to a particular stimulus, such as presentation of an antigen.
As used herein, the terms “candidate substance” and “candidate compound” are used interchangeably and refer to a substance that is believed to interact with another moiety as a biological response modifier. For example, a representative candidate compound is believed to interact with a complete Hsp90 protein, or fragment thereof, and which can be subsequently evaluated for such an interaction. Exemplary candidate compounds that can be investigated using the methods of the present invention include, but are not restricted to, agonists and antagonists of an Hsp90 protein, viral epitopes, peptides, enzymes, enzyme substrates, co-factors, lectins, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins, chemical compounds small molecules, and monoclonal antibodies.
As used herein, the term “modulate” means an increase, decrease, or other alteration of any or all chemical and biological activities or properties of a wild-type or mutant Hsp90 protein, preferably a wild-type or mutant GRP94 or HSP90 polypeptide. The term “modulation” as used herein refers to both upregulation (i.e., activation or stimulation) and downregulation (i.e. inhibition or suppression) of a response.
As used herein, the term “agonist” means an agent that supplements or potentiates the biological activity of a functional Hsp90 protein.
As used herein, the term “antagonist” means an agent that decreases or inhibits the biological activity of a functional Hsp90 protein, or that supplements or potentiates the biological activity of a naturally occurring or engineered non-functional Hsp90 protein.
As used herein, the terms “α-helix”, “alpha-helix” and “alpha helix” are used interchangeably and mean the conformation of a polypeptide chain wherein the polypeptide backbone is wound around the long axis of the molecule in a left-handed or right-handed direction, and the R groups of the amino acids protrude outward from the helical backbone, wherein the repeating unit of the structure is a single turnoff the helix, which extends about 0.56 nm along the long axis.
As used herein, the terms “β-sheet”, “beta-sheet” and “beta sheet” are used interchangeably and mean the conformation of a polypeptide chain stretched into an extended zig-zig conformation. Portions of polypeptide chains that run “parallel” all run in the same direction. Polypeptide chains that are “antiparallel” run in the opposite direction from the parallel chains.
As used herein, the term “crystal lattice” means the array of points defined by the vertices of packed unit cells.
As used herein, the term “unit cell” means a basic parallelipiped shaped block. The entire volume of a crystal can be constructed by regular assembly of such blocks. Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal. Thus, the term “unit cell” means the fundamental portion of a crystal structure that is repeated infinitely by translation in three dimensions. A unit cell is characterized by three vectors a, b, and c, not located in one plane, which form the edges of a parallelepiped. Angles α, β, and γ define the angles between the vectors: angle α is the angle between vectors b and c; angle β is the angle between vectors a and c; and angle γ is the angle between vectors a and b. The entire volume of a crystal can be constructed by regular assembly of unit cells; each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal.
As used herein, “orthorhombic unit cell” means a unit cell wherein a≠b≠c; and α=β=γ=90°. The vectors a, b and c describe the unit cell edges and the angles α, β, and γ describe the unit cell angles. In a first preferred embodiment of the present invention, the unit cell has lattice constants of a=99.889 Å, b=89.614 Å, c=60.066 Å, β=90.132°. In a second preferred embodiment of the present invention, the unit cell has lattice constants of the unit cell has lattice constants of a=89.200 Å, b=99.180 Å, c=63.071 Å; α=β=γ=90.0°. While preferred lattice constants are provided, a crystalline polypeptide of the present invention also comprises variations from the preferred lattice constants, wherein the variations range from about one to about two percent.
As used herein, the terms “cells,” “host cells” or “recombinant host cells” are used interchangeably and mean not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny might not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
As used herein, the terms “chimeric protein” or “fusion protein” are used interchangeably and mean a fusion of a first amino acid sequence encoding an Hsp90 polypeptide with a second amino acid sequence defining a polypeptide domain foreign to, and not homologous with, any domain of a Hsp90 polypeptide (preferably a GRP94 polypeptide). For example, a chimeric protein can include a foreign domain that is found in an organism that also expresses the first protein, or it can be an “interspecies” or “intergenic” fusion of protein structures expressed by different kinds of organisms. In general, a fusion protein can be represented by the general formula X-GRP94-Y, wherein GRP94 represents a portion of the protein which is derived from a GRP94 polypeptide, and X and Y are independently absent or represent amino acid sequences which are not related to a GRP94 sequence in an organism, which includes naturally occurring mutants.
As used herein, the term “detecting” means confirming the presence of a target entity by observing the occurrence of a detectable signal, such as a radiologic or spectroscopic signal that will appear exclusively in the presence of the target entity.
As used herein, the term “DNA segment” means a DNA molecule that has been isolated free of total genomic DNA of a particular species. In a preferred embodiment, a DNA segment encoding a GRP94 polypeptide refers to a DNA segment that comprises any of SEQ ID NOs:1, 3 or 5, but can optionally comprise fewer or additional nucleic acids, yet is isolated away from, or purified free from, total genomic DNA of a source species, such as Homo sapiens. Included within the term “DNA segment” are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. 
As used herein, the term “DNA sequence encoding a GRP94 polypeptide” can refer to one or more coding sequences within a particular individual. Moreover, certain differences in nucleotide sequences can exist between individual organisms, which are called alleles. It is possible that such allelic differences might or might not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity. As is well known, genes for a particular polypeptide can exist in single or multiple copies within the genome of an individual. Such duplicate genes can be identical or can have certain modifications, including nucleotide substitutions, additions or deletions, all of which still code for polypeptides having substantially the same activity.
As used herein, the terms “GRP94 gene product”, “GRP94 protein”, “GRP94 polypeptide”, and “GRP94 peptide” are used interchangeably and mean peptides having amino acid sequences which are substantially identical to native amino acid sequences from the organism of interest and which are biologically active in that they comprise all or a part of the amino acid sequence of a GRP94 polypeptide, or cross-react with antibodies raised against a GRP94 polypeptide, or retain all or some of the biological activity (e.g., DNA or ligand binding ability and/or transcriptional regulation) of the native amino acid sequence or protein. Such biological activity can include immunogenicity. A preferred embodiment in a GRP94 LBD polypeptide, and representative embodiments of a GRP94 LBD are set forth in SEQ ID NOs:4 and 6.
The terms “GRP94 gene product”, “GRP94 protein”, “GRP94 polypeptide”, and “GRP94 peptide” also include analogs of a GRP94 polypeptide. By “analog” is intended that a DNA or peptide sequence can contain alterations relative to the sequences disclosed herein, yet retain all or some of the biological activity of those sequences. Analogs can be derived from nucleotide sequences as are disclosed herein or from other organisms, or can be created synthetically. Those skilled in the art will appreciate that other analogs, as yet undisclosed or undiscovered, can be used to design and/or construct GRP94 analogs. There is no need for a “GRP94 gene product”, “GRP94 protein”, “GRP94 polypeptide”, or “GRP94 peptide” to comprise all or substantially all of the amino acid sequence of a GRP94 gene product. Shorter or longer sequences are anticipated to be of use in the invention; shorter sequences are herein referred to as “segments”. Thus, the terms “GRP94 gene product”, “GRP94 protein”, “GRP94 polypeptide”, and “GRP94 peptide” also include fusion or recombinant GRP94 polypeptides and proteins comprising sequences of the present invention. Methods of preparing such proteins are disclosed herein and are known in the art.
As used herein, the terms “GRP94 gene” and “recombinant GRP94 gene” mean a nucleic acid molecule comprising an open reading frame encoding a GRP94 polypeptide of the present invention, including both exon and (optionally) intron sequences.
As used herein, the term “interact” means detectable interactions between molecules, such as can be detected using, for example, a yeast two-hybrid assay. The term “interact” is also meant to include “binding” interactions between molecules. Interactions can, for example, be protein-protein or protein-nucleic acid in nature.
As used herein, the term “labeled” means the attachment of a moiety, capable of detection by spectroscopic, radiologic or other methods, to a probe molecule.
As used herein, the term “modified” means an alteration from an entity's normally occurring state. An entity can be modified by removing discrete chemical units or by adding discrete chemical units. The term “modified” encompasses detectable labels as well as those entities added as aids in purification.
As used herein, the term “molecular replacement” means a method that involves generating a preliminary model of the wild-type GRP94 ligand binding domain, or a GRP94 mutant crystal whose structure coordinates are unknown, by orienting and positioning a molecule or model whose structure coordinates are known (e.g., an Hsp) within the unit cell of the unknown crystal so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal. See, e.g., Lattman, (1985) Method Enzymol., 115: 55-77; Rossmann, ed, (1972) The Molecular Replacement Method, Gordon & Breach, New York. Using the structure coordinates of the ligand binding domain of GRP94 provided by this invention, molecular replacement can be used to determine the structure coordinates of a crystalline mutant or homologue (e.g., another Hsp90 polypeptide or Hsp90 LBD polypeptide) of the GRP94 ligand binding domain, or of a different crystal form of the GRP94 ligand binding domain.
As used herein, the term “mutation” carries its traditional connotation and means a change, inherited, naturally occurring or introduced, in a nucleic acid or polypeptide sequence, and is used in its sense as generally known to those of skill in the art.
As used herein, the term “partial agonist” means an entity that can bind to a target and induce only part of the changes in the target that are induced by agonists. The differences can be qualitative or quantitative. Thus, a partial agonist can induce some of the conformation changes induced by agonists, but not others, or it can only induce certain changes to a limited extent.
As used herein, the term “partial antagonist” means an entity that can bind to a target and inhibit only part of the changes in the target that are induced by antagonists. The differences can be qualitative or quantitative. Thus, a partial antagonist can inhibit some of the conformation changes induced by an antagonist, but not others, or it can inhibit certain changes to a limited extent.
As used herein, the term “polypeptide” means any polymer comprising any of the 20 protein amino acids, regardless of its size. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides and proteins, unless otherwise noted. As used herein, the terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to a gene product.
As used herein, the term “sequencing” means the determining the ordered linear sequence of nucleic acids or amino acids of a DNA or protein target sample, using conventional manual or automated laboratory techniques.
As used herein, the term “space group” means the arrangement of symmetry elements of a crystal.
As used herein, the terms “structure coordinates” and “structural coordinates” mean mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a molecule in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal.
Those of skill in the art understand that a set of coordinates determined by X-ray crystallography is not without standard error. In general, the error in the coordinates tends to be reduced as the resolution is increased, since more experimental diffraction data is available for the model fitting and refinement. Thus, for example, more diffraction data can be collected from a crystal that diffracts to a resolution of 2.8 angstroms than from a crystal that diffracts to a lower resolution, such as 3.5 angstroms. Consequently, the refined structural coordinates will usually be more accurate when fitted and refined using data from a crystal that diffracts to higher resolution. The design of ligands and modulators for GRP94 or any other Hsp90 polypeptide depends on the accuracy of the structural coordinates. If the coordinates are not sufficiently accurate, then the design process will be ineffective. In most cases, it is very difficult or impossible to collect sufficient diffraction data to define atomic coordinates precisely when the crystals diffract to a resolution of only 3.5 angstroms or poorer. Thus, in most cases, it is difficult to use X-ray structures in structure-based ligand design when the X-ray structures are based on crystals that diffract to a resolution of only 3.5 angstroms or poorer. However, common experience has shown that crystals diffracting to 2.8 angstroms or better can yield X-ray structures with sufficient accuracy to greatly facilitate structure-based drug design. Further improvement in the resolution can further facilitate structure-based design, but the coordinates obtained at 2.8 angstroms resolution are generally adequate for most purposes.
Also, those of skill in the art will understand that Hsp90 proteins can adopt different conformations when different ligands are bound. In particular, Hsp90 proteins will adopt substantially different conformations when agonists and antagonists are bound. Subtle variations in the conformation can also occur when different agonists are bound, and when different antagonists are bound. These variations can be difficult or impossible to predict from a single X-ray structure. Generally, structure-based design of GRP94 modulators depends to some degree on knowledge of the differences in conformation that occur when agonists and antagonists are bound. Thus, structure-based modulator design is most facilitated by the availability of X-ray structures of complexes with potent agonists as well as potent antagonists.
As used herein, the term “target cell” refers to a cell, into which it is desired to insert a nucleic acid sequence or polypeptide, or to otherwise effect a modification from conditions known to be standard in the unmodified cell. A nucleic acid sequence introduced into a target cell can be of variable length. Additionally, a nucleic acid sequence can enter a target cell as a component of a plasmid or other vector or as a naked sequence.
II. Description of Tables
Table 1 is a table of the atomic structure coordinate data obtained from X-ray diffraction from Form 1 of the ligand binding domain of canine GRP94 (residues 69-337337 of GENBANK® Accession No. AAA17708; residues 48-316 of SEQ ID NO: 6) in complex with NECA.
Table 2 is a table of the atomic structure coordinates of the NECA and GRP94 binding pocket within Form 1 of the canine GRP94 LBD.
Table 3 is a table of the atomic structure coordinate data obtained from X-ray diffraction from Form 2 of the ligand binding domain of canine GRP94 (residues 69-337 of GENBANK® Accession No. AAA17708; residues 48-316 of SEQ ID NO: 6) in complex with NECA.
III. General Considerations
As noted above, GRP94 (gp96, ERp99, endoplasmin) is the endoplasmic reticulum paralog of cytosolic HSP90, and as such, is an abundant resident ER lumenal protein that by virtue of its association with nascent polypeptides performs a chaperone function. The terms “GRP94” “GRP94 polypeptide”, and/or “GRP94 protein” also refer to biologically active fragments of a GRP94 protein. A preferred biologically active fragment is the GRP94 LBD. Consistent with this role, GRP94 expression is upregulated by stress conditions that promote protein misfolding or unfolding, such as glucose starvation, oxidative stress, and heavy metal poisoning. In addition to its role in the regulation of protein folding in the ER, GRP94 can function in the intercellular trafficking of peptides from the extracellular space to the major histocompatability complex (MHC) class I antigen processing pathway of professional antigen presenting cells. Thus, in addition to a homeostatic role in protein folding and assembly, GRP94 functions as a component of the MHC class I antigen processing and presentation pathways of mammalian cells.
GRP94 also contributes to the folding and assembly of immunoglobulins, MHC class II molecules, HSV-1 glycoproteins, thyroglobulin, collagen, and p185erbB2. (Melnick et al. (1992) J Biol Chem 267:21303-21306; Melnick et al. (1994) Nature 370:373-375; Schaiff et al. (1992) J Exp Med 176:657-666; Navarro et al. (1991) Virology 184:253-264; Kuznetsov et al. (1994) J Biol Chem 269:22990-22995; Ferreira et al. (1994) J Cell Biochem 56:518-26; Chavany et al. (1996) J Biol Chem 273:4974-4977). In addition to interactions with polypeptide folding substrates, GRP94 binds peptides, a subset of which is suitable for assembly on nascent MHC class 1 molecules. (Srivastava et al. (1986) Proc Natl Acad Sci USA 83:3407-3411; Nieland et al. (1996) Proc Natl Acad Sci USA 93:6135-6139; Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156; Ishii et al. (1999) J Immunol 162:1303-1309; Srivastava et al. (1998) Immunity 8:657-665; Sastry & Linderoth (1999) J Biol Chem 274:12023-12035). The peptide binding activity of GRP94 plays a role in its ability to elicit CD8+ T cell immune responses. (Udono et al. (1994) Proc Natl Acad Sci USA, 91:3077-30781; Suto & Srivastava (1995) Science 269:1585-1588; Arnold et al. (1995) J Exp Med 182:885-889; Nair et al. (1999) J Immunol 162:6426-6432; Blachere et al. (1997) J Exp Med 186:465-472; Heike et al. (1996) J Leukoc Biol 139:613-623; Srivastava et al. (1998) Immunity 8:657-665). Peptide binding activity is not, however, alone sufficient to impart immunogenic activity to a protein and thus GRP94 is among a limited subset of molecular chaperones that can function in the essential immunological process of cross-presentation. (Srivastava et al. (1998) Immunity 8:657-665; Nair et al. (1999) J Immunol 162:6426-6432; Basu and Srivastava (1999) J Exp Med 189:797-802; Schild et al. (1999) Curr Opin Immunol 11:109-113).
HSP90 has adenosine nucleotide-dependent modes of regulation. Additionally, amino acid side chains that participate in water-mediated hydrogen bonds with the N7 group of the purine ring of adenosine (N51 in human HSP90=N86 in GRP94) and the N1 group of the purine ring of adenosine (G97 in human HSP90=G130 of GRP94) are conserved between HSP90 and GRP94. The N6 group of the purine ring of adenosine (L48 in human HSP90=L83 in GRP94) that mediates direct nucleotide binding is also conserved between HSP90 and GRP94. In ATP binding with HSP90, the N6 group of the adenine purine is the sole direct hydrogen bond between the nucleotide and the nucleotide binding pocket (Prodromou et al. (1997) Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910), and N6 substituted adenosine analogs do not bind to GRP94. (Hutchison & Fox (1989) J Biol Chem 264:19898-903; Hutchison et al. (1990) Biochemistry 29:5138-5144). Thus, although ATP/ADP binding and hydrolysis are generally accepted as biological properties of HSP90, it is not known whether ATP/ADP serve an identical function(s) in the regulation of GRP94 activity. ATP and ADP bind with very low affinity to GRP94 and thus experimental limitations require that ATP/ADP interactions at the GRP94 nucleotide binding pocket be analyzed by indirect methods, including but not limited to ligand displacement assays. (Wearsch et al. (1998) Biochemistry 37(16):5709-5719; Csermely et al. (1995) J Biol Chem 270:6381-6388; Li & Srivastava (1993) EMBO J 12:3143-3151).
The peptide binding activity of GRP94 plays a role in its ability to elicit CD8+ T cell immune responses. Peptide binding activity is not, however, alone sufficient to impart immunogenic activity to a protein and thus GRP94 is among a limited subset of molecular chaperones that can function in the essential immunological process of cross-presentation. Until the disclosure of the present invention, a crystalline form providing a detailed look at a GRP94 ligand-interaction that modulates activity of GRP94 with respect to both polypeptide and peptide substrates remained to be determined.
HSP90 and GRP94 have been proposed as possible targets of several antitumor agents, principally radicicol and geldanamycin. Scheibel & Buckner (1998) Biochem Pharm 56:675-82. These compounds are believed to act by inhibiting the ability of the Hsp90 proteins to assist proto-oncogenic kinases, hormone receptors, and other signaling proteins assume their active folded states and appropriate subcellular location. Pratt (1998) Proc Soc Exp Biol Med 217:420-434.
GRP94 has also been found to elicit cytotoxic T cell responses, a reflection of its peptide binding activity (Nicchitta (1998) Curr Opin Immunol 10:103-109; Srivastava et al. (1998) Immunity 8:657-665). It is now established that GRP94-peptide complexes can be processed by professional antigen presenting cells, with the GRP94-bound peptides exchanged onto MHC class 1 molecules of the antigen presenting cell. The antigen presenting cells can then interact with naive CD8+ T cell responses against tissue(s) displaying peptide epitopes present in complex with GRP94 (Srivastava et al. (1998) Immunity 8:657-665).
A potential yet heretofore uncharacterized protective role of grp94 in ischemia is supported by the observation that expression of GRP94 is enhanced in hippocampus after transient forebrain ischemia of a duration known to result in neuronal death (Yagita et al. (1999) J Neurochem 72:1544-1551). grp94 is similarly induced following acute kidney ischemia (Kuznetsov (1996) Proc Natl Aced Sci USA 93:8584-8589). Heat-shock proteins, including HSP90, are overexpressed during the oxidative stress of reperfusion that generally follows ischemia (Sciandra et al. (1984) Proc Natl Aced Sci USA 81:4843-4847). HSP90 might also play a role in ischemic signaling by binding to the hypoxia-inducible factor 1-a (Gradin et al. (1996) Mol Cell Biol 16:5221-5231).
Summarily, in accordance with the present invention, GRP94 and HSP90 represent rational targets for chemotherapeutics, immunotherapeutics and vaccines relevant to the treatment of infections disease and cancer. In view of their function as molecular chaperones, GRP94 and HSP90 further represent rational targets for the development of therapeutics for tissue injury and stress, such as can occur in ischemic injuries including, but not limited to, organ (kidney, heart, lung, liver) transplantation, cerebral stroke, and myocardial infarct. Furthermore, Hsp90 and GRP94 represent rational targets for anti-tumor drug design.
Despite the aforementioned indirect characterization of the structure of GRP94, until the present disclosure, a detailed three-dimensional model of GRP94, including particularly the ligand binding domain of GRP94, has not been achieved.
Sequence analysis and X-ray crystallography, including the disclosure of the present invention, have confirmed that GRP94 has a modular architecture, with three domains, including a N-terminal ligand binding domain (LBD). The modularity of GRP94 permits different domains of each protein to separately accomplish certain functions. Some of the functions of a domain within the full-length protein are preserved when that particular domain is isolated from the remainder of the protein. Using conventional protein chemistry techniques, a modular domain can sometimes be separated from the parent protein. Using conventional molecular biology techniques, each domain can usually be separately expressed with its original function intact or, as discussed herein below, chimeras comprising two different proteins can be constructed, wherein the chimeras retain the properties of the individual functional domains of the protein from which the chimeras were generated.
As described herein, the LBD of a GRP94 can be mutated or engineered, expressed, crystallized, its three dimensional structure determined with a ligand bound as disclosed in the present invention, and computational methods can be used to design ligands to heat shock proteins, preferably to Hsp90 proteins, and more preferably to GRP94. Thus, the present invention will usually be applicable mutatis mutandis to heat shock proteins, more particularly to Hsp90 proteins and even more particularly to GRP94 proteins, including GRP94 isoforms, as discussed herein, based, in part, on the patterns of heat shock protein structure and modulation that have emerged as a consequence of the present disclosure, which in part discloses determining the three dimensional structure of the ligand binding domain of GRP94 in complex with NECA.
IV. The Neca Ligand
Ligand binding can induce biological functions in a variety of ways. In one aspect of the present invention, a GRP94 LBD is co-crystallized with a ligand, preferably a ligand that bears structural similarities to the purine nucleoside, adenosine. In a preferred embodiment, a crystalline form of the present invention comprises a GRP94 LBD in complex with N-ethylcarboxamidoadenosine (NECA). A crystalline form of the present invention can also comprise a ligand composition of the present invention as disclosed herein below. Thus, an aspect of the present invention is the definition of the protein fold and space occupied by the ligand provided by the crystalline form.
V. Atomic Structure of Hsp90 Polypeptides
A Hsp90 protein can be expressed, crystallized, its three dimensional structure determined with a ligand bound as disclosed in the present invention. Once crystallization has been accomplished, crystallographic data provides useful structural information that can assist the design of compounds that function as agonists or antagonists, as described herein below. In a preferred embodiment, the Hsp90 polypeptide is GRP94 or a fragment thereof.
The crystallization process also purifies the polypeptide, and satisfies one of the classical criteria for homogeneity. In fact, crystallization frequently provides unparalleled purification quality, removing impurities that are not removed by other purification methods such as HPLC, dialysis, conventional column chromatography, etc. Moreover, crystalline polypeptides are often stable at ambient temperatures and free of protease contamination and other degradation associated with solution storage. Crystallization techniques in general yield a pure polypeptide largely free of problems such as denaturation associated with other stabilization methods (e.g., lyophilization such crystallized polypeptides can be useful as pharmaceutical preparations).
V.A. Production of Hsp90 Polypeptides
According to the present invention, an Hsp90 polypeptide, preferably a GRP94 or GRP94 LBD polypeptide, can be expressed using an expression vector. An expression vector, as is well known to those of skill in the art, typically includes elements that permit autonomous replication in a host cell independent of the host genome, and one or more phenotypic markers for selection purposes. Either prior to or after insertion of the DNA sequences surrounding the desired Hsp90 or GRP94 (e.g., GRP94 LBD) coding sequence, an expression vector also will include control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes and a signal for termination. In some embodiments, where secretion of the produced polypeptide is desired, nucleotides encoding a “signal sequence” can be inserted prior to an Hsp90 or GRP94, or Hsp90 or GRP94 LBD, coding sequence. For expression under the direction of the control sequences, a desired DNA sequence must be operatively linked to the control sequences; that is, the sequence must have an appropriate start signal in front of the DNA sequence encoding the Hsp90 or GRP94, or Hsp90 or GRP94 LBD polypeptide, and the correct reading frame to permit expression of that sequence under the control of the control sequences and production of the desired product encoded by that Hsp90 or GRP94, or Hsp90 or GRP94 LBD, sequence must be maintained.
After a review of the disclosure of the present invention presented herein, any of a wide variety of well-known available expression vectors can be useful to express a mutated coding sequence of this invention. These include for example, vectors consisting of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40, known bacterial plasmids, e.g., plasmids from E. coli including col E1, pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM 989, and other DNA phages, e.g., M13 and filamentous single stranded DNA phages, yeast plasmids and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences.
In addition, any of a wide variety of expression control sequences—sequences that control the expression of a DNA sequence when operatively linked to it—can be used in these vectors to express the mutated DNA sequences according to this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40 for animal cells, the lac system, the trp system the TAC or TRC system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, all for E. coli, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors for yeast, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
A wide variety of hosts are also useful for producing Hsp90 or GRP94, or Hsp90 or GRP94 LBD, polypeptides according to this invention. These hosts include, for example, bacteria, such as E. coli, Bacillus and Streptomyces, fungi, such as yeasts, and animal cells, such as CHO and COS-1 cells, plant cells, insect cells, such as SF9 cells, and transgenic host cells.
It should be understood that not all expression vectors and expression systems function in the same way to express DNA sequences of this invention, and to produce Hsp90 or GRP94 polypeptide, Hsp90 or GRP94 LBD polypeptides, Hsp90 or GRP94 mutants, or Hsp90 or GRP94 LBD mutants. Neither do all hosts function equally well with the same expression system. One of skill in the art can, however, make a selection among these vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, an important consideration in selecting a vector will be the ability of the vector to replicate in a given host. The copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the system, its controllability and its compatibility with the DNA sequence encoding an Hsp90 or GRP94 polypeptide, or Hsp90 or GRP94 LBD polypeptide of this invention, with particular regard to the formation of potential secondary and tertiary structures.
Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of a modified polypeptide to them, their ability to express mature products, their ability to fold proteins correctly, their fermentation requirements, the ease of purification of an Hsp90 or GRP94, or Hsp90 or GRP94 LBD, and safety. Within these parameters, one of skill in the art can select various vector/expression control system/host combinations that will produce useful amounts of a polypeptide. A polypeptide produced in these systems can be purified, for example, via the approaches disclosed in the Examples.
Thus, it is envisioned, based upon the disclosure of the present invention, that purification of the unliganded or liganded Hsp90 or GRP94, or Hsp90 or GRP94 LBD, polypeptide can be obtained by conventional techniques, such as hydrophobic interaction chromatography (HPLC), ion exchange chromatography (HPLC), gel filtration chromatography, and heparin affinity chromatography. To achieve higher purification for improved crystals Hsp90 or GRP94, or Hsp90 or GRP94 LBD, polypeptide it is sometimes preferable to ligand shift purify the Hsp90 or GRP94, or Hsp90 or GRP94 LBD, polypeptide using a column that separates the polypeptide according to charge, such as an ion exchange or hydrophobic interaction column, and then bind the eluted polypeptide with a ligand. The ligand induces a change in the polypeptide's surface charge such that when re-chromatographed on the same column, the polypeptide then elutes at the position of the liganded polypeptide and is removed by the original column run with the unliganded polypeptide. Typically, saturating concentrations of ligand can be used in the column and the polypeptide can be preincubated with the ligand prior to passing it over the column.
More recently developed methods involve engineering a “tag” such as with histidine placed on the end of the protein, such as on the amino terminus, and then using a nickel chelation column for purification. See Janknecht, (1991) Proc. Natl. Acad. Sci. U.S.A. 88: 8972-8976 (1991), incorporated by reference.
In a preferred embodiment as disclosed in the Examples, canine GRP94 LBD (residues 69-337 of GENBANK® Accession No. AAA17708; residues 48-316 of SEQ ID NO: 6) was overexpressed as a GST fusion in E. coli and purified to homogeneity by affinity and ion-exchange chromatography. The protein was exchanged into 10 mM Tris-HCl, pH 7.6, 1 mM DTT, 100 mM NaCl and concentrated to 30 mg/mL.
V.B. Formation of Hsp90 Protein Crystals
In one embodiment, the present invention provides crystals of a GRP94 ligand binding domain (LBD) polypeptide. A preferred approach for obtaining the crystals is disclosed in the Examples. The GRP94 LBD crystals, which can be native crystals, derivative crystals or co-crystals, have c-centered orthorhombic unit cells. In a first preferred embodiment of the present invention, the unit cell has lattice constants of a=99.889 Å, b=89.614 Å, c=60.066 Å; α=β=90.132°; space group symmetry C2; and 2 GRP94+NECA complexes in the asymmetric unit. In a second preferred embodiment of the present invention, the unit cell has lattice constants of a=89.200 Å, b=99.180 Å, c=63.071 Å; α=β=γ=90.0°; space group symmetry C222(1); and 1 GRP94+NECA complex in the asymmetric unit.
The native and derivative co-crystals, and fragments thereof, disclosed in the present invention can be obtained by a variety of techniques, including batch, liquid bridge, dialysis, vapor diffusion and hanging drop methods. See, e.g., McPherson (1982) Preparation and Analysis of Protein Crystals, John Wiley, New York, N.Y.; McPherson (1990) Eur J Biochem 189:1-23; Weber (1991) Adv Protein Chem 41:1-36). In a preferred embodiment, hanging drop methods are used for the crystallization of Hsp90 proteins and fragments thereof. A more preferred hanging drop method is disclosed in the Examples.
In general, native crystals of the present invention are grown by dissolving a substantially pure Hsp90 protein or a fragment thereof in an aqueous buffer containing a precipitant at a concentration just below that necessary to precipitate the protein. Water is removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases.
In one embodiment of the invention, native crystals are grown by vapor diffusion (See, e.g., McPherson, (1982) Preparation and Analysis of Protein Crystals, John Wiley, New York; McPherson, (1990) Eur. J. Biochem. 189:1-23). In this method, the polypeptide/precipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir having a precipitant concentration optimal for producing crystals. Generally, less than about 25 μL of Hsp90 (preferably GRP94 LBD) polypeptide solution is mixed with an equal volume of reservoir solution, giving a precipitant concentration about half that required for crystallization. This solution is suspended as a droplet underneath a coverslip, which is sealed onto the top of the reservoir. The sealed container is allowed to stand, until crystals grow. Crystals generally form within two to six weeks, and are suitable for data collection within approximately seven to ten weeks. Of course, those of skill in the art will recognize that the above-described crystallization procedures and conditions can be varied.
V.C. Preparation of Derivative Crystals
Derivative crystals of the present invention, e.g. heavy atom derivative crystals, can be obtained by soaking native crystals in mother liquor containing salts of heavy metal atoms. Such derivative crystals are useful for phase analysis in the solution of crystals of the present invention. In a preferred embodiment of the present invention, for example, growing the GRP94 overexpressing bacteria in minimal media containing Se-Met provides protein containing Se-Met in place of Met and provides crystals suitable for use in multi-wavelength anomalous dispersion X-ray phasing methods. Additional reagents useful for the preparation of the derivative crystals of the present invention will be apparent to those of skill in the art after review of the disclosure of the present invention.
V.D. Preparation of Co-rystals
Co-crystals of the present invention can be obtained by soaking a native crystal in mother liquor containing compounds known or predicted to bind the Hsp90 protein, or a fragment thereof. Alternatively, co-crystals can be obtained by co-crystallizing a Hsp90 protein or a fragment thereof in the presence of one or more compounds known or predicted to bind the polypeptide. In a preferred embodiment, such a compound is NECA, and the Hsp90 protein is GRP94 or a fragment thereof.
V.E. Solving a Crystal Structure of the Present Invention
Crystal structures of the present invention can be solved using a variety of techniques including, but not limited to, isomorphous replacement anomalous scattering or molecular replacement methods. Computer software packages will also be helpful in solving a crystal structure of the present invention. Applicable software packages include but are not limited to X-PLORTM program (Brünger (1992) X-PLOR, Version 3.1. A System for X-ray Crystallography and NMR, Yale University Press, New Haven, Conn.; X-PLOR is available from Molecular Simulations, Inc., San Diego, Calif.), Xtal View (McRee (1992) J Mol Graphics 10:44-47; X-tal View is available from the San Diego Supercomputer Center), SHELXS 97 (Sheldrick (1990) Acta Cryst A46:467; SHELX 97 is available from the Institute of Inorganic Chemistry, Georg-August-Universität, Göttingen, Germany), HEAVY (Terwilliger, Los Alamos National Laboratory) and SHAKE-AND-BAKE (Hauptman (1997) Curr Opin Struct Biol 7:672-680; Weeks et al. (1993) Acta Cryst D49:179; available from the Hauptman-Woodward Medical Research Institute, Buffalo, N.Y.) can be used. See also, Ducruix & Geige (1992) Crystallization of Nucleic Acids and Proteins: A Practical Approach, IRL Press, Oxford, England, and references cited therein.
V.F. Characterization of the GRP94 LBD Crystal
The following amino acid residues of the GRP94 LBD form the pocket into which NECA binds and forms specific interactions with the protein: Met 85, Glu 103, Leu 104, Asn 107, Ala 108, Asp 110, Ala 111, Val 147, Asp 149, Val 152, Gly 153, Met 154, Glu 158, Lys 161, Asn 162, Leu 163, Ala 167, Lys 168, Thr 171, Gly 196, Val 197, Gly 198, Phe 199, Tyr 200, Val 211, Trp 223, Thr 245, Ile 247.
Water molecules, here given the designation Wat1, Wat2, Wat3, Wat9, Wat10, Wat12, Wat13, Wat17, and Wat23, are also part of the binding pocket and are considered to be key parts of the structure. Thus, an aspect of the present invention is the definition of the protein fold and space (or cavity) occupied by the ligand provided by the crystalline form.
Analysis of the crystalline forms of the present invention suggests that conformational shifts allow NECA binding. The residues in the following two ranges exhibit an en bloc movement, compared to similar residues in yeast Hsp90, and by their alternate conformation create part of the NECA binding pocket that discriminates against other substrates such as ADP. Range 1: Gln 79-Asn 96; Range 2: Gly 164-Gly 198.
The C-terminal amino acids also stabilize the GRP94 LBD. Amino acids Val 330 through Met 336 form a series of hydrogen bonds with amino acids Ile 279 through Lys 285 and stabilize the C-terminus of the GRP94 LBD.
V.G. Generation of Easily-Solved Hsp90 Crystals
The present invention discloses a substantially pure GRP94 LBD polypeptide in crystalline form. In a preferred embodiment, exemplified in the Examples, the GRP94 LBD is crystallized with bound ligand. Crystals can be formed from an Hsp90 or GRP94, or Hsp90 or GRP94 LBD, polypeptide that is usually expressed by a cell culture, such as E. coli. Se-Met substitutions can be introduced during the preparation of the protein from bacterial cultures and can act as heavy atom substitutions in crystals of an Hsp90 or GRP94, or Hsp90 or GRP94 LBD. This method can be advantageous for the phasing of the crystal, which is a crucial, and sometimes limiting, step in solving the three-dimensional structure of a crystallized entity. Thus, the need for generating the heavy metal derivatives traditionally employed in crystallography can be eliminated. After the three-dimensional structure of an Hsp90 or GRP94, or Hsp90 or GRP94 LBD, with or without a ligand bound is determined, the resultant three-dimensional structure can be used in computational methods to design synthetic ligands for an Hsp90 or GRP94, or Hsp90 or GRP94 LBD, polypeptide. Further activity structure relationships can be determined through routine testing, using assays disclosed herein and known in the art.
VI. Design and Development of Hsp90 Protein Modulators
The knowledge of the structure of Hsp90 proteins, an aspect of the present invention, provides a tool for investigating the mechanism of action of Hsp90 proteins in a subject. For example, various computer models, as described herein, can predict the binding of various substrate molecules to Hsp90 proteins. Upon discovering that such binding in fact takes place, knowledge of the protein structure then allows design and synthesis of small molecules that mimic the functional binding of the substrate to the Hsp90 proteins. This is the method of “rational” drug design, further described herein.
Use of the isolated and purified Hsp90 protein structures of the present invention in rational drug design is thus provided in accordance with the present invention. Additional rational drug design techniques are described in U.S. Pat. Nos. 5,834,228; 5,872,011; and 6,136,831.
Thus, in addition to the compounds described herein, other sterically similar compounds can be formulated to mimic the key structural regions of a Hsp90 proteins in general, or of GRP94 or HSP90 in particular. The generation of a structural functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art and described herein. It will be understood that all such sterically similar constructs fall within the scope of the present invention. Programs such as but not limited to RASMOL (Biomolecular Structures Group, Glaxo Wellcome Research & Development Stevenage, Hertfordshire, United Kingdom Version 2.6, August 1995, Version 2.6.4, December 1998, Copyright © Roger Sayle 1992-1999) can be used with the atomic structural coordinates from crystals generated by practicing the invention or used to practice the invention by generating three-dimensional models and/or determining the structures involved in ligand binding. Computer programs such as those sold under the registered trademark INSIGHT II® and such as GRASP (Nicholls et al. (1991) Proteins 11:282) allow for further manipulations and the ability to introduce new structures. In addition, high throughput binding and biological activity assays can be devised using purified recombinant protein and assays discussed herein and known to those of skill in the art in order to refine the activity of a designed ligand.
A method of identifying modulators of the activity of an Hsp90 protein using rational drug design is thus provided in accordance with the present invention. The method comprises designing a potential modulator for an Hsp90 protein of the present invention that will form non-covalent bonds with amino acids in the ligand binding pocket based upon the crystalline structure of the GRP94 LBD polypeptide; synthesizing the modulator; and determining whether the potential modulator modulates the activity of an Hsp90 protein. In a preferred embodiment, the modulator is designed for a GRP94 or HSP90 polypeptide. Modulators can be synthesized using techniques known to those of ordinary skill in the art.
In an alternative embodiment, a method of designing a modulator of a Hsp90 protein in accordance with the present invention comprises: (a) selecting a candidate ligand; (b) determining one or more amino acids of an Hsp90 protein that interact with the ligand using a three-dimensional model of a crystallized GRP94 LBD polypeptide; (c) identifying in a biological assay for Hsp90 protein activity a degree to which the ligand modulates the activity of the Hsp90 protein; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the Hsp90 protein and the ligand is predicted to be modulated by the chemical modification; (e) synthesizing the ligand to form a modified ligand; (f) contacting the modified ligand with the Hsp90 protein; (g) identifying in a biological assay for Hsp90 protein a degree to which the modified ligand modulates the biological activity of the Hsp90 protein; and (h) comparing the biological activity of the Hsp90 protein in the presence of modified ligand with the biological activity of the Hsp90 protein in the presence of the unmodified ligand, whereby a modulator of a Hsp90 protein is designed.
In another embodiment, the present invention also provides methods for the design of modulators that bind at a site other than the ligand binding pocket, also called an accessory binding site.
In accordance with a preferred embodiment of the present invention, the structure coordinates of a crystalline GRP94 LBD polypeptide can be used to design compounds that bind to a Hsp90 protein, more preferably a GRP94 or HSP90 polypeptide, and alter the properties of a Hsp90 protein by competitive inhibition, non-competitive inhibition, agonism, or other mechanism.
A second design approach is to probe a Hsp90 protein (preferably GRP94 or HSP90) crystal with molecules comprising a variety of different chemical entities to determine optimal sites for interaction between candidate Hsp90 protein modulators and the polypeptide. For example, high resolution X-ray diffraction data collected from crystals saturated with solvent allows the determination of the site where each type of solvent molecule adheres. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their capacity to modulate Hsp90 protein activity. Representative approaches are disclosed in PCT International Publication No. WO 99/26966.
Once a computationally-designed ligand is synthesized using the methods of the present invention or other methods known to those of skill in the art, assays can be used to establish its efficacy as a modulator of Hsp90 protein (preferably GRP94 or HSP90) activity. The ligands can be further refined by generating intact Hsp90 protein crystals bound to the synthetic ligand and determining atomic structural data therefrom. As discussed herein the structure of the ligand can then be further modified using methods known to those of skill in the art, in order to improve the modulation activity or the binding affinity of the ligand. This process can lead to second generation ligands with improved properties.
The design of candidate substances, also referred to as “compounds” or “candidate compounds”, that bind to or inhibit activity of an Hsp90 protein according to the present invention generally involves consideration of two factors. First, the compound must be capable of physically and structurally associating with an Hsp90 protein. Non-covalent molecular interactions important in the association of an Hsp90 protein with its substrate include hydrogen bonding, van der Waals interactions and hydrophobic interactions. Second, the compound must be able to assume a conformation that allows it to associate with an Hsp90 protein. Although certain portions of the compound will not directly participate in this association with an Hsp90 protein, those portions can still influence the overall conformation of the molecule. This, in turn, can have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., the ligand binding pocket or an accessory binding site of a Hsp90 protein, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with a Hsp90 protein.
The potential modulatory or binding effect of a chemical compound on an Hsp90 protein can be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques that employ the coordinates of a crystalline Hsp90 protein of the present invention. If the theoretical structure of the given compound suggests insufficient interaction and association with an Hsp90 protein, synthesis and testing of the compound is obviated. However, if computer modeling indicates a strong interaction, the molecule can then be synthesized and tested for its ability to bind and modulate the activity of an Hsp90 protein. In this manner, synthesis of unproductive or inoperative compounds can be minimized.
A modulatory or other binding compound of an Hsp90 protein (preferably GRP94 or HSP90) can be computationally evaluated and designed via a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the individual binding sites or other areas of a crystalline Hsp90 protein of the present invention.
One of several methods can be used to screen chemical entities or fragments for their ability to associate with a Hsp90 protein and, more particularly, with the individual binding sites of a GRP94 or HSP90 polypeptide, such as ligand binding pocket or an accessory binding site. This process can begin by visual inspection of, for example, the ligand binding pocket on a computer screen based on the GRP94 LBD atomic coordinates in Tables 1-2. Selected fragments or chemical entities can then be positioned in a variety of orientations, or docked, within an individual binding site of a Hsp90 protein as defined herein above. Docking can be accomplished using software programs such as those available under the tradenames QUANTATM (Molecular Simulations Inc., San Diego, Calif.) and SYBYLTM (Tripos, Inc., St. Louis, Mo.), followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARM (Brooks et al. (1983) J Comp Chem 8:132) and AMBER 5 (Case et al. (1997) AMBER 5, University of California, San Francisco, Calif.; Pearlman et al. (1995) Comput Phys Commun 91:1-41).
Specialized computer programs can also assist in the process of selecting fragments or chemical entities. These include:
1. GRP94IDTM program, version 17 (Goodford (1985) J Med Chem 28:849-857), which is available from Molecular Discovery Ltd., Oxford, United Kingdom;
2. MCSSTM program (Miranker & Karplus (1991) Proteins 11:29-34), which is available from Molecular Simulations, Inc., San Diego, Calif.;
3. AUTODOCKTM 3.0 program (Goodsell & Olsen (1990) Proteins 8:195-202), which is available from the Scripps Research Institute, La Jolla, Calif.;
4. DOCKTM 4.0 program (Kuntz et al. (1992) J Mol Biol 161:269-288), which is available from the University of California, San Francisco, Calif.;
5. FLEX-XTM program (See Rarey et al. (1996) J Comput Aid Mol Des 10:41-54), which is available from Tripos, Inc., St. Louis, Mo.;
6. MVP program (Lambert (1997) in Practical Application of Computer-Aided Drug Design, Charifson (ed), pp. 243-303, Marcel-Dekker, New York, N.Y.); and
7. LUDITM program (Bohm (1992) J Comput Aid Mol Des 6:61-78), which is available from Molecular Simulations, Inc., San Diego, Calif.
Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or modulator. Assembly can proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of a Hsp90 protein. Manual model building using software such as QUANTATM or SYBYLTM typically follows.
Useful programs to aid one of ordinary skill in the art in connecting the individual chemical entities or fragments include:
1. CAVEATTM program (Bartlett et al. (1989) Special Pub Royal Chem Soc 78: 182-196), which is available from the University of California, Berkeley, Calif.;
2. 3D Database systems, such as MACCS-3DTM system program, which is available from MDL Information Systems, San Leandro, Calif. This area is reviewed in Martin (1992) J Med Chem 35:2145-254; and
3. HOOKTM program (Eisen et al. (1994) Proteins 19:199-221), which is available from Molecular Simulations, Inc., San Diego, Calif.
Instead of proceeding to build a Hsp90 protein modulator (preferably a GRP94 or HSP90 modulator) in a step-wise fashion one fragment or chemical entity at a time as described above, modulatory or other binding compounds can be designed as a whole or de novo using the structural coordinates of a crystalline Hsp90 protein of the present invention and either an empty binding site or optionally including some portion(s) of a known modulator(s). Applicable methods can employ the following software programs:
1. LUDITM program (Bohm, 1992), which is available from Molecular Simulations, Inc., San Diego, Calif.;
2. LEGENDTM program (Nishibata & Itai (1991) Tetrahedron 47:8985); and
3. LEAPFROGTM, which is available from Tripos Associates, St. Louis, Mo.
Other molecular modeling techniques can also be employed in accordance with this invention. See, e.g., Cohen et al. (1990) J Med Chem 33:883-894, Navia & Murcko (1992) Curr Opin Struc Biol 2:202-210, and U.S. Pat. No. 6,008,033.
Once a compound has been designed or selected by the above methods, the efficiency with which that compound can bind to an Hsp90 protein can be tested and optimized by computational evaluation. By way of particular example, a compound that has been designed or selected to function as an Hsp90 protein modulator should also preferably traverse a volume not overlapping that occupied by the binding site when it is bound to a known ligand. Additionally, an effective Hsp90 protein modulator should preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient Hsp90 protein modulators should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, and preferably, not greater than 7 kcal/mole. It is possible for Hsp90 protein modulators to interact with the polypeptide in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free compound and the average energy of the conformations observed when the modulator binds to the polypeptide.
A compound designed or selected as binding to an Hsp90 protein (preferably GRP94 or HSP90) can be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target polypeptide. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the modulator and the polypeptide when the modulator is bound to an Hsp90 protein preferably make a neutral or favorable contribution to the enthalpy of binding.
Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include:
1. Gaussian 98TM, which is available from Gaussian, Inc., Pittsburgh, Pa.;
2. AMBERTM program, version 6.0, which is available from the University of California at San Francisco;
3. QUANTATM program, which is available from Molecular Simulations, Inc., San Diego, Calif.;
4. CHARMm® program, which is available from Molecular Simulations, Inc., San Diego, Calif.; and
5. Insight II® program, which is available from Molecular Simulations, Inc., San Diego, Calif.
These programs can be implemented using a suitable computer system. Other hardware systems and software packages will be apparent to those skilled in the art after review of the disclosure of the present invention.
Once an Hsp90 protein modulator has been optimally selected or designed, as described above, substitutions can then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds can then be analyzed for efficiency of fit to an Hsp90 protein binding site using the same computer-based approaches described in detail above.
VII. Additional Screening Methods Using a Crystalline Hsp90 Protein
The present invention further provides methods for identifying substances that modulate a Hsp90 protein wherein such methods employ a crystalline Hsp90 protein as disclosed herein.
VII.A. Method for Identifying Compounds that Stimulate Hsp90 Activity
In a cell-free system, the method comprises the steps of establishing a control system comprising a crystalline Hsp90 protein and a ligand which is capable of binding to the polypeptide; establishing a test system comprising a crystalline Hsp90 protein, the ligand, and a candidate compound; and determining whether the candidate compound binds the polypeptide by comparison of the test and control systems. A representative ligand comprises NECA, a substituted adenosine molecule, or a relevant mimetic as obtained through combinatorial chemistry. Thus, in this embodiment, the property screened includes binding affinity.
In another embodiment of the invention, a crystalline form of a Hsp90 protein or a catalytic or immunogenic fragment or oligopeptide thereof, can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such a screening can be affixed to a solid support. The formation of binding complexes, between a crystalline Hsp90 protein and the agent being tested, will be detected. In a preferred embodiment, the crystalline Hsp90 protein is a crystalline GRP94 LBD polypeptide.
Another technique for drug screening which can be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in International Publication No. WO 84/03564. According to this method, as applied to a crystalline polypeptide of the present invention, a multiplicity of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with the crystalline polypeptide, or fragments thereof. Bound polypeptide is then detected by methods known to those of skill in the art. The crystalline polypeptide can also be placed directly onto plates for use in the aforementioned drug screening techniques.
In yet another embodiment, a method of screening for a modulator of a GRP94 or HSP90 polypeptide comprises: providing a library of test samples; contacting a crystalline form of a GRP94 LBD polypeptide with each test sample; detecting an interaction between a test sample and a crystalline form of a GRP94 LBD polypeptide; identifying a test sample that interacts with a crystalline form of a GRP94 LBD polypeptide; and isolating a test sample that interacts with a crystalline form of a GRP94 LBD polypeptide.
In accordance with the present invention there is also provided a rapid and high throughput screening method that relies on the methods described above. This screening method comprises separately contacting each of a plurality of samples with a crystalline form of a GRP94 LBD polypeptide and detecting a resulting binding complex. In such a screening method, the plurality of samples preferably comprises more than about 104 samples, or more preferably comprises more than about 5×104 samples.
In each of the foregoing embodiments, an interaction can be detected spectrophotometrically, radiologically, or immunologically. An interaction between a crystalline form of a GRP94 LBD polypeptide and a test sample can also be quantified using methodology known to those of skill in the art. Finally, each of the foregoing embodiments can also find application in the screening of non-crystalline forms of an Hsp90 polypeptide, such as in the screening of a computationally designed modulator for the desired effect on the biological activity of the Hsp90 polypeptide, including a GRP94 LBD polypeptide of the present invention. Other screening methods pertaining to the biological activity of an Hsp90 protein and employing a GRP94 polypeptide are disclosed herein below.
VII.B. Method for Identifying Compounds that Inhibit Hsp90 Activity
The present invention further discloses an assay method for identifying a compound that inhibits Hsp90 protein transition to or stability of an active conformation. A ligand of a Hsp90 protein, for example NECA, a substituted adenosine molecule, related purine nucleoside derivatives, a relevant mimetic as obtained through combinatorial chemistry and/or those compounds bearing structural similarities to the natural product compounds geldanamycin and radicicol which bind the GRP94 NBD and inhibit GRP94 function, can be used in the assay method as the ligand against which the inhibition by a test compound is gauged. The method comprises (a) incubating a Hsp90 protein with a ligand in the presence of a test inhibitor compound; (b) determining an amount of ligand that is bound to the Hsp90 protein, wherein decreased binding of ligand to the Hsp90 protein in the presence of the test inhibitor compound relative to binding in the absence of the test inhibitor compound is indicative of inhibition; and (c) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed.
In another aspect of the present invention, the disclosed assay method can be used in the structural refinement of candidate Hsp90 ligands. For example, multiple rounds of optimization can be followed by incremental structural changes in a strategy of inhibitor design. A strategy such as this is made possible by the disclosure of the atomic coordinates of the GRP94 LBD polypeptide.
VIII. Design, Preparation, and Structural Analysis of Related Hsp90 Proteins
VII.A. Hsp90 Mutants
The present invention provides for the generation of Hsp90 mutants (preferably GRP94 or HSP90 mutants), and the ability to solve the crystal structures of Hsp90 mutant polypeptides that can be crystallized. More particularly, through the provision of the three-dimensional structure of GRP94 and HSP90 polypeptides, desirable sites for mutation can be identified.
The structure coordinates of the GRP94 LBD polypeptide provided in accordance with the present invention also facilitate the identification and characterization of other proteins that are analogous to Hsp90 proteins in function, structure or both. This homology-based understanding can contribute to the development of related therapeutic compositions and methods.
VIII.B. Hsp90 Protein Equivalents
A further aspect of the present invention is that sterically similar compounds can be formulated to mimic the key portions of a Hsp90 protein structure. Such compounds are functional equivalents. The generation of a structural functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art and described herein. Modeling and chemical design of Hsp90 protein structural equivalents can be based on the structure coordinates of a crystalline GRP94 LBD polypeptide of the present invention. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
VIII.C. Solving Crystalline Structures of Related Hsp90 Proteins
Because polypeptides can crystallize in more than one crystal form, the structural coordinates of a GRP94 LBD polypeptide, or portions thereof, as provided by the present invention, are particularly useful in solving the structure of other crystal forms of GRP94, HSP90, and the crystalline forms of other Hsp90 proteins. The coordinates provided in the present invention can also be used to solve the structure of Hsp90 protein mutants, Hsp90 protein co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to a functional domain of a Hsp90 protein.
One method that can be employed for the purpose of solving additional Hsp90 crystal structures is molecular replacement. See generally, Rossmann, ed, (1972) The Molecular Replacement Method, Gordon & Breach, New York, N.Y. In the molecular replacement method, the unknown crystal structure, whether it is another crystal form of a GRP94 or HSP90 polypeptide, (i.e. a GRP94 or HSP90 mutant), or a GRP94 or HSP90 polypeptide complexed with another compound (a “co-complex”), or the crystal of some other protein with significant amino acid sequence homology to any functional region of a GRP94 or HSP90 polypeptide, can be determined using the GRP94 and HSP90 structure coordinates provided in Tables 1-2. This method provides an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
In addition, in accordance with this invention, GRP94 or HSP90 mutants can be crystallized in complex with known modulators. The crystal structures of a series of such complexes can then be solved by molecular replacement and compared with that of wild type GRP94 or HSP90. Potential sites for modification within the various binding sites of the enzyme can thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between a wild type GRP94 or HSP90 polypeptide and a ligand when compared to the interaction between a mutant GRP94 or HSP90 polypeptide and a ligand.
All of the complexes referred to in the present disclosure can be studied using X-ray diffraction techniques. See, e.g., Blundell & Johnson (1985) Method Enzymol 114A & 115B and can be refined using computer software, such as the X-PLORTM program (Brünger, 1992). This information is thus used to optimize known classes of Hsp90 protein modulators, and more importantly, to design and synthesize novel classes of Hsp90 protein modulators.
IX. Design, Preparation and Structural Analysis of GRP94 and GRP94 LBD Polypeptides and Structural Equivalents
The present invention also provides novel purified and isolated Hsp90 and GRP94 polypeptides, Hsp90 and GRP94 LBD polypeptides, and mutants and structural equivalents thereof (preferably GRP94 and GRP94 LBD mutants), and the ability to solve the crystal structures of those that crystallize. Thus, an aspect of the present invention involves the production of a recombinant protein for, among other things, the purpose of crystallization, characterization of biologically relevant protein-protein interactions, and compound screening assays, or for the production of a recombinant protein having other desirable characteristic(s). Polypeptide products produced by the methods of the present invention are also disclosed herein.
The structure coordinates of a Hsp90 or GRP94, or Hsp90 or GRP94 LBD provided in accordance with the present invention also facilitate the identification of related proteins or enzymes analogous to GRP94 in function, structure or both, (for example, a GRP94 that can lead to novel therapeutic modes for treating or preventing a range of disease states).
IX.A. GRP94 Nucleic Acids
The nucleic acid molecules provided by the present invention include the isolated nucleic acid molecules of any one of SEQ ID NOs:1, 3, or 5, sequences substantially similar to sequences of any one of SEQ ID NOs: 1, 3, or 5, conservative variants thereof, subsequences and elongated sequences thereof, complementary DNA molecules, and corresponding RNA molecules. The present invention also encompasses genes, cDNAs, chimeric genes, and vectors comprising disclosed GRP94 nucleic acid sequences. In a preferred embodiment, a nucleic acid molecule of the present invention encodes a GRP94 LBD polypeptide. Thus, in a more preferred embodiment, a nucleic acid molecule of the present invention is set forth as SEQ ID NO: 3 or 5.
The term “nucleic acid molecule” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference natural nucleic acid. Unless otherwise indicated, a particular nucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions), complementary sequences, subsequences, elongated sequences, as well as the sequence explicitly indicated. The terms “nucleic acid molecule” or “nucleotide sequence” can also be used in place of “gene”, “cDNA”, or “mRNA”. Nucleic acids can be derived from any source, including any organism.
The term “isolated”, as used in the context of a nucleic acid molecule, indicates that the nucleic acid molecule exists apart from its native environment and is not a product of nature. An isolated DNA molecule can exist in a purified form or can exist in a non-native environment such as a transgenic host cell.
The term “purified”, when applied to a nucleic acid, denotes that the nucleic acid is essentially free of other cellular components with which it is associated in the natural state. Preferably, a purified nucleic acid molecule is a homogeneous dry or aqueous solution. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.
The term “substantially identical”, the context of two nucleotide or amino acid sequences, can also be defined as two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90-95%, and most preferably at least 99% nucleotide or amino acid sequence identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms (described herein below) or by visual inspection. Preferably, the substantial identity exists in nucleotide sequences of at least 50 residues, more preferably in nucleotide sequence of at least about 100 residues, more preferably in nucleotide sequences of at least about 150 residues, and most preferably in nucleotide sequences comprising complete coding sequences. In one aspect, polymorphic sequences can be substantially identical sequences. The term “polymorphic” refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. An allelic difference can be as small as one base pair.
Another indication that two nucleotide sequences are substantially identical is that the two molecules specifically or substantially hybridize to each other under stringent conditions. In the context of nucleic acid hybridization, two nucleic acid sequences being compared can be designated a “probe” and a “target”. A “probe” is a reference nucleic acid molecule, and a “target” is a test nucleic acid molecule, often found within a heterogenous population of nucleic acid molecules. A “target sequence” is synonymous with a “test sequence”.
A preferred nucleotide sequence employed for hybridization studies or assays includes probe sequences that are complementary to or mimic at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule of the present invention. Preferably, probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of any of those set forth as SEQ ID NOs: 1, 3, or 5. Such fragments can be readily prepared by, for example, directly synthesizing the fragment by chemical synthesis, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production.
The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA). The phrase “binds substantially to” refers to complementary hybridization between a probe nucleic acid molecule and a target nucleic acid molecule and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired hybridization.
“Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern blot analysis are both sequence- and environment-dependent. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, Elsevier, New York, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize specifically to its target subsequence, but to no other sequences.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for Southern or Northern Blot analysis of complementary nucleic acids having more than about 100 complementary residues is overnight hybridization in 50% formamide with 1 mg of heparin at 42° C. An example of highly stringent wash conditions is 15 minutes in 0.1 5 M NaCl at 65° C. An example of stringent wash conditions is 15 minutes in 0.2×SSC buffer at 65° C. (See Sambrook et al. eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of medium stringency wash conditions for a duplex of more than about 100 nucleotides, is 15 minutes in 1×SSC at 45° C. An example of low stringency wash for a duplex of more than about 100 nucleotides, is 15 minutes in 4-6×SSC at 40° C. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0-8.3, and the temperature is typically at least about 30° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2-fold (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
The following are examples of hybridization and wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a probe nucleotide sequence preferably hybridizes to a target nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. followed by washing in 2×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. followed by washing in 1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. followed by washing in 0.5×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at 65° C.
A further indication that two nucleic acid sequences are substantially identical is that proteins encoded by the nucleic acids are substantially identical, share an overall three-dimensional structure, are biologically functional equivalents, or are immunologically cross-reactive. These terms are defined further under the heading GRP94 Polypeptides herein below. Nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially identical if the corresponding proteins are substantially identical. This can occur, for example, when two nucleotide sequences are significantly degenerate as permitted by the genetic code.
The term “conservatively substituted variants” refers to nucleic acid sequences having degenerate codon substitutions wherein the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acids Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Rossolini et al. (1994) Mol Cell Probes 8:91-98).
The term “subsequence” refers to a sequence of nucleic acids that comprises a part of a longer nucleic acid sequence. An exemplary subsequence is a probe, described herein above, or a primer. The term “primer” as used herein refers to a contiguous sequence comprising about 8 or more deoxyribonucleotides or ribonucleotides, preferably 10-20 nucleotides, and more preferably 20-30 nucleotides of a selected nucleic acid molecule. The primers of the invention encompass oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a nucleic acid molecule of the present invention.
The term “elongated sequence” refers to an addition of nucleotides (or other analogous molecules) incorporated into the nucleic acid. For example, a polymerase (e.g., a DNA polymerase), e.g., a polymerase that adds sequences at the 3′ terminus of the nucleic acid molecule can be used to provide an elongated sequence. In addition, the nucleotide sequence can be combined with other DNA sequences, such as promoters, promoter regions, enhancers, polyadenylation signals, intronic sequences, additional restriction enzyme sites, multiple cloning sites, and other coding segments.
The term “complementary sequence”, as used herein, indicates two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between base pairs. As used herein, the term “complementary sequences” means nucleotide sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein. A particular example of a complementary nucleic acid segment is an antisense oligonucleotide.
The term “gene” refers broadly to any segment of DNA associated with a biological function. A gene encompasses sequences including but not limited to a coding sequence, a promoter region, a cis-regulatory sequence, a non-expressed DNA segment is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof. A gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation of an existing sequence.
The term “gene expression” generally refers to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence.
The present invention also encompasses chimeric genes comprising the disclosed GRP94 sequences. The term “chimeric gene”, as used herein, refers to a promoter region operably linked to a GRP94 coding sequence, a nucleotide sequence producing an antisense RNA molecule, a RNA molecule having tertiary structure, such as a hairpin structure, or a double-stranded RNA molecule.
The term “operably linked”, as used herein, refers to a promoter region that is connected to a nucleotide sequence in such a way that the transcription of that nucleotide sequence is controlled and regulated by that promoter region. Techniques for operatively linking a promoter region to a nucleotide sequence are well known in the art.
The terms “heterologous gene”, “heterologous DNA sequence”, “heterologous nucleotide sequence”, “exogenous nucleic acid molecule”, or “exogenous DNA segment”, as used herein, each refer to a sequence that originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example by mutagenesis or by isolation from native cis-regulatory sequences. The terms also include non-naturally occurring multiple copies of a naturally occurring nucleotide sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid wherein the element is not ordinarily found.
The term “promoter region” defines a nucleotide sequence within a gene that is positioned 5′ to a coding sequence of a same gene and functions to direct transcription of the coding sequence. The promoter region includes a transcriptional start site and at least one cis-regulatory element. The present invention encompasses nucleic acid sequences that comprise a promoter region of a GRP94 gene, or functional portion thereof.
The term “cis-acting regulatory sequence” or “cis-regulatory motif” or “response element”, as used herein, each refer to a nucleotide sequence that enables responsiveness to a regulatory transcription factor. Responsiveness can encompass a decrease or an increase in transcriptional output and is mediated by binding of the transcription factor to the DNA molecule comprising the response element.
As used herein, the term “transcription” means a cellular process involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene. The process includes, but is not limited to the following steps: (a) the transcription initiation, (b) transcript elongation, (c) transcript splicing, (d) transcript capping, (e) transcript termination, (f) transcript polyadenylation, (g) nuclear export of the transcript, (h) transcript editing, and (i) stabilizing the transcript.
The term “transcription factor” generally refers to a protein that modulates gene expression by interaction with the cis-regulatory element and cellular components for transcription, including RNA Polymerase, Transcription Associated Factors (TAFs), chromatin-remodeling proteins, and any other relevant protein that impacts gene transcription.
A “functional portion” of a promoter gene fragment is a nucleotide sequence within a promoter region that is required for normal gene transcription. To determine nucleotide sequences that are functional, the expression of a reporter gene is assayed when variably placed under the direction of a promoter region fragment.
Promoter region fragments can be conveniently made by enzymatic digestion of a larger fragment using restriction endonucleases or DNAse I. Preferably, a functional promoter region fragment comprises about 5000 nucleotides, more preferably 2000 nucleotides, more preferably about 1000 nucleotides. Even more preferably a functional promoter region fragment comprises about 500 nucleotides, even more preferably a functional promoter region fragment comprises about 100 nucleotides, and even more preferably a functional promoter region fragment comprises about 20 nucleotides.
The terms “reporter gene” or “marker gene” or “selectable marker” each refer to a heterologous gene encoding a product that is readily observed and/or quantitated. A reporter gene is heterologous in that it originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form. Non-limiting examples of detectable reporter genes that can be operably linked to a transcriptional regulatory region can be found in Alam & Cook (1990) Anal Biochem 188:245-254 and PCT International Publication No. WO 97/47763. Preferred reporter genes for transcriptional analyses include the lacZ gene (See, e.g., Rose & Botstein (1983) Meth Enzymol 101:167-180), Green Fluorescent Protein (GFP) (Cubitt et al. (1995) Trends Biochem Sci 20:448-455), luciferase, or chloramphenicol acetyl transferase (CAT). Preferred reporter genes for methods to produce transgenic animals include but are not limited to antibiotic resistance genes, and more preferably the antibiotic resistance gene confers neomycin resistance. Any suitable reporter and detection method can be used, and it will be appreciated by one of skill in the art that no particular choice is essential to or a limitation of the present invention.
An amount of reporter gene can be assayed by any method for qualitatively or preferably, quantitatively determining presence or activity of the reporter gene product. The amount of reporter gene expression directed by each test promoter region fragment is compared to an amount of reporter gene expression to a control construct comprising the reporter gene in the absence of a promoter region fragment. A promoter region fragment is identified as having promoter activity when there is significant increase in an amount of reporter gene expression in a test construct as compared to a control construct. The term “significant increase”, as used herein, refers to an quantified change in a measurable quality that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater relative to a control measurement, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.
The present invention further includes vectors comprising the disclosed GRP94 sequences, including plasmids, cosmids, and viral vectors. The term “vector”, as used herein refers to a DNA molecule having sequences that enable its replication in a compatible host cell. A vector also includes nucleotide sequences to permit ligation of nucleotide sequences within the vector, wherein such nucleotide sequences are also replicated in a compatible host cell. A vector can also mediate recombinant production of a GRP94 polypeptide, as described further herein below. Preferred vectors are listed above under the heading Production of Hsp90 Polypeptide and also include but are not limited to pBluescript (Stratagene), pUC18, pBLCAT3 (Luckow & Schutz (1987) Nucleic Acids Res 15:5490), pLNTK (Gorman et al. (1996) Immunity 5:241-252), and pBAD/gIII (Stratagene). A preferred host cell is a mammalian cell; more preferably the cell is a Chinese hamster ovary cell, a HeLa cell, a baby hamster kidney cell, or a mouse cell; even more preferably the cell is a human cell.
Nucleic acids of the present invention can be cloned, synthesized, recombinantly altered, mutagenized, or combinations thereof. Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are well known in the art. Exemplary, non-limiting methods are described by Sambrook et al., eds. (1989); by Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; by Ausubel et al. (1992) Current Protocols in Molecular Biology, John Wylie and Sons, Inc., New York, N.Y.; and by Glover, ed. (1985) DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, United Kingdom. Site-specific mutagenesis to create base pair changes, deletions, or small insertions are also well known in the art as exemplified by publications, see, e.g., Adelman et al., (1983) DNA 2:183; Sambrook et al. (1989).
Sequences detected by methods of the invention can be detected, subcloned, sequenced, and further evaluated by any measure well known in the art using any method usually applied to the detection of a specific DNA sequence including but not limited to dideoxy sequencing, PCR, oligomer restriction (Saiki et al. (1985) Bio/Technology 3:1008-1012), allele-specific oligonucleotide (ASO) probe analysis (Conner et al. (1983) Proc Natl Acad Sci USA 80:278), and oligonucleotide ligation assays (OLAs) (Landgren et. al. (1988) Science 241:1007). Molecular techniques for DNA analysis have been reviewed (Landgren et. al. (1988) Science 242:229-237).
IX.B. GRP94 Polypeptides
The polypeptides provided by the present invention include the isolated polypeptides set forth as SEQ ID NOs:2, 4 or 6, polypeptides substantially identical to SEQ ID NOs:2, 4 or 6, GRP94 polypeptide fragments, fusion proteins comprising GRP94 amino acid sequences, biologically functional analogs, and polypeptides that cross-react with an antibody that specifically recognizes a GRP94 polypeptide. In a preferred embodiment the GRP94 polypeptide is a GRP94 LBD polypeptide. Thus, in a more preferred embodiment, a GRP94LBD comprises the amino acid sequence of any of SEQ ID NOS:4 or 6.
The term “isolated”, as used in the context of a polypeptide, indicates that the polypeptide exists apart from its native environment and is not a product of nature. An isolated polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.
The term “purified”, when applied to a polypeptide, denotes that the polypeptide is essentially free of other cellular components with which it is associated in the natural state. Preferably, a polypeptide is a homogeneous solid or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. The term “purified” denotes that a polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polypeptide is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.
The term “substantially identical” in the context of two or more polypeptides sequences is measured by (a) polypeptide sequences having about 35%, or 45%, or preferably from 45-55%, or more preferably 55-65%, or most preferably 65% or greater amino acids that are identical or functionally equivalent. Percent “identity” and methods for determining identity are defined herein below.
Substantially identical polypeptides also encompass two or more polypeptides sharing a conserved three-dimensional structure. Computational methods can be used to compare structural representations, and structural models can be generated and easily tuned to identify similarities around important active sites or ligand binding sites. See Henikoff et al. (2000) Electrophoresis 21(9):1700-1706; Huang et al. (2000) Pac Symp Biocomput 230-241; Saqi et al. (1999) Bioinformatics 15(6):521-522; and Barton (1998) Acta Crystallogr D Biol Crystallogr 54:1139-1146.
The term “functionally equivalent” in the context of amino acid sequences is well known in the art and is based on the relative similarity of the amino acid side-chain substituents. See Henikoff & Henikoff (2000) Adv Protein Chem 54:73-97. Relevant factors for consideration include side-chain hydrophobicity, hydrophilicity, charge, and size. For example, arginine, lysine, and histidine are all positively charged residues; that alanine, glycine, and serine are all of similar size; and that phenylalanine, tryptophan, and tyrosine all have a generally similar shape. By this analysis, described further herein below, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine; are defined herein as biologically functional equivalents.
In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al. (1982) J Mol Biol 157:105.). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 of the original value is preferred, those which are within ±1 of the original value are particularly preferred, and those within ±0.5 of the original value are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 of the original value is preferred, those which are within ±1 of the original value are particularly preferred, and those within ±0.5 of the original value are even more particularly preferred.
The present invention also encompasses GRP94 polypeptide fragments or functional portions of a GRP94 polypeptide. Such functional portion need not comprise all or substantially all of the amino acid sequence of a native GRP94 gene product. The term “functional” includes any biological activity or feature of GRP94, including immunogenicity. Preferred embodiments include a GRP94 LBD and a fragment thereof defining the ligand binding pocket.
The present invention also includes longer sequences of a GRP94 polypeptide, or portion thereof. For example, one or more amino acids can be added to the N-terminus or C-terminus of a GRP94 polypeptide. Fusion proteins comprising GRP94 polypeptide sequences are also provided within the scope of the present invention. Methods of preparing such proteins are known in the art.
The present invention also encompasses functional analogs of a GRP94 polypeptide. Functional analogs share at least one biological function with a GRP94 polypeptide. An exemplary function is immunogenicity. In the context of amino acid sequence, biologically functional analogs, as used herein, are peptides in which certain, but not most or all, of the amino acids can be substituted. Functional analogs can be created at the level of the corresponding nucleic acid molecule, altering such sequence to encode desired amino acid changes. In one embodiment, changes can be introduced to improve the antigenicity of the protein. In another embodiment, a GRP94 polypeptide sequence is varied so as to assess the activity of a mutant GRP94 polypeptide.
The present invention also encompasses recombinant production of the disclosed GRP94 polypeptides. Briefly, a nucleic acid sequence encoding a GRP94 polypeptide, or portion thereof, is cloned into a expression cassette, the cassette is introduced into a host organism, where it is recombinantly produced.
The term “expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest can be chimeric. The expression cassette can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. Exemplary promoters include Simian virus 40 early promoter, a long terminal repeat promoter from retrovirus, an action promoter, a heat shock promoter, and a metallothien protein. In the case of a multicellular organism, the promoter and promoter region can direct expression to a particular tissue or organ or stage of development. Exemplary tissue-specific promoter regions include a GRP94 promoter, described herein. Suitable expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus, yeast vectors, bacteriophage vectors (e.g., lambda phage), and plasmid and cosmids DNA vectors.
The term “host cell”, as used herein, refers to a cell into which a heterologous nucleic acid molecule has been introduced. Transformed cells, tissues, or organisms are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
A host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. For example, different host cells have characteristic and specific mechanisms for the translational and post-transactional processing and modification (e.g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. Expression in a bacterial system can be used to produce a non-glycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in animal cells can be used to ensure “native” glycosylation of a heterologous protein.
Expression constructs are transfected into a host cell by any standard method, including electroporation, calcium phosphate precipitation, DEAE-Dextran transfection, liposome-mediated transfection, and infection using a retrovirus. The GRP94-encoding nucleotide sequence carried in the expression construct can be stably integrated into the genome of the host or it can be present as an extrachromosomal molecule.
Isolated polypeptides and recombinantly produced polypeptides can be purified and characterized using a variety of standard techniques that are well known to the skilled artisan. See, e.g. Ausubel et al. (1992), Bodanszky, et al. (1976) Peptide Synthesis, John Wiley and Sons, Second Edition, New York, N.Y. and Zimmer et al. (1993) Peptides, pp. 393-394, ESCOM Science Publishers, B. V.
IX.C. Nucleotide and Amino Acid Sequence Comparisons
The terms “identical” or percent “identity” in the context of two or more nucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms disclosed herein or by visual inspection.
The term “substantially identical” in regards to a nucleotide or polypeptide sequence means that a particular sequence varies from the sequence of a naturally occurring sequence by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the natural gene, gene product, or sequence. Such sequences include “mutant” sequences, or sequences wherein the biological activity is altered to some degree but retains at least some of the original biological activity. The term “naturally occurring”, as used herein, is used to describe a composition that can be found in nature as distinct from being artificially produced by man. For example, a protein or nucleotide sequence present in an organism, which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer program, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are selected. The sequence comparison algorithm then calculates the percent sequence identity for the designated test sequence(s) relative to the reference sequence, based on the selected program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman (1981) Adv Appl Math 2:482, by the homology alignment algorithm of Needleman & Wunsch (1970) J Mol Biol 48:443, by the search for similarity method of Pearson & Lipman (1988) Proc Natl Acad Sci USA 85:2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.), or by visual inspection. See generally, Ausubel et al., 1992.
A preferred algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J Mol Biol 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information <www.ncbi.nlm.nih.gov/>. This algorithm involves first identifying high scoring sequence pairs by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer high scoring sequence pairs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength W=11, an expectation E=10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See Henikoff & Henikoff (1989) Proc Natl Aced Sci USA 89:10915.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See, e.g., Karlin and Altschul (1993) Proc Natl Aced Sci USA 90:5873-5887. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
IX.D. Hsp90 and GRP94 Mutant Polypeptides
The generation of chimeric GRP94 polypeptides is also an aspect of the present invention. Such a chimeric polypeptide can comprise a GRP94, or a GRP94 LBD polypeptide or a portion of a GRP94 or a GRP94 LBD, (e.g. a binding pocket of a GRP94 LBD) that is fused to a candidate polypeptide or a suitable region of the candidate polypeptide. Throughout the present disclosure it is intended that the term “mutant” encompass not only mutants of a polypeptide but chimeric proteins generated using a GRP94 or a GRP94 LBD, as well. It is thus intended that the following discussion of a mutant GRP94 or GRP94 LBD apply mutatis mutandis to chimeric GRP94 and GRP94 LBD polypeptides and to structural equivalents thereof.
In accordance with the present invention, a mutation can be directed to a particular site or combination of sites of a wild-type GRP94 or GRP94 LBD polypeptide. For example, an accessory binding site or the binding pocket can be chosen for mutagenesis. Similarly, a residue having a location on, at or near the surface of the polypeptide can be replaced, resulting in an altered surface charge of one or more charge units, as compared to the wild-type GRP94 or GRP94 LBD polypeptide. Alternatively, an amino acid residue in a GRP94 or GRP94 LBD polypeptide can be chosen for replacement based on its hydrophilic or hydrophobic characteristics.
Such mutants can be characterized by any one of several different properties, i.e. a “desired” or “predetermined” characteristic as compared with the wild type GRP94 or a GRP94 LBD polypeptide. For example, such mutants can have an altered surface charge of one or more charge units, or can have an increase in overall stability. Other mutants can have altered substrate specificity in comparison with, or a higher specific activity than, a wild-type GRP94 or a GRP94 LBD polypeptide.
GRP94 or GRP94 LBD polypeptide mutants of the present invention can be generated in a number of ways. For example, the wild-type sequence of a GRP94 or a GRP94 LBD polypeptide can be mutated at those sites identified using this invention as desirable for mutation, by the approach of oligonucleotide-directed mutagenesis or other conventional methods, such as deletion. Alternatively, mutants of a GRP94 or a GRP94 LBD polypeptide can be generated by the site-specific replacement of a particular amino acid with an unnaturally occurring amino acid. In addition, GRP94 or GRP94 LBD polypeptide mutants can be generated through replacement of an amino acid residue, for example, a particular cysteine or methionine residue, with selenocysteine or selenomethionine. This can be achieved by growing a host organism capable of expressing either the wild-type or mutant polypeptide on a growth medium depleted of either natural cysteine or methionine (or both) but enriched in selenocysteine or selenomethionine (or both).
Mutations can be introduced into a DNA sequence coding for a GRP94 or GRP94 LBD polypeptide using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites. Mutations can be generated in the full-length DNA sequence of a GRP94 or a GRP94 LBD polypeptide or in any sequence coding for polypeptide fragments of a GRP94 or a GRP94 LBD polypeptide.
According to the present invention, a mutated GRP94 or a GRP94 LBD polypeptide-encoding DNA sequence produced by the methods described above, or any alternative methods known in the art, can be expressed using an expression vector in accordance with techniques disclosed herein above. Subsequently, the polypeptide can be purified and/or crystallized in accordance with techniques disclosed herein.
Once a mutation(s) has been generated in the desired location, such as an active site, the mutants can be tested for any one of several properties of interest, i.e. “desired” or “predetermined” positions. For example, mutants can be screened for an altered charge at physiological pH. This property can be determined by measuring the mutant polypeptide isoelectric point (pI) and comparing the observed value with that of the wild-type parent. Isoelectric point can be measured by gel-electrophoresis according to the method of Wellner (Wellner, (1971) Anal. Chem. 43: 597). A mutant polypeptide containing a replacement amino acid located at the surface of the enzyme, as provided by the structural information of this invention, can lead to an altered surface charge and an altered pI.
IX.E. Antibodies to a GRP94 Polypeptide of the Present Invention
The present invention also provides an antibody that specifically binds a GRP94 or a GRP94 LBD polypeptide and methods to generate same. The term “antibody” indicates an immunoglobulin protein, or functional portion thereof, including a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, Fab fragments, and a Fab expression library. “Functional portion” refers to the part of the protein that binds a molecule of interest. In a preferred embodiment, an antibody of the invention is a monoclonal antibody. Techniques for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). A monoclonal antibody of the present invention can be readily prepared through use of well-known techniques such as the hybridoma techniques exemplified in U.S. Pat. No. 4,196,265 and the phage-displayed techniques disclosed in U.S. Pat. No. 5,260,203.
The phrase “specifically (or selectively) binds to an antibody”, or “specifically (or selectively) immunoreactive with”, when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological materials. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not show significant binding to other proteins present in the sample. Specific binding to an antibody under such conditions can require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to a protein with an amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with unrelated proteins.
The use of a molecular cloning approach to generate antibodies, particularly monoclonal antibodies, and more particularly single chain monoclonal antibodies, are also provided. The production of single chain antibodies has been described in the art. See, e.g., U.S. Pat. No. 5,260,203. For this approach, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning on endothelial tissue. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by heavy (H) and light (L) chain combinations in a single chain, which further increases the chance of finding appropriate antibodies. Thus, an antibody of the present invention, or a “derivative” of an antibody of the present invention, pertains to a single polypeptide chain binding molecule which has binding specificity and affinity substantially similar to the binding specificity and affinity of the light and heavy chain aggregate variable region of an antibody described herein.
The term “immunochemical reaction”, as used herein, refers to any of a variety of immunoassay formats used to detect antibodies specifically bound to a particular protein, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. See Harlow & Lane (1988) for a description of immunoassay formats and conditions.
IX.F. Methods for Detecting a GRP94 or a GRP94 LBD Polypeptide or for Detecting a Nucleic Acid Molecule Encoding the Same
In another aspect of the invention, a method is provided for detecting a level of a GRP94 or a GRP94 LBD polypeptide using an antibody that specifically recognizes a GRP94 or a GRP94 LBD polypeptide, or portion thereof. In a preferred embodiment, biological samples from an experimental subject and a control subject are obtained, and a GRP94 or GRP94 LBD polypeptide is detected in each sample by immunochemical reaction with the antibody. More preferably, the antibody recognizes amino acids of any one of SEQ ID NOs:2, 4 and 6, and is prepared according to a method of the present invention for producing such an antibody.
In one embodiment, an antibody is used to screen a biological sample for the presence of a GRP94 or a GRP94 LBD polypeptide. A biological sample to be screened can be a biological fluid such as extracellular or intracellular fluid, or a cell or tissue extract or homogenate. A biological sample can also be an isolated cell (e.g., in culture) or a collection of cells such as in a tissue sample or histology sample. A tissue sample can be suspended in a liquid medium or fixed onto a solid support such as a microscope slide. In accordance with a screening assay method, a biological sample is exposed to an antibody immunoreactive with a GRP94 or a GRP94 LBD polypeptide whose presence is being assayed, and the formation of antibody-polypeptide complexes is detected. Techniques for detecting such antibody-antigen conjugates or complexes are well known in the art and include but are not limited to centrifugation, affinity chromatography and the like, and binding of a labeled secondary antibody to the antibody-antigen complex.
In another aspect of the invention, a method is provided for detecting a nucleic acid molecule that encodes a GRP94 or a GRP94 LBD polypeptide. According to the method, a biological sample having nucleic acid material is procured and hybridized under stringent hybridization conditions to a GRP94 or a GRP94 LBD polypeptide-encoding nucleic acid molecule of the present invention. Such hybridization enables a nucleic acid molecule of the biological sample and a GRP94 or a GRP94 LBD polypeptide-encoding nucleic acid molecule to form a detectable duplex structure. Preferably, the GRP94 or a GRP94 LBD polypeptide-encoding nucleic acid molecule includes some or all nucleotides of any one of SEQ ID NOs:3 or 5. Also preferably, the biological sample comprises human nucleic acid material.
X. Ligand Compositions
In one embodiment the present invention pertains to a composition of matter that acts as a ligand for GRP94. Such a ligand can be identified using the methods disclosed herein and can also be used in the preparation of a crystalline structure of the present invention. The ligand can comprise a purified and isolated natural ligand for GRP94, or can comprise a synthetic compound, such as are identified by the screening and rational drug design techniques disclosed herein. Preferably, the ligand is a small molecule mimetic. More preferably, the ligand has activity in the modulation of GRP94 biological activity. Thus, ligands having such activity are also referred to herein as “modulators”. Representative ligand compositions are preferably about 500-1000 daltons, polycyclic molecules that can show structural resemblance to radicicol, geldanamycin, or adenosine derivatives. Optionally, a ligand is hydrophobic.
A representative ligand or modulator composition of matter comprises an adenosine moiety or structural mimetic thereof having any of a variety of substitutions at the 2′, 3′, and 5′ positions, in the case of adenosine, as deemed appropriate by high resolution structural analyses of ligand-GRP94 interactions. Optionally, 5′ position alkyl extensions can be included, preferably as a carboxamido linkage to the parent adenosine and, to facilitate stable chemical linkage to a solid support for the purposes of affinity-based purification, terminating in any of a subset of chemically reactive groups including, but not limited to vinyl, maleimide and/or succinimide esters, or substituents suitable for chemical coupling to solid phase supports, such as amino or sulphydryl groups. The composition acts as a ligand for GRP94 and has application in the purification, screening and therapeutic methods disclosed herein.
Additional ligands can be identified through combinatorial chemistry of a parent precursor molecule bearing a hydrogen bond mimetic, preferably corresponding to the ribose of adenosine, and a benzimidazole or structurally related scaffold, corresponding to the adenine base of adenosine.
A representative ligand or modulator composition comprises a compound of the formula (I):




where:

X and Y are the same or different and X and Y═C, N, O or S; and X and Y can be substituted with hydrogen, hydroxyl, or oxygen, including double-bonded oxygen;
R1=hydrogen, hydroxyl, C1 to C6 alkyl, C1 to C6 branched alkyl, C1 to C6 hydroxyalkyl, branched C1 to C6 hydroxyalkyl, C4 to C8 cycloalkyl, C1 to C6 alkenyl, branched C1 to C6 alkenyl, C4 to C8 cycloalkenyl, C4 to C8 aryl, C4 to C8 aroyl, C4 to C8 aryl-substituted C1 to C6 alkyl, C1 to C6 alkoxy, C1 to C6 branched alkoxy, C4 to C8 aryloxy, primary, secondary or tertiary C1 to C6 alkylamino, primary, secondary or tertiary branched C1 to C6 alkylamino, primary, secondary or tertiary cycloalkylamino, primary, secondary or tertiary C4 to C8 arylamino, C1 to C6 alkylcarboxylic acid, branched C1 to C6 alkylcarboxylic acid, C1 to C6alkylester, branched C1 to C6 alkylester, C4 to C8 arylcarboxylic acid, C4 to C8 arlyester, C4 to C8 aryl substituted C1 to C6 alkyl, C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring, alkyl-substituted or aryl-substituted C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-, amino-, or halo-substituted versions thereof; or R1 is halo where halo is chloro, fluoro, bromo, or iodo;
R2=hydrogen, hydroxyl, C1 to C6 alkyl, C1 to C6 branched alkyl, C1 to C6 hydroxyalkyl, branched C1 to C6 hydroxyalkyl, C4 to C8 cycloalkyl, C1 to C6 alkenyl, branched C1 to C6 alkenyl, C4 to C8 cycloalkenyl, C4 to C8 aryl, C4 to C8 aroyl, C4 to C8 aryl-substituted C1 to C6 alkyl, C1 to C6 alkoxy, C1 to C6 branched alkoxy, C4 to C8 aryloxy, primary, secondary or tertiary C1 to C6 alkylamino, primary, secondary or tertiary branched C1 to C6 alkylamino, primary, secondary or tertiary cycloalkylamino, primary, secondary or tertiary C4 to C8 arylamino, C1 to C6 alkylcarboxylic acid, branched C1 to C6 alkylcarboxylic acid, C1 to C6 alkylester, branched C1 to C6 alkylester, C4 to C8 arylcarboxylic acid, C4 to C8 arlyester, C4 to C8 aryl substituted C1 to C6 alkyl, C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring, alkyl-substituted or aryl-substituted C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-, amino-, or halo-substituted versions thereof; or R2 is halo where halo is chloro, fluoro, bromo, or iodo; and
R3=hydrogen, hydroxyl, C1 to C6 alkyl, C1 to C6 branched alkyl, C1 to C6 hydroxyalkyl, branched C1 to C6 hydroxyalkyl, C4 to C8 cycloalkyl, C1 to C6 alkenyl, branched C1 to C6 alkenyl, C4 to C8 cycloalkenyl, C4 to C8 aryl, C4 to C8 aroyl, C4 to C8 aryl-substituted C1 to C6 alkyl, C1 to C6 alkoxy, C1 to C6 branched alkoxy, C4 to C8 aryloxy, primary, secondary or tertiary C1 to C6 alkylamino, primary, secondary or tertiary branched C1 to C6 alkylamino, primary, secondary or tertiary cycloalkylamino, primary, secondary or tertiary C4 to C8 arylamino, C1 to C6 alkylcarboxylic acid, branched C1 to C6 alkylcarboxylic acid, C1 to C6 alkylester, branched C1 to C6 alkylester, C4 to C8 arylcarboxylic acid, C4 to C8 arlyester, C4 to C8 aryl substituted C1 to C6 alkyl, C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring, alkyl-substituted or aryl-substituted C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-, amino-, or halo-substituted versions thereof; or R3 is halo where halo is chloro, fluoro, bromo, or iodo.
Where the ligand composition further comprises a compound of the formula (II):




where:

X and Y are the same or different and X and Y═C, N, O or S; and X and Y can be substituted with hydrogen, hydroxyl, or oxygen, including double-bonded oxygen;
R1=hydrogen, hydroxyl, C1 to C6 alkyl, C1 to C6 branched alkyl, C1 to C6 hydroxyalkyl, branched C1 to C6 hydroxyalkyl, C4 to C8 cycloalkyl, C1 to C6 alkenyl, branched C1 to C6 alkenyl, C4 to C8 cycloalkenyl, C4 to C8 aryl, C4 to C8 aroyl, C4 to C8 aryl-substituted C1 to C6 alkyl, C1 to C6 alkoxy, C1 to C6 branched alkoxy, C4 to C8 aryloxy, primary, secondary or tertiary C1 to C6 alkylamino, primary, secondary or tertiary branched C1 to C6 alkylamino, primary, secondary or tertiary cycloalkylamino, primary, secondary or tertiary C4 to C8 arylamino, C1 to C6 alkylcarboxylic acid, branched C1 to C6 alkylcarboxylic acid, C1 to C6 alkylester, branched C1 to C6 alkylester, C4 to C8 arylcarboxylic acid, C4 to C8 arlyester, C4 to C8 aryl substituted C1 to C6 alkyl, C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring, alkyl-substituted or aryl-substituted C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-, amino-, or halo-substituted versions thereof; or R1 is halo where halo is chloro, fluoro, bromo, or iodo;
R2=hydrogen, hydroxyl, C1 to C6 alkyl, C1 to C6 branched alkyl, C1 to C6 hydroxyalkyl, branched C1 to C6 hydroxyalkyl, C4 to C8 cycloalkyl, C1 to C6 alkenyl, branched C1 to C6 alkenyl, C4 to C8 cycloalkenyl, C4 to C8 aryl, C4 to C8 aroyl, C4 to C8 aryl-substituted C1 to C6 alkyl, C1 to C6 alkoxy, C1 to C6 branched alkoxy, C4 to C8 aryloxy, primary, secondary or tertiary C1 to C6 alkylamino, primary, secondary or tertiary branched C1 to C6 alkylamino, primary, secondary or tertiary cycloalkylamino, primary, secondary or tertiary C4 to C8 arylamino, C1 to C6 alkylcarboxylic acid, branched C1 to C6 alkylcarboxylic acid, C1 to C6 alkylester, branched C1 to C6 alkylester, C4 to C8 arylcarboxylic acid, C4 to C8 arlyester, C4 to C8 aryl substituted C1 to C6 alkyl, C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring, alkyl-substituted or aryl-substituted C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-, amino-, or halo-substituted versions thereof; or R2 is halo where halo is chloro, fluoro, bromo, or iodo;
R3=hydrogen, hydroxyl, C1 to C6 alkyl, C1 to C6 branched alkyl, C1 to C6 hydroxyalkyl, branched C1 to C6 hydroxyalkyl, C4 to C8 cycloalkyl, C1 to C6 alkenyl, branched C1 to C6 alkenyl, C4 to C8 cycloalkenyl, C4 to C8 aryl, C4 to C8 aroyl, C4 to C8 aryl-substituted C1 to C6 alkyl, C1 to C6 alkoxy, C1 to C6 branched alkoxy, C4 to C8 aryloxy, primary, secondary or tertiary C1 to C6 alkylamino, primary, secondary or tertiary branched C1 to C6 alkylamino, primary, secondary or tertiary cycloalkylamino, primary, secondary or tertiary C4 to C8 arylamino, C1 to C6 alkylcarboxylic acid, branched C1 to C6 alkylcarboxylic acid, C1 to C6 alkylester, branched C1 to C6 alkylester, C4 to C8 arylcarboxylic acid, C4 to C8 arlyester, C4 to C8 aryl substituted C1 to C6 alkyl, C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring, alkyl-substituted or aryl-substituted C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-, amino-, or halo-substituted versions thereof; or R3 is halo where halo is chloro, fluoro, bromo, or iodo; and
R4═C1 to C6 alkyl, C1 to C6 branched alkyl, C4 to C8 cycloalkyl with or without O, N or S in the ring, C1 to C6 alkenyl, branched C1 to C6 alkenyl, C4 to C8 cycloalkenyl with or without O, N or S in the ring, C4 to C8 aroyl, C4 to C8 aryl, C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring, C4 to C8 aryl-substituted C1 to C6 alkyl, alkyl-substituted or aryl-substituted C4 to C12 heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in the ring, alkyl-substituted C4 to C8 aroyl, or alkyl-substituted C4 to C8 aryl; or hydroxyl-, amino-, or halo-substituted versions thereof where halo is chloro, bromo, fluoro or iodo.
XI. Purification Methods
In accordance with the present invention, a method for purifying a complex comprising GRP94, or in some instances HSP90, by affinity chromatography is provided. The complex preferably comprises GRP94 bound to an antigenic molecule. More preferably, the complex comprises GRP94 non-covalently bound to an antigenic molecule. In one embodiment, the method comprises contacting a sample comprising a GRP94 complex with a binding agent that preferentially binds GRP94, the binding agent immobilized to a solid phase support, to immobilize the complex to the solid phase support; collecting the remaining sample; and eluting the GRP94 complex from the solid phase support to give purified GRP94 complex in the eluate. By the phrase “a binding agent that preferentially binds GRP94” it is meant an agent that preferentially binds GRP94 as compared to other molecular entities, including but not limited to other heat shock proteins.
The binding agent preferably comprises an adenosine moiety or structural mimetic thereof having any of a variety of substitutions at the 2′, 3′, and 5′ positions, in the case of adenosine, as deemed appropriate by high resolution structural analyses of ligand-GRP94 interactions. Optionally, 5′ position alkyl extensions can be included, preferably as a carboxamido linkage to the parent adenosine and, to facilitate stable chemical linkage to a solid support for the purposes of affinity-based purification, terminating in any of a subset of chemically reactive groups including, but not limited to vinyl, maleimide and/or succinimide esters, or substituents suitable for chemical coupling to solid phase supports, such as amino or sulphydryl groups. More preferably, the binding agent is free of ATP or ADP. A representative binding agent comprises a compound of the formula (I) or a compound of formula (II). Another representative binding agent comprises N-ethylcarboxamidoadenosine (NECA). Additional ligands can be identified through combinatorial chemistry of a parent precursor molecule bearing a hydrogen bond mimetic, preferably corresponding to the ribose of adenosine, and a benzimidazole or structurally related scaffold, corresponding to the adenine base of adenosine.
Optionally, the complex bound to the immobilized binding agent is eluted by washing the solid phase support with a buffer comprising a physiological salts solution containing appropriate concentrations of the parent ligand (i.e., the binding agent) to give complex in the eluate. Hence, a complex further comprising the binding agent or eluting ligand is also provided in accordance with the present invention. The eluting ligand will then be removed from the eluate solution by dialysis in buffers appropriate for GMP production including, but not limited to, physiological salts and volatile salts.
The affinity methods disclosed herein above can be used to isolate GRP94-peptide complexes or GRP94 alone, or in some instances, HSP90-peptide complexes, or the HSP90 protein alone, from any eukaryotic cell. For example, tissues, isolated cells, or immortalized eukaryote cell lines infected with a preselected intracellular pathogen, tumor cells or tumor cell lines can be used. The complex can also be obtained from a vertebrate subject, such as a warm-blooded vertebrate, including mammals and bird. Optionally, the mammal includes, but is not limited to, human, mouse, pig, rat, ape, monkey, cat, guinea pig, cow, goat and horse.
In one embodiment, the complex is “autologous” to the vertebrate subject; that is, the complex is isolated from either from the infected cells or the cancer cells or precancerous cells of the vertebrate subject (e.g., preferably prepared from infected tissues or tumor biopsies of a vertebrate subject).
Alternatively, the complex is produced in vitro (e.g., wherein a complex with an exogenous antigenic molecule is desired). Alternatively, GRP94 and/or the antigenic molecule can be isolated from a particular vertebrate subject, or from others, or by recombinant production methods using a cloned GRP94 originally derived from a particular vertebrate subject or from others. Exogenous antigens and fragments and derivatives (both peptide and non-peptide) thereof for use in complexing with GRP94 (or in some instances HSP90), can be selected from among those known in the art, as well as those readily identified by standard immunoassays know in the art by the ability to bind antibody or MHC molecules (antigenicity) or generate immune response (immunogenicity). Complexes of GRP94 and antigenic molecules can be isolated from cancer or precancerous tissue of a subject, or from a cancer cell line, or can be produced in vitro (as is necessary in the embodiment in which an exogenous antigen is used as the antigenic molecule).
XI.A. Isolation of Antigenic/Immunogenic Components
A method for isolating or purifying an antigenic molecule associated with a complex comprising GRP94, or in some instances HSP90, is also provided in accordance with the present invention. In one embodiment, the method comprises: contacting a sample comprising a complex comprising an antigenic molecule and GRP94 with a binding agent that preferentially binds GRP94, the binding agent immobilized to a solid phase support, to immobilize the complex to the solid phase support; collecting the remaining sample; eluting the complex from the solid phase support to give purified complex in the eluate; and isolating the antigenic molecule from the eluate.
The binding agent preferably comprises an adenosine moiety or structural mimetic thereof having any of a variety of substitutions at the 2′, 3′, and 5′ positions, in the case of adenosine, as deemed appropriate by high resolution structural analyses of ligand-GRP94 interactions. Optionally, 5′ position alkyl extensions can be included, preferably as a carboxamido linkage to the parent adenosine and, to facilitate stable chemical linkage to a solid support for the purposes of affinity-based purification, terminating in any of a subset of chemically reactive groups including, but not limited to vinyl, maleimide and/or succinimide esters, or substituents suitable for chemical coupling to solid phase supports, such as amino or sulphydryl groups. More preferably, the binding agent is free of ATP or ADP. A representative binding agent comprises a compound of formula (I) or a compound of formula (II). Another representative binding agent comprises N-ethylcarboxamidoadenosine (NECA). Additional ligands can be identified through combinatorial chemistry of a parent precursor molecule bearing a hydrogen bond mimetic, preferably corresponding to the ribose of adenosine, and a benzimidazole or structurally related scaffold, corresponding to the adenine base of adenosine.
Optionally, the complex bound to the immobilized binding agent is eluted by washing the solid phase support with a buffer comprising a physiological salts solution containing appropriate concentrations of the parent ligand (i.e. the binding agent) to give complex in the eluate. Hence, a complex further comprising the binding agent or eluting ligand is also provided in accordance with the present invention. The eluting ligand will then be removed from the eluate solution by dialysis in buffers appropriate for GMP production including, but not limited to, physiological salts and volatile salts.
It has been found that antigenic peptides and/or components can be eluted from GRP94-complexes under low pH conditions. These experimental conditions can be used to isolate peptides and/or antigenic components from cells that can contain potentially useful antigenic determinants. Once isolated, the amino acid sequence of each antigenic peptide can be determined using conventional amino acid sequencing methodologies. Such antigenic molecules can then be produced by chemical synthesis or recombinant methods; purified; and complexed to GRP94, or alternatively HSP90, in vitro. Additionally, antigenic peptide sequences can be obtained by mass spectrometry using, but not limited to, electrospray and MALDI-TOF instrumentation, coupled with quadrapole detection and CAD-based sequencing.
XI.B. Elution of Peptides From GRP94-Peptide Complexes
Several methods can be used to elute a peptide from a GRP94-peptide complex or from a HSP90-peptide complex. The approaches involve incubating the complex in a low pH buffer and/or in guanidinium/HCl (3-6 M), 0.1-1% TFA or acetic acid. Briefly, the complex of interest is centrifuged through a CENTRICON®10 assembly (Amicon of Beverly, Mass.) to remove any low molecular weight material loosely associated with the complex. The large molecular weight fraction can be removed and analyzed by SDS-PAGE while the low molecular weight material is fractionated by capillary and/or nanoscale HPLC, with a flow rate of 0.5 mL/min, with monitoring at 210/220 nm.
In the low pH protocol, acetic acid or trifluoroacetic acid (TFA) is added to the complex to give a final concentration of 10% (vol/vol) and the mixture incubated at room temperature or other suitable temperature, for 10 minutes (Van Bleek et al. (1990) Nature 348:213-216; Li et al. (1993) EMBO J 12:3143-3151).
The resulting samples are centrifuged through a CENTRICON®10 assembly as mentioned previously. The high and low molecular weight fractions are recovered. The remaining large molecular weight complexes can be reincubated with guanidinium or low pH to remove any remaining peptides. The resulting lower molecular weight fractions are pooled, concentrated by evaporation and dissolved in 0.1% trifluoroacetic acid (TFA). The dissolved material is fractionated by microbore HPLC, with a flow rate of 0.5 ml/min. The elution of the peptides can be monitored by OD210/220 nm and the fractions containing the peptides collected.
XI.C. Sequencing and Synthesis of Peptides
The amino acid sequences of the eluted peptides can be determined either by manual or automated amino acid sequencing techniques well known in the art. Once the amino acid sequence of a potentially protective peptide has been determined the peptide can be synthesized in any desired amount using conventional peptide synthesis or other protocols well known in the art.
A subject peptide can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a solid-phase Merrifield-type synthesis, are preferred for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production and the like. Many techniques for peptide synthesis are available and can be found in Steward et al. (1969) Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco, Calif.; Bodanszky, et al. (1976) Peptide Synthesis, John Wiley & Sons, Second Edition; Meienhofer (1983) Hormonal Proteins and Peptides, Vol. 2, p. 46, Academic Press, New York, N.Y.; Merrifield (1969) Adv Enzymol 32:221-296; Fields et al. (1990) Int J Peptide Protein Res 35:161-214; and U.S. Pat. No. 4,244,946 for solid phase peptide synthesis; and Schroder et al. (1965) The Peptides, Vol. 1, Academic Press, New York, N.Y. for classical solution synthesis, each of which is incorporated herein by reference. Appropriate protective groups usable in such synthesis are described in the above texts and in McOmie (1973) Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y., which is incorporated herein by reference.
In general, the solid-phase synthesis methods contemplated comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.
Using a solid phase synthesis as exemplary, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to afford the final linear polypeptide.
The resultant linear polypeptides prepared for example as described above can be reacted to form their corresponding cyclic peptides. An exemplary method for cyclizing peptides is described by Zimmer et al. (1993) Peptides, pp. 393-394, ESCOM Science Publishers, B. V. Typically, tertbutoxycarbonyl protected peptide methyl ester is dissolved in methanol and sodium hydroxide solution are added and the admixture is reacted at 20° C. to hydrolytically remove the methyl ester protecting group. After evaporating the solvent, the tertbutoxycarbonyl protected peptide is extracted with ethyl acetate from acidified aqueous solvent. The tertbutoxycarbonyl protecting group is then removed under mildly acidic conditions in dioxane cosolvent. The unprotected linear peptide with free amino and carboxy termini so obtained is converted to its corresponding cyclic peptide by reacting a dilute solution of the linear peptide, in a mixture of dichloromethane and dimethylformamide, with dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazole and N-methylmorpholine. The resultant cyclic peptide is then purified by chromatography.
Purification of the resulting peptides is accomplished using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography. The choice of appropriate matrices and buffers are well known in the art and so are not described in detail herein.
XI.D. Detection Methods
A method for detecting a complex comprising GRP94, or in some instances HSP90, in a sample suspected of containing such a complex is also provided in accordance with the present invention. In one embodiment, the method comprises: contacting the sample with a binding substance that preferentially binds GRP94 under conditions favorable to binding a complex comprising GRP94 to the binding substance to form a second complex there between; and detecting the second complex via a label conjugated to the binding substance or via a labeled reagent that specifically binds to the second complex subsequent to its formation.
The binding substance preferably comprises an adenosine moiety or structural mimetic thereof having any of a variety of substitutions at the 2′, 3′, and 5′ positions, in the case of adenosine, as deemed appropriate by high resolution structural analyses of ligand-GRP94 interactions. Optionally, 5′ position alkyl extensions can be included, preferably as a carboxamido linkage to the parent adenosine and, to facilitate stable chemical linkage to a solid support for the purposes of affinity-based purification, terminating in any of a subset of chemically reactive groups including, but not limited to vinyl, maleimide and/or succinimide esters, or substituents suitable for chemical coupling to solid phase supports, such as amino or sulphydryl groups. More preferably, the binding substance is free of ATP or ADP. A representative binding substance comprises a compound of formula (I) or a compound of formula (II). Another representative binding substance comprises N-ethylcarboxamidoadenosine (NECA). Additional ligands can be identified through combinatorial chemistry of a parent precursor molecule bearing a hydrogen bond mimetic, preferably corresponding to the ribose of adenosine, and a benzimidazole or structurally related scaffold, corresponding to the adenine base of adenosine.
Optionally, the complex bound to the immobilized binding agent is eluted by washing the solid phase support with a buffer comprising a physiological salts solution containing appropriate concentrations of the parent ligand (i.e. the binding substance or agent) to give complex in the eluate. Hence, a complex further comprising the binding agent or eluting ligand is also provided in accordance with the present invention. The eluting ligand will then be removed from the eluate solution by dialysis in buffers appropriate for GMP production including, but not limited to, physiological salts and volatile salts.
The binding substance can be conjugated with a detectable label and in this case, the detecting step comprises: separating the complex from unbound labeled binding substance; and detecting the detectable label which is present in the complex or which is unbound.
XI.E. Kits for Purification or Detection
In another aspect, the present invention pertains to a kit for isolating or purifying a peptide complex, preferably a GRP94 complex, and an antigenic molecule. In one embodiment, the kit comprises a binding agent that preferentially binds GRP94, the binding agent contained in a first container. The binding agent preferably comprises an adenosine moiety or structural mimetic thereof having any of a variety of substitutions at the 2′, 3′, and 5′ positions, in the case of adenosine, as deemed appropriate by high resolution structural analyses of ligand-GRP94 interactions. Optionally, 5′ position alkyl extensions can be included, preferably as a carboxamido linkage to the parent adenosine and, to facilitate stable chemical linkage to a solid support for the purposes of affinity-based purification, terminating in any of a subset of chemically reactive groups including, but not limited to vinyl, maleimide and/or succinimide esters, or substituents suitable for chemical coupling to solid phase supports, such as amino or sulphydryl groups. More preferably, the binding agent is free of ATP or ADP.
A representative binding agent comprises a compound of formula (I) or a compound of formula (II). Another representative binding agent comprises N-ethylcarboxamidoadenosine (NECA). Additional ligands can be identified through combinatorial chemistry of a parent precursor molecule bearing a hydrogen bond mimetic, preferably corresponding to the ribose of adenosine, and a benzimidazole or structurally related scaffold, corresponding to the adenine base of adenosine. Optionally, the binding agent can be immobilized to a solid phase support, or the kit can also comprise a solid phase support contained in a second container.
The kit can further comprise an elution buffer for use in eluting a complex from the binding agent, the elution buffer contained in a third container. Optionally, the elution buffer comprises a physiological salts solution containing appropriate concentrations of the parent ligand to give complex in the eluate. The kit can further comprise dialysis buffers appropriate for GMP production including, but not limited to, physiological salts and volatile salts. The kit can also further comprise an elution buffer for use in eluting an antigenic molecule from a complex, the elution buffer contained in a fourth container. Suitable elution buffers are disclosed herein above.
In the case of a kit used for detecting a complex comprising GRP94, or alternatively a complex comprising the kit can further comprise a reagent or indicator that comprises a detectable label, the indicator containing in a fifth container. Alternatively, the binding agent can comprise a detectable label or indicator. The indicator can comprise a radioactive label or an enzyme, or other indicator as disclosed herein.
XI.F. Determination of Immunogenicity of GRP94-Peptide Complexes
Purified GRP94-antigenic molecule complexes can be assayed for immunogenicity using the mixed lymphocyte tumor culture assay (MLTC) well known in the art. By way of example but not limitation, the following procedure can be used. Briefly, mice are injected subcutaneously with the candidate GRP94-antigenic molecule complexes. Other mice are injected with either other GRP94-antigenic molecule complexes or whole infected cells which act as positive controls for the assay. The mice are injected twice, 7-10 days apart. Ten days after the last immunization, the spleens are removed and the lymphocytes released. The released lymphocytes can be re-stimulated subsequently in vitro by the addition of dead cells that expressed the complex of interest.
For example, 8×106 immune spleen cells can be stimulated with 4×104 mitomycin C treated or γ-irradiated (5-10,000 rads) infected cells (or cells transfected with an appropriate gene, as the case can be) in 3 ml RPMI medium containing 10% fetal calf serum. In certain cases 33% secondary mixed lymphocyte culture supernatant can be included in the culture medium as a source of T cell growth factors, such as is described by Glasebrook et al. (1980) J Exp Med 151:876. To test the primary cytotoxic T cell response after immunization, spleen cells can be cultured without stimulation. In some experiments spleen cells of the immunized mice can also be re-stimulated with antigenically distinct cells, to determine the specificity of the cytotoxic T cell response.
Six days later the cultures are tested for cytotoxicity in a 4 hour 51Cr-release assay as is described by Palladino et al. (1987) Cancer Res 47:5074-5079 and Blachere et al. (1993) J Immunotherapy 14:352-356. In this assay, the mixed lymphocyte culture is added to a target cell suspension to give different effector:target (E:T) ratios (usually 1:1 to 40:1). The target cells are prelabeled by incubating 1×106 target cells in culture medium containing 200 mCi 51Cr/ml for one hour at 37° C. The cells are washed three times following labeling. Each assay point (E:T ratio) is performed in triplicate and the appropriate controls incorporated to measure spontaneous 51Cr release (no lymphocytes added to assay) and 100% release (cells lysed with detergent). After incubating the cell mixtures for 4 hours, the cells are pelleted by centrifugation at 200 g for 5 minutes. The amount of 51Cr released into the supernatant is measured by a gamma counter. The percent cytotoxicity is measured as cpm in the test sample minus spontaneously released cpm divided by the total detergent released cpm minus spontaneously released cpm.
In order to block the MHC class I cascade a concentrated hybridoma supernatant derived from K-44 hybridoma cells (an anti-MHC class I hybridoma) is added to the test samples to a final concentration of 12.5%.
XII. Screening Methods
Disclosed herein is the molecular basis, as well as a high throughput screen, for chemical compounds that elicit or inhibit conformational changes in the molecular chaperone GRP94, or in some instances HSP90, thereby regulating the chaperone and peptide binding activities of these proteins.
Also disclosed herein are several new and unique aspects of the regulation of GRP94 structure and function that can be readily exploited for purposes of identifying agonists and antagonists (“modulators”) of GRP94 function. GRP94 expression is upregulated by cellular stresses such as nutrient deprivation, oxidative stress, heavy metal poisoning, hypoxia/anoxia, and other conditions related to ischemia. However, until the disclosure of the present invention, the molecular mechanism underlying this activity remained unknown. Thus, disclosed herein is a functional correlation to heat shock in the observation that heat shock stimulates the peptide binding and chaperone activity of GRP94. The heat shock response of GRP94, which is responsible for its increased peptide binding and chaperone activity, is a result of a change in the conformational state of the protein from a closed form to an open, active form.
The heat shock induced conformational change can be blocked by the antitumor drugs geldanamycin and radicicol, thus providing a mechanism of their antitumor activity, namely that geldanamycin and radicicol block GRP94 conformational transitions, and hence chaperone activity. The functional consequence of such inhibition is that oncogenic signaling proteins, such as growth factor receptor kinases are not processed properly and thus, the cell does not receive the proliferative signals necessary for transformation. Thus, a chemical compound that modulates the conformation of GRP94 can be used to treat a disease state, such as cancer, wherein a therapeutic benefit can be provided by inhibiting or blocking the egress of proteins (e.g., growth factors) from the endoplasmic reticulum.
The present invention provides the theoretical and structural basis for the identification of low molecular weight molecules that bind to a recently crystallized conserved N-terminal domain of HSP90, which previously was identified as the binding site for the anti-tumor drug geldanamycin, and elicit a conformation change that yields a dramatic and substantial increase in (poly)peptide binding activity of GRP94, and in some cases, HSP90. In an alternative embodiment, the identified molecules inhibit conformational activation of GRP94, and in some cases HSP90, similar to the observed modulation of GRP94 and HSP90 by geldanamycin and/or radicicol.
The present invention is markedly distinguished from current perception in the art as to the mechanism of regulation of GRP94 and HSP90 function. In current views, the Hsp90 family of molecular chaperones are thought to be regulated by cycles of ATP binding and hydrolysis (Prodromou et al. (1997) Cell 90:65-75). This view of Hsp90 function is based on the observations that the highly conserved N-terminal domain of the protein contains a binding site for ATP and ADP and that X-ray crystallographic structures of the domain in complex with ATP and/or ADP can be obtained.
In accordance with the present invention, data are provided demonstrating that the related and relevant domain of the HSP90 paralog GRP94 does not display a specific structural preference for ATP or ADP. In a series of function-directed studies, applicants have further determined that ATP, ADP, geldanamycin and radicicol block or inhibit the ability of GRP94 to assume a conformation necessary for chaperone activity and/or peptide binding. Thus, ATP and ADP, rather than being physiological ligands agonising the activity of GRP94, act as inhibitory agents for this chaperone.
The identified conformational change in GRP94 is a component of the regulatory cycle of GRP94, as demonstrated in the Examples wherein bis-ANS, which bears structural similarities to adenosine nucleotides, was demonstrated to elicit a tertiary conformational change in GRP94 that was accompanied by an activation of molecular chaperone and peptide binding activity.
Structure of bis-ANS




In accordance with the present invention, also disclosed herein are the primary structural determinants that define low molecular weight compounds that bind to the conserved N-terminal domain of GRP94 and either A) elicit a conformational change in GRP94 that is accompanied by an activation of either peptide binding and/or molecular chaperone activity, or B) block or inhibit the ability of GRP94 to access or acquire the described conformation. In the present invention, and as would be apparent to one of ordinary skill in the art of the regulation of protein structure/function after reviewing the disclosure presented herein, cells and tissues originating from higher eukaryotes contain a native ligand compound bearing structural similarities to adenosine, yet may bear substituents at the 2′ and 5′ positions, but lack substituents at the N6 adenine.
Thus, a native ligand, as well an embodiment of a mimetic thereof, bears an adenosine moiety or moieties and the adenosine moiety(s) function in the binding of the ligand to the conserved N-terminal domain of GRP94 previously identified as an ATP/ADP binding pocket. Representative ligand compositions are disclosed herein above as formulas (I) and (II). Additional ligands can be identified through combinatorial chemistry of a parent precursor molecule bearing a hydrogen bond mimetic, preferably corresponding to the ribose of adenosine, and a benzimidazole or structurally related scaffold, corresponding to the adenine base of adenosine. Additional ligands can also be identified via the method disclosed herein above that employ a three-dimensional structure of the GRP94 LBD.
The binding of a ligand elicits the conformational change that is accompanied by an activation of chaperone and peptide binding activity. Furthermore, synthesis of the native ligand is likely stimulated by conditions that elicit a disruption in the efficiency of protein folding and assembly in the ER. These conditions include, but are not limited to, heat shock, oxidative stress, nutrient deprivation, disruptions in oligosaccharide synthesis and covalent assembly on to nascent glycoproteins, and the presence of excessive levels of heavy metals.
Coincident with the discovery of the functional role for GRP94 structural transitions in determining the chaperone activity and the mechanism of geldanamycin and radicicol action, a simple and rapid method for assaying the conformational state of GRP94 (or alternatively, HSP90) is disclosed herein. A preferred embodiment of this method is based on the preferential binding of the small synthetic fluorescent probe, bis-ANS, to the open, or active, conformation of GRP94. bis-ANS binding yields a dramatic increase in probe fluorescence intensity. bis-ANS is identified herein as a highly sensitive indicator of the heat shock induced conformational change of GRP94. Furthermore, bis-ANS itself can elicit the conformational change in GRP94 necessary for the activation of peptide binding and chaperone function. Thus, bis-ANS is both an agonist for GRP94 activation as well as an indicator for the relative state of activation. bis-ANS induces these changes on a slow time scale, thereby enabling it to be used both as an inducer for a heat shock-like conformational change as well as a probe for conformational changes induced by other compounds. Conversely, and as disclosed in the Examples, bis-ANS can be used to identify compounds that block the heat shock-induced conformational changes. Indeed, the screening system of the present invention showed that radicicol and geldanamycin, two anti-tumor agents known to act through GRP94/HSP90, block the conversion of these proteins to the conformation necessary for function.
Another preferred embodiment of this method employs a related synthetic fluorescent probe, 8-ANS. 8-ANS also displays preferential binding to the active conformation of GRP94. However, unlike bis-ANS, 8-ANS functions solely as an indicator and lacks agonist activity. 8-ANS is also useful in screening assays for discovery of GRP94 modulators.
Therefore, in accordance with the present invention, a method of screening candidate compounds for an ability to modulate the biological activity is provided. The screening methods are also used to identify a native or endogenous ligand or ligands for GRP94.
In one embodiment, a candidate substance is a substance which potentially can modulate the biological activity of GRP94 by binding or other intermolecular interaction with GRP94. By “modulate” is intended an increase, decrease, or other alteration of any or all biological activities or properties of GRP94. Thus, a native or endogenous ligand or ligands of GRP94 is also a “candidate substance”. A biological sample suspected of containing a native or endogenous ligand or ligands is also a “candidate substance”. Small molecules and combinatorial libraries of small molecules are also candidate “substances”. A candidate substance identified according to a screening assay described herein has the ability to modulate GRP94 biological activity. Such a candidate substance has utility in the treatment of disorders and conditions wherein modulation of the biological activity of GRP94 is desirable, as well as in the purification and screening methods disclosed herein.
The present invention thus pertains to the molecular basis for as well as a high throughput screen for chemical compounds that elicit or inhibit conformational changes in the molecular chaperone GRP94, or in some instances HSP90, thereby regulating the chaperone and peptide binding activities of these proteins.
XII.A. General Screening Methods
A method of screening candidate substances for an ability to modulate GRP94 and/or HSP90 biological activity is thus provided in accordance with the present invention. In one embodiment, the method comprises (a) establishing a test sample comprising GRP94 and a ligand for GRP94; (b) administering a candidate substance or a sample suspected of containing a candidate substance to the test sample; and (c) measuring an effect on binding of GRP94 and the ligand for GRP94 in the test sample to thereby determine the ability of the candidate substance to modulate GRP94 biological activity. Preferably, the GRP94 is a GRP94 LBD polypeptide of the present invention as disclosed herein above.
The test sample can further comprise an indicator. The term “indicator” is meant to refer to a chemical species or compound that is readily detectable using a standard detection technique, such as dark versus light detection, fluorescence or chemiluminescence spectrophotometry, scintillation spectroscopy, chromatography, liquid chromatography/mass spectroscopy (LC/MS), colorimetry, and the like. Representative indicator compounds thus include, but are not limited to, fluorogenic or fluorescent compounds, chemiluminescent compounds, colorimetric compounds, UV/VIS absorbing compounds, radionucleotides and combinations thereof. In a preferred embodiment, the ligand further comprises an indicator. In a more preferred embodiment, the ligand/indicator comprises 1,8-anilinonapthalenesulfonate (8-ANS).
The ability of the candidate substance to modulate GRP94 and/or HSP90 biological activity can determined in any suitable manner. For example, the ability of the candidate substance to modulate GRP94 and/or HSP90 biological activity can determined by: (i) detecting a signal produced by the indicator upon an effect of the candidate substance on binding of GRP94 and/or HSP90 and the ligand for GRP94 and/or HSP90; and (ii) identifying the candidate substance as a modulator of GRP94 and/or HSP90 biological activity based upon an amount of signal produced as compared to a control sample.
In a preferred embodiment, a simple and effective fluorescence based screening methodology is provided to identify inhibitors and activators of the conformational transitions of GRP94 that are responsible for its activity. The method is readily amenable to both robotic and very high throughput systems.
Thus, in one embodiment, a screening method of the present invention pertains to a method for a identifying a candidate substance as an activator of the biological activity of an Hsp90 protein. In a preferred embodiment, the Hsp90 protein is GRP94 or HSP90. The method comprises establishing a test sample comprising an Hsp90 protein and a candidate substance; administering 8-ANS to the test sample; and detecting a fluorescence signal produced by the 8-ANS; and identifying the candidate substance as an activator of the biological activity of the Hsp90 protein based upon an amount of fluorescence signal produced by the 8-ANS as compared to a control sample.
The method can further comprise incubating the Hsp90 protein with the candidate substance at 37° C. for about one hour prior to adding the 8-ANS. Optionally, the 8-ANS can be added in an approximately equimolar amount to the Hsp90 protein. Additionally, the candidate substance is identified as an activator of the biological activity of an Hsp90 protein by detection of an increased 8-ANS fluorescence signal as compared to a control sample.
In another embodiment, a screening method of the present invention pertains to a method for a identifying a candidate substance as an inhibitor of the biological activity of a Hsp90 protein. The method comprises establishing a test sample comprising an Hsp90 protein and a candidate substance; heat-shocking the test sample to induce a conformational change to the Hsp90 protein; administering 8-ANS to the test sample; detecting a fluorescence signal produced by the 8-ANS; and identifying the candidate substance as an inhibitor of the biological activity of an Hsp90 protein based upon an amount of fluorescence signal produced by the 8-ANS as compared to a control sample. In a preferred embodiment, the Hsp90 protein is GRP94 or HSP90.
Optionally, the method can further comprise incubating the test sample at 37° C. for about one hour prior to heat-shocking the test sample. The heat-shocking can be carried out at 50° C. for about 15 minutes. Preferably, the 8-ANS is added in an approximately equimolar amount to the Hsp90 protein. The candidate substance can also be identified as an inhibitor of the biological activity of an Hsp90 protein by detection of a decreased 8-ANS fluorescence signal as compared to a control sample.
XII.B. Cell Based Screening Assays
A screening assay of the present invention may also involve determining the ability of a candidate substance to modulate, i.e. inhibit or promote the biological activity of an Hsp90 protein such as GRP94 and preferably, to thereby modulate the biological activity of an Hsp90 protein such as GRP94 in target cells. Target cells can be either naturally occurring cells known to contain a polypeptide of the present invention or transformed cells produced in accordance with a process of transformation set forth herein above. The test samples can further comprise a cell or cell line that expresses an Hsp90 polypeptide; the present invention also contemplates a recombinant cell line suitable for use in the exemplary method. Such cell lines may be mammalian, or human, or they may from another organism, including but not limited to yeast. Preferably, the cells express a GRP94 LBD polypeptide of the present invention as disclosed herein above.
Representative assays including genetic screening assays and molecular biology screens such as a yeast two-hybrid screen that will effectively identify Hsp90-interacting genes important for Hsp90 or other Hsp90-mediated cellular process, including a native Hsp90 ligand or ligands. One version of the yeast two-hybrid system has been described (Chien et al. (1991) Proc Natl Acad Sci USA 88:9578-9582) and is commercially available from Clontech (Palo Alto, Calif.). Thus, in accordance with one embodiment of a screening assay of the present invention, the candidate substance is further characterized as a candidate polypeptide, and the screening method can further comprise the step of purifying and isolating a nucleic acid molecule encoding the candidate polypeptide.
Thus, enzymes in the cells of higher eukaryotes that mediate the steady state and stress-elicited production of a GRP94 and/or HSP90 ligand can also be modulated in accordance with the present invention. Such catabolic enzymes also represent appropriate and rational targets for the design of compounds that elicit an increase in the steady state levels of a native Hsp90 ligand (e.g., a native GRP94 or HSP90 ligand) and thereby lead to the elicitation of the structural and functional activation of chaperone and peptide binding activity of an Hsp90 protein, preferably GRP94, disclosed herein.
A screening assay can provide a cell under conditions suitable for testing the modulation of biological activity of an Hsp90 protein such as GRP94. These conditions include but are not limited to pH, temperature, tonicity, the presence of relevant metabolic factors (e.g., metal ions such as for example Ca++, growth factor, interleukins, or colony stimulating factors), and relevant modifications to the polypeptide such as glycosylation or prenylation. A polypeptide of the present invention can be expressed and utilized in a prokaryotic or eukaryotic cell. The host cell can also be fractionated into sub-cellular fractions where the receptor can be found. For example, cells expressing the polypeptide can be fractionated into the nuclei, the endoplasmic reticulum, vesicles, or the membrane surfaces of the cell. U.S. Pat. Nos. 5,837,479; 5,645,999; 5,786,152; 5,739,278; and 5,352,660 also describe exemplary screening assays, and the entire contents of each are herein incorporated by reference.
XII.C. High Throughput Screening
In another embodiment of the screening method of the present invention, an Hsp90 polypeptide (e.g., human GRP94) or active fragment or oligopeptide thereof (e.g., GRP94 LBD disclosed herein), can be used for screening libraries of compounds in any of a variety of high throughput drug screening techniques. The fragment employed in such screening can be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between the Hsp90 polypeptide, preferably a GRP94 polypeptide, more preferably a GRP94 LBD polypeptide, and the candidate substance being tested, can be measured as described herein.
XIII. Modulation of Hsp90 Biological Activity
Because Hsp90 proteins are found in essentially every cell of the human body and are involved in the processing of many different cellular proteins as well as the presentation of tumor and foreign antigens to the immune system, compounds identified through the screening method of the present invention and disclosed herein (referred to as “ligand compositions” or “modulators”) have wide ranging value as therapeutics and in vaccine development. Representative ligand compositions or modulators are described herein above as formula (I). Modulators that do not structurally resemble adenosine are also provided, and include those designed and/or identified by the rational drug design and combinatorial screening methods disclosed hereinabove.
In a preferred embodiment, the Hsp90 modulator elicits a conformational change in an Hsp90 protein. Even more preferably, the Hsp90 protein activity modulator is identified according to a screening assay described herein. A modulator can modulate the biological activity of an Hsp90 protein such as GRP94. Relevant to the antigen-presenting activity of GRP94 and HSP90, activators thereof can be applied in vitro to assist in peptide loading onto these proteins for the production of vaccines directed against the tissues or invasive organisms possessing those specific peptide epitopes. Activators of GRP94/HSP90 biological activity can be applied to tumor cells excised from cancer patients to increase the antigenicity of the tumor cells prior to lethal inactivation of the cells and their re-injection into the body as immunostimulatory agents. Activators of GRP94/HSP90 biological activity can be administered directly into the body of a vertebrate for increasing the antigenicity of tumors in situ. Activators of GRP94/HSP90 biological activity can also have antibiotic action against bacteria, viruses, or internal parasites by increasing the antigenicity of the bacteria, virus, or parasites and recognition of same by the adaptive immune system. Activators of GRP94/HSP90 biological activity can be used in further screens to identify peptides from combinatorial libraries that represent specific anti-tumor, anti-viral, or anti-bacterial epitopes. Relevant to the chaperone activity of GRP94 and HSP90, activators thereof can also ameliorate or prevent cellular damage resulting from ischemic conditions.
Inhibitors of GRP94/HSP90 function can possess anti-tumor activity. Inhibitors of GRP94/HSP90 function can also interfere with the processing of viral or bacterial proteins in infectious states and slow the progress of these infections. Inhibitors of GRP94/HSP90 function can also be administered to a vertebrate subject to decrease the antigenicity of tissues to alleviate transplanted tissue rejection or even slow the progression of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythramatosis. Inhibitors of GRP94 activity can also be used for treatment of diseases, such as cancer, by inhibiting or blocking the egress of proteins (e.g., growth factors) from the endoplasmic reticulum.
A biological activity of a Hsp90 protein such as GRP94 that is modulated in accordance with the present invention can include, but is not limited to, loading activity in the formation of a complex with antigenic molecules, eliciting an immune response in a subject; treating or preventing a type of cancer in a subject; treating or preventing an infectious disease in a subject; sensitizing antigen presenting cells (APC), particularly with respect to a type of cancer or an infectious disease; and enhancing protein transport along the endoplasmic reticulum.
Another modulatable biological activity of a Hsp90 protein comprises preventing or ameliorating cellular damage arising from conditions of ischemia/reperfusion including but not limited to cardiac arrest, asystole and sustained ventricular arrythmias, cardiac surgery, cardiopulmonary bypass surgery, organ transplantation, spinal cord injury, head trauma, stroke, thromboembolic stroke, hemorrhagic stroke, cerebral vasospasm, hypotension, hypoglycemia, status epilepticus, an epileptic seizure, anxiety, schizophrenia, a neurodegenerative disorder, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or neonatal stress. In this case, a ligand can modulate an endogenous Hsp90 protein by promoting conformational activation of the Hsp90 protein. Preferably, the ligand was identified according to a screening or rational drug design method disclosed herein and is relevant for the modulation of GRP94 or HSP90.
XIII.A. In vitro Production of GRP94-Antigenic Molecule Complexes
In accordance with the present invention, complexes of an Hsp90 protein, such as GRP94, to antigenic molecules are produced in vitro using an Hsp90 protein activity modulator. As will be appreciated by those skilled in the art, the peptides either isolated by procedures disclosed herein, chemically synthesized or recombinantly produced, can be reconstituted with a variety of naturally purified or recombinant Hsp90 proteins in vitro to generate, for example, immunogenic non-covalent GRP94-antigenic molecule complexes. Alternatively, exogenous antigens or antigenic/immunogenic fragments or derivatives thereof can be non-covalently complexed to an Hsp90 protein for use in the immunotherapeutic or prophylactic vaccines of the invention. The complexes can then be purified using any suitable method, and are preferably purified via the affinity purification methods of the present invention disclosed herein above.
In a representative approach, antigenic molecules (1 μg) and GRP94 (9 μg) are admixed to give an approximately 5 antigenic molecule: 1 GRP94 molar ratio. Then, the mixture is incubated for 15 minutes to 3 hours at 4° C. to 45° C. with bis-ANS in a quantity equimolar to GRP94 in a suitable binding buffer such as one containing 20 mM sodium phosphate, pH 7.2, 350 mM NaCl, 3 mM MgCl2 and 1 mM phenyl methyl sulfonyl fluoride (PMSF). The preparations are centrifuged through CENTRICON®10 assembly (Amicon of Beverly, Mass.) to remove any unbound peptide. The association of the peptides with GRP94 can be assayed by SDS-PAGE. Additional representative approaches are disclosed in the Examples.
Following complexing, the immunogenic GRP94-antigenic molecule complexes can optionally be assayed in vitro using, for example, the mixed lymphocyte tumor cell assay (MLTC) described herein. Once immunogenic complexes have been isolated they can be optionally characterized further in animal models using the preferred administration protocols and excipients discussed herein.
XIII.A.1. Exogenous Antigenic Molecules
Antigens or antigenic portions thereof can be selected for use as antigenic molecules, for complexing to an Hsp90 protein, such as GRP94, from among those known in the art or determined by immunoassay to be able to bind to antibody or MHC molecules (antigenicity) or generate immune response (immunogenicity). To determine immunogenicity or antigenicity by detecting binding to antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in vivo immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, immunoprecipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immuno-electrophoresis assays, etc.
In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods and techniques are known in the art for detecting binding in an immunoassay and can be used. In one embodiment for detecting immunogenicity, T cell-mediated responses can be assayed by standard methods, e.g., in vitro cytotoxicity assays or in vivo delayed-type hypersensitivity assays.
Potentially useful antigens or derivatives thereof for use as antigenic molecules can also be identified by various criteria, such as the antigen's involvement in neutralization of a pathogen's infectivity (wherein it is desired to treat or prevent infection by such a pathogen) (Norrby (1985) “Summary” in Vaccines 85, Lerner et al. (eds.), pp. 388-389, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), type or group specificity, recognition by subjects' antisera or immune cells, and/or the demonstration of protective effects of antisera or immune cells specific for the antigen. In addition, where it is desired to treat or prevent a disease caused by a pathogen, the antigen's encoded epitope should preferably display a small or no degree of antigenic variation in time or amongst different isolates of the same pathogen.
Preferably, where it is desired to treat or prevent cancer, known tumor-specific antigens or fragments or derivatives thereof are used. For example, such tumor specific or tumor-associated antigens include but are not limited to KS 1/4 pan-carcinoma antigen (Perez & Walker (1990) J Immunol 142:3662-3667; Bumal (1988) Hybridoma 7(4):407-415); ovarian carcinoma antigen (CA125) (Yu et al. (1991) Cancer Res 51(2):468-475); prostatic acid phosphate (Tailer et al. (1990) Nuc Acids Res 18(16):4928); prostate specific antigen (Henttu & Vihko (1989) Biochem Biophys Res Comm 160(2):903-910; Israeli et al. (1993) Cancer Res 53:227-230); melanoma-associated antigen p97 (Estin et al. (1989) J Natl Cancer Inst 81(6):445-446); melanoma antigen gp75 (Vijayasardahl et al. (1990) J Exp Med 171(4):1375-1380); high molecular weight melanoma antigen (Natali et al. (1987) Cancer 59:55-63) and prostate specific membrane antigen. In a specific embodiment, an antigen or fragment or derivative thereof specific to a certain tumor is selected for complexing to an Hsp90 protein, such as GRP94, and subsequent administration to a subject having that tumor. The term “specific” can refer to an antigen found in or on a tumor cell, but not in or on a non-tumorous or non-cancerous cell.
Preferably, where it is desired to treat or prevent viral diseases, molecules comprising epitopes of known viruses are used. For example, such antigenic epitopes can be prepared from viruses including, but not limited to, hepatitis type A hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus (RSV), papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HIV-II). Preferably, where it is desired to treat or prevent bacterial infections, molecules comprising epitopes of known bacteria are used. For example, such antigenic epitopes can be prepared from bacteria including, but not limited to, Mycobacteria, Mycoplasma, Neisseria, and Legionella. 
Preferably, where it is desired to treat or prevent protozoal infectious, molecules comprising epitopes of known protozoa are used. For example, such antigenic epitopes can be prepared from protozoa including, but not limited to, Leishmania, Kokzidioa, and Trypanosoma. Preferably, where it is desired to treat or prevent parasitic infectious, molecules comprising epitopes of known parasites are used. For example, such antigenic epitopes can be from parasites including, but not limited to, Chlamydia and Rickettsia. 
XIII.A.2. Peptides from MHC Complexes
Candidate immunogenic or antigenic peptides can be isolated from either endogenous Hsp90-peptide complexes as described above or from endogenous MHC-peptide complexes for use subsequently as antigenic molecules, by complexing in vitro to an Hsp90 protein, such as GRP94. The isolation of potentially immunogenic peptides from MHC molecules is well known in the art and so is not described in detail herein. See Falk et al. (1990) Nature 348:248-251; Rotzsche et al. (1990) Nature 348:252-254; Elliott et al. (1990) Nature 348:191-197; Falk et al. (1991) Nature 351:290-296; Demotz et al. (1989) Nature 343:682-684; Rotzsche et al. (1990) Science 249:283-287, the disclosures of which are incorporated herein by reference. Briefly, MHC-peptide complexes can be isolated by a conventional immuno-affinity procedure. The peptides can then be eluted from the MHC-peptide complex by incubating the complexes in the presence of about 0.1% TFA in acetonitrile. The eluted peptides can be fractionated and purified by HPLC as described herein.
XIII.B. Therapeutic Methods for Modulating Hsp90 Biological Activity
A therapeutic method according to the present invention comprises administering to a subject in need thereof a substance that modulates, i.e., inhibits or promotes, biological activity of an Hsp90 protein, such as GRP94. Representative substances, also referred to as “ligand compositions” or “modulators”, are disclosed herein (e.g., compounds of formula (I)) and can also be identified according to any of the screening assays set forth herein. The method comprises treating a subject suffering from a disorder wherein modulation of the biological activity of an Hsp90 protein is desirable by administering to the subject an effective amount of an Hsp90 modulator. Preferably, the Hsp90 protein is GRP94. More preferably, the modulator elicits a conformational change in an Hsp90 protein. Even more preferably, the modulator is identified according to a screening assay described herein.
By the term “modulating”, it is meant that the substance can either promote or inhibit the biological activity of an Hsp90 protein, depending on the disorder to be treated, and can affect one or several of the Hsp90 proteins, including GRP94. Administration can provide treatment of disorders that can be exacerbated by GRP94/HSP90-mediated mechanisms, including but not limited to, cancer, infectious diseases, and ischemic conditions.
The subject treated in the present invention in its many embodiments is desirably a human subject, although it is to be understood that the principles of the invention indicate that the invention is effective with respect to invertebrate and to all vertebrate species, including mammals, which are intended to be included in the term “subject”. This is particularly the case in view of the phylogenetically ubiquitous nature of Hsp90 proteins. Moreover, a mammal is understood to include any mammalian species in which treatment or prevention of cancer or infectious diseases is desirable, particularly agricultural and domestic mammalian species.
The methods of the present invention are particularly useful in the treatment of warm-blooded vertebrates. Therefore, the invention concerns mammals and birds.
More particularly, contemplated is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also contemplated is the treatment of birds, including the treatment of those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans. Thus, contemplated is the treatment of livestock, including, but not limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.
In one embodiment, a ligand composition or modulator is administered in conjunction with a complex comprising an Hsp90 protein (preferably GRP94 or HSP90) and an antigenic molecule. Preferably, the complex is “autologous” to the subject; that is, the complex is isolated from either from the infected cells or the cancer cells or precancerous cells of the subject (e.g., preferably prepared from infected tissues or tumor biopsies of a subject). More preferably, the complex is purified in accordance with a purification method of the present invention disclosed herein above.
Alternatively, the complex is produced in vitro (e.g., wherein a complex with an exogenous antigenic molecule is desired). Alternatively, the Hsp90 protein (preferably GRP94 or HSP90) and/or the antigenic molecule can be isolated from a particular subject or from others or by recombinant production methods using a cloned Hsp90 protein (preferably GRP94 or HSP90) originally derived from a particular subject or from others. Exogenous antigens and fragments and derivatives (both peptide and non-peptide) thereof for use in complexing with an Hsp90 protein, can be selected from among those known in the art, as well as those readily identified by standard immunoassays know in the art by the ability to bind antibody or MHC molecules (antigenicity) or generate immune response (immunogenicity). Complexes of an Hsp90 protein (preferably GRP94 or HSP90) and antigenic molecules can be isolated from cancer or precancerous tissue of a subject, or from a cancer cell line, or can be produced in vitro (as is necessary in the embodiment in which an exogenous antigen is used as the antigenic molecule). Preferably, the complex is purified in accordance with a purification method of the present invention disclosed herein above.
The invention also provides a method for measuring tumor rejection in vivo in a subject, preferably a human subject, comprising measuring the generation by the subject of MHC Class I-restricted CD8+ cytotoxic T lymphocytes specific to the tumor after administering a complex comprising GRP94 and antigenic molecules specific to the tumor in conjunction with an GRP94 biological activity modulator. Preferably, GRP94 comprises human GRP94. The immunogenic GRP94-peptide complexes of the invention can include any complex containing a GRP94 and a peptide that is capable of inducing an immune response in a subject. The peptides are preferably non-covalently associated with the GRP94.
Although the Hsp90 protein can be allogenic to the subject (e.g., isolated from cancerous tissue from a second vertebrate subject that is the same type as a cancerous tissue present in a first vertebrate subject to be treated), in a preferred embodiment, the Hsp90 protein is autologous to (derived from) the subject to whom they are administered. The Hsp90 protein and/or antigenic molecules can be purified from natural sources, chemically synthesized, or recombinantly produced. Preferably, the complex and/or antigenic molecule is purified in accordance with a purification method of the present invention disclosed herein above. The invention provides methods for determining doses for human cancer immunotherapy by evaluating the optimal dose of an Hsp90 protein non-covalently bound to peptide complexes in experimental tumor models and extrapolating the data. Specifically, a scaling factor not exceeding a fifty-fold increase over the effective dose estimated in animals, is used as the optimal prescription method for cancer immunotherapy or vaccination in human subjects. Preferably, the Hsp90 protein is GRP94.
The invention provides combinations of compositions that enhance the immunocompetence of the host individual and elicit specific immunity against infectious agents or specific immunity against preneoplastic and neoplastic cells. The therapeutic regimens and pharmaceutical compositions of the invention are described below. These compositions have the capacity to prevent the onset and progression of infectious diseases and prevent the development of tumor cells and to inhibit the growth and progression of tumor cells, indicating that such compositions can induce specific immunity in infectious diseases and cancer immunotherapy. For example, Hsp90-antigenic molecule complexes can be administered in combination with other complexes, such as calreticulin, and antigenic molecules in accordance with the methods of the present invention.
Accordingly, the invention provides methods of preventing and treating cancer in a subject. A representative method comprises administering a therapeutically effective amount of an Hsp90 modulator (preferably a GRP94 modulator) to a subject in need thereof. Such a subject can include but is not limited to a subject suffering from cancer or at risk to develop cancer. Representative modulators that can be employed in the method comprise ligands that inhibit GRP94 (Hsp90) function. Such ligands are designed and identified using the screening methods disclosed herein and are thus employed as anti-tumor drugs, and/or anti-neoplastic agents. Characterization of these activities can be accomplished via techniques disclosed herein and known in the art.
In another embodiment, the method comprises administering a complex comprising an Hsp90 protein and pertinent antigenic molecule in conjunction with a modulator that stimulates the immunocompetence of the host individual and elicits specific immunity against the preneoplastic and/or neoplastic cells. Preferably, the Hsp90 protein is GRP94.
As used herein, “preneoplastic” cell refers to a cell which is in transition from a normal to a neoplastic form; and morphological evidence, increasingly supported by molecular biologic studies, indicates that preneoplasia progresses through multiple steps. Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia, or most particularly, dysplasia (for review of such abnormal growth conditions. See Robbins & Angell (1976) Basic Pathology, 2d Ed., pp. 68-79, W. B. Saunders Co., Philadelphia, Pa.).
Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder. Although preneoplastic lesions can progress to neoplasia, they can also remain stable for long periods and can even regress, particularly if the inciting agent is removed or if the lesion succumbs to an immunological attack by its host.
The therapeutic regimens and pharmaceutical compositions of the invention can be used with additional adjuvants or biological response modifiers including, but not limited to, the cytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF, or other cytokine affecting immune cells. In accordance with this aspect of the invention, a complex of an Hsp90 protein and an antigenic molecule along with a modulator are administered in combination therapy with one or more of these cytokines. Preferably, the Hsp90 protein is GRP94.
The invention also pertains to administration of a complex of an Hsp90 protein and an antigenic molecule and a modulator to individuals at enhanced risk of cancer due to familial history or environmental risk factors. Preferably, the Hsp90 protein is GRP94.
Enzymes in the cells of higher eukaryotes that mediate the steady state and stress-elicited production of a native GRP94 ligand can also be modulated in accordance with the present invention. Particularly, such catabolic enzymes represent appropriate and rational targets for modulation to elicit an increase in the steady state levels of a native GRP94 ligand and thereby lead to the elicitation of the structural and functional activation of chaperone and peptide binding activity of GRP94 disclosed herein.
Protein misfolding disorders are a common component of numerous genetic disease states including, but not limited to, cystic fibrosis, familial hypercholesterolemia, retinitis pigmentosa and α1-antitrypsin misfolding. Compounds that modulate the activity of the Hsp90 family of molecular chaperones can thus be used in accordance with a therapeutic method of the present invention for reversing the protein folding defects that identify the disease state or for enhancing protein transport from the endoplasmic reticulum of a cell. Thus, a compound that modulates the conformation of GRP94 can be used to treat a disease state resulting from defects in protein transport into or from the endoplasmic reticulum. Compounds that abrogate GRP94 activity can be used for the treatment of a disease state, such as cancer, wherein a therapeutic benefit can be provided by blocking the egress of proteins (e.g., growth factors) from the endoplasmic reticulum. conversely, compounds that promote GRP94 activity can be used to treat a disease wherein a therapeutic benefit can be provided by enhancing protein export from the endoplasmic reticulum.
The present invention also pertains to administration of compounds for the prevention or amelioration of cellular damage arising from conditions of ischemia/reperfusion including but not limited to cardiac arrest, asystole and sustained ventricular arrythmias, cardiac surgery, cardiopulmonary bypass surgery, organ transplantation, spinal cord injury, head trauma, stroke, thromboembolic stroke, hemorrhagic stroke, cerebral vasospasm, hypotension, hypoglycemia, status epilepticus, an epileptic seizure, anxiety, schizophrenia, a neurodegenerative disorder, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or neonatal stress. In one embodiment, a composition comprising a Hsp90 ligand is administered to promote conformational activation of a Hsp90 protein, thereby promoting its cellular protective function relevant to recovery following a injury or onset of a disease state associated with ischemia. In another embodiment, administration of a composition comprising a Hsp90 ligand can alter a subsequent cellular response to an ischemic condition at a tissue location in a subject. Cells at the tissue location are contacted with a Hsp90 protein ligand, whereby Hsp90 activity in the cells is enhanced to a degree effective to alter a subsequent cellular response to an ischemic condition. Preferably, the therapeutic composition comprises a ligand identified according to a screening or rational drug design method disclosed herein. Also preferably, the therapeutic composition modulates the activity of GRP94 or HSP90.
XIII.C. Dosage Regimens
Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration, e.g., two to four separate doses per day. It will be understood, however, that the specific dose level for any particular subject will depend upon a variety of factors including the body weight, general health, diet, time and route of administration, combination with other drugs and the severity of the particular disease being treated.
The dosage ranges for the administration of a modulator depend upon the form of the modulator, and its potency, as described further herein, and are amounts large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
The therapeutic compositions can be administered as a unit dose. The term “unit dose” when used in reference to a therapeutic composition employed in the method of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier or vehicle.
The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies can also be administered.
A therapeutically effective amount is an amount of a modulator sufficient to produce a measurable modulation of Hsp90 protein (preferably GRP94) biological activity in a subject being treated, i.e., an Hsp90 protein biological activity-modulating amount. Modulation of Hsp90 protein biological activity can be measured using the screening methods disclosed herein, via the method disclosed in the Examples, or by other methods known to one skilled in the art.
The potency of a modulator can vary, and therefore a “therapeutically effective” amount can vary. However, as shown by the present assay methods, one skilled in the art can readily assess the potency and efficacy of a candidate modulator of this invention and adjust the therapeutic regimen accordingly. A modulator of Hsp90 protein (preferably GRP94) biological activity can be evaluated by a variety of methods and techniques including the screening assays disclosed herein.
A preferred modulator has the ability to substantially bind an Hsp90 protein in solution at modulator concentrations of less than one (1) micromolar (μM), preferably less than 0.1 μM, and more preferably less than 0.01 μM. By “substantially” is meant that at least a 50 percent reduction in biological activity is observed by modulation in the presence of the modulator, and at 50% reduction is referred to herein as an “IC50 value”.
In one embodiment, the therapeutically effective amount of a modulator can respectively range from about 0.01 mg to about 10,000 mg per day. Alternatively, the therapeutically effective amount of a modulator can respectively range from about 0.1 mg to about 1,000 mg per day. Alternatively, the therapeutically effective amount of a modulator can respectively range from about 1 mg to about 300 mg per day. In a preferred embodiment, the therapeutically effective amount of a modulator can respectively range from about 15 mg per kg body weight per day to about 35 mg per kg body weight per day.
It was established in experimental tumor models (Blachere et al., 1993) that the lowest dose of heat shock proteins noncovalently bound to peptide complexes which produced tumor regression in mice was between 10 and 25 microgram/mouse weighing 20-25 g which is equal to 25 mg/25 g=1 mg/kg. Conventional methods extrapolate to human dosages based on body weight and surface area. For example, conventional methods of extrapolating human dosage based on body weight can be carried out as follows: since the conversion factor for converting the mouse dosage to human dosage is Dose Human per kg=Dose Mouse per kg×12 (Freireich et al. (1966) Cancer Chemotherap Rep 50:219-244), the effective dose of Hsp90-peptide complexes in humans weighing 70 kg should be 1 mg/kg÷12×70, i.e., about 6 mg (5.8 mg).
Drug doses are also given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and excretionary functions (Shirkey (1965) JAMA 193:443). Moreover, body surface area can be used as a common denominator for drug dosage in adults and children as well as in different animal species as described by Freireich et al. (1966) Cancer Chemotherap Rep 50:219-244. Briefly, to express a mg/kg dose in any given species as the equivalent mg/sq m dose, multiply the dose by the appropriate km factor. In adult human, 100 mg/kg is equivalent to 100 mg/kg×37 kg/sq m=3700 mg/sq m.
International Publication Nos. WO 95/24923, WO 97/10000, WO 97/10002, and WO 98/34641, as well as U.S. Pat. Nos. 5,750,119, 5,830,464, and 5,837,251, each provide dosages of the purified complexes of heat shock proteins and antigenic molecules, and the entire contents of each of these documents are herein incorporated by reference. Briefly, and as applied to the present invention, an amount of Hsp90 protein (preferably GRP94)-antigenic molecule complexes is administered that is in the range of about 10 microgram to about 600 micrograms for a human subject, the preferred human dosage being the same as used in a 25 g mouse, i.e., in the range of 10-100 micrograms. The dosage for Hsp90 protein (preferably GRP94)-peptide complexes in a human subject provided by the present invention is in the range of about 50 to 5,000 micrograms, the preferred dosage being 100 micrograms.
In a series of preferred and more preferred embodiments, the Hsp90-peptide complex is administered in an amount of less than about 50 micrograms. In this case, the Hsp90 protein (preferably GRP94)-peptide complex is preferably administered in an amount of ranging from about 5 to about 49 micrograms. In a preferred embodiment, a GRP94-peptide complex is administered in an amount of less than about 10 micrograms. In this case, the GRP94-peptide complex is preferably administered in an amount ranging from about 0.1 to about 9.0 micrograms. More preferably, the GRP94-peptide complexes is administered in an amount ranging from about 0.5 to about 2.0 micrograms. In accordance with one aspect of the present invention, administration of a lower dosage of complex is facilitated and preferred when a modulator is also administered.
The doses recited above are preferably given once weekly for a period of about 4-6 weeks, and the mode or site of administration is preferably varied with each administration. In a preferred example, subcutaneous administrations are given, with each site of administration varied sequentially. For example, half the dose can be given in one site and the other half on an other site on the same day.
Alternatively, the mode of administration is sequentially varied. For example, weekly injections are given in sequence subcutaneously, intramuscularly, intravenously or intraperitoneally. After 4-6 weeks, further injections are preferably given at two-week intervals over a period of time of one month. Later injections can be given monthly. The pace of later injections can be modified, depending upon the subject's clinical progress and responsiveness to the immunotherapy.
XIII.D. Therapeutic Compositions for Immune Responses to Cancer
Compositions comprising an Hsp90 protein bound (e.g., GRP94-preferably non-covalently bound) to antigenic molecules are administered to elicit an effective specific immune response to the complexed antigenic molecules (and preferably not to the HSP90 protein). In a preferred embodiment, non-covalent complexes of the Hsp90 protein with peptides are prepared and purified postoperatively from tumor cells obtained from the cancer patient that have also been treated with an Hsp90 protein biological activity modulator in accordance with the present invention. A preferred Hsp90 protein is GRP94. In a more preferred embodiment, the complexes are purified using an affinity purification method of the present invention, as disclosed herein above.
In accordance with the methods described herein, immunogenic or antigenic peptides that are endogenously complexed to Hsp90 (e.g. GRP94) or MHC antigens can be used as antigenic molecules. For example, such peptides can be prepared that stimulate cytotoxic T cell responses against different tumor antigens (e.g., tyrosinase, gp100, melan-A, gp75, mucins, etc.) and viral proteins including, but not limited to, proteins of immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus (RSV), papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus and polio virus. In the embodiment wherein the antigenic molecules are peptides noncovalently complexed to GRP94 in vivo, the complexes can be isolated from cells, or alternatively, produced in vitro from purified preparations each of GRP94 and antigenic molecules. The complexes can be further purified using an affinity purification method of the present invention, as disclosed herein above.
In another specific embodiment, antigens of cancers (e.g., tumors) or infectious agents (e.g., viral antigen, bacterial antigens, etc.) can be obtained by purification from natural sources, by chemical synthesis, or recombinantly, and, through in vitro procedures such as those described herein, complexed to GRP94. The complexes can also be further purified using an affinity purification method of the present invention, as disclosed herein above.
XIII.E. Formulation
In accordance with the present invention, modulators as well as antigenic molecule complexes can be formulated into pharmaceutical preparations for administration to a subject for treatment or prevention of cancer or infectious diseases. Compositions comprising a complex prepared in accordance with the present invention are formulated in a compatible pharmaceutical carrier can be prepared, packaged, and labeled for treatment of the indicated disorder (e.g. cancer or infectious disease).
If the modulator or complex is water-soluble, then it can be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if a modulator or a resulting complex has poor solubility in aqueous solvents, then it can be formulated with a non-ionic surfactant, such as TWEENTM, or polyethylene glycol. Thus, the compounds and their physiologically acceptable solvates can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration or, in the case of tumors, directly injected into a solid tumor.
For oral administration, the pharmaceutical preparation can be in liquid form, for example, solutions, syrups or suspensions, or can be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well-known in the art. Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compositions can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
The compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.
The invention also provides kits for carrying out the therapeutic regimens of the invention. Such kits comprise in one or more containers therapeutically or prophylactically effective amounts of a modulator and/or a antigenic molecule complex in pharmaceutically acceptable form. The modulator and the antigenic molecule complex in a vial of a kit of the invention can be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid. Alternatively, the modulator or complex can be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the modulator complex to form a solution for injection purposes.
In another embodiment, a kit of the invention further comprises needles or syringes, preferably packaged in sterile form, for injecting the modulator and complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of antigenic molecule complexes by a clinician or by the subject.
XIV. Target Infectious Diseases
Infectious diseases that can be treated or prevented by the methods of the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa and parasites. In one embodiment of the present invention wherein it is desired to treat a subject having an infectious disease, the above-described affinity purification methods are used to isolate GRP94-peptide complexes from cells infected with an infectious organism, e.g., of a cell line or from a subject. Thus, preferably, the peptides are found in cells infected with an infectious organism and not in cells that are not infected.
Viral diseases that can be treated or prevented by the methods of the present invention include, but are not limited to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus (RSV), papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HIV-II).
Bacterial diseases that can be treated or prevented by the methods of the present invention are caused by bacteria including, but not limited to, Mycobacteria, Mycoplasma, Neisseria, and Legionella. 
Protozoal diseases that can be treated or prevented by the methods of the present invention are caused by protozoa including, but not limited to, Leishmania, Kokzidioa, and Trypanosoma. Parasitic diseases that can be treated or prevented by the methods of the present invention are caused by parasites including, but not limited to, Chlamydia and Rickettsia. 
XV. Target Cancers
Cancers that can be treated or prevented by the methods of the present invention include, but not limited to human sarcomas and carcinomas, including but not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenströom's macroglobulinemia, and heavy chain disease.
In a specific embodiment the cancer is metastatic. In another specific embodiment, the subject having a cancer is immunosuppressed by reason of having undergone anti-cancer therapy (e.g., chemotherapy radiation) prior to administration of the GRP94-antigenic molecule complexes and a GRP94 modulator in accordance with the present invention.
XVI. Combination with Adoptive Immunotherapy
Adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to tumor cells and/or antigenic components or regression of the tumor or treatment of infectious diseases, as the case can be. In accordance with the methods described herein, APC are sensitized with GRP94 preferably noncovalently complexed with antigenic (or immunogenic) molecules in conjunction with a GRP94 biological activity modulator and used in adoptive immunotherapy.
According to one embodiment of the present invention, therapy by administration of GRP94-peptide complexes and a GRP94 biological activity modulator, using any desired route of administration, is combined with adoptive immunotherapy using APC sensitized with GRP94-antigenic molecule complexes and a modulator. The sensitized APC can be administered concurrently with GRP94-peptide complexes and the modulator, or before or after administration of GRP94-peptide complexes and the modulator. Furthermore, the mode of administration can be varied, including but not limited to, e.g., subcutaneously, intravenously, intraperitoneally, intramuscularly, intradermally or mucosally.
XVI.A. Obtaining Macrophages and Antigen-Presenting Cells
The antigen-presenting cells, including but not limited to macrophages, dendritic cells and B-cells, are preferably obtained by production in vitro from stem and progenitor cells from human peripheral blood or bone marrow as described by Inaba (1992) J Exp Med 176:1693-1702.
APC can be obtained by any of various methods known in the art. In a preferred aspect human macrophages are used, obtained from human blood cells. By way of example but not limitation, macrophages can be obtained as follows: mononuclear cells are isolated from peripheral blood of a subject (preferably the subject to be treated), by Ficoll-Hypaque gradient centrifugation and are seeded on tissue culture dishes which are pre-coated with the subject's own serum or with other AB+ human serum. The cells are incubated at 37° C. for 1 hr, then non-adherent cells are removed by pipetting. To the adherent cells left in the dish, is added cold (4° C.) 1 mM EDTA in phosphate-buffered saline and the dishes are left at room temperature for 15 minutes. The cells are harvested, washed with RPMI buffer and suspended in RPMI buffer. Increased numbers of macrophages can be obtained by incubating at 37° C. with macrophage-colony stimulating factor (M-CSF); increased numbers of dendritic cells can be obtained by incubating with granulocyte-macrophage-colony stimulating factor (GM-CSF) as described in detail by Inaba, et al. (1992).
XVI.B. Sensitization of Macrophages and Antigen Presenting Cells with GRP94-Peptide Complexes
APC are sensitized with GRP94 (preferably noncovalently) bound to antigenic molecules by incubating the cells in vitro with the complexes and a modulator. The APC are sensitized with complexes of GRP94 and antigenic molecules preferably by incubating in vitro with the GRP94-complex and a modulator at 37° C. for 15 minutes to 24 hours. By way of example but not limitation, 4×107 macrophages can be incubated with 10 microgram GRP94-peptide complexes per ml or 100 microgram GRP94-peptide complexes per mL and a modulator in an equimolar amount with respect to the GRP94-peptide complex at 37° C. for 15 minutes-24 hours in 1 mL plain RPMI medium. The cells are washed three times and resuspended in a physiological medium preferably sterile, at a convenient concentration (e.g., 1×107/ml) for injection in a subject. Preferably, the subject into which the sensitized APCs are injected is the subject from which the APC were originally isolated (autologous embodiment).
Optionally, the ability of sensitized APC to stimulate, for example, the antigen-specific, class I-restricted cytotoxic T-lymphocytes (CTL) can be monitored by their ability to stimulate CTLs to release tumor necrosis factor, and by their ability to act as targets of such CTLs.
XVI.C. Reinfusion of Sensitized APC
The sensitized APC are reinfused into the subject systemically, preferably intravenously, by conventional clinical procedures. These activated cells are reinfused, preferentially by systemic administration into the autologous subject. Subjects generally receive from about 106 to about 1012 sensitized macrophages, depending on the condition of the subject. In some regimens, subjects can optionally receive in addition a suitable dosage of a biological response modifier including but not limited to the cytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF or other cytokine growth factor.
XVII. Autologous Embodiment
The specific immunogenicity of an Hsp90 protein derives not from Hsp90 protein per se, but from the peptides bound to them. In a preferred embodiment of the invention directed to the use of autologous complexes of GRP94-peptides as cancer vaccines wherein the immunogenicity has been enhanced with a modulator in accordance with the present invention, two of the most intractable hurdles to cancer immunotherapy are circumvented. First is the possibility that human cancers, like some cancers of experimental animals, are antigenically distinct. Thus, in an embodiment of the present invention, GRP94 chaperones antigenic peptides of the cancer cells from which they are derived and circumvent this hurdle.
Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines. This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. Thus, in an embodiment of the present invention directed to autologous complexes of GRP94 and peptides, preferably wherein the immunogenicity has been enhanced with a modulator of the present invention, cancer immunotherapy does not depend on the availability of cell lines or CTLs nor does it require definition of the antigenic epitopes of cancer cells. These advantages make autologous Hsp90 proteins (e.g., GRP94) noncovalently bound to peptide complexes attractive and novel immunogens against cancer.
XVIII. Prevention and Treatment of Primary and Metastatic Neoplastic Diseases
There are many reasons why immunotherapy as provided by the present invention is desired for use in cancer patients. First, if cancer patients are immunosuppressed and surgery, with anesthesia, and subsequent chemotherapy, can worsen the immunosuppression, then with appropriate immunotherapy in the preoperative period, this immunosuppression can be prevented or reversed. This could lead to fewer infectious complications and to accelerated wound healing. Second, tumor bulk is minimal following surgery and immunotherapy is most likely to be effective in this situation. A third reason is the possibility that tumor cells are shed into the circulation at surgery and effective immunotherapy applied at this time can eliminate these cells.
The preventive and therapeutic methods of the invention are directed at enhancing the immunocompetence of the cancer patient either before surgery, at or after surgery, and to induce tumor-specific immunity to cancer cells (i.e., an immune response against the cancer cells but not a non-cancerous or normal cell), with the objective being inhibition of cancer, and with the ultimate clinical objective being total cancer regression and eradication.
XIX. Monitoring of Effects During Cancer Prevention and Immunotherapy with Hsp90 Protein-Antigenic Molecule Complexes
The effect of immunotherapy with GRP94-antigenic molecule complexes on development and progression of neoplastic diseases can be monitored by any methods known to one skilled in the art, including but not limited to measuring: 1) delayed hypersensitivity as an assessment of cellular immunity; 2) activity of cytolytic T-lymphocytes in vitro; 3) levels of tumor specific antigens, e.g., carcinoembryonic (CEA) antigens; 4) changes in the morphology of tumors using techniques such as a computed tomographic (CT) scan; 5) changes in levels of putative biomarkers of risk for a particular cancer in individuals at high risk, and 6) changes in the morphology of tumors using a sonogram.
Delayed Hypersensitivity Skin Test. Delayed hypersensitivity skin tests are of great value in the overall immunocompetence and cellular immunity to an antigen. Inability to react to a battery of common skin antigens is termed anergy (Sato et al. (1995) Clin Immunol Pathol 74:35-43). Proper technique of skin testing requires that the antigens be stored sterile at 4° C., protected from light and reconstituted shortly before use. A 25- or 27-gauge needle ensures intradermal, rather than subcutaneous, administration of antigen. Twenty-four and forty-eight hours after intradermal administration of the antigen, the largest dimensions of both erythema and induration are measured with a ruler. Hypoactivity to any given antigen or group of antigens is confirmed by testing with higher concentrations of antigen or, in ambiguous circumstances, by a repeat test with an intermediate concentration.
Activity of Cytolytic T-lymphocytes In vitro. 8×106 peripheral blood derived T lymphocytes isolated by the Ficoll-Hypaque centrifugation gradient technique, are restimulated with 4×104 mitomycin C treated tumor cells in 3 ml RPMI medium containing 10% fetal calf serum. In some experiments, 33% secondary mixed lymphocyte culture supernatant or IL-2, is included in the culture medium as a source of T cell growth factors.
In order to measure the primary response of cytolytic T-lymphocytes after immunization, T cells are cultured without the stimulator tumor cells. In other experiments, T cells are restimulated with antigenically distinct cells. After six days, the cultures are tested for cytotoxicity in a 4 hour 51Cr-release assay. The spontaneous 51Cr-release of the targets should reach a level less than 20%. For the anti-MHC class I blocking activity, a tenfold concentrated supernatant of W6/32 hybridoma is added to the test at a final concentration of about 12.5% (Heike et al. (1994) J Immunotherapy 15:165-174).
Levels of Tumor Specific Antigens. Although it can not be possible to detect unique tumor antigens on all tumors, many tumors display antigens that distinguish them from normal cells. Monoclonal antibody reagents have permitted the isolation and biochemical characterization of the antigens and have been invaluable diagnostically for distinction of transformed from nontransformed cells and for definition of the cell lineage of transformed cells. The best-characterized human tumor-associated antigens are the oncofetal antigens. These antigens are expressed during embryogenesis, but are absent or very difficult to detect in normal adult tissue. The prototype antigen is carcinoembryonic antigen (CEA), a glycoprotein found on fetal gut an human colon cancer cells, but not on normal adult colon cells. Since CEA is shed from colon carcinoma cells and found in the serum, it was originally thought that the presence of this antigen in the serum could be used to screen subjects for colon cancer. However, subjects with other tumors, such as pancreatic and breast cancer, also have elevated serum levels of CEA. Therefore, monitoring the fall and rise of CEA levels in cancer patients undergoing therapy has proven useful for predicting tumor progression and responses to treatment.
Several other oncofetal antigens have been useful for diagnosing and monitoring human tumors, e.g., alpha-fetoprotein, an alpha-globulin normally secreted by fetal liver and yolk sac cells, is found in the serum of subjects with liver and germinal cell tumors and can be used as a matter of disease status.
Computed Tomographic (CT) Scan. CT remains the choice of techniques for the accurate staging of cancers. CT has proved more sensitive and specific than any other imaging techniques for the detection of metastases.
Measurement of Putative Biomarkers. The levels of a putative biomarker for risk of a specific cancer are measured to monitor the effect of GRP94 noncovalently bound to peptide complexes. For example, in individuals at enhanced risk for prostate cancer, serum prostate-specific antigen (PSA) is measured by the procedure described by Brawer et al. (1992) J Urol 147:841-845 and Catalona et al. (1993) JAMA 270:948-958; or in individuals at risk for colorectal cancer CEA is measured as described above; and in individuals at enhanced risk for breast cancer, 16-hydroxylation of estradiol is measured by the procedure described by Schneider et al. (1982) Proc Natl Acad Sci USA 79:3047-3051. The references cited above are incorporated by reference herein in their entirety.
Sonogram. A Sonogram remains an alternative choice of technique for the accurate staging of cancers.
XX. Target Disorders/Traumas Associated with Ischemia
The present invention provides methods for treating and preventing ischemia-induced damage comprising administering a Hsp90 protein modulator to a subject wherein Hsp90 activity modulation is desired. The term “ischemia”, as used herein, is a loss of blood flow to a tissue. Blood loss is characterized by deprivation of both oxygen and glucose, and leads to ischemic necrosis or infarction. Thus, the term “ischemia” refers to both conditions of oxygen deprivation and of nutrient deprivation. Loss of blood flow to a particular vascular region is described as “focal ischemia”. Loss of blood flow to an entire tissue or body is referred to as “global ischemia”.
The present invention provides therapeutic compositions and methods to ameliorate cellular damage arising from conditions of ischemia/reperfusion including but not limited to cardiac arrest, asystole and sustained ventricular arrythmias, cardiac surgery, cardiopulmonary bypass surgery, organ transplantation, spinal cord injury, head trauma, stroke, thromboembolic stroke, hemorrhagic stroke, cerebral vasospasm, hypotension, hypoglycemia, status epilepticus, an epileptic seizure, anxiety, schizophrenia, a neurodegenerative disorder, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), neonatal stress, and any condition in which a neuroprotectant composition that prevents or ameliorates ischemic cerebral damage is indicated, useful, recommended, or prescribed.
The destructive effects of ischemia/reperfusion are manifest as a cascade of deleterious events that lead to cell death and ultimately organ failure. The metabolic events underlying ischemia-induced cell death include energy failure through ATP depletion, cellular acidosis, glutamate release, calcium ion influx, stimulation of membrane phospholipid degradation and subsequent free-fatty-acid accumulation, and free radical degeneration. Further, in contrast to apoptotic cell death, ischemia-induced cell death is characterized by degeneration of the most distal cell regions, and subsequent progressive degeneration of the cell soma and nucleus (Yamamoto et al. (1986) Brain Res 384:1-10; Yamamoto et al. (1990) Acta Neuropathol 80:487-492). Consistent with this degeneration profile, cells that bear extended processes, such as neuronal cells, are particularly sensitive to ischemic damage. Although not intended to be limited according to any particular theory, these observations suggest that intracellular transport and protein availability are essential components of cellular response to stress, and further implicate molecular components of such function, including Hsp90 proteins, as targets for ischemic response.
Thus, in one embodiment, the present invention pertains to the treatment of central nervous system ischemia. Examples of central nervous system ischemia include cerebral ischemic and spinal column ischemia. “Cerebral ischemia” is the interruption or reduction of blood flow in the arteries in or leading to the brain, usually as a result of a blood clot (thrombus) or other matter (embolus) occluding the artery.
A therapeutic composition of the present invention for the prevention or amelioration of ischemia-induced damage comprises a Hsp90 protein ligand. Preferably, such modulators promote or stabilize an active structural conformation of an endogenous Hsp90 protein. Also preferably, the Hsp90 ligand modulates the activity of GRP94 or HSP90. Desired properties of a composition having a cellular protectant effect include the following: (1) easy administration by oral or injectable routes (e.g., not significantly degraded in the stomach, intestine, or vascular system such that it reaches the tissues to be treated in a therapeutically effective amount), (2) therapeutic activity (e.g., efficacy) when administered following an ischemic insult, and (3) minimal or no side effects including impairment of cognition, disruption of motor performance, sedation, hyperexcitability, neuronal vacuolization, and impaired cardiovascular activity.
Compositions comprising Hsp90 protein ligands can be administered immediately following a trauma or other event that induces an ischemic condition. Alternatively, such a composition may be administered continuously or intermittently following detection of a progressive disorder, including but not limited to neurodegenerative diseases. In still another embodiment, such a composition may be administered to prevent or improve recovery from a subsequent ischemic condition. In each case, effective dose and administration profiles can be determined using standard experiments directed at such determination in animal models of ischemic conditions as disclosed in, for example, Tacchini et al. (1997) Hepatology 26(1):186-191 and U.S. Pat. Nos. 4,968,671, 5,504,090, and 5,733,916. Exemplary animal models are described herein below.
In another embodiment, the present invention pertains to treatment of tissue prior to transplantation. Such tissue is entirely devascularized following removal from the donor body. A therapeutic composition comprising a Hsp90 protein ligand can promote recovery and health of the transplanted tissue. Several methods for providing such a compound to donor or transplanted tissue are known in the art, including but not limited to administering the therapeutic compound that promotes organ preservation and health to a donor subject prior to procurance of the organ, perfusing an isolated organ with the therapeutic composition, and administering the composition to a transplant recipient prior, concurrent, or following tissue transplantation. See Mizoe et al. (1997) J Surg Res 73(2):160-165 and U.S. Pat. Nos. 5,066,578; 5,756,492; and 6,080,730.
In still another embodiment, a composition comprising a Hsp90 protein modulator can be repetitiously provided to a subject in the absence of an ischemic condition, whereby the ability of the subject to tolerate a subsequent ischemic condition is enhanced. Therapeutic compositions comprising a Hsp90 ligand of the present invention can provide such a cellular protectant effect. Preferably, a dose of the therapeutic composition intended to induce ischemic tolerance would effect a mild ischemic condition as disclosed, for example, in Chen et al. (1996) J Cereb Blood Flow Metab 16:566-577 and U.S. Pat. Nos. 5,504,090 and 5,733,916.
XX.A. In vivo Models of Ischemia
Numerous models of ischemic injury and disease are available for evaluating the therapeutic capacity of compositions comprising Hsp90 protein modulators. In addition to animal models described herein below, see also Massa et al. (1996) “The Stress Gene Response in Brain” in Cerebrovascular and Brain Metabolism Reviews, pp. 95-158, Lippincott-Raven Publishers, Philadelphia, Pa. and references cited therein. One skilled in the art will appreciate that alternative models can be used as disclosed. To assess therapeutic capacity, candidate compounds can be administered, for example, as a single dose given intraperitoneally immediately or 30 minutes after reinstating blood flow.
Transient Global Cerebral Ischemia. U.S. Pat. No. 5,571,840 discloses a dog model of cardiac arrest. According to this model, adult dogs are anesthetised and mechanically ventilated to maintain surgical anesthesia and suppression of corneal reflexes. Expired CO2 tension and esophageal temperature are stably maintained before arrest and for at least one hour after resuscitation. Two venous catheters are inserted; one passed by way of the left external jugular vein to the right atrium for administration of resuscitation drugs, and the other into a muscular branch of the left femoral vein for fluid administration. Arterial blood pressure is measured through a catheter placed in a muscular branch of the left femoral vein for fluid administration. Arterial blood pressure is measured through a catheter placed in a muscular branch of the left femoral artery. Subcutaneous disk electrodes are placed to monitor an electrocardiogram (ECG).
Each animal is intravenously hydrated before arrest and during recovery. All catheters and electrical leads are passed subcutaneously to exit the skin in the dorsal midscapular region for later attachment to a dog jacket and hydraulic/electric swivel. Pulsatile and mean arterial blood pressure (MAP), ECG, and end-expiratory CO2 can be continuously recorded on a six-channel oscillograph. At the conclusion of surgical instrumentation, anesthesia is discontinued and ventilation proceeds with room air. When corneal reflexes are apparent, the heart is fibrillated by delivering a 10-15 second, 60 Hz, 2 msec square-wave stimulus to the left ventricular epicardium. Ventilation is discontinued and circulatory arrest is confirmed by ECG, MAP, and direct observation of the heart. After 9 minutes of normothermic ventricular fibrillation, ventilation is restored and direct cardiac massage is maintained MAP above 75 mmHg. Mechanical ventilation is continued until spontaneous ventilation ensues, but for not longer than 6 hours (typically only 30 minutes).
Conditions of stroke can be approximated by occlusion of the primary arteries to the brain. In one model, a bilateral common carotid artery occlusion is performed in the gerbil as further disclosed in Karpiak et al. (1989) Ann Rev Pharmacol Toxicol 29:403, Ginsberg & Busto (1989) Stroke 20:1627, and U.S. Pat. No. 6,017,965. Briefly, blood flow to the brain is interrupted for 7 minutes by clamping the carotid arteries. During the course of these experiments, the core body temperature of the animals is maintained at 37° C. to prevent a hypothermic reaction.
Permanent Focal Cerebral Ischemia. In another model of cerebral ischemia, the middle cerebral artery is occluded in rat as disclosed in Karpiak et al. (1989) Ann Rev Pharmacol Toxicol 29:403, Ginsberg & Busto (1989) Stroke 20:1627, Chen et al. (1996) Mol Endocrinol 10:682-693, and U.S. Pat. No. 6,017,965. According to this model, the middle cerebral artery is permanently occluded by passing a small piece of suture thread through the carotid artery to the region of the middle cerebral artery. Core body temperature is maintained at 37° C. This model is different from the bilateral common carotid artery occlusion in gerbil in eliciting a more restricted brain infarct, and thereby approximating a different kind of stroke (focal thrombotic stroke).
Transient Focal Cerebral Ischemia. In another model of focal cerebral ischemia in the rat, the middle cerebral artery is temporarily occluded by passing a small piece of suture thread through the carotid artery to the region of the middle cerebral artery. The suture thread is withdrawn after an ischemic period of 2 hours. Core body temperature is maintained at 37° C.
Additional models of focal ischemia include, but are not limited to, photochemically induced focal cerebral thrombosis, blood clot embolization, microsphere embolization and related methods. See McAuley (1995) Cerebrovasc Brain Metab Review 7:153-180.
Renal Ischemia. Adult male rats are anesthetized with phenobarbital (50 mg/kg) and the body temperature of rats is maintained between 36-37° C. Renal ischemia is induced by clamping the left renal artery for 15 minutes (mild ischemia) or 45 minutes (severe ischemia), followed by reperfusion for 5 hours, as disclosed in Kuznetsov (1996) Proc Natl Acad Sci USA 93:8584-8589.
XX.B. In vitro Models of Ischemia
Cell Culture Model of Epithelial Ischemia. Canine kidney (MDCK) cells are grown in Dulbecco's minimal essential medium supplemented with 5% fetal bovine serum. Rat thyroid (PCC13) cells are grown in Coon's modified Ham's F-12 medium (Sigma of St. Louis, Mo.) supplemented with 5% bovine calf serum and a hormone mixture as described in Grollman et al. (1993) J Biol Chem 268:3604-3609. Cultured MDCK or PCC13 cells are subjected to inhibition of oxidative metabolism by treatment with antimycin A, a specific inhibitor of mitochondrial oxidative phosphorylation as disclosed in Ramachandran & Gottlieb (1961) Biochim Biophys Acta 53:396-402. Alternatively, or in addition, the cells can be treated with 2-deoxyglucose, a nonhydrolyzble analog of glucose, to inhibit glycolytic metabolism. See Bacalloa et al. (1994) J Cell Sci 107:3301-3313, Mandel et al. (1994) J Cell Sci 107:3315-224, and Kuznetsov (1996) Proc Natl Acad Sci USA 93:8584-8589.
Cell Culture Model of Oxygen and Glucose Deprivation. Chinese hamster ovary (CHO) cells are grown in Ham's F-10 medium containing 15% newborn calf serum (GibcoBRL of Gaithersburg, Md.). Cells (5 ml) are seeded at a density of 150,000 cells per ml to T25 flasks (Corning of Acton, Mass.) and are used for experiments in a subconfluent state approximately 48 hours later. To achieve glucose deprivation, 15% serum is added to F-10 medium prepared without glucose, resulting in a partially glucose deficient broth. During incubation, cells use the remaining glucose after about 20 hours, as can be determined using a Sigma glucose colorimetric assay kit. Glucose-deprived cells are harvested after an additional 24 hours of incubation.
To achieve anoxia, cultures in fell medium (or in full medium containing 50% additional glucose) were placed in a sealed Brewer jar (Baltimore Biological Laboratory, Microbiology Systems of Baltimore, Md.) and anaerobiosis was initiated by using a hydrogen generator in a 4-7% carbon dioxide atmosphere as described previously by Anderson & Matovcik (1977) Science 197:1371-1374 and Seip & Evans (1980) J Clin Microbiol 11:226-233. The oxygen concentration in the jar is decreased to <0.4% in 100 minutes, and the concentration of oxygen at cell depth in a nonagitated solution is calculated to be within 1% of the environmental value within 30 minutes. Such a calculation can be made according to the methods described in Gerweck et al. (1979) Cancer Res 39:966-972. The formation of water vapor from hydrogen and oxygen causes a brief (about 15 minute) temperature increase to about 38.6° C. in the culture medium soon after initiation of anaerobiosis. This increase is insufficient to elicit a heat-shock response.
Anoxia can be verified using a methylene blue indicator solution. This solution becomes colorless (indicating the absence of oxygen) 5-6 hours after the initiation of anaerobiosis. A constant glucose concentration (1 g/L) can be maintained by changing the medium at 24 hours prior to and immediately prior to the initiation of anaerobiosis.
Cell Culture Model of Cerebral Ischemia. Isolated neurons can be cultured on a monolayer comprising a growth-permissive substrate, such as an immobilized monolayer of a purified, growth-promoting factor, such a monolayer comprising collagen, fibronectin, of the L1 glycoprotein. As an exemplary procedure, neurons (post-natal days 2-7) are dissociated by trypsinization essentially as described, for example, in U.S. Pat. No. 5,932,542. Neurons are added to a well coated with a growth-promoting factor, followed by addition of either a single concentration or increasing concentrations of the candidate composition. Neurons are cultured overnight (about 16 hours) at 37° C., and then neurite outgrowth is measured. Hypoxia/anoxia can be achieved as described herein above. Neurite outgrowth of cells subjected to ischemic conditions and to which a candidate therapeutic composition was administered can then be compared to neurite outgrowth on control cells also subjected to ischemic conditions without administration of a therapeutic composition.
Cell Culture Model of Glutamate-induced Oxidative Toxicity in Hippocampus. Glutamate is the major excitatory transmitter in the brain, and is proposed to play a role in epileptic pathogenesis and seizure activity. Numerous in vivo models involving different kinds of seizures and behavioral effects that are relevant for clinically distinct forms of epilepsy are known. In vitro models of glutamate-induced oxidative toxicity are also known, an exemplary procedure described herein. The mouse hippocampal cell line (Davis & Maher (1994) Brain Res 652(1):169-173) is maintained in Dulbecco's modified Eagles' medium (GibcoBRL of Gaithersburg, Md.) with 10% fetal bovine serum (Atlanta Biologicals of Atlanta, Ga.). HT22 cells are seeded onto 96-well plates at 20,000 cells per well and cultured overnight at 37° C. in normal growth medium. Glutamate-induced oxidative toxicity is elicited by administration of 2-10 mM glutamate or NMDA. Further methods are disclosed in Su et al. (1998) J Mol Cell Cardiol 30(3):587-598; Xiao et al. (1999) J Neurochem 72:95-101, and U.S. Pat. No. 6,017,965.
XX.C. Assays for Recovery Following Ischemia or Other Stress Conditions
The effects of therapeutic compositions disclosed herein, may be examined to determine potential therapeutic strategies for mitigating and/or reversing cellular damage in these animal models. Exemplary, although not limiting, measures to assess therapeutic efficacy as disclosed herein below.
Neurological Assessment Assay. Neurological deficit and recovery can be monitored using standardized scores that represent careful observation of consciousness, respiration, cranial nerve activity, spinal nerve activity, and motor function, as disclosed in U.S. Pat. No. 5,571,840. Interobserver variability can be resolved by consultation of the detailed description of each neurological function. Additional assays of cognitive, sensory, and motor impairment are disclosed in U.S. Pat. No. 6,017,965.
Infarct Size Assay. The efficacy of candidate compounds disclosed herein can also be evaluated by determination of infarct size following administration of the composition to an animal subjected to ischemic conditions. At a selected timepoint(s) following initiation of ischemic conditions, such an animal is sacrificed and processed for routine histology suitable for the tissue of interest and according to methods well-known in the art Image processing software (e.g. Bio Scan OPTIMAS of Edmonds, Wash.) can be utilized to facilitate accurate calculation of infarct volume.
Detection of Molecular Markers for Cell Degeneration. In another embodiment, damaged tissue can be identified in brain sections by immunolabeling with antibodies that recognize antigens such as Alz-50, tau, A2B5, neurofilaments, neuron-specific enolase, and others that are characteristic of neurodegeneration as disclosed in U.S. Pat. No. 6,046,381. Immunolabeled cells can be quantified using computer-aided semiquantitative analysis of confocal images.
Cell Viability Assay. When in vitro models of ischemia are employed, cell viability can be assessed by measuring cell ability to metabolize 3-(4,5-dimethyldiazol-2-yl)-2,5-dipehnyltetrazolium bromide (MTT) as described in Hansen et al. (1989) Electrophoresis 10:645-652. Briefly 10 μl of MTT solution (5 mg/ml) is added to cell cultures is 96-well plates and the cells are maintained in normal growth medium for 4 hours at 37° C. Solubilization solution (100 μl; 50% dimethylformamide and 20% sodium dodecyl sulfate, pH 4.8) is then added directly to each culture in individual wells of the 96-well plate. After an overnight incubation at room temperature, absorbance is measured.
Alternatively, cell viability can be assessed by measuring the release of lactate dehydrogenase, a cytoplasmic enzyme that is released from dying cells as disclosed in Choi et al. (1987) J Neurosci 7:357 and U.S. Pat. No. 6,017,965.
Neuronal Growth Assays. A cell culture model of neural ischemia as described herein above can be evaluated by visual examination of labeled neuronal processes, and quantitation of the length, density, and dynamicism of neuronal processes (e.g. dendrites and spines) as disclosed in Horch et al. (1999) Neuron 23:353-364 and McAllister et al. (1997) Neuron 18:767-778.
In another embodiment, molecular markers can be used to evaluate neurite growth in fixed brain tissue section. For example, brain sections derived from an animal model of ischemia can labeled using antibodies that recognize MAP-2 (a marker of neuronal cell bodies and dendrites) and for synaptophysin (a marker of presynaptic terminals). Labeled sections can be viewed on a confocal microscope and documented using computer-aided semiquantitative analysis of confocal images. The area of the neuropil occupied by MAP-2-immunolabeled dendrites or by synaptophysin-immunolabeled terminals can be quantified and expressed as a percentage of the total image area. See Masliah et al. (1992) Exp Neurol 136:107-122 and Toggas et al. (1994) Nature 367:188-193.
Additional methods for assaying neuronal growth are disclosed in Doherty et al. (1995) Neuron 14:57-66, Schnell et al. (1990) Nature 343:269-272, U.S. Pat. Nos. 5,250,414 and 5,898,066, and International PCT Publication WO 99/61585.
XXI. Disorders of Protein Transport
Protein misfolding disorders are a common component of numerous genetic disease states including, but not limited to, cystic fibrosis, familial hypercholesterolemia, retinitis pigmentosa and α1-antitrypsin misfolding. Compounds that modulate the activity of the Hsp90 family of molecular chaperones can thus be used in accordance with a therapeutic method of the present invention for reversing the protein folding defects that identify the disease state or for enhancing protein transport from the endoplasmic reticulum of a cell. Thus, a compound that modulates the conformation of GRP94 can be used to treat a disease state resulting from defects in protein transport into or from the endoplasmic reticulum. Compounds that abrogate GRP94 activity can be used for the treatment of a disease state, such as cancer, wherein a therapeutic benefit can be provided by blocking the egress of proteins (e.g., growth factors) from the endoplasmic reticulum. conversely, compounds that promote GRP94 activity can be used to treat a disease wherein a therapeutic benefit can be provided by enhancing protein export from the endoplasmic reticulum.
To assess misregulation of protein transport, a model system that measures epidermal growth factor receptor (EGF-R) levels and/or intracellular localization can be employed (Supino-Rosin et al. (2000) J Biol Chem 275(29):21850-21855). For example, the benzoquinone ansamaycin geldanamycin targets two Hsp90 molecular chaperones (Hsp90 and GRP94) and by inhibiting their activities, blocks and promotes its subsequent proteolytic degradation. In response to geldanamycin treatment, EGF-R is unable to traffic to the plasma membrane and the cell becomes refractory to stimulation by EGF.
EXAMPLES
The following Laboratory Examples have been included to illustrate preferred modes of the invention. Certain aspects of the following Laboratory Examples are described in terms of techniques and procedures found or contemplated by the present inventors to work well in the practice of the invention. These Laboratory Examples are exemplified through the use of standard laboratory practices of the inventors. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Laboratory Examples are intended to be exemplary only and that numerous changes, modifications and alterations can be employed without departing from the spirit and scope of the invention. It will be understood that various details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation—the invention being defined by the claims.
Examples 1-8
Ligand-Mediated Activation of GRP94 Molecular Chaperone Activity
The amino terminal domain of Hsp90 chaperones contains an adenosine nucleotide binding pocket that binds the Hsp90 inhibitors geldanamycin and radicicol. The following Examples 1-8 demonstrate that bis-ANS (1-1′ bis(4-anilino-5-napthalenesulfonic acid)), an environment-sensitive fluorophore that interacts with nucleotide binding sites, binds to the adenosine nucleotide binding domain of GRP94 and activates its peptide binding and molecular chaperone activities. Bis-ANS, like heat shock, elicits a tertiary conformational change in GRP94 which activates GRP94 function and is inhibited by radicicol. Confirmation of the N-terminal nucleotide-binding domain as the bis-ANS binding site was obtained by sequencing of bis-ANS-labeled GRP94 protease digestion products. These data identify a ligand-dependent, allosteric regulation of GRP94 and suggest a model for ligand-mediated regulation of GRP94 function.
Materials and Methods for Examples 1-8
Materials. Fluorescent probes were obtained from Molecular Probes (Eugene, Oreg.). Bis-ANS concentration was determined by absorbance at 385 nm (ε385=16,790 cm−1 M−1 in water). Citrate synthase (E.C. 4.1.3.7) was purchased from Boehringer Mannheim (Mannheim, Germany). Radicicol was obtained from Dr. Len Neckers, National Cancer Institute, Frederick, Md. Peptide VSV8 (RGYVYQGL—SEQ ID NO:7) was synthesized by the University of North Carolina at Chapel Hill Peptide Synthesis Facility (Chapel Hill, N.C.). Na [125I] was purchased from Amersham Pharmacia (Piscataway, N.J.). All other reagents were obtained from Sigma Chemical Co. (St. Louis, Mo.) unless otherwise indicated. GRP94 was purified from porcine pancreas as described by Wearsch & Nicchitta (1996b) Biochemistry 35:16760-16769. The concentration of GRP94 was determined by absorbance at 280 nm (1 mg/ml=1.18A280).
Fluorophore Binding Reactions. All binding reactions, with the exception of the indicated circular dichroism and citrate synthase aggregation experiments, were conducted in buffer A (110 mM KOAc, 20 mM NaCl, 2 mM Mg(OAc)2, 25 mM K-HEPES pH 7.2, 100 μM CaCl2). Fluorescent probe and radicicol stocks were prepared in dimethyl formamide at 5 mM final concentration. For all assays, control reactions at solvent dilutions identical to experimental conditions were performed to correct for any solvent effects. Where indicated, GRP94 was heat shocked by incubation in a 50° C. water bath for 15 minutes followed by cooling to 37° C.
Fluorescence Measurements. Emission spectra were obtained in a FLUOROMAXTM spectrofluorometer (SPEX Industries Inc. of Edison, N.J.) operating in photon counting mode. Spectra were recorded and processed with DM3000f operating software, version 2.1 (SPEX Industries Inc. of Edison, N.J.). For emission scans, slit width was set at 1 nm. Excitation wavelengths were as follows: Prodan, 360 nm; ANS, 372 nm; bis-ANS, 393 nm; tryptophan, 295 nm. All spectra were background corrected.
Circular Dichroism Measurements. Far-UV CD spectrometry was performed on an AVIV Associates 62DSTM circular dichroism spectrometer (AVIV Associates of Lakewood, N.J.). Samples were analyzed in a 1 mm path length quartz cuvette at 37° C. GRP94 samples (1 μM) were prepared in standard phosphate buffered saline solution as buffer A produced unacceptable dynode voltages in the relevant region of the spectrum. GRP94 was incubated with 10 M bis-ANS for 2 hours at 37° C. prior to obtaining spectra. Spectra were recorded from 300 to 195 nm. The α-helical content of GRP94 was calculated from the molar ellipticity at 222 nm. See Myers & Jakoby (1975) J Biol Chem 250:3785-3789.
Conformational Analysis by Proteolysis. The conformational state of GRP94 was assessed by tryptic digestion of the protein and subsequent SDS-PAGE analysis. For simple proteolysis experiments, 10 μl of a 0.5 mg/ml GRP94 stock, with or without prior heat shock, was combined with 1 μl bis-ANS and/or radicicol stock solutions and incubated for the indicated times at 37° C. Samples were then combined with 0.1% trypsin and digested for 30 minutes at 37° C. An equal volume of SDS-PAGE sample buffer was added and the samples were snap frozen in liquid nitrogen. Immediately prior to gel analysis, samples were thawed and boiled for 5 minutes. Samples were then separated on 12.5% SDS-polyacrylamide gels. Gels were fixed and stained with Coomassie Blue. For time course experiments, excess free bis-ANS was removed immediately prior to trypsinization by gel filtration on 0.5 ml G-25 SEPHADEX® spin columns.
Identification of the bis-ANS binding site. The bis-ANS binding region of GRP94 was identified by covalent incorporation of bis-ANS into GRP94 following bis-ANS photolysis procedures described by Sharma et al. (1998) J Biol Chem 273(25):15474-78 and Seale et al. (1998) Methods Enzymol 290:318-323. Briefly, 50 μg of GRP94 was combined with 50 M bis-ANS in a final volume of 100 μl and photo-crosslinked for 15 minutes on ice with a 366 nm hand-held UV lamp (Ultra-violet Products, Inc. of San Gabriel, Calif.). Following photocrosslinking, GRP94-bis-ANS complexes were digested with trypsin for one hour at 37° C. The trypsin-derived limit digestion products were then separated by C-18 reverse phase HPLC using a continuous acetonitrile/water gradient in 20 mM ammonium bicarbonate, with sequential detection by UV absorbance (220 nm) and fluorescence emission (excitation 418 nm; emission 498 nm). The major resultant fluorescent peak was collected and the corresponding peptide sequenced by Edman degradation on an Applied Biosystems PROCISETM model 492 automated protein sequencer.
Native Blue Electrophoresis. The oligomeric state of GRP94 was assayed by blue native polyacrylamide gel electrophoresis (BN-PAGE) as described by Schagger et al. (1994) Anal Biochem 217:220-230. GRP94 was either heat shocked or exposed to a 10-fold molar excess of bis-ANS for the indicated times. Samples were then dissolved in 15% glycerol and loaded onto 5-18% gradient gels with 0.02% Coomassie Brilliant Blue in the cathode buffer. Gels were run at 4° C., stained with Coomassie Blue, de-stained and dried.
Citrate Synthase Aggregation Assays. The effects of GRP94 on the thermal aggregation of citrate synthase were assayed by the methods described by Buchner et al. (1998) Methods Enzymol 290:323-338. Samples containing no protein, or GRP94 (1 μM), were incubated in 40 mM HEPES pH 7.5 for two hours at 37° C. with either 0.2% DMF or 10 M bis-ANS. The samples were then warmed to 43° C. for five minutes and placed in a spectrofluorometer thermostatted at 43° C. Citrate synthase was then added to 0.15 μM final concentration and the thermal aggregation of the enzyme followed by light scattering. Excitation and emission wavelengths were both 500 nm with 2 nm slit width. The time course of citrate synthase aggregation was followed for 1000 seconds.
Peptide Binding to GRP94. Iodination of VSV8 was performed by the IODOBEADSTM procedure (Pierce Chemical Co. of Chicago, Ill.), and unincorporated [125I] was removed by fractionation on a SEP-PAKTM C18 reverse-phase cartridge. Iodinated peptide was mixed with unlabeled peptide to yield a final specific activity of 6.0 Ci/mg. GRP94 (4.7 μg, final concentration 0.5 μM) was incubated with an equimolar quantity of bis-ANS in 0.1% DMF in 100 μL buffer A for 3.5 hr at 37° C. Samples were then incubated for an additional 30 min at 37° C., or heat shocked for 15 min at 50° C. and allowed to recover for 15 min at 37° C. A ten-fold molar excess of [125I]VSV8 was added (final concentration 5 μM) and the mixture incubated for 30 min at 37° C. All incubations were performed in the dark to prevent bis-ANS degradation. Samples were then eluted on 1.2-mL SEPHADEX® G-75 spin columns pre-blocked with 75 μg BSA, and [125I] was quantitated by gamma counting.
Example 1
Binding of Polarity-sensitive Fluorescent Probes to GRP94
Recent studies on the conformational regulation of GRP94 have identified a tertiary structural change that occurs in response to heat shock and is associated with an activation of peptide binding activity. See Wearsch et al. (1998) Biochemistry 37(16):5709-16, Sastry & Linderoth (1999) J Biol Chem 274:12023-12035. Coincident with the heat shock-elicited conformational change, GRP94 displays enhanced binding of environment sensitive fluorescent probes such as Nile Red, which preferentially bind to hydrophobic domains (Wearsch et al., 1998). GRP94 contains two domains of significant hydrophobicity, a C-terminal assembly domain and a highly conserved N-terminal region, which corresponds to the Hsp90 geldanamycin and adenosine nucleotide binding site. See Stebbins et al. (1997) Cell 89:239-250; and Prodromou et al. (1997) Cell 90:65-75.
To characterize the structural basis for the heat shock dependent activation of GRP94 activity, the interaction of polarity-sensitive fluorophores with native and heat shocked GRP94 was examined. The three probes tested, Prodan (6-propionyl-2-(dimethylamino)naphthalene), 8-ANS (1,8-anilinonaphthalenesulfonate) and bis-ANS (bis(1,8-anilino-naphthalenesulfonate) are structurally related probes that bind to hydrophobic sites on proteins and undergo substantial fluorescence spectrum changes upon introduction into nonpolar environments, as discussed by Rosen & Weber (1969) Biochemistry 8:3915-3920; Weber & Farris (1979) Biochemistry 18:3075-3078; Takashi et al. (1977) Proc Natl Acad Sci USA 74:2334-2338; Shi et al. (1994) Biochemistry 33:7536-7546. The following experimental protocol was utilized. GRP94 was warmed to 37° C. and either maintained at 37° C. or heat shocked for 15 minutes at 50° C., followed by incubation at 37° C. Subsequently, probe stocks were added to the GRP94 stocks and emission spectra recorded after 30 min at 37° C.
As depicted in FIG. 1A, the emission maxima of Prodan in the presence of native or heat shocked GRP94 were essentially identical, indicating that Prodan does not interact with the hydrophobic binding pocket(s) displayed by heat shocked GRP94. In contrast, the structurally related probe, 8-ANS, displays weak interactions with native GRP94, yet binds avidly following heat shock (FIG. 1B).
The interaction of bis-ANS with GRP94 was complex, and displayed a clear time dependence. As depicted in FIGS. 1C and 1D, the initial bis-ANS binding to native GRP94 was bi-phasic and following extended incubations in the presence of bis-ANS, a level of fluorophore binding similar to that seen with heat shocked GRP94 was observed. These data suggest that maximal bis-ANS binding to GRP94 required a slow structural transition. This transition further suggests a bis-ANS elicited conformational change in GRP94 and/or the bis-ANS dependent stabilization of a conformation state accessed at low frequency by the native protein.
Example 2
Analysis of bis-ANS Binding to Heat Shocked GRP94
To determine the affinity of bis-ANS for GRP94, bis-ANS was added to increasing concentrations of heat shocked GRP94, the fluorescence spectrum was determined, and the emission intensity at 475 nm plotted as a function of GRP94 concentration (FIGS. 2A and 2B). Under the experimental conditions used in this series of experiments, bis-ANS binding to GRP94 was near maximal at a 20-fold molar excess of GRP94 monomer over bis-ANS, with half maximal binding observed at 110 nM GRP94 (FIG. 2B). Importantly, these data indicate that bis-ANS binds in a saturable manner to heat shocked GRP94 and that the site(s) of bis-ANS binding to GRP94 displayed similar relative affinities for bis-ANS.
Example 3
Structural Consequences of bis-ANS Binding to GRP94
Following an extended incubation period, the emission spectra of bis-ANS bound to native GRP94 bears substantial similarity to that emission spectra of bis-ANS bound to heat shocked GRP94. Because heat shock is known to elicit a stable tertiary conformational change in GRP94 (Wearsch et al. (1998) Biochemistry 37(16):5709-16) these data suggest that the binding of bis-ANS to GRP94 induces, or stabilizes, a conformational change similar to that occurring in response to heat shock. To determine whether the GRP94 conformation seen upon addition of bis-ANS is similar to that observed following heat shock, a series of structural studies on the bis-ANS/GRP94 complex was performed.
In one series of experiments, the proteolysis patterns of native, heat shocked and bis-ANS treated GRP94 were examined. As shown in FIG. 3A, lanes 2 and 3, incubation of native GRP94 with low levels of trypsin yields two prominent proteolysis products, representing known structural domains of the protein, as described by Stebbins et al. (1997); Prodromou et al. (1997) Cell 90:65-75; Wearsch & Nicchitta (1996b) Biochemistry 35:16760-16769. In contrast, proteolysis of either bis-ANS treated or heat shocked GRP94 yields a substantially reduced recovery of the prominent proteolysis products, with the concomitant appearance of a diverse array of proteolytic fragments of higher SDS-PAGE mobility. Essentially identical proteolysis patterns were observed following either heat shock or bis-ANS treatment of HSP90.
These data provide evidence that bis-ANS binding to GRP94 elicits or stabilizes GRP94 in a conformation similar to that occurring in response to heat shock, suggesting that there exists a GRP94 conformation state that can be readily accessed and/or stabilized by either heat shock or ligand (bis-ANS) binding.
Example 4
Effects of bis-ANS Binding on GRP94 Quaternary and Secondary Structure
When purified from tissue, GRP94 exists as a homodimer, as described by Wearsch & Nicchitta (1996a) Prot Express Purif 7(1):114-21; Nemoto et al. (1996) J Biochem 120:249-256. Following heat shock however, GRP94 forms higher molecular weight complexes, as described by Wearsch et al. (1998) Biochemistry 37:5709-5719. To further characterize the effects of bis-ANS on GRP94 structure, the oligomerization states of native, heat shocked and bis-ANS treated GRP94 were assayed by the blue native polyacrylamide gel electrophoresis (BN-PAGE) technique described by Schagger et al. (1994). In these experiments, GRP94 was incubated with bis-ANS or briefly heat shocked and subsequently incubated at 37° C. The samples were then analyzed by BN-PAGE. As seen in FIG. 4, in the absence of heat shock or bis-ANS treatment the majority of GRP94 exists as a dimer with an apparent molecular weight of approximately 200 kDa. However, exposure to heat shock causes a relatively rapid formation of tetramers, hexamers, and octamers (FIG. 4, lanes 2-4). Incubation of GRP94 with a ten-fold molar excess of bis-ANS induces changes in the quaternary structure of GRP94 that mimic those seen upon heat shock (FIG. 4, lanes 4, 5). These data lend further support to the hypothesis that bis-ANS induces or stabilizes a structural transition in GRP94 that is similar to that occurring in response to heat shock.
To gain further insight into the nature of the bis-ANS dependent conformational change, GRP94 was subjected to heat shocked or treated with bis-ANS and far-UV CD spectra obtained (FIG. 5). As shown in FIG. 5, the CD spectra for native, heat shocked, and bis-ANS treated GRP94 were identical, indicating that bis-ANS binding does not alter GRP94 secondary structure.
Example 5
Radicicol Inhibits Temperature and bis-ANS Induced GRP94 Conformational Changes
Radicicol, a macrocyclic antibiotic, binds to the highly conserved N-terminal nucleotide binding pocket of HSP90 and thereby blocks HSP90 function. (Sharma et al. (1998) Oncogene 16(20):2639-45; Roe et al. (1999) J Med Chem 42:260-266). To determine if radicicol binding also influenced the structural dynamics of GRP94, the following experiments were performed. GRP94 was incubated with increasing concentrations of radicicol, heat shocked, cooled, and digested with trypsin. Subsequent SDS-PAGE analysis of the samples showed that in the presence of radicicol, GRP94 was unable to undergo the heat shock-induced structural transition, as assayed by the similarities in proteolysis patterns between native GRP94 and radicicol-treated, heat shocked GRP94. Similar inhibition of the heat shock induced structural transition of HSP90 by radicicol was also observed.
To determine if radicicol could also inhibit the bis-ANS dependent GRP94 structural transition, GRP94 was incubated with increasing concentrations of radicicol, bis-ANS was then added, and the samples were incubated for one hour. Samples were subsequently digested with trypsin and the proteolysis patterns determined by SDS-PAGE. As is depicted in FIG. 6A, radicicol, when present at a ten-fold molar excess over bis-ANS, efficiently blocked the bis-ANS-dependent GRP94 conformation change.
Though the experiment depicted in FIG. 6A indicated that radicicol was able to inhibit the appearance of the bis-ANS-dependent conformational state, it was necessary to determine if bis-ANS binding to GRP94 was blocked by radicicol treatment. To this end, the following experiment was performed. GRP94 was incubated in the presence of increasing concentrations of radicicol, subsequently heat treated under conditions sufficient to elicit efficient bis-ANS binding, and bis-ANS binding assayed. As shown in FIG. 6B, radicicol, in a dose-dependent manner, inhibited bis-ANS binding to heat-treated GRP94.
Because radicicol itself blocks the heat shock-induced conformation change, these data present two models of bis-ANS action. In one model, bis-ANS binds to the nucleotide binding domain and directly elicits the observed conformational change. Radicicol, by binding to the adenosine nucleotide binding pocket, would then be predicted to inhibit the bis-ANS-dependent conformational change. In an alternative model, GRP94 interconverts, in a temperature sensitive manner, between two conformational states, arbitrarily referred to as the open or the closed state. In the open state, bis-ANS bind and thereby stabilizes the open conformation whereas radicicol binding would stabilize the closed conformation. For both models, bis-ANS binding to the N-terminal adenosine nucleotide binding domain was predicted and was subsequently examined.
Example 6
bis-ANS Binds to the N-terminal Adenosine Nucleotide/Radicicol/Geldanamycin Binding Domain
Having determined that bis-ANS can alter the conformation of GRP94, the site of bis-ANS binding to GRP94 was targeted for identification. Irradiation of bis-ANS with UV light allows the covalent incorporation of the probe into protein binding sites, as described by Sharma et al. (1998) J Biol Chem 273(25):15474-78 and Seale et al. (1998) Methods Enzymol 290:318-323. As described in Materials and Methods, GRP94 was combined with an excess of bis-ANS and photo-crosslinked on ice for 15 minutes. GRP94 was subsequently digested with trypsin, the fluorescent peptides purified by HPLC, and the sequence of the labeled peptides determined by Edman sequencing. The major resultant fluorescent peptide yielded the sequence YSQFINFPIYV (SEQ ID NO:8), which mapped to residues 271-281 of the N-terminal domain of GRP94. This segment is homologous to the human HSP90 sequence HSQFIGYPITLFV (SEQ ID NO:9) from amino acids 210-222, and overlaps with the C-terminal region of the adenosine nucleotide/geldanamycin/radicicol binding domain (Stebbins et al. (1997) Cell 89:239-250; Prodromou et al. (1997) Cell 90:65-75).
Example 7
Bis-ANS Activates GRP94 Chaperone Activity
To determine if the bis-ANS-dependent conformational changes in GRP94 were of functional significance, the molecular chaperone activities of native, heat shocked and bis-ANS treated GRP94 were evaluated in a thermal aggregation assay, as described by Jakob et al. (1995) J Biol Chem 270:7288-7294 and Buchner et al. (1998) Methods Enzymol 290:323-338. In these experiments, citrate synthase aggregation was assayed in the presence of buffer, native GRP94, heat shocked GRP94 or GRP94 that had been previously exposed to bis-ANS for two hours. Following experimental treatment of the GRP94, reactions were equilibrated at 43° C., citrate synthase then added and aggregation, as represented by light scattering, was measured.
In the absence of GRP94, citrate synthase undergoes rapid thermal aggregation and under the experimental conditions depicted in FIG. 7A, reaches a plateau level within 15 min. In the presence of native GRP94, the degree of aggregation is reduced, suggesting that at least a fraction of the population of native GRP94 molecules are in an active conformation. Under these experimental conditions, approximately 50% of the citrate synthase aggregated. At the concentration of GRP94 used in these experiments, and assuming a stoichiometric interaction, these results indicate that roughly 8% of the native GRP94 is in the active conformation. In the presence of heat shocked or bis-ANS treated GRP94, no thermal aggregation of citrate synthase was detectable (FIG. 7A). These data indicate that the ability of GRP94 to bind to substrate proteins is enhanced by prior heat shock or bis-ANS treatment and suggest that the GRP94 conformation elicited by heat shock or bis-ANS binding represents an active state of the molecule.
Example 8
bis-ANS Activates Peptide Binding Activity to GRP94
To assess the effects of bis-ANS treatment on the peptide binding activity of GRP94, GRP94 was either treated with bis-ANS, or briefly heat shocked. A ten-fold molar excess of [125I]-VSV8 was then added and the mixture incubated for 30 min at 37° C. Free peptide was separated from bound peptide by SEPHADEX® G75 spin column chromatography and the bound peptide was quantitated by gamma counting. As shown in FIG. 7B, treatment of GRP94 with bis-ANS significantly enhanced the peptide binding activity of GRP94, yielding approximately a four to five-fold stimulation over native protein. Under similar conditions, heat shocked GRP94 displayed approximately a ten-fold stimulation of binding. From the data presented in FIGS. 7A and 7B, it is apparent that bis-ANS elicits or stabilizes a GRP94 conformation that displays markedly enhanced molecular chaperone and peptide binding activities.
Summary of Examples 1-8
Examples 1-8 demonstrate that bis-ANS binds to the conserved, N-terminal adenosine nucleotide binding domain of GRP94 and elicits a tertiary conformational change yielding markedly enhanced molecular chaperone and peptide binding activities. The binding of bis-ANS to GRP94 is bi-phasic, with an initial rapid binding phase followed by a slow, extended binding phase. In accord with these data, bis-ANS binds to and stabilizes a low abundance GRP94 conformation, referred to as the open state. In this model, GRP94 molecular chaperone and peptide binding activity is intimately coupled to such a conformation change. While it is not applicants' desire to be bound by any particularly theory or act, in the absence of regulatory ligands, access to this conformation is believed to occur in a time and temperature-dependent manner through intrinsic structural fluctuations. Inhibitory ligands, such as geldanamycin and radicicol, function by binding to and stabilizing GRP94 in a closed, or inactive, conformation.
Summarily, Examples 1-8 disclose the identification of a ligand elicited conformational change in GRP94 that is accompanied by a marked activation of molecular chaperone and peptide binding activities. The similarities between the conformations of GRP94 following heat shock activation and bis-ANS binding support the conclusion that GRP94 conformation and activity can be regulated by ligand binding to the N-terminal adenosine nucleotide binding domain and that the conformation of the protein in the bis-ANS liganded state is physiologically relevant.
Examples 9-13
Allosteric Ligand Interactions in the Adenosine Nucleotide Binding Domain of the Hsp90 Chaperone, GRP94
Examples 9-13 disclose that GRP94 and HSP90 differ in their interactions with adenosine-based ligands. GRP94 displayed high affinity saturable binding of the adenosine derivative N-ethylcarboxamido-adenosine (NECA), whereas HSP90 did not. In NECA displacement assays, GRP94 exhibited weak binding affinities for ATP, ADP, AMP, adenosine and cAMP. GRP94 ATPase activity, though present, was non-saturable with respect to ATP concentration and thus could not be characterized by traditional enzymatic criteria. Radioligand and calorimetric studies of NECA binding to GRP94 revealed that ligand binding to the nucleotide binding domain is under allosteric regulation. GRP94 is thus regulated through a ligand-based allosteric mechanism and through regulatory adenosine-based ligand(s) other than ATP.
Materials and Methods for Examples 9-13
Purification of GRP94, BiP and Hsp90. GRP94 was purified from porcine pancreas rough microsomes as described by Wearsch & Nicchitta (1996a) Prot Express Purif 7:114-121 with the following modifications. Rough microsomes were washed after the initial isolation by 10-fold dilution in 0.25M sucrose, 20 mM KOAc, 25 mM K-Hepes, pH 7.2, 5 mM Mg(OAc)2 and subsequent re-isolation by centrifugation (30 min, 40K rpm, 4° C., Ti50.2 rotor). To release the lumenal contents from the isolated rough microsomes, the microsomes were permeabilized by addition of 5 mM CHAPS and the lumenal contents were subsequently isolated by centrifugation for 2 hours at 45,000 RPM (4° C., Ti50.2 rotor).
BiP was purified by the following procedure. A lumenal protein fraction obtained from porcine pancreas rough microsomes was cycled overnight through a 1 ml ADP-agarose and a 1 ml ATP-agarose (Sigma Chemical Co. of St. Louis, Mo.) column coupled in series. The columns were then washed with 2×5 ml of a buffer containing 350 mM NaCl, 25 mM Tris, pH 7.8, 5 mM Mg2+ and the BiP was eluted from the nucleotide affinity columns with 3×5 ml of the identical buffer supplemented with 10 mM ATP and ADP. The BiP containing fractions were identified by SDS-PAGE, and dialyzed against 2×4 L of buffer A (110 mM KOAc, 20 mM NaCl, 25 mM K-Hepes, pH 7.2, 2 mM Mg(OAc)2 0.1 mM CaCl2). The protein sample was then applied to a SUPERDEX® 26/60 column (Amersham Pharmacia Biotech of Piscataway, N.J.) equilibrated in buffer A, and the BiP containing fractions, again identified by SDS-PAGE, were pooled and concentrated by centrifugal ultrafiltration (CENTRICON-30®; Amicon of Beverly, Mass.).
Hsp90 was purified from rat liver cytosol as follows. Cytosol was adjusted to 30% ammonium sulfate and stirred for 60 min on ice. The solution was centrifuged at 20,000×g in a Sorvall SS34 rotor for 15 minutes and the supernatant collected and filtered through a 0.22 μm filter. The filtered supernatant was supplemented with protease inhibitors (1 μg/ml pepstatin, 1 μg/ml leupeptin, 20 μg/ml SBTI, and 0.5 mM PMSF) and loaded onto a phenyl-SUPEROSETM HR10/10 column (Amersham Pharmacia Biotech of Piscataway, N.J.). After washing, the bound proteins were eluted with a gradient of 30-0% saturated ammonium sulfate in 10 mM Tris/HCl, pH 7.5, 1 mM EGTA, 0.5 mM DTT and the Hsp90 containing fractions were identified by SDS-PAGE. The Hsp90 containing fractions were then pooled and dialyzed 2×3 hr against 2 L of low salt buffer (10 mM NaCl, 25 mM Tris, pH 7.8). The dialyzed sample was then filtered through a 0.22 μm filter, and injected onto a MONO-QTM HR 10/10 column (Amersham Pharmacia Biotech of Piscataway, N.J.) equilibrated in low salt buffer. The column was eluted with a gradient of 10 mM-750 mM NaCl in 25 mM Tris, pH 7.8. The Hsp90-containing fractions were identified by SDS-PAGE and pooled.
Further purification was achieved by applying the MONO-QTM pool to a 4 mL hydroxylapatite column (Bio-Rad HTP of Hercules, Calif.) equilibrated in buffer B (10 mM NaH2PO4, pH 6.8, 10 mM KCl and 90 mM NaCl). The hydroxylapatite column was eluted with a 10 mM NaH2PO4 to 250 mM NaH2PO4, gradient and the Hsp90 fractions were identified by SDS-PAGE. The Hsp90 pool, in 225 mM NaH2PO4, 10 mM KCl, and 90 mM NaCl, was concentrated by centrifugal ultrafiltration (CENTRICON®-30; Amicon, Beverly, Mass.) and stored at −80° C.
[3H]-NECA Binding Assay. Nine μg of GRP94 was incubated with 20 nM [3H]-NECA (Amersham Pharmacia Biotech of Piscataway, N.J.), and various concentrations of competitors for one hour on ice in a final volume of 250 μl of 50 mM Tris, pH 7.5. Where indicated, binding reactions were performed in either buffer C (10 mM Tris, pH 7.5, 50 mM KCl, 5 mM MgCl2, 2 mM DTT, 0.01% NP-40, 20 mM Na2MoO4) or 50 mM Tris, pH 7.5, 10 mM Mg(OAc)2. Bound versus free [3H]-NECA was assayed by vacuum filtration of the binding reactions on #32 glass fiber filters (Schleicher and Schuell of Keene, N.H.), pre-treated with 0.3% polyethyleneimine (Sigma Chemical Co. of St. Louis, Mo.). Vacuum filtration was performed with an Amersham Pharmacia Biotech (Piscataway, N.J.) vacuum filtration manifold.
Filters were rapidly washed with 3×4 ml of ice cold 50 mM Tris, pH 7.5, placed in 5 ml of scintillation fluid (SAFETYSOLVETM, RPI of Mt. Prospect, Ill.), vortexed, and counted by liquid scintillation spectrometry. In experiments in which the kinetic parameters of [3H]-NECA binding to GRP94 were determined, the chemical concentration and specific activity of NECA was adjusted by addition of unlabeled NECA. All binding reactions were performed in triplicate and corrected by subtraction of background values, determined in binding reactions lacking GRP94.
ATP Binding Assay. Six μg of GRP94, BiP, and Hsp90 was incubated with 50 μM yZ[32P] ATP (1000 μCi/μmol) (Amersham Pharmacia Biotech of Piscataway, N.J.) in buffer B on ice for 1 hour. Nitrocellulose filters (BA85) (Schleicher & Schuell of Keene, N.H.) were individually wet in buffer B before use, and bound versus free [32P]-ATP was separated by vacuum filtration. Filters were washed with 3×2 mL of ice cold buffer B, placed in 5 mL of scintillation fluid, vortexed, and counted.
Isothermal Titration Calorimetry. Isothermal calorimetry experiments were performed at 25° C. using a MSC calorimeter (MicroCal Inc. of Northampton, Mass.). To determine the NECA binding parameters, two 5 μl injections were followed by twenty-three 10 μL injections from a 152 μM NECA stock. The reaction chamber (1.3 mL) contained 5 μM GRP94. Necessary corrections were made by subtracting the heats of dilution resulting from buffer addition to protein solution and ligand solution into buffer. The corrected data were then fit by the ORIGINTM software (Microcal Software, 1998) to obtain the binding parameters. The radicicol binding parameters were obtained in a similar manner with 5 μM GRP94 and 115 μM radicicol.
Phosphorylation Assays. To assay for GRP94 autophosphorylation, 1 μM GRP94 was incubated with γ-[32P]ATP (6000 cpm/pmol) (Amersham Pharmacia Biotech of Piscataway, N.J.), diluted with cold ATP to yield a final concentration of 0.15 mM ATP in a buffer containing 10 mM Mg(OAc)2 and 50 mM K-Hepes, pH 7.4. For the casein kinase assay, 1 unit of casein kinase II was incubated as described above, with the addition of 4 μM casein. Competitors were added to the appropriate samples to yield final concentrations of 180 μM NECA in 3.6% DMSO, 180 μM radicicol in 3.6% DMSO, 5 μg/ml heparin, 5 mM GTP, or 3.6% DMSO. The 25 μl reaction mixtures were incubated at 37° C. for 1 hour and quenched by addition of 10% trichloroacetic acid. Samples were analyzed by 10% SDS-PAGE gels and the phosphorylated species were quantitated using a Fuji MACBAS1000TM phosphorimaging system (Fuji Medical Systems of Stamford, Conn.).
ATPase Assay. 100 μl reactions consisting of 1 μM GRP94 monomer, various concentrations of MgATP (pH 7.0), and 50 mM K-Hepes, pH 7.4, were incubated for two hours at 37° C. Samples were then spun through a CENTRICON®-30 (Amicon of Beverly, Mass.) at 10,000 rpm, 4° C. to separate protein from nucleotide. A final concentration of 50 mM (NH4)2HPO4, pH 7.0, and 4 μM AMP, pH 7.0, was added to dilutions of the above samples and centrifuged at 15,200 rpm for 5 minutes at 4° C. 100 μL of supernatant was then fractionated on a PARTISILTM SAX column (Alltech of Deerfield, Ill.), using a Series 1050 Hewlett Packard HPLC system. Elution of nucleotides was performed by step gradient elution using a mobile phase of 150 mM (NH4)2HPO4, pH 5.2, at 1.2 ml/min for the first ten minutes, followed by 300 mM (NH4)2HPO4, pH 5.2, at a flow rate of 2 ml/min for the remainder of the elution. In this protocol, ADP and ATP were well resolved, with ADP eluting at 7 minutes and ATP at 12 minutes. Peak height values were used in calculations of percent hydrolysis and ADP formation. Spontaneous hydrolysis was determined for each ATP concentration in paired incubations lacking GRP94. The AMP was used as an internal reference standard to control for equivalent sample loading.
Tryptophan Fluorescence. Tryptophan fluorescence measurements were conducted in a FLUOROMAXTM spectrofluorometer (Spex Industries, Inc. of Edison, N.J.) with the slit widths set to 1 nm for both excitation and emission. Samples were excited at a wavelength of 295 nm and the emission spectra were recorded from 300-400 nm. All spectra were corrected by subtraction of buffer or buffer plus ligand samples. GRP94 (50 μg/ml) was incubated in buffer A supplemented with 10 mM Mg(OAc)2 and the following concentrations of ligands for 1 hour at 37° C. (50 μM NECA, 50 μM geldanamycin, 2.5 mM ATP, or 2.5 mM ADP). Samples were then cooled to room temperature, transferred to a quartz cuvette, and the spectra collected. In control experiments, free tryptophan fluorescence was not significantly influenced by the presence of any of the assayed ligands.
Example 9
Hsp90 Proteins Differ in Adenosine-based Ligand Binding Properties
To determine whether Hsp90 and GRP94 displayed distinct adenosine-ligand binding properties, the relative NECA and ATP binding activities of GRP94, Hsp90 and BiP, the endoplasmic reticulum Hsp70 paralog, were compared (FIG. 8). In these assays, purified GRP94, Hsp90 or BiP were incubated on ice for 60 min in the presence of 20 nM [3H]-NECA and the bound versus free NECA resolved by vacuum filtration. As is evident in FIG. 8, whereas GRP94 displayed readily detectable [3H]-NECA binding activity, [3H]-NECA binding was not observed for Hsp90 or BiP. In similar experiments, [3H]-NECA binding to Hsp90 was evaluated in the presence of molybdate and NP-40, which are known to stabilize the Hsp90 conformation associated with ATP binding, as described by Sullivan et al. (1997). Under these conditions, [3H]-NECA binding to Hsp90 was again not observed.
When ATP binding was assayed, BiP displayed the expected ATP binding activity whereas no ATP binding was observed to Hsp90 or GRP94. As discussed below, the inability to detect ATP binding to Hsp90 is likely a consequence of the low affinity of Hsp90 for ATP (Prodromou et al. (1997) Cell 90:65-75; Scheibel et al. (1997) J Biol Chem 272:18608-18613). In summary, these data indicate that GRP94 and Hsp90 differ in their ability to bind the adenosine-based ligand NECA, and suggest that the ligand specificity of the adenosine nucleotide binding pocket of GRP94 differs from that of Hsp90.
Example 10
Kinetic Analysis of NECA Binding to GRP94
A kinetic analysis of [3H]-NECA binding to mammalian GRP94 is depicted in FIGS. 9A and 9B. [3H]-NECA binding to GRP94 was saturable, with a Kd of 200 nM and displayed a binding stoichiometry of 0.5 mol [3]-NECA/mol GRP94 monomer. These values are similar to those observed with placental GRP94 (adenotin) by Hutchison et al. (1990) Biochemistry 29:5138-5144. A Hill plot of the binding data yielded a slope of 1.2, indicating that [3H]-NECA binding to GRP94 was not cooperative.
Structurally, GRP94 exists as a dimer of identical subunits as described by Wearsch & Nicchitta (1996a) Prot Express Purif 7:114-121; Wearsch & Nicchitta (1996b) Biochemistry 35:16760-16769; Nemoto et al. (1996) J Biochem 120:249-256). Given that the two subunits are identical, a 50% ligand occupancy at binding saturation was unexpected. The dissociation rate of NECA from GRP94 is rapid (Huttemann et al. (1984) Naunyn Schmiedebergs Arch Pharmacol. 325:226-33) and so it was considered that the observed fractional occupancy level could reflect an artifact of the method used to separate bound vs. free [3H]-NECA.
To evaluate the accuracy of the half-site occupancy value, the kinetics of NECA-GRP94 interaction were evaluated by isothermal titration calorimetry, a method that does not require the physical separation of bound and free ligand. In these experiments, illustrated in FIG. 9C, the binding stoichiometries of GRP94 for NECA and radicicol were determined. Radicicol is an antibiotic inhibitor of Hsp90 function that binds to the N-terminal nucleotide binding pocket of Hsp90 with high affinity (19 nM) and the expected binding stoichiometry of 2 mol radicicol/mol Hsp90 dimer, as proposed by Roe et al. (1999) J Med Chem 42:260-266. Analysis of NECA binding to GRP94 by isothermal titration calorimetry yielded a binding stoichiometry of 1.1 mol NECA/mol GRP94 dimer. (FIG. 9C).
Radicicol, in contrast, bound at a stoichiometry of 2 mol radicicol/mol GRP94 dimer, as shown in FIG. 9C. These data indicate that while radicicol can achieve full occupancy of the two nucleotide binding sites present in the native GRP94 dimer, other ligands, such as NECA, either bind to a single unique site on GRP94, or upon binding to one of the nucleotide binding sites, elicit a conformational change in the paired site that prevents further ligand binding.
Example 11
Specificity of Ligand Binding to the Nucleotide Binding Pocket of GRP94
To determine whether NECA bound to a single unique site on GRP94 or, alternatively, displayed half-site occupancy of the N-terminal adenosine nucleotide binding pockets, experiments were first performed to determine if NECA binds to the adenosine nucleotide binding pocket. [3H]-NECA competition assays were performed with geldanamycin and radicicol, both of which are known to bind with high affinities to the nucleotide binding pocket of Hsp90 (Roe et al. (1999) J Med Chem 42:260-266, Lawson et al. (1998) J Cell Physiol 174:170-8). The data depicted in FIG. 10A indicate that both geldanamycin and radicicol compete with [3H]-NECA for binding to GRP94 and do so with high relative affinities and in the following rank order, radicicol >geldanamycin.
As described Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156, it is difficult to detect stable binding of ATP to GRP94. Should GRP94 display a similar and quite low affinity for ATP, as reported for Hsp90 (Kd=132 μM) by Prodromou et al. (1997) Cell 90:65-75, it would be very unlikely that ATP binding could be detected by standard techniques. Given the high affinity of GRP94 for NECA, however, potential interactions of NECA with the nucleotide binding domain could be addressed by competitive displacement assays. To determine the nucleotide binding specificity of GRP94, the ability of ATP, ADP or AMP to compete with NECA binding to GRP94 was examined. In these experiments, GRP94 was incubated with 20 nM [3H]-NECA in the presence of increasing concentrations of ATP, ADP or AMP and the relative [3H]-NECA binding determined by vacuum filtration. In the presence of nominal (80 μM) Mg2+, it was observed that ATP, ADP and AMP effectively competed with [3H]-NECA for binding to GRP94.
Three points are evident from these experiments. One, because NECA binding to GRP94 can be effectively inhibited by geldanamycin, radicicol, and adenosine nucleotides, it can be concluded that NECA binds to the analogous N-terminal adenosine nucleotide binding domain of GRP94 (FIG. 10A). Two, the relative affinities of GRP94 for ATP, ADP and AMP are quite low (FIG. 10B). Thus, a 50% inhibition of [3H]-NECA binding required approximately a 1000-fold molar excess of ATP. Three, the relatively high binding affinity of GRP94 for NECA, when viewed with respect to the established molecular interactions of the adenine and ribose moieties of adenosine in the adenosine nucleotide binding pocket of Hsp90, suggest that a principal selection for ligands is made on the basis of the adenosine moiety. For this reason, the interaction of other adenosine-bearing ligands with the N-terminal nucleotide binding pocket was examined (FIG. 10C). These data indicated that cAMP and free adenosine also bound to the N-terminal adenosine nucleotide binding pocket of GRP94, with the relative displacement activity approximating that observed for ADP.
Because the data indicated that GRP94 bound adenosine, adenosine derivatives, and adenosine nucleotides with an unusually broad specificity, additional studies were performed to confirm the nucleoside specificity of these binding phenomena. In the experiment depicted in FIG. 11, the [3H]-NECA competitive displacement assay was used to address the nucleoside base specificity directly. Though GRP94 could bind both ATP and deoxyATP, little to no binding of GTP, CTP or UTP was observed. The nucleotide binding pocket of GRP94 thus appears to be strict in its selection of adenosine-bearing ligands.
In comparing the relative affinities of GRP94 for ATP and ADP, as displayed in NECA competition assays, clear differences between the ATP/ADP binding properties of GRP94 and those previously reported for Hsp90 were noted. Regarding GRP94, ATP was found to compete NECA binding with an eight-fold higher efficacy than ADP. In contrast, the N-terminal domain of Hsp90 binds ADP with a four-fold higher affinity than that observed for ATP (Prodromou et al. (1997) Cell 90:65-75). It was hypothesized that this difference was due to a lack of Mg2+ ions in the assay buffer, as Mg2+ has been demonstrated to be essential for ATP/ADP binding to recombinant forms of the Hsp90 N-terminal nucleotide binding domain by Prodromou et al. (1997) Cell 90:65-75 and Obermann et al. (1998) J Cell Biol 143:901-910.
This hypothesis was examined in experiments where the relative affinity of GRP94 for NECA, adenosine, ATP, ADP and AMP were compared in the presence and absence of excess Mg2+ (FIG. 12). In these experiments, it was observed that although excess Mg2+ was without effect on the binding of NECA or adenosine to GRP94, Mg2+ markedly stimulated the binding of ATP, ADP and AMP. These data are consistent with recent crystal structure data identifying Mg2+ interactions with the α and β phosphates as being requisite for ATP/ADP binding to the N-terminal domain of Hsp90. See Prodromou et al. (1997) Cell 90:65-75. However, unlike the N-terminal domain of Hsp90, MgATP and MgADP bind to GRP94 with nearly identical relative affinities. It should also be noted that the presence of excess Mg2+ was without effect on the relative binding affinities of cAMP and geldanamycin for GRP94.
Example 12
Nucleotide Requirement for Autophosphorylation and ATP Hydrolysis
To test whether binding to the nucleotide binding pocket is directly responsible for the observed GRP94 autophosphorylation activity, NECA and radicicol were utilized as inhibitors of ATP binding to GRP94. Data regarding autophosphorylation activities are shown in FIG. 13A. In this experiment, the autophosphorylation activity of GRP94 was assayed in the presence of NECA, radicicol, heparin and GTP. Heparin and GTP were included on the basis of previous studies indicating a casein kinase II-like contaminant in purified preparations of GRP94 (Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156; Riera et al. (1999) Mol Cell Biochem 191:97-104; and Ramakrishnan et al. (1997) J Cell Physiol 170:115-29). By similar logic, the relative effects of these compounds on GRP94 kinase activity were compared in parallel with purified casein kinase II, with casein kinase II activity measured with purified casein.
As is evident from the data presented in FIG. 13A, neither NECA nor radicicol, both of which bind to the N-terminal nucleotide binding domain of GRP94, significantly inhibit GRP94 derived or casein kinase II activity below the solvent background. Because of the relatively high hydrophobicity of NECA and radicicol, incubations containing these compounds contained significant concentrations of the ligand solvent, dimethylsulfoxide, which itself significantly reduced both the GRP94-derived and casein kinase II activities. Heparin and GTP markedly attenuated GRP94-derived and casein kinase II activity. In summary, blocking nucleotide access to the N-terminal adenosine nucleotide GRP94 binding pocket does not significantly inhibit GRP94 autophosphorylation activity.
The findings that cycles of ATP binding and hydrolysis function in the regulation of Hsp90 activity, and that GRP94 exhibits an ATPase activity suggest that GRP94 and Hsp90 are indeed regulated by a similar mechanism. To further evaluate this suggestion, the ATPase activity of GRP94 was assayed as a function of ATP concentration (FIG. 13B). Two points are immediately evident from the observed data. First, the ATPase activity does not display saturation; no evidence for a Vmax could be obtained and so traditional criteria for enzymatic function (i.e., Km/Kcat/Vmax) could not be applied. Secondly, the absolute magnitude of the ATPase activity exceeded the spontaneous rate of ATP hydrolysis by only a small factor. The observed ATPase activity was sensitive to inhibition by NECA, and thus is likely generated upon binding of ATP to the N-terminal nucleotide binding domain.
Example 13
Conformational Consequences of Adenosine Nucleotide Binding to GRP94
Having been unable to identify a functional correlate of ATP binding to GRP94, the effects of ATP, ADP, NECA and geldanamycin on GRP94 conformation were assessed. In these studies, the tryptophan emission spectra of GRP94, complexed with the indicated ligands, was examined as a measure of tertiary conformational state in accordance with techniques described by Guilbault (1967) Fluoresence: Theory, Instrumentation, and Practice, Marcel Dekker, Inc., New York, N.Y. As shown in FIG. 14, high concentrations of ATP or ADP elicited near identical changes in the GRP94 tryptophan fluorescence spectra. Significantly, in the presence of ATP or ADP, the tryptophan fluorescence was decreased, as was observed in the presence of geldanamycin. These data indicate that ATP and ADP elicit a conformational change similar to that occurring in the presence of the inhibitory ligand geldanamycin and that the conformation of GRP94 in the ATP and ADP-bound state, as assessed by tryptophan fluorescence, are essentially identical. In contrast, the addition of NECA increased the tryptophan fluorescence, indicating that ligands can elicit different conformational states in GRP94. As demonstrated in Examples 1-8 above, such changes in GRP94 conformation can have dramatic effects on GRP94 chaperone function.
Summary of Examples 9-13
Examples 9-13 disclose that Hsp90 paralogs GRP94 and HSP90 display distinct structural and functional interactions with adenosine nucleotides. Unlike HSP90, GRP94 displays specific, high affinity binding interactions with substituted adenosine derivatives such as N-ethylcarboxamidoadenosine (NECA). In analyzing such interactions, the occupancy states of the N-terminal ATP/ADP binding domains of GRP94 are communicated between the two identical subunits. This conclusion is drawn from the observation that at saturation NECA is bound to GRP94 at a stoichiometry of 1 mol NECA:mol GRP94 dimer. In contrast to NECA, the GRP94 inhibitory ligand, radicicol, binds at a stoichiometry of 2 mol:mol GRP94. Thus, although the relevant structural components of the adenosine nucleotide binding pocket are conserved between GRP94 and Hsp90, the ligand specificities of the two binding sites differ. Thus, while it is not applicants' desire to be bound by a particularly mechanistic theory, it is envisioned that the specificity of ligand binding to the N-terminal adenosine nucleotide binding pocket is influenced by the domains C and N-terminal to the binding pocket, where significant sequence divergence between HSP90 and GRP94 can be identified.
The data obtained from both traditional ligand binding studies (FIG. 9) and isothermal titration calorimetry demonstrate that GRP94 binds NECA at a stoichiometry of 1 mol NECA: mol GRP94 dimer. In addition, competition studies indicate that NECA binding to GRP94 can be wholly competed by geldanamycin, radicicol, ATP, and ADP (FIGS. 10A-10C), indicating that NECA is binding to the conserved, N-terminal adenosine nucleotide binding domain. Because GRP94 contains two such sites per molecule (Wearsch & Nicchitta (1996b) Biochemistry 35:16760-16769), it then follows that GRP94 subunits communicate with one another to confer single site occupancy.
The identification of ATP and ADP as the native ligands for the Hsp90 proteins is based on crystallographic studies identifying an N-terminal, highly conserved nucleotide binding pocket (Prodromou et al. (1997) Cell 90:65-75), complementary in vivo studies demonstrating that the amino acids that participate in ATP/ADP binding are essential for Hsp90 function in vivo and lastly (Obermann et al. (1998) J Cell Biol 143:901-910; Panaretou et al. (1998) EMBO J 17:4829-4836), that the Hsp90 proteins display ATPase activity (Grenert et al. (1999) J Biol Chem 274:17525-17533; Nadeau et al. (1993) J Biol Chem 268:1479-1487; Obermann et al. (1998) J Cell Biol 143:901-910). That HSP90 and GRP94 differ in NECA binding activity, despite the high homologies in the N-terminal nucleotide binding pockets of the two protein, suggests that differences might also exist in the ability of the two proteins to catalyze ATP hydrolysis. In fact, when the GRP94 ATPase activity was investigated at ATP concentrations appropriate for such a low affinity interaction it was observed that the GRP94 ATPase activity barely exceeded the rate of spontaneous hydrolysis and, more importantly, did not saturate at increasing ATP concentrations.
Further studies of the binding properties of the conserved domain indicated that it displays poor selectivity between adenosine nucleotides, and will bind ATP, dATP, ADP, AMP, cAMP and free adenosine. On the basis of these and other data, GRP94 conformation is regulated in an allosteric manner by an adenosine-bearing ligand other than ATP/ADP, based on ligand-mediated conformational regulation.
GRP94-dependent ATP hydrolysis, as displayed by the purified protein in the absence of any, as yet unidentified co-factors, is non-enzymatic, and therefore unlikely to contribute to the regulation of GRP94 function. Further confounding the assignment of ATP and ADP as the physiological ligands for GRP94 are the following observations. First, neither ATP nor ADP has been demonstrated to regulate GRP94 activity, as described by Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156. Secondly, that by virtue of its insensitivity to NECA and radicicol, the GRP94 autophosphorylation activity does not reflect adenosine nucleotide binding to the N-terminal nucleotide binding domain (FIG. 13). Thirdly, and perhaps most importantly, ATP, ADP, and the inhibitor geldanamycin elicit similar conformational changes in GRP94. Interestingly, in the presence of NECA, a different conformational change from that occurring in the presence of ATP, ADP, or geldanamycin was observed (FIG. 14). These data are consistent with ATP and ADP binding to GRP94 and stabilizing the protein in an inactive conformation, as is observed in the presence of geldanamycin.
In evaluating these data, the inability to identify an enzymatic basis for the ATPase activity and the conformation data suggesting that ATP/ADP would serve as inhibitory agent, either unidentified accessory proteins interact with GRP94 to substantively alter the kinetic and thermodynamic basis for its interaction with ATP/ADP or an adenosine-based ligand, other than ATP/ADP, serves as the physiological ligand. The ligand is produced during times of cell stress, such as anoxia, nutrient deprivation or heat shock, to activate GRP94 function. The ligand elicits a conformational change in GRP94 that substantively alters its interaction with substrate (poly)peptides.
Example 14
Preparation of GRP94 Ligand Binding Domain Polypeptide
Canine GRP94 69-337 (amino acids 69-337 of GENBANK® Accession No. AAA17708; residues 48-316 of SEQ ID NO: 6) was overexpressed as a GST fusion in E. coli and purified to homogeneity by affinity and ion-exchange chromatography. The protein was exchanged into 10 mM Tris-HCl, pH 7.6, 1 mM DTT, 100 mM NaCl and concentrated to 30 mg/mL. A three fold molar excess of N-ethylcarboxamidoadenosine (NECA) was added to the protein prior to crystallization.
Example 15
Crystallization of GRP94 Ligand Binding Domain Polypeptide
Crystals were obtained by the hanging drop vapor diffusion method. Two microliters of GRP94 domain complexed with a three-fold molar excess of NECA were mixed with two microliters of a reservoir solution, which typically comprised 100 mM Tris-HCl, pH 7.6, 150-250 mM MgCl2, and 33-38% Polyethylene glycol 400 (PEG 400). Drops were incubated at 18° C. Crystals typically formed in 2-3 days. Similar crystals could be obtained substituting PEG 1500 or PEG 4000 for the PEG 400. Two crystalline forms, designated herein as Crystal Form 1 and Crystal Form 2, were obtained.
Example 16
Crystal Data and Data Collection Statistics Crystal Form














Form 1
Form 2


















Space group
C2
C222(1)


Unit cell
a = 99.899 Å
a = 89.200 Å



b = 89.614 Å
b = 99.180 Å



c = 63.066 Å
c = 63.071 Å



β = 90.132
β = 90.0


asymmetric unit
2 GRP94 + NECA
1 GRP94 + NECA



complexes
complex


Resolution
1.90 Å
1.75 Å



(last shell 2.0 − 1.9 Å)
(1.81 − 1.75 Å)


Completeness
97.6% (93.4%)
99.7% (97.9%)


Rmerge
0.117 (0.293)
0.043 (0.293)


l/s(l)
17.1 (3.11)
46.9 (4.45)









Example 17
Structure Solution
Crystal Forms 1 and 2 were solved by the molecular replacement method. The search model was yeast Hsp90+ADP(PDB code 1AMW). Side chains in the search model that were not identical to the equivalent position in GRP94 were truncated to alanines.
Coordinates for Form 1 are listed in Table 1. The coordinates of the NECA and GRP94 binding pocket are listed in Table 2. Coordinates for Form 2 are listed in Table 3.
REFERENCES
The references listed below as well as all references cited in the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques and/or compositions employed herein.

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TABLE 1





ATOMIC STRUCTURE COORDINATE DATA OBTAINED FROM X-RAY


DIFFRACTION FROM FORM 1 OF THE LIGAND BINDING DOMAIN OF GRP94


(RESIDUES 48-316 OF SEQ ID NO: 6) IN COMPLEX WITH NECA


























ATOM
1
CB
LYS
A
72
16.433
13.813
40.193
1.00
72.11
A


ATOM
2
CG
LYS
A
72
15.697
13.996
41.523
1.00
73.17
A


ATOM
3
CD
LYS
A
72
14.969
12.738
41.966
1.00
74.15
A


ATOM
4
CE
LYS
A
72
14.239
12.957
43.285
1.00
74.93
A


ATOM
5
NZ
LYS
A
72
15.159
13.355
44.391
1.00
76.86
A


ATOM
6
C
LYS
A
72
15.008
12.263
38.820
1.00
70.47
A


ATOM
7
O
LYS
A
72
15.763
11.359
38.463
1.00
70.77
A


ATOM
8
N
LYS
A
72
14.412
14.682
39.050
1.00
71.64
A


ATOM
9
CA
LYS
A
72
15.525
13.694
38.960
1.00
71.44
A


ATOM
10
N
SER
A
73
13.719
12.076
39.102
1.00
68.65
A


ATOM
11
CA
SER
A
73
13.064
10.771
39.014
1.00
67.10
A


ATOM
12
CB
SER
A
73
12.943
10.149
40.409
1.00
67.99
A


ATOM
13
OG
SER
A
73
12.307
8.884
40.354
1.00
70.18
A


ATOM
14
C
SER
A
73
11.672
10.963
38.411
1.00
64.75
A


ATOM
15
O
SER
A
73
10.948
11.878
38.810
1.00
66.63
A


ATOM
16
N
GLU
A
74
11.294
10.112
37.458
1.00
59.74
A


ATOM
17
CA
GLU
A
74
9.988
10.238
36.813
1.00
56.73
A


ATOM
18
CB
GLU
A
74
10.126
11.049
35.515
1.00
59.39
A


ATOM
19
CG
GLU
A
74
8.809
11.498
34.890
1.00
63.49
A


ATOM
20
CD
GLU
A
74
8.144
12.641
35.648
1.00
67.43
A


ATOM
21
OE1
GLU
A
74
7.003
13.013
35.285
1.00
67.96
A


ATOM
22
OE2
GLU
A
74
8.762
13.172
36.599
1.00
69.08
A


ATOM
23
C
GLU
A
74
9.352
8.876
36.513
1.00
53.34
A


ATOM
24
O
GLU
A
74
10.030
7.938
36.087
1.00
48.79
A


ATOM
25
N
ALA
A
75
8.046
8.781
36.739
1.00
50.06
A


ATOM
26
CA
ALA
A
75
7.307
7.542
36.510
1.00
48.67
A


ATOM
27
CB
ALA
A
75
6.332
7.298
37.666
1.00
48.93
A


ATOM
28
C
ALA
A
75
6.551
7.586
35.188
1.00
47.67
A


ATOM
29
O
ALA
A
75
6.008
8.630
34.802
1.00
46.57
A


ATOM
30
N
PHE
A
76
6.518
6.454
34.489
1.00
43.49
A


ATOM
31
CA
PHE
A
76
5.817
6.381
33.214
1.00
42.64
A


ATOM
32
CB
PHE
A
76
6.795
6.398
32.035
1.00
43.68
A


ATOM
33
CG
PHE
A
76
7.708
7.593
31.986
1.00
43.05
A


ATOM
34
CD1
PHE
A
76
8.843
7.656
32.793
1.00
44.22
A


ATOM
35
CD2
PHE
A
76
7.456
8.630
31.088
1.00
44.40
A


ATOM
36
CE1
PHE
A
76
9.726
8.740
32.704
1.00
46.28
A


ATOM
37
CE2
PHE
A
76
8.331
9.719
30.989
1.00
44.49
A


ATOM
38
CZ
PHE
A
76
9.468
9.772
31.798
1.00
43.57
A


ATOM
39
C
PHE
A
76
4.994
5.104
33.063
1.00
43.02
A


ATOM
40
O
PHE
A
76
5.291
4.076
33.678
1.00
41.13
A


ATOM
41
N
ALA
A
77
3.971
5.181
32.222
1.00
42.30
A


ATOM
42
CA
ALA
A
77
3.153
4.021
31.909
1.00
40.65
A


ATOM
43
CB
ALA
A
77
1.682
4.393
31.872
1.00
41.85
A


ATOM
44
C
ALA
A
77
3.633
3.659
30.513
1.00
40.75
A


ATOM
45
O
ALA
A
77
4.072
4.544
29.761
1.00
36.27
A


ATOM
46
N
PHE
A
78
3.593
2.374
30.164
1.00
38.04
A


ATOM
47
CA
PHE
A
78
4.000
1.977
28.819
1.00
37.01
A


ATOM
48
CB
PHE
A
78
4.184
0.456
28.702
1.00
32.60
A


ATOM
49
CG
PHE
A
78
5.427
−0.073
29.350
1.00
31.73
A


ATOM
50
CD1
PHE
A
78
5.405
−0.544
30.668
1.00
33.46
A


ATOM
51
CD2
PHE
A
78
6.606
−0.171
28.624
1.00
30.57
A


ATOM
52
CE1
PHE
A
78
6.548
−1.115
31.246
1.00
28.34
A


ATOM
53
CE2
PHE
A
78
7.750
−0.736
29.188
1.00
28.60
A


ATOM
54
CZ
PHE
A
78
7.719
−1.211
30.503
1.00
29.55
A


ATOM
55
C
PHE
A
78
2.877
2.376
27.868
1.00
36.00
A


ATOM
56
O
PHE
A
78
1.728
2.522
28.281
1.00
36.79
A


ATOM
57
N
GLN
A
79
3.209
2.549
26.595
1.00
36.10
A


ATOM
58
CA
GLN
A
79
2.204
2.876
25.597
1.00
38.02
A


ATOM
59
CB
GLN
A
79
2.871
3.230
24.274
1.00
38.53
A


ATOM
60
CG
GLN
A
79
1.920
3.686
23.185
1.00
42.76
A


ATOM
61
CD
GLN
A
79
2.622
3.875
21.855
1.00
45.14
A


ATOM
62
OE1
GLN
A
79
3.818
4.172
21.813
1.00
45.16
A


ATOM
63
NE2
GLN
A
79
1.879
3.718
20.760
1.00
44.85
A


ATOM
64
C
GLN
A
79
1.384
1.590
25.444
1.00
38.00
A


ATOM
65
O
GLN
A
79
1.910
0.488
25.642
1.00
36.49
A


ATOM
66
N
ALA
A
80
0.109
1.732
25.110
1.00
37.16
A


ATOM
67
CA
ALA
A
80
−0.774
0.578
24.953
1.00
38.95
A


ATOM
68
CB
ALA
A
80
−2.124
1.029
24.389
1.00
38.25
A


ATOM
69
C
ALA
A
80
−0.179
−0.512
24.058
1.00
39.42
A


ATOM
70
O
ALA
A
80
−0.120
−1.684
24.444
1.00
41.54
A


ATOM
71
N
GLU
A
81
0.256
−0.126
22.862
1.00
39.30
A


ATOM
72
CA
GLU
A
81
0.820
−1.077
21.914
1.00
39.92
A


ATOM
73
CB
GLU
A
81
1.234
−0.370
20.622
1.00
43.06
A


ATOM
74
CG
GLU
A
81
0.054
0.097
19.776
1.00
48.76
A


ATOM
75
CD
GLU
A
81
−0.613
1.358
20.313
1.00
51.87
A


ATOM
76
OE1
GLU
A
81
−1.641
1.779
19.734
1.00
53.98
A


ATOM
77
OE2
GLU
A
81
−0.108
1.930
21.307
1.00
52.54
A


ATOM
78
C
GLU
A
81
1.994
−1.854
22.480
1.00
38.84
A


ATOM
79
O
GLU
A
81
2.175
−3.034
22.159
1.00
38.13
A


ATOM
80
N
VAL
A
82
2.791
−1.203
23.319
1.00
35.57
A


ATOM
81
CA
VAL
A
82
3.934
−1.869
23.925
1.00
34.33
A


ATOM
82
CB
VAL
A
82
4.868
−0.851
24.623
1.00
33.22
A


ATOM
83
CG1
VAL
A
82
5.948
−1.579
25.385
1.00
32.87
A


ATOM
84
CG2
VAL
A
82
5.513
0.069
23.575
1.00
32.40
A


ATOM
85
C
VAL
A
82
3.453
−2.936
24.921
1.00
34.76
A


ATOM
86
O
VAL
A
82
4.066
−4.003
25.047
1.00
33.56
A


ATOM
87
N
ASN
A
83
2.362
−2.658
25.628
1.00
33.27
A


ATOM
88
CA
ASN
A
83
1.832
−3.655
26.553
1.00
34.85
A


ATOM
89
CB
ASN
A
83
0.635
−3.119
27.344
1.00
34.31
A


ATOM
90
CG
ASN
A
83
1.039
−2.186
28.475
1.00
35.50
A


ATOM
91
OD1
ASN
A
83
2.087
−2.353
29.100
1.00
33.31
A


ATOM
92
ND2
ASN
A
83
0.183
−1.210
28.762
1.00
32.10
A


ATOM
93
C
ASN
A
83
1.390
−4.874
25.731
1.00
36.41
A


ATOM
94
O
ASN
A
83
1.693
−6.016
26.086
1.00
36.00
A


ATOM
95
N
ARG
A
84
0.690
−4.623
24.629
1.00
36.92
A


ATOM
96
CA
ARG
A
84
0.223
−5.693
23.741
1.00
38.05
A


ATOM
97
CB
ARG
A
84
−0.626
−5.118
22.604
1.00
40.19
A


ATOM
98
CG
ARG
A
84
−1.773
−4.231
23.077
1.00
44.63
A


ATOM
99
CD
ARG
A
84
−2.814
−3.974
21.989
1.00
47.27
A


ATOM
100
NE
ARG
A
84
−3.925
−3.180
22.520
1.00
49.29
A


ATOM
101
CZ
ARG
A
84
−4.066
−1.868
22.348
1.00
50.38
A


ATOM
102
NH1
ARG
A
84
−3.175
−1.184
21.642
1.00
50.19
A


ATOM
103
NH2
ARG
A
84
−5.088
−1.236
22.910
1.00
50.47
A


ATOM
104
C
ARG
A
84
1.414
−6.449
23.145
1.00
39.01
A


ATOM
105
O
ARG
A
84
1.393
−7.684
23.042
1.00
38.93
A


ATOM
106
N
MET
A
85
2.451
−5.703
22.762
1.00
36.19
A


ATOM
107
CA
MET
A
85
3.652
−6.287
22.182
1.00
36.75
A


ATOM
108
CB
MET
A
85
4.609
−5.184
21.693
1.00
38.82
A


ATOM
109
CG
MET
A
85
5.974
−5.703
21.210
1.00
41.23
A


ATOM
110
SD
MET
A
85
6.976
−4.519
20.233
1.00
43.17
A


ATOM
111
CE
MET
A
85
7.035
−3.128
21.369
1.00
45.81
A


ATOM
112
C
MET
A
85
4.374
−7.186
23.177
1.00
36.50
A


ATOM
113
O
MET
A
85
4.917
−8.229
22.797
1.00
37.23
A


ATOM
114
N
MET
A
86
4.393
−6.778
24.443
1.00
34.68
A


ATOM
115
CA
MET
A
86
5.052
−7.557
25.477
1.00
36.95
A


ATOM
116
CB
MET
A
86
5.041
−6.811
26.822
1.00
36.65
A


ATOM
117
CG
MET
A
86
6.019
−5.622
26.896
1.00
37.30
A


ATOM
118
SD
MET
A
86
6.348
−5.091
28.589
1.00
39.30
A


ATOM
119
CE
MET
A
86
5.046
−3.884
28.822
1.00
41.95
A


ATOM
120
C
MET
A
86
4.352
−8.905
25.617
1.00
37.51
A


ATOM
121
O
MET
A
86
5.007
−9.944
25.651
1.00
38.48
A


ATOM
122
N
ALA
A
87
3.025
−8.876
25.694
1.00
37.56
A


ATOM
123
CA
ALA
A
87
2.227
−10.097
25.833
1.00
38.50
A


ATOM
124
CB
ALA
A
87
0.752
−9.736
25.976
1.00
36.26
A


ATOM
125
C
ALA
A
87
2.429
−11.057
24.651
1.00
39.16
A


ATOM
126
O
ALA
A
87
2.533
−12.278
24.839
1.00
40.78
A


ATOM
127
N
LEU
A
88
2.485
−10.507
23.442
1.00
39.19
A


ATOM
128
CA
LEU
A
88
2.682
−11.310
22.232
1.00
41.17
A


ATOM
129
CB
LEU
A
88
2.613
−10.429
20.984
1.00
43.80
A


ATOM
130
CG
LEU
A
88
1.256
−9.839
20.609
1.00
46.16
A


ATOM
131
CD1
LEU
A
88
1.421
−8.945
19.379
1.00
48.50
A


ATOM
132
CD2
LEU
A
88
0.255
−10.968
20.337
1.00
48.36
A


ATOM
133
C
LEU
A
88
4.030
−12.015
22.254
1.00
41.23
A


ATOM
134
O
LEU
A
88
4.136
−13.199
21.935
1.00
40.24
A


ATOM
135
N
ILE
A
89
5.065
−11.276
22.629
1.00
39.12
A


ATOM
136
CA
ILE
A
89
6.410
−11.825
22.691
1.00
38.88
A


ATOM
137
CB
ILE
A
89
7.439
−10.714
22.993
1.00
37.36
A


ATOM
138
CG2
ILE
A
89
8.788
−11.323
23.330
1.00
33.69
A


ATOM
139
CG1
ILE
A
89
7.533
−9.765
21.798
1.00
36.49
A


ATOM
140
CD1
ILE
A
89
8.545
−8.630
21.990
1.00
35.13
A


ATOM
141
C
ILE
A
89
6.508
−12.893
23.766
1.00
40.48
A


ATOM
142
O
ILE
A
89
6.997
−14.004
23.523
1.00
41.38
A


ATOM
143
N
ILE
A
90
6.032
−12.551
24.953
1.00
39.66
A


ATOM
144
CA
ILE
A
90
6.071
−13.463
26.078
1.00
44.50
A


ATOM
145
CB
ILE
A
90
5.397
−12.825
27.299
1.00
45.01
A


ATOM
146
CG2
ILE
A
90
5.148
−13.876
28.374
1.00
46.20
A


ATOM
147
CG1
ILE
A
90
6.266
−11.674
27.814
1.00
47.20
A


ATOM
148
CD1
ILE
A
90
5.599
−10.830
28.891
1.00
48.97
A


ATOM
149
C
ILE
A
90
5.387
−14.789
25.744
1.00
46.87
A


ATOM
150
O
ILE
A
90
5.985
−15.857
25.896
1.00
46.46
A


ATOM
151
N
ASN
A
91
4.142
−14.709
25.283
1.00
48.34
A


ATOM
152
CA
ASN
A
91
3.369
−15.900
24.931
1.00
51.26
A


ATOM
153
CB
ASN
A
91
1.936
−15.504
24.562
1.00
53.61
A


ATOM
154
CG
ASN
A
91
1.047
−15.322
25.779
1.00
57.42
A


ATOM
155
OD1
ASN
A
91
0.704
−16.292
26.460
1.00
60.21
A


ATOM
156
ND2
ASN
A
91
0.668
−14.079
26.062
1.00
58.87
A


ATOM
157
C
ASN
A
91
3.995
−16.685
23.782
1.00
50.90
A


ATOM
158
O
ASN
A
91
3.879
−17.908
23.723
1.00
51.42
A


ATOM
159
N
SER
A
92
4.664
−15.978
22.877
1.00
50.08
A


ATOM
160
CA
SER
A
92
5.306
−16.601
21.726
1.00
49.59
A


ATOM
161
CB
SER
A
92
5.700
−15.528
20.710
1.00
52.19
A


ATOM
162
OG
SER
A
92
6.570
−16.055
19.721
1.00
55.91
A


ATOM
163
C
SER
A
92
6.540
−17.425
22.091
1.00
48.23
A


ATOM
164
O
SER
A
92
6.792
−18.476
21.495
1.00
48.01
A


ATOM
165
N
LEU
A
93
7.302
−16.946
23.071
1.00
44.77
A


ATOM
166
CA
LEU
A
93
8.523
−17.612
23.513
1.00
44.30
A


ATOM
167
CB
LEU
A
93
9.667
−16.585
23.569
1.00
44.17
A


ATOM
168
CG
LEU
A
93
9.955
−15.801
22.285
1.00
45.55
A


ATOM
169
CD1
LEU
A
93
10.928
−14.665
22.576
1.00
45.11
A


ATOM
170
CD2
LEU
A
93
10.521
−16.746
21.221
1.00
46.15
A


ATOM
171
C
LEU
A
93
8.383
−18.286
24.885
1.00
40.97
A


ATOM
172
O
LEU
A
93
9.378
−18.660
25.500
1.00
39.89
A


ATOM
173
N
TYR
A
94
7.155
−18.451
25.358
1.00
41.99
A


ATOM
174
CA
TYR
A
94
6.929
−19.049
26.670
1.00
43.74
A


ATOM
175
CB
TYR
A
94
5.430
−19.199
26.930
1.00
46.24
A


ATOM
176
CG
TYR
A
94
5.109
−19.509
28.371
1.00
51.80
A


ATOM
177
CD1
TYR
A
94
5.315
−18.555
29.372
1.00
53.42
A


ATOM
178
CE1
TYR
A
94
5.046
−18.842
30.707
1.00
55.59
A


ATOM
179
CD2
TYR
A
94
4.622
−20.762
28.742
1.00
53.62
A


ATOM
180
CE2
TYR
A
94
4.347
−21.061
30.075
1.00
55.21
A


ATOM
181
CZ
TYR
A
94
4.562
−20.099
31.053
1.00
56.87
A


ATOM
182
OH
TYR
A
94
4.304
−20.393
32.378
1.00
57.87
A


ATOM
183
C
TYR
A
94
7.621
−20.402
26.875
1.00
44.70
A


ATOM
184
O
TYR
A
94
8.070
−20.710
27.981
1.00
42.57
A


ATOM
185
N
LYS
A
95
7.723
−21.193
25.808
1.00
45.14
A


ATOM
186
CA
LYS
A
95
8.345
−22.518
25.886
1.00
46.47
A


ATOM
187
CB
LYS
A
95
7.562
−23.504
25.020
1.00
48.12
A


ATOM
188
CG
LYS
A
95
6.164
−23.818
25.523
1.00
52.83
A


ATOM
189
CD
LYS
A
95
6.210
−24.688
26.770
1.00
57.04
A


ATOM
190
CE
LYS
A
95
4.848
−25.304
27.080
1.00
59.01
A


ATOM
191
NZ
LYS
A
95
3.827
−24.288
27.471
1.00
60.76
A


ATOM
192
C
LYS
A
95
9.818
−22.558
25.484
1.00
46.81
A


ATOM
193
O
LYS
A
95
10.442
−23.628
25.479
1.00
45.94
A


ATOM
194
N
ASN
A
96
10.368
−21.396
25.147
1.00
44.69
A


ATOM
195
CA
ASN
A
96
11.766
−21.282
24.740
1.00
44.51
A


ATOM
196
CB
ASN
A
96
11.855
−21.178
23.216
1.00
47.19
A


ATOM
197
CG
ASN
A
96
11.311
−22.410
22.519
1.00
49.13
A


ATOM
198
OD1
ASN
A
96
11.959
−23.458
22.492
1.00
51.66
A


ATOM
199
ND2
ASN
A
96
10.114
−22.295
21.961
1.00
49.94
A


ATOM
200
C
ASN
A
96
12.333
−20.022
25.385
1.00
44.78
A


ATOM
201
O
ASN
A
96
12.886
−19.153
24.712
1.00
41.88
A


ATOM
202
N
LYS
A
97
12.198
−19.949
26.703
1.00
43.79
A


ATOM
203
CA
LYS
A
97
12.638
−18.792
27.471
1.00
43.72
A


ATOM
204
CB
LYS
A
97
12.351
−19.027
28.960
1.00
42.66
A


ATOM
205
CG
LYS
A
97
10.877
−19.256
29.288
1.00
43.52
A


ATOM
206
CD
LYS
A
97
10.671
−19.362
30.800
1.00
45.04
A


ATOM
207
CE
LYS
A
97
9.211
−19.237
31.205
1.00
47.13
A


ATOM
208
NZ
LYS
A
97
8.324
−20.293
30.652
1.00
46.59
A


ATOM
209
C
LYS
A
97
14.089
−18.362
27.292
1.00
43.92
A


ATOM
210
O
LYS
A
97
14.387
−17.166
27.357
1.00
43.02
A


ATOM
211
N
GLU
A
98
14.990
−19.313
27.060
1.00
41.72
A


ATOM
212
CA
GLU
A
98
16.402
−18.979
26.904
1.00
42.63
A


ATOM
213
CB
GLU
A
98
17.242
−20.245
26.693
1.00
43.53
A


ATOM
214
CG
GLU
A
98
16.859
−21.079
25.483
1.00
49.13
A


ATOM
215
CD
GLU
A
98
15.763
−22.095
25.779
1.00
51.40
A


ATOM
216
OE1
GLU
A
98
15.433
−22.891
24.872
1.00
54.63
A


ATOM
217
OE2
GLU
A
98
15.234
−22.102
26.911
1.00
51.95
A


ATOM
218
C
GLU
A
98
16.712
−17.983
25.787
1.00
40.21
A


ATOM
219
O
GLU
A
98
17.764
−17.350
25.788
1.00
40.18
A


ATOM
220
N
ILE
A
99
15.803
−17.847
24.834
1.00
40.55
A


ATOM
221
CA
ILE
A
99
16.009
−16.924
23.727
1.00
41.11
A


ATOM
222
CB
ILE
A
99
14.856
−17.053
22.703
1.00
43.78
A


ATOM
223
CG2
ILE
A
99
14.851
−15.871
21.735
1.00
44.38
A


ATOM
224
CG1
ILE
A
99
15.022
−18.364
21.921
1.00
45.95
A


ATOM
225
CD1
ILE
A
99
13.993
−18.570
20.823
1.00
46.27
A


ATOM
226
C
ILE
A
99
16.145
−15.475
24.216
1.00
40.18
A


ATOM
227
O
ILE
A
99
16.583
−14.589
23.467
1.00
37.76
A


ATOM
228
N
PHE
A
100
15.794
−15.234
25.475
1.00
38.91
A


ATOM
229
CA
PHE
A
100
15.905
−13.881
26.009
1.00
39.16
A


ATOM
230
CB
PHE
A
100
15.387
−13.800
27.455
1.00
36.23
A


ATOM
231
CG
PHE
A
100
16.407
−14.179
28.495
1.00
37.78
A


ATOM
232
CD1
PHE
A
100
16.516
−15.497
28.936
1.00
36.00
A


ATOM
233
CD2
PHE
A
100
17.264
−13.218
29.034
1.00
35.49
A


ATOM
234
CE1
PHE
A
100
17.456
−15.851
29.894
1.00
34.24
A


ATOM
235
CE2
PHE
A
100
18.210
−13.563
29.995
1.00
36.56
A


ATOM
236
CZ
PHE
A
100
18.309
−14.880
30.427
1.00
39.89
A


ATOM
237
C
PHE
A
100
17.364
−13.449
25.958
1.00
38.09
A


ATOM
238
O
PHE
A
100
17.664
−12.292
25.679
1.00
37.97
A


ATOM
239
N
LEU
A
101
18.269
−14.390
26.216
1.00
37.51
A


ATOM
240
CA
LEU
A
101
19.693
−14.087
26.219
1.00
38.15
A


ATOM
241
CB
LEU
A
101
20.480
−15.213
26.901
1.00
39.85
A


ATOM
242
CG
LEU
A
101
21.961
−14.918
27.171
1.00
41.99
A


ATOM
243
CD1
LEU
A
101
22.091
−13.710
28.100
1.00
42.72
A


ATOM
244
CD2
LEU
A
101
22.624
−16.130
27.795
1.00
43.76
A


ATOM
245
C
LEU
A
101
20.227
−13.854
24.808
1.00
37.64
A


ATOM
246
O
LEU
A
101
21.167
−13.079
24.607
1.00
36.64
A


ATOM
247
N
ARG
A
102
19.640
−14.537
23.833
1.00
35.76
A


ATOM
248
CA
ARG
A
102
20.050
−14.369
22.442
1.00
36.99
A


ATOM
249
CB
ARG
A
102
19.245
−15.298
21.533
1.00
37.21
A


ATOM
250
CG
ARG
A
102
19.738
−15.335
20.092
1.00
41.86
A


ATOM
251
CD
ARG
A
102
18.773
−16.104
19.196
1.00
41.14
A


ATOM
252
NE
ARG
A
102
17.589
−15.320
18.862
1.00
40.14
A


ATOM
253
CZ
ARG
A
102
16.555
−15.785
18.167
1.00
45.54
A


ATOM
254
NH1
ARG
A
102
16.560
−17.039
17.732
1.00
44.26
A


ATOM
255
NH2
ARG
A
102
15.515
−14.998
17.903
1.00
43.91
A


ATOM
256
C
ARG
A
102
19.770
−12.922
22.032
1.00
36.16
A


ATOM
257
O
ARG
A
102
20.607
−12.266
21.414
1.00
34.66
A


ATOM
258
N
GLU
A
103
18.584
−12.439
22.390
1.00
35.80
A


ATOM
259
CA
GLU
A
103
18.172
−11.084
22.033
1.00
36.77
A


ATOM
260
CB
GLU
A
103
16.667
−10.927
22.244
1.00
38.94
A


ATOM
261
CG
GLU
A
103
15.845
−11.797
21.293
1.00
44.14
A


ATOM
262
CD
GLU
A
103
16.277
−11.613
19.843
1.00
48.10
A


ATOM
263
OE1
GLU
A
103
16.200
−10.471
19.344
1.00
47.73
A


ATOM
264
OE2
GLU
A
103
16.706
−12.603
19.205
1.00
48.75
A


ATOM
265
C
GLU
A
103
18.933
−9.987
22.764
1.00
37.18
A


ATOM
266
O
GLU
A
103
19.260
−8.956
22.167
1.00
38.35
A


ATOM
267
N
LEU
A
104
19.213
−10.181
24.052
1.00
36.53
A


ATOM
268
CA
LEU
A
104
19.967
−9.168
24.782
1.00
34.96
A


ATOM
269
CB
LEU
A
104
20.051
−9.514
26.266
1.00
36.16
A


ATOM
270
CG
LEU
A
104
18.724
−9.575
27.021
1.00
34.61
A


ATOM
271
CD1
LEU
A
104
18.994
−9.853
28.490
1.00
36.29
A


ATOM
272
CD2
LEU
A
104
17.971
−8.254
26.855
1.00
37.01
A


ATOM
273
C
LEU
A
104
21.369
−9.081
24.183
1.00
36.79
A


ATOM
274
O
LEU
A
104
21.956
−7.995
24.098
1.00
35.24
A


ATOM
275
N
ILE
A
105
21.916
−10.223
23.765
1.00
34.90
A


ATOM
276
CA
ILE
A
105
23.241
−10.217
23.164
1.00
35.92
A


ATOM
277
CB
ILE
A
105
23.819
−11.649
23.074
1.00
36.84
A


ATOM
278
CG2
ILE
A
105
24.983
−11.688
22.095
1.00
37.43
A


ATOM
279
CG1
ILE
A
105
24.260
−12.106
24.474
1.00
39.98
A


ATOM
280
CD1
ILE
A
105
24.758
−13.546
24.536
1.00
41.86
A


ATOM
281
C
ILE
A
105
23.184
−9.559
21.781
1.00
36.00
A


ATOM
282
O
ILE
A
105
24.082
−8.795
21.415
1.00
36.27
A


ATOM
283
N
SER
A
106
22.118
−9.833
21.032
1.00
36.13
A


ATOM
284
CA
SER
A
106
21.944
−9.231
19.715
1.00
37.12
A


ATOM
285
CB
SER
A
106
20.715
−9.822
19.029
1.00
38.68
A


ATOM
286
OG
SER
A
106
20.542
−9.258
17.740
1.00
45.19
A


ATOM
287
C
SER
A
106
21.785
−7.704
19.866
1.00
37.64
A


ATOM
288
O
SER
A
106
22.343
−6.921
19.085
1.00
35.27
A


ATOM
289
N
ASN
A
107
21.038
−7.279
20.880
1.00
35.94
A


ATOM
290
CA
ASN
A
107
20.841
−5.846
21.110
1.00
38.70
A


ATOM
291
CB
ASN
A
107
19.829
−5.623
22.241
1.00
37.51
A


ATOM
292
CG
ASN
A
107
18.406
−5.947
21.826
1.00
36.75
A


ATOM
293
OD1
ASN
A
107
17.509
−6.028
22.667
1.00
38.88
A


ATOM
294
ND2
ASN
A
107
18.188
−6.120
20.531
1.00
32.53
A


ATOM
295
C
ASN
A
107
22.168
−5.161
21.457
1.00
38.73
A


ATOM
296
O
ASN
A
107
22.425
−4.029
21.030
1.00
38.05
A


ATOM
297
N
ALA
A
108
23.010
−5.854
22.224
1.00
37.25
A


ATOM
298
CA
ALA
A
108
24.306
−5.319
22.616
1.00
37.11
A


ATOM
299
CB
ALA
A
108
24.944
−6.218
23.689
1.00
35.32
A


ATOM
300
C
ALA
A
108
25.208
−5.220
21.384
1.00
38.35
A


ATOM
301
O
ALA
A
108
25.949
−4.248
21.219
1.00
36.53
A


ATOM
302
N
SER
A
109
25.136
−6.232
20.524
1.00
37.79
A


ATOM
303
CA
SER
A
109
25.918
−6.266
19.290
1.00
39.56
A


ATOM
304
CB
SER
A
109
25.660
−7.580
18.544
1.00
40.09
A


ATOM
305
OG
SER
A
109
26.349
−7.615
17.309
1.00
43.33
A


ATOM
306
C
SER
A
109
25.542
−5.078
18.397
1.00
38.52
A


ATOM
307
O
SER
A
109
26.413
−4.450
17.787
1.00
40.46
A


ATOM
308
N
ASP
A
110
24.249
−4.776
18.322
1.00
37.55
A


ATOM
309
CA
ASP
A
110
23.765
−3.645
17.523
1.00
38.97
A


ATOM
310
CB
ASP
A
110
22.236
−3.578
17.545
1.00
39.76
A


ATOM
311
CG
ASP
A
110
21.580
−4.651
16.686
1.00
42.87
A


ATOM
312
OD1
ASP
A
110
20.344
−4.769
16.743
1.00
41.85
A


ATOM
313
OD2
ASP
A
110
22.290
−5.373
15.956
1.00
46.31
A


ATOM
314
C
ASP
A
110
24.327
−2.326
18.060
1.00
36.57
A


ATOM
315
O
ASP
A
110
24.750
−1.469
17.284
1.00
37.95
A


ATOM
316
N
ALA
A
111
24.315
−2.173
19.385
1.00
36.03
A


ATOM
317
CA
ALA
A
111
24.827
−0.969
20.047
1.00
35.99
A


ATOM
318
CB
ALA
A
111
24.577
−1.047
21.560
1.00
34.35
A


ATOM
319
C
ALA
A
111
26.315
−0.808
19.779
1.00
36.64
A


ATOM
320
O
ALA
A
111
26.801
0.310
19.586
1.00
34.89
A


ATOM
321
N
LEU
A
112
27.043
−1.926
19.787
1.00
35.89
A


ATOM
322
CA
LEU
A
112
28.472
−1.904
19.518
1.00
37.87
A


ATOM
323
CB
LEU
A
112
29.104
−3.260
19.871
1.00
37.98
A


ATOM
324
CG
LEU
A
112
29.277
−3.464
21.385
1.00
38.68
A


ATOM
325
CD1
LEU
A
112
29.498
−4.929
21.706
1.00
40.90
A


ATOM
326
CD2
LEU
A
112
30.442
−2.617
21.877
1.00
38.61
A


ATOM
327
C
LEU
A
112
28.734
−1.540
18.052
1.00
38.56
A


ATOM
328
O
LEU
A
112
29.636
−0.752
17.763
1.00
38.58
A


ATOM
329
N
ASP
A
113
27.944
−2.096
17.132
1.00
38.47
A


ATOM
330
CA
ASP
A
113
28.105
−1.775
15.710
1.00
40.19
A


ATOM
331
CB
ASP
A
113
27.090
−2.523
14.836
1.00
41.72
A


ATOM
332
CG
ASP
A
113
27.414
−3.997
14.665
1.00
44.88
A


ATOM
333
OD1
ASP
A
113
28.606
−4.350
14.569
1.00
46.86
A


ATOM
334
OD2
ASP
A
113
26.463
−4.804
14.592
1.00
47.83
A


ATOM
335
C
ASP
A
113
27.866
−0.279
15.509
1.00
40.20
A


ATOM
336
O
ASP
A
113
28.556
0.376
14.724
1.00
40.35
A


ATOM
337
N
LYS
A
114
26.880
0.251
16.227
1.00
39.26
A


ATOM
338
CA
LYS
A
114
26.517
1.656
16.109
1.00
41.87
A


ATOM
339
CB
LYS
A
114
25.252
1.939
16.927
1.00
45.64
A


ATOM
340
CG
LYS
A
114
24.537
3.228
16.533
1.00
51.03
A


ATOM
341
CD
LYS
A
114
23.068
3.219
16.963
1.00
54.21
A


ATOM
342
CE
LYS
A
114
22.332
4.453
16.434
1.00
56.69
A


ATOM
343
NZ
LYS
A
114
20.871
4.458
16.760
1.00
58.33
A


ATOM
344
C
LYS
A
114
27.648
2.591
16.523
1.00
40.73
A


ATOM
345
O
LYS
A
114
27.992
3.512
15.781
1.00
40.09
A


ATOM
346
N
ILE
A
115
28.233
2.361
17.696
1.00
39.71
A


ATOM
347
CA
ILE
A
115
29.333
3.214
18.138
1.00
39.52
A


ATOM
348
CB
ILE
A
115
29.692
2.974
19.630
1.00
38.85
A


ATOM
349
CG2
ILE
A
115
30.385
1.614
19.809
1.00
39.36
A


ATOM
350
CG1
ILE
A
115
30.589
4.113
20.133
1.00
40.22
A


ATOM
351
CD1
ILE
A
115
29.897
5.482
20.136
1.00
38.97
A


ATOM
352
C
ILE
A
115
30.561
2.976
17.252
1.00
40.05
A


ATOM
353
O
ILE
A
115
31.352
3.890
17.018
1.00
40.80
A


ATOM
354
N
ARG
A
116
30.728
1.761
16.739
1.00
40.94
A


ATOM
355
CA
ARG
A
116
31.874
1.517
15.864
1.00
45.55
A


ATOM
356
CB
ARG
A
116
32.011
0.038
15.494
1.00
47.82
A


ATOM
357
CG
ARG
A
116
33.361
−0.259
14.851
1.00
53.66
A


ATOM
358
CD
ARG
A
116
33.292
−1.334
13.780
1.00
58.47
A


ATOM
359
NE
ARG
A
116
33.135
−2.680
14.322
1.00
63.09
A


ATOM
360
CZ
ARG
A
116
33.059
−3.775
13.570
1.00
64.30
A


ATOM
361
NH1
ARG
A
116
33.128
−3.676
12.248
1.00
64.89
A


ATOM
362
NH2
ARG
A
116
32.914
−4.968
14.136
1.00
64.89
A


ATOM
363
C
ARG
A
116
31.728
2.353
14.580
1.00
44.91
A


ATOM
364
O
ARG
A
116
32.699
2.927
14.099
1.00
45.70
A


ATOM
365
N
LEU
A
117
30.516
2.432
14.039
1.00
45.28
A


ATOM
366
CA
LEU
A
117
30.284
3.221
12.832
1.00
45.31
A


ATOM
367
CB
LEU
A
117
28.890
2.944
12.262
1.00
47.48
A


ATOM
368
CG
LEU
A
117
28.578
1.500
11.848
1.00
48.18
A


ATOM
369
CD1
LEU
A
117
27.137
1.415
11.360
1.00
49.37
A


ATOM
370
CD2
LEU
A
117
29.544
1.045
10.760
1.00
50.59
A


ATOM
371
C
LEU
A
117
30.426
4.714
13.144
1.00
45.31
A


ATOM
372
O
LEU
A
117
30.942
5.485
12.331
1.00
42.68
A


ATOM
373
N
ILE
A
118
29.970
5.125
14.320
1.00
43.80
A


ATOM
374
CA
ILE
A
118
30.083
6.528
14.690
1.00
44.18
A


ATOM
375
CB
ILE
A
118
29.377
6.823
16.026
1.00
43.51
A


ATOM
376
CG2
ILE
A
118
29.654
8.267
16.449
1.00
42.87
A


ATOM
377
CG1
ILE
A
118
27.870
6.596
15.878
1.00
43.94
A


ATOM
378
CD1
ILE
A
118
27.123
6.562
17.205
1.00
44.35
A


ATOM
379
C
ILE
A
118
31.552
6.924
14.807
1.00
43.76
A


ATOM
380
O
ILE
A
118
31.924
8.055
14.479
1.00
42.32
A


ATOM
381
N
SER
A
119
32.390
5.990
15.257
1.00
44.58
A


ATOM
382
CA
SER
A
119
33.817
6.267
15.426
1.00
44.85
A


ATOM
383
CB
SER
A
119
34.538
5.074
16.073
1.00
45.06
A


ATOM
384
OG
SER
A
119
34.815
4.048
15.132
1.00
46.75
A


ATOM
385
C
SER
A
119
34.485
6.602
14.092
1.00
45.54
A


ATOM
386
O
SER
A
119
35.589
7.140
14.070
1.00
44.02
A


ATOM
387
N
LEU
A
120
33.824
6.268
12.986
1.00
45.74
A


ATOM
388
CA
LEU
A
120
34.370
6.559
11.663
1.00
49.20
A


ATOM
389
CB
LEU
A
120
33.709
5.678
10.594
1.00
48.81
A


ATOM
390
CG
LEU
A
120
33.841
4.154
10.658
1.00
50.97
A


ATOM
391
CD1
LEU
A
120
32.957
3.545
9.586
1.00
51.32
A


ATOM
392
CD2
LEU
A
120
35.289
3.727
10.460
1.00
51.93
A


ATOM
393
C
LEU
A
120
34.145
8.027
11.297
1.00
49.95
A


ATOM
394
O
LEU
A
120
34.862
8.578
10.461
1.00
50.69
A


ATOM
395
N
THR
A
121
33.147
8.648
11.922
1.00
51.96
A


ATOM
396
CA
THR
A
121
32.809
10.045
11.653
1.00
54.50
A


ATOM
397
CB
THR
A
121
31.312
10.212
11.324
1.00
54.43
A


ATOM
398
OG1
THR
A
121
30.526
9.930
12.491
1.00
55.71
A


ATOM
399
CG2
THR
A
121
30.906
9.273
10.203
1.00
54.95
A


ATOM
400
C
THR
A
121
33.123
10.985
12.814
1.00
56.48
A


ATOM
401
O
THR
A
121
33.056
12.204
12.663
1.00
57.04
A


ATOM
402
N
ASP
A
122
33.451
10.421
13.971
1.00
57.38
A


ATOM
403
CA
ASP
A
122
33.771
11.230
15.143
1.00
58.24
A


ATOM
404
CB
ASP
A
122
32.552
11.301
16.070
1.00
59.87
A


ATOM
405
CG
ASP
A
122
32.836
12.036
17.369
1.00
61.77
A


ATOM
406
OD1
ASP
A
122
33.541
13.071
17.343
1.00
62.10
A


ATOM
407
OD2
ASP
A
122
32.337
11.577
18.419
1.00
62.22
A


ATOM
408
C
ASP
A
122
34.978
10.632
15.858
1.00
57.50
A


ATOM
409
O
ASP
A
122
34.886
9.570
16.474
1.00
56.22
A


ATOM
410
N
ALA
A
123
36.109
11.328
15.757
1.00
56.69
A


ATOM
411
CA
ALA
A
123
37.371
10.892
16.352
1.00
56.90
A


ATOM
412
CB
ALA
A
123
38.488
11.853
15.946
1.00
57.71
A


ATOM
413
C
ALA
A
123
37.359
10.733
17.870
1.00
57.14
A


ATOM
414
O
ALA
A
123
38.174
9.994
18.420
1.00
57.29
A


ATOM
415
N
ASN
A
124
36.454
11.429
18.550
1.00
57.40
A


ATOM
416
CA
ASN
A
124
36.370
11.336
20.009
1.00
57.97
A


ATOM
417
CB
ASN
A
124
36.222
12.732
20.625
1.00
60.18
A


ATOM
418
CG
ASN
A
124
37.458
13.592
20.429
1.00
62.71
A


ATOM
419
OD1
ASN
A
124
37.791
13.980
19.308
1.00
64.70
A


ATOM
420
ND2
ASN
A
124
38.148
13.890
21.522
1.00
63.57
A


ATOM
421
C
ASN
A
124
35.204
10.461
20.466
1.00
56.61
A


ATOM
422
O
ASN
A
124
34.810
10.496
21.627
1.00
56.62
A


ATOM
423
N
ALA
A
125
34.664
9.666
19.550
1.00
55.85
A


ATOM
424
CA
ALA
A
125
33.533
8.799
19.856
1.00
54.44
A


ATOM
425
CB
ALA
A
125
33.096
8.065
18.598
1.00
54.60
A


ATOM
426
C
ALA
A
125
33.774
7.795
20.982
1.00
53.61
A


ATOM
427
O
ALA
A
125
32.885
7.556
21.795
1.00
52.10
A


ATOM
428
N
LEU
A
126
34.969
7.213
21.035
1.00
53.75
A


ATOM
429
CA
LEU
A
126
35.281
6.210
22.059
1.00
54.69
A


ATOM
430
CB
LEU
A
126
36.083
5.069
21.428
1.00
54.14
A


ATOM
431
CG
LEU
A
126
35.413
4.371
20.242
1.00
55.12
A


ATOM
432
CD1
LEU
A
126
36.371
3.362
19.625
1.00
54.89
A


ATOM
433
CD2
LEU
A
126
34.136
3.687
20.708
1.00
55.04
A


ATOM
434
C
LEU
A
126
36.031
6.729
23.285
1.00
54.49
A


ATOM
435
O
LEU
A
126
36.515
5.939
24.098
1.00
54.80
A


ATOM
436
N
ALA
A
127
36.111
8.050
23.417
1.00
54.59
A


ATOM
437
CA
ALA
A
127
36.813
8.696
24.525
1.00
55.79
A


ATOM
438
CB
ALA
A
127
36.791
10.214
24.328
1.00
55.81
A


ATOM
439
C
ALA
A
127
36.283
8.352
25.918
1.00
56.37
A


ATOM
440
O
ALA
A
127
37.032
8.393
26.897
1.00
56.95
A


ATOM
441
N
GLY
A
128
34.998
8.024
26.012
1.00
56.51
A


ATOM
442
CA
GLY
A
128
34.410
7.695
27.302
1.00
56.45
A


ATOM
443
C
GLY
A
128
34.758
6.303
27.794
1.00
56.02
A


ATOM
444
O
GLY
A
128
34.720
6.031
28.992
1.00
56.77
A


ATOM
445
N
ASN
A
129
35.095
5.421
26.861
1.00
56.46
A


ATOM
446
CA
ASN
A
129
35.460
4.046
27.175
1.00
56.64
A


ATOM
447
CB
ASN
A
129
34.206
3.251
27.569
1.00
56.17
A


ATOM
448
CG
ASN
A
129
34.530
1.877
28.136
1.00
56.13
A


ATOM
449
OD1
ASN
A
129
33.748
1.311
28.898
1.00
56.76
A


ATOM
450
ND2
ASN
A
129
35.677
1.330
27.755
1.00
54.99
A


ATOM
451
C
ASN
A
129
36.093
3.491
25.904
1.00
57.41
A


ATOM
452
O
ASN
A
129
35.448
3.416
24.861
1.00
58.38
A


ATOM
453
N
GLU
A
130
37.359
3.104
25.991
1.00
58.34
A


ATOM
454
CA
GLU
A
130
38.081
2.605
24.826
1.00
59.34
A


ATOM
455
CB
GLU
A
130
39.585
2.730
25.075
1.00
62.83
A


ATOM
456
CG
GLU
A
130
40.046
4.156
25.346
1.00
66.60
A


ATOM
457
CD
GLU
A
130
39.839
5.077
24.158
1.00
68.82
A


ATOM
458
OE1
GLU
A
130
40.121
6.289
24.286
1.00
70.09
A


ATOM
459
OE2
GLU
A
130
39.398
4.590
23.094
1.00
70.56
A


ATOM
460
C
GLU
A
130
37.766
1.190
24.353
1.00
57.95
A


ATOM
461
O
GLU
A
130
38.109
0.827
23.230
1.00
59.19
A


ATOM
462
N
ALA
A
131
37.112
0.389
25.184
1.00
56.39
A


ATOM
463
CA
ALA
A
131
36.802
−0.986
24.795
1.00
54.28
A


ATOM
464
CB
ALA
A
131
36.816
−1.888
26.031
1.00
55.68
A


ATOM
465
C
ALA
A
131
35.478
−1.160
24.050
1.00
52.74
A


ATOM
466
O
ALA
A
131
34.568
−0.340
24.172
1.00
52.73
A


ATOM
467
N
LEU
A
132
35.393
−2.238
23.271
1.00
49.97
A


ATOM
468
CA
LEU
A
132
34.193
−2.592
22.511
1.00
48.60
A


ATOM
469
CB
LEU
A
132
34.429
−2.403
21.010
1.00
49.50
A


ATOM
470
CG
LEU
A
132
34.704
−0.959
20.585
1.00
50.36
A


ATOM
471
CD1
LEU
A
132
35.029
−0.908
19.098
1.00
52.14
A


ATOM
472
CD2
LEU
A
132
33.491
−0.091
20.914
1.00
48.99
A


ATOM
473
C
LEU
A
132
33.927
−4.065
22.822
1.00
48.59
A


ATOM
474
O
LEU
A
132
34.354
−4.960
22.082
1.00
46.69
A


ATOM
475
N
THR
A
133
33.217
−4.308
23.922
1.00
45.79
A


ATOM
476
CA
THR
A
133
32.937
−5.672
24.368
1.00
45.37
A


ATOM
477
CB
THR
A
133
33.929
−6.090
25.464
1.00
46.45
A


ATOM
478
OG1
THR
A
133
33.769
−5.206
26.583
1.00
47.94
A


ATOM
479
CG2
THR
A
133
35.372
−6.010
24.974
1.00
48.42
A


ATOM
480
C
THR
A
133
31.554
−5.867
24.986
1.00
44.45
A


ATOM
481
O
THR
A
133
30.838
−4.903
25.279
1.00
41.44
A


ATOM
482
N
VAL
A
134
31.213
−7.138
25.196
1.00
43.48
A


ATOM
483
CA
VAL
A
134
29.969
−7.550
25.842
1.00
43.09
A


ATOM
484
CB
VAL
A
134
29.083
−8.419
24.926
1.00
43.69
A


ATOM
485
CG1
VAL
A
134
27.858
−8.883
25.696
1.00
44.19
A


ATOM
486
CG2
VAL
A
134
28.652
−7.632
23.691
1.00
42.76
A


ATOM
487
C
VAL
A
134
30.424
−8.421
27.014
1.00
44.48
A


ATOM
488
O
VAL
A
134
31.087
−9.450
26.812
1.00
43.22
A


ATOM
489
N
LYS
A
135
30.081
−8.012
28.231
1.00
43.24
A


ATOM
490
CA
LYS
A
135
30.475
−8.756
29.418
1.00
43.72
A


ATOM
491
CB
LYS
A
135
31.421
−7.912
30.273
1.00
44.84
A


ATOM
492
CG
LYS
A
135
32.751
−7.639
29.585
1.00
46.03
A


ATOM
493
CD
LYS
A
135
33.645
−6.710
30.389
1.00
47.51
A


ATOM
494
CE
LYS
A
135
35.024
−6.599
29.741
1.00
48.30
A


ATOM
495
NZ
LYS
A
135
35.943
−5.750
30.545
1.00
48.86
A


ATOM
496
C
LYS
A
135
29.269
−9.190
30.234
1.00
43.79
A


ATOM
497
O
LYS
A
135
28.378
−8.394
30.525
1.00
41.89
A


ATOM
498
N
ILE
A
136
29.251
−10.463
30.603
1.00
44.62
A


ATOM
499
CA
ILE
A
136
28.140
−11.008
31.366
1.00
45.53
A


ATOM
500
CB
ILE
A
136
27.408
−12.072
30.535
1.00
46.56
A


ATOM
501
CG2
ILE
A
136
26.293
−12.717
31.354
1.00
47.11
A


ATOM
502
CG1
ILE
A
136
26.849
−11.426
29.264
1.00
46.22
A


ATOM
503
CD1
ILE
A
136
26.351
−12.418
28.240
1.00
49.64
A


ATOM
504
C
ILE
A
136
28.601
−11.627
32.678
1.00
46.73
A


ATOM
505
O
ILE
A
136
29.626
−12.306
32.726
1.00
46.05
A


ATOM
506
N
LYS
A
137
27.850
−11.374
33.744
1.00
47.82
A


ATOM
507
CA
LYS
A
137
28.169
−11.943
35.045
1.00
50.85
A


ATOM
508
CB
LYS
A
137
29.052
−10.997
35.871
1.00
53.80
A


ATOM
509
CG
LYS
A
137
28.679
−9.522
35.844
1.00
56.43
A


ATOM
510
CD
LYS
A
137
29.643
−8.729
36.725
1.00
58.40
A


ATOM
511
CE
LYS
A
137
29.651
−7.238
36.394
1.00
60.49
A


ATOM
512
NZ
LYS
A
137
28.329
−6.579
36.598
1.00
61.87
A


ATOM
513
C
LYS
A
137
26.924
−12.326
35.838
1.00
51.75
A


ATOM
514
O
LYS
A
137
25.897
−11.648
35.791
1.00
49.24
A


ATOM
515
N
CYS
A
138
27.028
−13.440
36.553
1.00
52.27
A


ATOM
516
CA
CYS
A
138
25.933
−13.944
37.366
1.00
53.85
A


ATOM
517
CB
CYS
A
138
25.862
−15.474
37.269
1.00
54.76
A


ATOM
518
SG
CYS
A
138
25.881
−16.147
35.591
1.00
57.29
A


ATOM
519
C
CYS
A
138
26.204
−13.551
38.815
1.00
53.98
A


ATOM
520
O
CYS
A
138
27.355
−13.475
39.241
1.00
53.90
A


ATOM
521
N
ASP
A
139
25.145
−13.286
39.565
1.00
54.37
A


ATOM
522
CA
ASP
A
139
25.290
−12.938
40.971
1.00
55.78
A


ATOM
523
CB
ASP
A
139
25.045
−11.446
41.197
1.00
56.31
A


ATOM
524
CG
ASP
A
139
25.455
−11.002
42.579
1.00
57.02
A


ATOM
525
OD1
ASP
A
139
25.008
−11.636
43.556
1.00
56.63
A


ATOM
526
OD2
ASP
A
139
26.227
−10.026
42.690
1.00
57.45
A


ATOM
527
C
ASP
A
139
24.258
−13.754
41.733
1.00
56.37
A


ATOM
528
O
ASP
A
139
23.184
−13.256
42.079
1.00
56.43
A


ATOM
529
N
ALA
A
140
24.586
−15.020
41.974
1.00
56.65
A


ATOM
530
CA
ALA
A
140
23.689
−15.925
42.673
1.00
57.35
A


ATOM
531
CB
ALA
A
140
24.371
−17.280
42.879
1.00
57.61
A


ATOM
532
C
ALA
A
140
23.238
−15.353
44.009
1.00
57.65
A


ATOM
533
O
ALA
A
140
22.055
−15.380
44.331
1.00
58.06
A


ATOM
534
N
GLU
A
141
24.183
−14.825
44.779
1.00
59.43
A


ATOM
535
CA
GLU
A
141
23.875
−14.261
46.088
1.00
60.59
A


ATOM
536
CB
GLU
A
141
25.145
−13.691
46.731
1.00
63.85
A


ATOM
537
CG
GLU
A
141
26.252
−14.723
46.938
1.00
67.61
A


ATOM
538
CD
GLU
A
141
27.452
−14.163
47.687
1.00
69.93
A


ATOM
539
OE1
GLU
A
141
27.928
−13.065
47.323
1.00
72.18
A


ATOM
540
OE2
GLU
A
141
27.926
−14.824
48.638
1.00
71.05
A


ATOM
541
C
GLU
A
141
22.795
−13.184
46.039
1.00
59.63
A


ATOM
542
O
GLU
A
141
21.989
−13.067
46.965
1.00
60.53
A


ATOM
543
N
ALA
A
142
22.772
−12.403
44.962
1.00
56.79
A


ATOM
544
CA
ALA
A
142
21.781
−11.339
44.824
1.00
53.70
A


ATOM
545
CB
ALA
A
142
22.471
−10.032
44.453
1.00
53.90
A


ATOM
546
C
ALA
A
142
20.691
−11.660
43.803
1.00
51.59
A


ATOM
547
O
ALA
A
142
19.819
−10.834
43.545
1.00
52.00
A


ATOM
548
N
ALA
A
143
20.740
−12.859
43.229
1.00
48.22
A


ATOM
549
CA
ALA
A
143
19.750
−13.282
42.236
1.00
45.39
A


ATOM
550
CB
ALA
A
143
18.365
−13.366
42.881
1.00
47.05
A


ATOM
551
C
ALA
A
143
19.701
−12.352
41.018
1.00
42.69
A


ATOM
552
O
ALA
A
143
18.626
−12.042
40.509
1.00
41.09
A


ATOM
553
N
LEU
A
144
20.866
−11.923
40.547
1.00
41.05
A


ATOM
554
CA
LEU
A
144
20.936
−11.025
39.397
1.00
41.10
A


ATOM
555
CB
LEU
A
144
21.505
−9.665
39.820
1.00
38.84
A


ATOM
556
CG
LEU
A
144
20.902
−8.925
41.015
1.00
40.65
A


ATOM
557
CD1
LEU
A
144
21.687
−7.632
41.248
1.00
41.07
A


ATOM
558
CD2
LEU
A
144
19.430
−8.629
40.769
1.00
41.05
A


ATOM
559
C
LEU
A
144
21.793
−11.560
38.255
1.00
40.77
A


ATOM
560
O
LEU
A
144
22.739
−12.322
38.470
1.00
42.44
A


ATOM
561
N
LEU
A
145
21.434
−11.164
37.038
1.00
39.26
A


ATOM
562
CA
LEU
A
145
22.180
−11.522
35.838
1.00
40.45
A


ATOM
563
CB
LEU
A
145
21.353
−12.432
34.924
1.00
41.08
A


ATOM
564
CG
LEU
A
145
21.999
−12.780
33.571
1.00
44.82
A


ATOM
565
CD1
LEU
A
145
23.352
−13.454
33.801
1.00
44.44
A


ATOM
566
CD2
LEU
A
145
21.069
−13.689
32.761
1.00
44.40
A


ATOM
567
C
LEU
A
145
22.426
−10.170
35.161
1.00
39.40
A


ATOM
568
O
LEU
A
145
21.484
−9.403
34.958
1.00
38.82
A


ATOM
569
N
HIS
A
146
23.682
−9.866
34.845
1.00
38.79
A


ATOM
570
CA
HIS
A
146
24.022
−8.582
34.212
1.00
38.60
A


ATOM
571
CB
HIS
A
146
25.085
−7.817
35.015
1.00
37.52
A


ATOM
572
CG
HIS
A
146
24.761
−7.621
36.464
1.00
38.83
A


ATOM
573
CD2
HIS
A
146
25.291
−8.184
37.576
1.00
38.65
A


ATOM
574
ND1
HIS
A
146
23.847
−6.688
36.906
1.00
38.20
A


ATOM
575
CE1
HIS
A
146
23.833
−6.681
38.227
1.00
39.86
A


ATOM
576
NE2
HIS
A
146
24.701
−7.579
38.659
1.00
37.50
A


ATOM
577
C
HIS
A
146
24.616
−8.802
32.825
1.00
36.94
A


ATOM
578
O
HIS
A
146
25.496
−9.644
32.661
1.00
37.49
A


ATOM
579
N
VAL
A
147
24.143
−8.039
31.844
1.00
35.57
A


ATOM
580
CA
VAL
A
147
24.662
−8.102
30.478
1.00
36.33
A


ATOM
581
CB
VAL
A
147
23.599
−8.553
29.478
1.00
35.23
A


ATOM
582
CG1
VAL
A
147
24.188
−8.592
28.082
1.00
37.63
A


ATOM
583
CG2
VAL
A
147
23.096
−9.941
29.858
1.00
37.09
A


ATOM
584
C
VAL
A
147
25.087
−6.669
30.160
1.00
36.34
A


ATOM
585
O
VAL
A
147
24.250
−5.778
29.980
1.00
34.90
A


ATOM
586
N
THR
A
148
26.395
−6.455
30.120
1.00
37.82
A


ATOM
587
CA
THR
A
148
26.956
−5.131
29.881
1.00
38.60
A


ATOM
588
CB
THR
A
148
27.940
−4.750
31.010
1.00
39.96
A


ATOM
589
OG1
THR
A
148
27.259
−4.791
32.267
1.00
39.52
A


ATOM
590
CG2
THR
A
148
28.517
−3.339
30.786
1.00
41.58
A


ATOM
591
C
THR
A
148
27.702
−5.012
28.571
1.00
38.59
A


ATOM
592
O
THR
A
148
28.513
−5.873
28.224
1.00
37.69
A


ATOM
593
N
ASP
A
149
27.425
−3.938
27.840
1.00
37.49
A


ATOM
594
CA
ASP
A
149
28.112
−3.694
26.593
1.00
37.69
A


ATOM
595
CB
ASP
A
149
27.166
−3.865
25.388
1.00
38.06
A


ATOM
596
CG
ASP
A
149
26.100
−2.779
25.299
1.00
39.30
A


ATOM
597
OD1
ASP
A
149
26.449
−1.599
25.056
1.00
39.14
A


ATOM
598
OD2
ASP
A
149
24.905
−3.107
25.471
1.00
37.40
A


ATOM
599
C
ASP
A
149
28.687
−2.286
26.637
1.00
37.48
A


ATOM
600
O
ASP
A
149
28.236
−1.442
27.412
1.00
37.39
A


ATOM
601
N
THR
A
150
29.710
−2.048
25.832
1.00
38.71
A


ATOM
602
CA
THR
A
150
30.323
−0.733
25.771
1.00
38.68
A


ATOM
603
CB
THR
A
150
31.868
−0.834
25.879
1.00
40.35
A


ATOM
604
OG1
THR
A
150
32.365
−1.798
24.940
1.00
40.36
A


ATOM
605
CG2
THR
A
150
32.268
−1.257
27.289
1.00
39.00
A


ATOM
606
C
THR
A
150
29.904
−0.088
24.449
1.00
39.32
A


ATOM
607
O
THR
A
150
30.683
0.617
23.799
1.00
39.69
A


ATOM
608
N
GLY
A
151
28.648
−0.338
24.075
1.00
38.91
A


ATOM
609
CA
GLY
A
151
28.089
0.199
22.845
1.00
39.06
A


ATOM
610
C
GLY
A
151
27.774
1.683
22.904
1.00
38.81
A


ATOM
611
O
GLY
A
151
28.286
2.401
23.764
1.00
37.68
A


ATOM
612
N
VAL
A
152
26.921
2.139
21.990
1.00
38.14
A


ATOM
613
CA
VAL
A
152
26.539
3.547
21.916
1.00
36.85
A


ATOM
614
CB
VAL
A
152
25.663
3.800
20.660
1.00
36.97
A


ATOM
615
CG1
VAL
A
152
24.359
3.030
20.784
1.00
34.47
A


ATOM
616
CG2
VAL
A
152
25.398
5.312
20.473
1.00
36.38
A


ATOM
617
C
VAL
A
152
25.797
4.071
23.150
1.00
38.24
A


ATOM
618
O
VAL
A
152
25.862
5.264
23.457
1.00
37.41
A


ATOM
619
N
GLY
A
153
25.090
3.188
23.854
1.00
35.89
A


ATOM
620
CA
GLY
A
153
24.344
3.613
25.023
1.00
33.35
A


ATOM
621
C
GLY
A
153
23.074
4.371
24.641
1.00
33.66
A


ATOM
622
O
GLY
A
153
22.769
4.519
23.458
1.00
33.60
A


ATOM
623
N
MET
A
154
22.339
4.844
25.645
1.00
33.73
A


ATOM
624
CA
MET
A
154
21.098
5.583
25.435
1.00
35.02
A


ATOM
625
CB
MET
A
154
19.881
4.687
25.713
1.00
35.45
A


ATOM
626
CG
MET
A
154
19.672
3.579
24.711
1.00
36.62
A


ATOM
627
SD
MET
A
154
18.279
2.497
25.176
1.00
39.95
A


ATOM
628
CE
MET
A
154
19.165
1.378
26.258
1.00
38.04
A


ATOM
629
C
MET
A
154
20.991
6.802
26.353
1.00
34.74
A


ATOM
630
O
MET
A
154
21.330
6.734
27.532
1.00
34.79
A


ATOM
631
N
THR
A
155
20.503
7.907
25.801
1.00
36.95
A


ATOM
632
CA
THR
A
155
20.296
9.127
26.580
1.00
37.93
A


ATOM
633
CB
THR
A
155
20.060
10.334
25.670
1.00
35.53
A


ATOM
634
OG1
THR
A
155
18.860
10.115
24.917
1.00
36.10
A


ATOM
635
CG2
THR
A
155
21.228
10.526
24.714
1.00
34.17
A


ATOM
636
C
THR
A
155
19.015
8.932
27.392
1.00
39.62
A


ATOM
637
O
THR
A
155
18.270
7.965
27.180
1.00
38.97
A


ATOM
638
N
ARG
A
156
18.738
9.859
28.303
1.00
40.35
A


ATOM
639
CA
ARG
A
156
17.531
9.767
29.111
1.00
38.63
A


ATOM
640
CB
ARG
A
156
17.416
10.973
30.042
1.00
40.13
A


ATOM
641
CG
ARG
A
156
16.189
10.930
30.923
1.00
42.27
A


ATOM
642
CD
ARG
A
156
16.056
12.189
31.769
1.00
45.98
A


ATOM
643
NE
ARG
A
156
15.014
12.046
32.780
1.00
48.94
A


ATOM
644
CZ
ARG
A
156
13.709
12.121
32.539
1.00
50.12
A


ATOM
645
NH1
ARG
A
156
13.264
12.348
31.312
1.00
50.52
A


ATOM
646
NH2
ARG
A
156
12.846
11.951
33.532
1.00
52.90
A


ATOM
647
C
ARG
A
156
16.289
9.688
28.224
1.00
39.01
A


ATOM
648
O
ARG
A
156
15.381
8.874
28.478
1.00
36.95
A


ATOM
649
N
ALA
A
157
16.245
10.529
27.185
1.00
35.34
A


ATOM
650
CA
ALA
A
157
15.111
10.549
26.267
1.00
35.84
A


ATOM
651
CB
ALA
A
157
15.255
11.700
25.252
1.00
36.22
A


ATOM
652
C
ALA
A
157
14.952
9.216
25.523
1.00
35.61
A


ATOM
653
O
ALA
A
157
13.839
8.763
25.300
1.00
35.81
A


ATOM
654
N
GLU
A
158
16.061
8.597
25.142
1.00
35.85
A


ATOM
655
CA
GLU
A
158
16.003
7.314
24.438
1.00
38.21
A


ATOM
656
CB
GLU
A
158
17.372
6.982
23.852
1.00
37.42
A


ATOM
657
CG
GLU
A
158
17.828
8.013
22.815
1.00
42.18
A


ATOM
658
CD
GLU
A
158
19.127
7.649
22.141
1.00
41.21
A


ATOM
659
OE1
GLU
A
158
20.131
7.435
22.843
1.00
42.08
A


ATOM
660
OE2
GLU
A
158
19.153
7.587
20.897
1.00
47.22
A


ATOM
661
C
GLU
A
158
15.511
6.174
25.346
1.00
37.73
A


ATOM
662
O
GLU
A
158
14.792
5.283
24.895
1.00
38.81
A


ATOM
663
N
LEU
A
159
15.896
6.200
26.618
1.00
37.15
A


ATOM
664
CA
LEU
A
159
15.453
5.176
27.561
1.00
37.47
A


ATOM
665
CB
LEU
A
159
16.058
5.412
28.949
1.00
37.32
A


ATOM
666
CG
LEU
A
159
17.538
5.060
29.108
1.00
38.03
A


ATOM
667
CD1
LEU
A
159
18.033
5.485
30.493
1.00
38.93
A


ATOM
668
CD2
LEU
A
159
17.727
3.564
28.894
1.00
37.21
A


ATOM
669
C
LEU
A
159
13.939
5.216
27.649
1.00
38.76
A


ATOM
670
O
LEU
A
159
13.262
4.172
27.614
1.00
37.86
A


ATOM
671
N
VAL
A
160
13.409
6.432
27.766
1.00
36.40
A


ATOM
672
CA
VAL
A
160
11.974
6.634
27.839
1.00
36.76
A


ATOM
673
CB
VAL
A
160
11.627
8.115
28.159
1.00
36.24
A


ATOM
674
CG1
VAL
A
160
10.112
8.323
28.072
1.00
37.44
A


ATOM
675
CG2
VAL
A
160
12.131
8.486
29.546
1.00
34.35
A


ATOM
676
C
VAL
A
160
11.266
6.248
26.536
1.00
36.92
A


ATOM
677
O
VAL
A
160
10.315
5.463
26.548
1.00
37.95
A


ATOM
678
N
ALA
A
161
11.730
6.786
25.413
1.00
34.61
A


ATOM
679
CA
ALA
A
161
11.091
6.516
24.128
1.00
35.52
A


ATOM
680
CB
ALA
A
161
11.512
7.573
23.110
1.00
35.95
A


ATOM
681
C
ALA
A
161
11.304
5.129
23.523
1.00
34.49
A


ATOM
682
O
ALA
A
161
10.349
4.476
23.115
1.00
36.23
A


ATOM
683
N
ASN
A
162
12.555
4.698
23.457
1.00
34.89
A


ATOM
684
CA
ASN
A
162
12.898
3.422
22.839
1.00
36.90
A


ATOM
685
CB
ASN
A
162
14.414
3.331
22.691
1.00
37.47
A


ATOM
686
CG
ASN
A
162
14.961
4.415
21.785
1.00
42.03
A


ATOM
687
OD1
ASN
A
162
14.383
5.505
21.688
1.00
43.34
A


ATOM
688
ND2
ASN
A
162
16.075
4.134
21.125
1.00
40.97
A


ATOM
689
C
ASN
A
162
12.354
2.196
23.551
1.00
37.70
A


ATOM
690
O
ASN
A
162
12.039
1.186
22.908
1.00
37.59
A


ATOM
691
N
LEU
A
163
12.232
2.277
24.869
1.00
37.09
A


ATOM
692
CA
LEU
A
163
11.722
1.135
25.624
1.00
38.10
A


ATOM
693
CB
LEU
A
163
12.587
0.906
26.864
1.00
35.47
A


ATOM
694
CG
LEU
A
163
14.093
0.725
26.645
1.00
34.53
A


ATOM
695
CD1
LEU
A
163
14.792
0.602
28.003
1.00
34.16
A


ATOM
696
CD2
LEU
A
163
14.361
−0.506
25.770
1.00
37.46
A


ATOM
697
C
LEU
A
163
10.262
1.297
26.035
1.00
38.71
A


ATOM
698
O
LEU
A
163
9.541
0.309
26.209
1.00
38.36
A


ATOM
699
N
GLY
A
164
9.814
2.542
26.163
1.00
37.47
A


ATOM
700
CA
GLY
A
164
8.455
2.783
26.602
1.00
36.44
A


ATOM
701
C
GLY
A
164
7.408
3.033
25.541
1.00
37.47
A


ATOM
702
O
GLY
A
164
6.217
3.032
25.845
1.00
37.44
A


ATOM
703
N
THR
A
165
7.838
3.241
24.302
1.00
37.94
A


ATOM
704
CA
THR
A
165
6.910
3.508
23.213
1.00
38.31
A


ATOM
705
CB
THR
A
165
6.899
5.002
22.821
1.00
39.27
A


ATOM
706
OG1
THR
A
165
8.014
5.265
21.954
1.00
36.14
A


ATOM
707
CG2
THR
A
165
6.993
5.893
24.058
1.00
39.57
A


ATOM
708
C
THR
A
165
7.353
2.775
21.960
1.00
38.40
A


ATOM
709
O
THR
A
165
8.438
2.197
21.906
1.00
36.47
A


ATOM
710
N
ILE
A
166
6.505
2.837
20.945
1.00
42.95
A


ATOM
711
CA
ILE
A
166
6.811
2.250
19.655
1.00
47.73
A


ATOM
712
CB
ILE
A
166
5.526
1.926
18.881
1.00
48.55
A


ATOM
713
CG2
ILE
A
166
4.566
3.088
18.963
1.00
52.33
A


ATOM
714
CG1
ILE
A
166
5.857
1.594
17.425
1.00
50.22
A


ATOM
715
CD1
ILE
A
166
4.660
1.157
16.620
1.00
50.44
A


ATOM
716
C
ILE
A
166
7.588
3.384
18.982
1.00
49.91
A


ATOM
717
O
ILE
A
166
7.013
4.269
18.349
1.00
49.34
A


ATOM
718
N
ALA
A
167
8.904
3.353
19.167
1.00
53.09
A


ATOM
719
CA
ALA
A
167
9.809
4.375
18.658
1.00
55.59
A


ATOM
720
CB
ALA
A
167
11.139
4.277
19.400
1.00
55.40
A


ATOM
721
C
ALA
A
167
10.062
4.404
17.155
1.00
57.83
A


ATOM
722
O
ALA
A
167
10.185
5.483
16.575
1.00
57.75
A


ATOM
723
N
LYS
A
168
10.148
3.237
16.522
1.00
61.19
A


ATOM
724
CA
LYS
A
168
10.418
3.186
15.085
1.00
63.46
A


ATOM
725
CB
LYS
A
168
11.717
2.421
14.825
1.00
64.87
A


ATOM
726
CG
LYS
A
168
12.978
3.188
15.198
1.00
67.57
A


ATOM
727
CD
LYS
A
168
13.103
4.488
14.403
1.00
68.54
A


ATOM
728
CE
LYS
A
168
13.123
4.234
12.898
1.00
69.62
A


ATOM
729
NZ
LYS
A
168
14.290
3.409
12.479
1.00
70.51
A


ATOM
730
C
LYS
A
168
9.318
2.600
14.213
1.00
64.19
A


ATOM
731
O
LYS
A
168
8.276
2.159
14.701
1.00
64.53
A


ATOM
732
N
SER
A
169
9.571
2.606
12.908
1.00
64.45
A


ATOM
733
CA
SER
A
169
8.632
2.080
11.922
1.00
64.31
A


ATOM
734
CB
SER
A
169
9.032
2.537
10.517
1.00
65.39
A


ATOM
735
OG
SER
A
169
9.367
3.914
10.499
1.00
69.31
A


ATOM
736
C
SER
A
169
8.659
0.558
11.973
1.00
62.99
A


ATOM
737
O
SER
A
169
7.621
−0.099
11.892
1.00
62.36
A


ATOM
738
N
GLY
A
170
9.864
0.012
12.105
1.00
62.18
A


ATOM
739
CA
GLY
A
170
10.033
−1.427
12.152
1.00
61.11
A


ATOM
740
C
GLY
A
170
9.173
−2.103
13.199
1.00
60.68
A


ATOM
741
O
GLY
A
170
8.496
−3.088
12.902
1.00
60.52
A


ATOM
742
N
THR
A
171
9.191
−1.575
14.421
1.00
59.30
A


ATOM
743
CA
THR
A
171
8.412
−2.158
15.505
1.00
59.38
A


ATOM
744
CB
THR
A
171
8.724
−1.453
16.850
1.00
58.69
A


ATOM
745
OG1
THR
A
171
7.737
−1.810
17.824
1.00
60.23
A


ATOM
746
CG2
THR
A
171
8.750
0.041
16.675
1.00
61.04
A


ATOM
747
C
THR
A
171
6.911
−2.127
15.221
1.00
58.48
A


ATOM
748
O
THR
A
171
6.161
−2.973
15.707
1.00
58.04
A


ATOM
749
N
SER
A
172
6.479
−1.162
14.415
1.00
58.58
A


ATOM
750
CA
SER
A
172
5.070
−1.042
14.056
1.00
58.03
A


ATOM
751
CB
SER
A
172
4.812
0.325
13.407
1.00
59.05
A


ATOM
752
OG
SER
A
172
3.428
0.545
13.201
1.00
58.95
A


ATOM
753
C
SER
A
172
4.700
−2.174
13.086
1.00
57.97
A


ATOM
754
O
SER
A
172
3.637
−2.788
13.205
1.00
57.78
A


ATOM
755
N
ALA
A
173
5.587
−2.447
12.132
1.00
57.28
A


ATOM
756
CA
ALA
A
173
5.362
−3.510
11.157
1.00
57.64
A


ATOM
757
CB
ALA
A
173
6.391
−3.424
10.027
1.00
57.77
A


ATOM
758
C
ALA
A
173
5.459
−4.863
11.856
1.00
57.34
A


ATOM
759
O
ALA
A
173
4.733
−5.799
11.519
1.00
56.73
A


ATOM
760
N
PHE
A
174
6.356
−4.965
12.833
1.00
56.78
A


ATOM
761
CA
PHE
A
174
6.512
−6.212
13.571
1.00
56.41
A


ATOM
762
CB
PHE
A
174
7.542
−6.062
14.693
1.00
54.27
A


ATOM
763
CG
PHE
A
174
7.475
−7.157
15.717
1.00
51.64
A


ATOM
764
CD1
PHE
A
174
6.842
−6.947
16.943
1.00
49.88
A


ATOM
765
CD2
PHE
A
174
8.012
−8.416
15.442
1.00
51.59
A


ATOM
766
CE1
PHE
A
174
6.743
−7.973
17.881
1.00
51.52
A


ATOM
767
CE2
PHE
A
174
7.920
−9.454
16.373
1.00
50.03
A


ATOM
768
CZ
PHE
A
174
7.284
−9.235
17.595
1.00
51.03
A


ATOM
769
C
PHE
A
174
5.181
−6.647
14.170
1.00
56.94
A


ATOM
770
O
PHE
A
174
4.803
−7.813
14.075
1.00
57.11
A


ATOM
771
N
LEU
A
175
4.469
−5.708
14.782
1.00
56.79
A


ATOM
772
CA
LEU
A
175
3.186
−6.023
15.395
1.00
58.46
A


ATOM
773
CB
LEU
A
175
2.595
−4.777
16.057
1.00
59.35
A


ATOM
774
CG
LEU
A
175
3.267
−4.350
17.364
1.00
61.12
A


ATOM
775
CD1
LEU
A
175
2.691
−3.020
17.834
1.00
61.82
A


ATOM
776
CD2
LEU
A
175
3.055
−5.433
18.419
1.00
62.48
A


ATOM
777
C
LEU
A
175
2.170
−6.623
14.425
1.00
58.54
A


ATOM
778
O
LEU
A
175
1.367
−7.476
14.810
1.00
58.31
A


ATOM
779
N
ASN
A
176
2.196
−6.177
13.174
1.00
59.10
A


ATOM
780
CA
ASN
A
176
1.263
−6.697
12.182
1.00
59.32
A


ATOM
781
CB
ASN
A
176
1.096
−5.711
11.026
1.00
61.65
A


ATOM
782
CG
ASN
A
176
0.373
−4.447
11.442
1.00
63.45
A


ATOM
783
OD1
ASN
A
176
−0.610
−4.499
12.188
1.00
64.07
A


ATOM
784
ND2
ASN
A
176
0.844
−3.304
10.954
1.00
63.36
A


ATOM
785
C
ASN
A
176
1.736
−8.037
11.641
1.00
58.64
A


ATOM
786
O
ASN
A
176
0.940
−8.962
11.474
1.00
58.77
A


ATOM
787
N
ALA
A
177
3.032
−8.136
11.364
1.00
57.09
A


ATOM
788
CA
ALA
A
177
3.600
−9.372
10.846
1.00
56.72
A


ATOM
789
CB
ALA
A
177
5.067
−9.164
10.488
1.00
56.36
A


ATOM
790
C
ALA
A
177
3.458
−10.452
11.907
1.00
56.28
A


ATOM
791
O
ALA
A
177
3.154
−11.606
11.602
1.00
55.03
A


ATOM
792
N
MET
A
178
3.663
−10.062
13.162
1.00
56.61
A


ATOM
793
CA
MET
A
178
3.560
−10.983
14.287
1.00
58.30
A


ATOM
794
CB
MET
A
178
3.887
−10.256
15.594
1.00
55.27
A


ATOM
795
CG
MET
A
178
3.969
−11.164
16.797
1.00
52.13
A


ATOM
796
SD
MET
A
178
5.240
−12.416
16.581
1.00
49.31
A


ATOM
797
CE
MET
A
178
5.117
−13.294
18.122
1.00
47.13
A


ATOM
798
C
MET
A
178
2.164
−11.587
14.374
1.00
60.81
A


ATOM
799
O
MET
A
178
2.006
−12.810
14.450
1.00
61.92
A


ATOM
800
N
THR
A
179
1.151
−10.727
14.356
1.00
62.45
A


ATOM
801
CA
THR
A
179
−0.233
−11.176
14.436
1.00
65.49
A


ATOM
802
CB
THR
A
179
−1.198
−9.974
14.570
1.00
66.54
A


ATOM
803
OG1
THR
A
179
−1.012
−9.083
13.462
1.00
68.60
A


ATOM
804
CG2
THR
A
179
−0.936
−9.221
15.865
1.00
66.93
A


ATOM
805
C
THR
A
179
−0.639
−12.003
13.212
1.00
66.44
A


ATOM
806
O
THR
A
179
−1.332
−13.010
13.339
1.00
66.28
A


ATOM
807
N
GLU
A
180
−0.203
−11.576
12.031
1.00
67.47
A


ATOM
808
CA
GLU
A
180
−0.533
−12.281
10.797
1.00
68.76
A


ATOM
809
CB
GLU
A
180
−0.221
−11.404
9.588
1.00
70.17
A


ATOM
810
CG
GLU
A
180
−1.273
−10.346
9.302
1.00
72.96
A


ATOM
811
CD
GLU
A
180
−0.925
−9.502
8.091
1.00
74.23
A


ATOM
812
OE1
GLU
A
180
−1.792
−8.727
7.629
1.00
74.78
A


ATOM
813
OE2
GLU
A
180
0.222
−9.613
7.605
1.00
75.19
A


ATOM
814
C
GLU
A
180
0.195
−13.612
10.654
1.00
69.31
A


ATOM
815
O
GLU
A
180
−0.123
−14.411
9.773
1.00
69.53
A


ATOM
816
N
ALA
A
181
1.175
−13.844
11.519
1.00
69.22
A


ATOM
817
CA
ALA
A
181
1.937
−15.083
11.488
1.00
69.33
A


ATOM
818
CB
ALA
A
181
3.418
−14.790
11.667
1.00
68.64
A


ATOM
819
C
ALA
A
181
1.448
−15.984
12.608
1.00
69.85
A


ATOM
820
O
ALA
A
181
1.150
−17.159
12.399
1.00
69.63
A


ATOM
821
N
GLN
A
182
1.363
−15.410
13.801
1.00
70.54
A


ATOM
822
CA
GLN
A
182
0.926
−16.131
14.988
1.00
71.47
A


ATOM
823
CB
GLN
A
182
1.080
−15.218
16.208
1.00
71.17
A


ATOM
824
CG
GLN
A
182
0.727
−15.838
17.541
1.00
71.33
A


ATOM
825
CD
GLN
A
182
1.202
−14.988
18.707
1.00
71.75
A


ATOM
826
OE1
GLN
A
182
2.396
−14.928
19.000
1.00
71.32
A


ATOM
827
NE2
GLN
A
182
0.268
−14.314
19.369
1.00
72.54
A


ATOM
828
C
GLN
A
182
−0.516
−16.608
14.851
1.00
71.82
A


ATOM
829
O
GLN
A
182
−0.973
−17.462
15.613
1.00
71.86
A


ATOM
830
N
GLU
A
183
−1.223
−16.063
13.866
1.00
72.03
A


ATOM
831
CA
GLU
A
183
−2.614
−16.426
13.635
1.00
72.86
A


ATOM
832
CB
GLU
A
183
−3.348
−15.277
12.932
1.00
74.17
A


ATOM
833
CG
GLU
A
183
−4.826
−15.540
12.667
1.00
76.24
A


ATOM
834
CD
GLU
A
183
−5.548
−14.326
12.106
1.00
77.73
A


ATOM
835
OE1
GLU
A
183
−6.730
−14.460
11.716
1.00
78.21
A


ATOM
836
OE2
GLU
A
183
−4.936
−13.235
12.061
1.00
78.20
A


ATOM
837
C
GLU
A
183
−2.758
−17.710
12.822
1.00
72.67
A


ATOM
838
O
GLU
A
183
−3.694
−18.481
13.039
1.00
72.75
A


ATOM
839
N
ASP
A
184
−1.835
−17.948
11.893
1.00
72.01
A


ATOM
840
CA
ASP
A
184
−1.909
−19.151
11.068
1.00
71.44
A


ATOM
841
CB
ASP
A
184
−2.078
−18.772
9.592
1.00
73.01
A


ATOM
842
CG
ASP
A
184
−0.847
−18.113
9.014
1.00
74.32
A


ATOM
843
OD1
ASP
A
184
−0.483
−17.016
9.484
1.00
76.20
A


ATOM
844
OD2
ASP
A
184
−0.244
−18.697
8.088
1.00
75.11
A


ATOM
845
C
ASP
A
184
−0.726
−20.111
11.230
1.00
69.97
A


ATOM
846
O
ASP
A
184
−0.269
−20.725
10.263
1.00
69.71
A


ATOM
847
N
GLY
A
185
−0.235
−20.226
12.460
1.00
67.94
A


ATOM
848
CA
GLY
A
185
0.858
−21.136
12.761
1.00
65.21
A


ATOM
849
C
GLY
A
185
2.221
−20.990
12.101
1.00
63.26
A


ATOM
850
O
GLY
A
185
2.910
−21.995
11.914
1.00
62.89
A


ATOM
851
N
GLN
A
186
2.624
−19.774
11.739
1.00
59.88
A


ATOM
852
CA
GLN
A
186
3.945
−19.581
11.137
1.00
56.96
A


ATOM
853
CB
GLN
A
186
3.924
−18.417
10.145
1.00
57.15
A


ATOM
854
CG
GLN
A
186
3.203
−18.723
8.843
1.00
58.88
A


ATOM
855
CD
GLN
A
186
3.159
−17.531
7.905
1.00
59.29
A


ATOM
856
OE1
GLN
A
186
4.195
−16.975
7.538
1.00
61.02
A


ATOM
857
NE2
GLN
A
186
1.955
−17.132
7.512
1.00
59.93
A


ATOM
858
C
GLN
A
186
4.926
−19.298
12.276
1.00
54.13
A


ATOM
859
O
GLN
A
186
4.524
−18.755
13.306
1.00
54.33
A


ATOM
860
N
SER
A
187
6.195
−19.670
12.107
1.00
49.48
A


ATOM
861
CA
SER
A
187
7.189
−19.455
13.162
1.00
47.88
A


ATOM
862
CB
SER
A
187
8.553
−20.008
12.751
1.00
46.11
A


ATOM
863
OG
SER
A
187
9.389
−20.159
13.889
1.00
46.52
A


ATOM
864
C
SER
A
187
7.312
−17.968
13.495
1.00
46.56
A


ATOM
865
O
SER
A
187
7.303
−17.122
12.604
1.00
45.29
A


ATOM
866
N
THR
A
188
7.452
−17.665
14.779
1.00
46.91
A


ATOM
867
CA
THR
A
188
7.523
−16.279
15.237
1.00
46.94
A


ATOM
868
CB
THR
A
188
6.366
−15.992
16.211
1.00
48.10
A


ATOM
869
OG1
THR
A
188
6.361
−16.989
17.240
1.00
47.90
A


ATOM
870
CG2
THR
A
188
5.029
−16.020
15.482
1.00
48.15
A


ATOM
871
C
THR
A
188
8.816
−15.810
15.905
1.00
46.41
A


ATOM
872
O
THR
A
188
9.011
−14.599
16.084
1.00
45.96
A


ATOM
873
N
SER
A
189
9.691
−16.735
16.290
1.00
43.65
A


ATOM
874
CA
SER
A
189
10.940
−16.336
16.940
1.00
43.00
A


ATOM
875
CB
SER
A
189
11.678
−17.555
17.525
1.00
44.76
A


ATOM
876
OG
SER
A
189
12.138
−18.428
16.505
1.00
45.08
A


ATOM
877
C
SER
A
189
11.854
−15.600
15.964
1.00
43.67
A


ATOM
878
O
SER
A
189
12.667
−14.760
16.373
1.00
41.00
A


ATOM
879
N
ALA
A
190
11.721
−15.908
14.673
1.00
41.06
A


ATOM
880
CA
ALA
A
190
12.542
−15.255
13.657
1.00
41.18
A


ATOM
881
CB
ALA
A
190
12.401
−15.967
12.315
1.00
42.02
A


ATOM
882
C
ALA
A
190
12.111
−13.798
13.518
1.00
42.09
A


ATOM
883
O
ALA
A
190
12.938
−12.919
13.278
1.00
38.77
A


ATOM
884
N
LEU
A
191
10.814
−13.555
13.668
1.00
42.41
A


ATOM
885
CA
LEU
A
191
10.281
−12.204
13.560
1.00
44.99
A


ATOM
886
CB
LEU
A
191
8.751
−12.219
13.583
1.00
44.92
A


ATOM
887
CG
LEU
A
191
8.003
−12.719
12.346
1.00
46.34
A


ATOM
888
CD1
LEU
A
191
6.507
−12.683
12.630
1.00
46.59
A


ATOM
889
CD2
LEU
A
191
8.335
−11.850
11.142
1.00
46.99
A


ATOM
890
C
LEU
A
191
10.789
−11.296
14.680
1.00
45.19
A


ATOM
891
O
LEU
A
191
11.202
−10.155
14.423
1.00
45.12
A


ATOM
892
N
ILE
A
192
10.759
−11.790
15.915
1.00
44.61
A


ATOM
893
CA
ILE
A
192
11.219
−10.979
17.036
1.00
46.52
A


ATOM
894
CB
ILE
A
192
11.044
−11.699
18.409
1.00
47.46
A


ATOM
895
CG2
ILE
A
192
9.594
−12.162
18.583
1.00
46.74
A


ATOM
896
CG1
ILE
A
192
12.002
−12.880
18.513
1.00
51.59
A


ATOM
897
CD1
ILE
A
192
12.338
−13.257
19.948
1.00
55.04
A


ATOM
898
C
ILE
A
192
12.688
−10.604
16.842
1.00
45.97
A


ATOM
899
O
ILE
A
192
13.085
−9.476
17.118
1.00
44.40
A


ATOM
900
N
GLY
A
193
13.484
−11.551
16.351
1.00
45.58
A


ATOM
901
CA
GLY
A
193
14.893
−11.287
16.117
1.00
45.18
A


ATOM
902
C
GLY
A
193
15.117
−10.342
14.946
1.00
45.44
A


ATOM
903
O
GLY
A
193
15.962
−9.446
15.018
1.00
45.12
A


ATOM
904
N
GLN
A
194
14.358
−10.542
13.869
1.00
44.42
A


ATOM
905
CA
GLN
A
194
14.472
−9.709
12.672
1.00
46.80
A


ATOM
906
CB
GLN
A
194
13.493
−10.188
11.594
1.00
48.06
A


ATOM
907
CG
GLN
A
194
13.938
−11.435
10.851
1.00
52.72
A


ATOM
908
CD
GLN
A
194
13.004
−11.798
9.701
1.00
54.82
A


ATOM
909
OE1
GLN
A
194
13.268
−12.739
8.951
1.00
58.37
A


ATOM
910
NE2
GLN
A
194
11.909
−11.054
9.560
1.00
53.81
A


ATOM
911
C
GLN
A
194
14.212
−8.228
12.947
1.00
46.26
A


ATOM
912
O
GLN
A
194
14.953
−7.359
12.483
1.00
45.58
A


ATOM
913
N
PHE
A
195
13.149
−7.948
13.689
1.00
45.59
A


ATOM
914
CA
PHE
A
195
12.785
−6.573
14.012
1.00
47.81
A


ATOM
915
CB
PHE
A
195
11.262
−6.470
14.112
1.00
48.72
A


ATOM
916
CG
PHE
A
195
10.571
−6.578
12.783
1.00
52.11
A


ATOM
917
CD1
PHE
A
195
10.450
−5.463
11.955
1.00
53.36
A


ATOM
918
CD2
PHE
A
195
10.084
−7.806
12.333
1.00
53.76
A


ATOM
919
CE1
PHE
A
195
9.855
−5.565
10.693
1.00
54.61
A


ATOM
920
CE2
PHE
A
195
9.485
−7.925
11.072
1.00
54.49
A


ATOM
921
CZ
PHE
A
195
9.371
−6.800
10.249
1.00
55.89
A


ATOM
922
C
PHE
A
195
13.452
−6.043
15.281
1.00
45.89
A


ATOM
923
O
PHE
A
195
13.224
−4.898
15.678
1.00
46.00
A


ATOM
924
N
GLY
A
196
14.281
−6.880
15.898
1.00
43.45
A


ATOM
925
CA
GLY
A
196
14.991
−6.496
17.105
1.00
43.03
A


ATOM
926
C
GLY
A
196
14.139
−6.021
18.269
1.00
41.78
A


ATOM
927
O
GLY
A
196
14.488
−5.043
18.935
1.00
39.12
A


ATOM
928
N
VAL
A
197
13.032
−6.717
18.529
1.00
39.22
A


ATOM
929
CA
VAL
A
197
12.139
−6.353
19.629
1.00
37.80
A


ATOM
930
CB
VAL
A
197
10.659
−6.282
19.155
1.00
37.78
A


ATOM
931
CG1
VAL
A
197
10.498
−5.196
18.101
1.00
39.35
A


ATOM
932
CG2
VAL
A
197
10.212
−7.633
18.592
1.00
39.35
A


ATOM
933
C
VAL
A
197
12.228
−7.321
20.821
1.00
36.08
A


ATOM
934
O
VAL
A
197
11.379
−7.292
21.708
1.00
33.97
A


ATOM
935
N
GLY
A
198
13.264
−8.159
20.838
1.00
35.72
A


ATOM
936
CA
GLY
A
198
13.445
−9.133
21.910
1.00
34.04
A


ATOM
937
C
GLY
A
198
13.722
−8.639
23.324
1.00
35.00
A


ATOM
938
O
GLY
A
198
13.688
−9.427
24.269
1.00
32.54
A


ATOM
939
N
PHE
A
199
14.009
−7.349
23.500
1.00
34.83
A


ATOM
940
CA
PHE
A
199
14.267
−6.841
24.841
1.00
33.95
A


ATOM
941
CB
PHE
A
199
14.400
−5.308
24.804
1.00
35.31
A


ATOM
942
CG
PHE
A
199
14.375
−4.663
26.159
1.00
35.09
A


ATOM
943
CD1
PHE
A
199
15.521
−4.614
26.946
1.00
34.35
A


ATOM
944
CD2
PHE
A
199
13.183
−4.130
26.660
1.00
35.32
A


ATOM
945
CE1
PHE
A
199
15.490
−4.044
28.225
1.00
34.68
A


ATOM
946
CE2
PHE
A
199
13.131
−3.561
27.929
1.00
34.37
A


ATOM
947
CZ
PHE
A
199
14.295
−3.518
28.720
1.00
35.36
A


ATOM
948
C
PHE
A
199
13.165
−7.250
25.829
1.00
33.31
A


ATOM
949
O
PHE
A
199
13.449
−7.652
26.961
1.00
31.85
A


ATOM
950
N
TYR
A
200
11.913
−7.178
25.395
1.00
33.78
A


ATOM
951
CA
TYR
A
200
10.799
−7.515
26.270
1.00
35.65
A


ATOM
952
CB
TYR
A
200
9.479
−7.090
25.621
1.00
35.72
A


ATOM
953
CG
TYR
A
200
9.432
−5.610
25.293
1.00
36.99
A


ATOM
954
CD1
TYR
A
200
9.404
−4.645
26.304
1.00
35.32
A


ATOM
955
CE1
TYR
A
200
9.413
−3.267
25.990
1.00
37.74
A


ATOM
956
CD2
TYR
A
200
9.465
−5.177
23.971
1.00
38.74
A


ATOM
957
CE2
TYR
A
200
9.474
−3.826
23.654
1.00
40.33
A


ATOM
958
CZ
TYR
A
200
9.449
−2.877
24.665
1.00
38.60
A


ATOM
959
OH
TYR
A
200
9.463
−1.542
24.326
1.00
43.22
A


ATOM
960
C
TYR
A
200
10.716
−8.982
26.707
1.00
35.83
A


ATOM
961
O
TYR
A
200
10.067
−9.284
27.712
1.00
32.96
A


ATOM
962
N
SER
A
201
11.379
−9.880
25.978
1.00
35.53
A


ATOM
963
CA
SER
A
201
11.332
−11.301
26.345
1.00
36.28
A


ATOM
964
CB
SER
A
201
11.969
−12.185
25.257
1.00
34.75
A


ATOM
965
OG
SER
A
201
13.364
−11.984
25.134
1.00
35.49
A


ATOM
966
C
SER
A
201
12.022
−11.544
27.682
1.00
36.13
A


ATOM
967
O
SER
A
201
11.854
−12.604
28.299
1.00
36.56
A


ATOM
968
N
ALA
A
202
12.805
−10.563
28.131
1.00
35.31
A


ATOM
969
CA
ALA
A
202
13.490
−10.680
29.404
1.00
33.89
A


ATOM
970
CB
ALA
A
202
14.315
−9.413
29.692
1.00
36.62
A


ATOM
971
C
ALA
A
202
12.495
−10.909
30.527
1.00
32.57
A


ATOM
972
O
ALA
A
202
12.838
−11.516
31.546
1.00
33.66
A


ATOM
973
N
PHE
A
203
11.274
−10.409
30.361
1.00
30.62
A


ATOM
974
CA
PHE
A
203
10.263
−10.574
31.396
1.00
32.83
A


ATOM
975
CB
PHE
A
203
9.056
−9.656
31.144
1.00
34.52
A


ATOM
976
CG
PHE
A
203
9.312
−8.195
31.498
1.00
37.83
A


ATOM
977
CD1
PHE
A
203
9.629
−7.825
32.808
1.00
36.70
A


ATOM
978
CD2
PHE
A
203
9.241
−7.205
30.521
1.00
37.21
A


ATOM
979
CE1
PHE
A
203
9.878
−6.478
33.144
1.00
39.70
A


ATOM
980
CE2
PHE
A
203
9.485
−5.853
30.841
1.00
39.06
A


ATOM
981
CZ
PHE
A
203
9.807
−5.492
32.156
1.00
35.63
A


ATOM
982
C
PHE
A
203
9.820
−12.033
31.524
1.00
33.89
A


ATOM
983
O
PHE
A
203
9.065
−12.369
32.428
1.00
33.98
A


ATOM
984
N
LEU
A
204
10.296
−12.894
30.623
1.00
35.14
A


ATOM
985
CA
LEU
A
204
9.964
−14.324
30.710
1.00
37.59
A


ATOM
986
CB
LEU
A
204
10.352
−15.061
29.424
1.00
37.82
A


ATOM
987
CG
LEU
A
204
9.396
−14.947
28.240
1.00
37.22
A


ATOM
988
CD1
LEU
A
204
10.077
−15.471
26.997
1.00
41.61
A


ATOM
989
CD2
LEU
A
204
8.113
−15.715
28.533
1.00
38.03
A


ATOM
990
C
LEU
A
204
10.744
−14.922
31.875
1.00
37.39
A


ATOM
991
O
LEU
A
204
10.290
−15.867
32.516
1.00
38.71
A


ATOM
992
N
VAL
A
205
11.918
−14.357
32.162
1.00
36.51
A


ATOM
993
CA
VAL
A
205
12.744
−14.863
33.248
1.00
36.53
A


ATOM
994
CB
VAL
A
205
14.147
−15.325
32.736
1.00
37.09
A


ATOM
995
CG1
VAL
A
205
13.984
−16.446
31.708
1.00
35.55
A


ATOM
996
CG2
VAL
A
205
14.918
−14.146
32.108
1.00
34.89
A


ATOM
997
C
VAL
A
205
12.960
−13.883
34.396
1.00
35.28
A


ATOM
998
O
VAL
A
205
13.476
−14.265
35.440
1.00
34.02
A


ATOM
999
N
ALA
A
206
12.563
−12.627
34.214
1.00
36.36
A


ATOM
1000
CA
ALA
A
206
12.775
−11.635
35.261
1.00
35.50
A


ATOM
1001
CB
ALA
A
206
13.787
−10.567
34.773
1.00
35.42
A


ATOM
1002
C
ALA
A
206
11.521
−10.945
35.783
1.00
34.37
A


ATOM
1003
O
ALA
A
206
10.611
−10.598
35.021
1.00
35.56
A


ATOM
1004
N
ASP
A
207
11.497
−10.758
37.099
1.00
33.09
A


ATOM
1005
CA
ASP
A
207
10.411
−10.065
37.791
1.00
33.92
A


ATOM
1006
CB
ASP
A
207
10.434
−10.442
39.268
1.00
35.69
A


ATOM
1007
CG
ASP
A
207
9.768
−11.784
39.545
1.00
37.34
A


ATOM
1008
OD1
ASP
A
207
10.273
−12.521
40.418
1.00
40.55
A


ATOM
1009
OD2
ASP
A
207
8.739
−12.085
38.908
1.00
36.54
A


ATOM
1010
C
ASP
A
207
10.635
−8.548
37.655
1.00
32.95
A


ATOM
1011
O
ASP
A
207
9.698
−7.749
37.764
1.00
36.47
A


ATOM
1012
N
LYS
A
208
11.889
−8.180
37.428
1.00
33.18
A


ATOM
1013
CA
LYS
A
208
12.292
−6.782
37.261
1.00
35.39
A


ATOM
1014
CB
LYS
A
208
12.688
−6.156
38.611
1.00
36.31
A


ATOM
1015
CG
LYS
A
208
13.332
−4.759
38.481
1.00
41.32
A


ATOM
1016
CD
LYS
A
208
12.372
−3.622
38.833
1.00
44.97
A


ATOM
1017
CE
LYS
A
208
12.173
−3.496
40.338
1.00
46.96
A


ATOM
1018
NZ
LYS
A
208
11.480
−2.222
40.715
1.00
48.18
A


ATOM
1019
C
LYS
A
208
13.471
−6.682
36.301
1.00
32.95
A


ATOM
1020
O
LYS
A
208
14.414
−7.472
36.353
1.00
31.45
A


ATOM
1021
N
VAL
A
209
13.408
−5.693
35.420
1.00
31.85
A


ATOM
1022
CA
VAL
A
209
14.469
−5.468
34.461
1.00
30.22
A


ATOM
1023
CB
VAL
A
209
13.948
−5.621
33.018
1.00
30.60
A


ATOM
1024
CG1
VAL
A
209
15.079
−5.326
32.016
1.00
32.72
A


ATOM
1025
CG2
VAL
A
209
13.393
−7.061
32.816
1.00
31.30
A


ATOM
1026
C
VAL
A
209
14.967
−4.045
34.691
1.00
33.58
A


ATOM
1027
O
VAL
A
209
14.170
−3.104
34.743
1.00
31.38
A


ATOM
1028
N
ILE
A
210
16.274
−3.910
34.863
1.00
33.27
A


ATOM
1029
CA
ILE
A
210
16.889
−2.612
35.086
1.00
37.08
A


ATOM
1030
CB
ILE
A
210
17.563
−2.558
36.478
1.00
38.79
A


ATOM
1031
CG2
ILE
A
210
18.181
−1.181
36.715
1.00
39.96
A


ATOM
1032
CG1
ILE
A
210
16.521
−2.856
37.557
1.00
37.52
A


ATOM
1033
CD1
ILE
A
210
17.077
−2.837
38.964
1.00
42.42
A


ATOM
1034
C
ILE
A
210
17.915
−2.353
33.990
1.00
37.29
A


ATOM
1035
O
ILE
A
210
18.724
−3.228
33.650
1.00
36.88
A


ATOM
1036
N
VAL
A
211
17.875
−1.148
33.430
1.00
34.49
A


ATOM
1037
CA
VAL
A
211
18.791
−0.793
32.366
1.00
32.75
A


ATOM
1038
CB
VAL
A
211
18.038
−0.519
31.036
1.00
33.29
A


ATOM
1039
CG1
VAL
A
211
19.032
−0.186
29.942
1.00
32.50
A


ATOM
1040
CG2
VAL
A
211
17.211
−1.759
30.608
1.00
32.87
A


ATOM
1041
C
VAL
A
211
19.558
0.456
32.786
1.00
34.30
A


ATOM
1042
O
VAL
A
211
18.958
1.504
33.015
1.00
31.53
A


ATOM
1043
N
THR
A
212
20.870
0.318
32.937
1.00
34.12
A


ATOM
1044
CA
THR
A
212
21.709
1.457
33.311
1.00
36.70
A


ATOM
1045
CB
THR
A
212
22.734
1.060
34.372
1.00
38.16
A


ATOM
1046
OG1
THR
A
212
23.592
0.039
33.844
1.00
47.00
A


ATOM
1047
CG2
THR
A
212
22.030
0.527
35.617
1.00
35.54
A


ATOM
1048
C
THR
A
212
22.404
1.830
32.017
1.00
35.20
A


ATOM
1049
O
THR
A
212
22.942
0.956
31.324
1.00
33.56
A


ATOM
1050
N
SER
A
213
22.395
3.116
31.671
1.00
33.86
A


ATOM
1051
CA
SER
A
213
22.980
3.500
30.403
1.00
32.52
A


ATOM
1052
CB
SER
A
213
21.891
3.448
29.328
1.00
29.90
A


ATOM
1053
OG
SER
A
213
22.465
3.348
28.047
1.00
31.83
A


ATOM
1054
C
SER
A
213
23.671
4.859
30.364
1.00
32.91
A


ATOM
1055
O
SER
A
213
23.199
5.825
30.966
1.00
34.92
A


ATOM
1056
N
LYS
A
214
24.776
4.912
29.627
1.00
34.54
A


ATOM
1057
CA
LYS
A
214
25.560
6.143
29.462
1.00
35.37
A


ATOM
1058
CB
LYS
A
214
26.862
6.056
30.260
1.00
35.40
A


ATOM
1059
CG
LYS
A
214
27.799
7.265
30.062
1.00
36.79
A


ATOM
1060
CD
LYS
A
214
27.173
8.547
30.588
1.00
35.02
A


ATOM
1061
CE
LYS
A
214
28.148
9.733
30.441
1.00
39.66
A


ATOM
1062
NZ
LYS
A
214
28.384
10.085
29.003
1.00
39.15
A


ATOM
1063
C
LYS
A
214
25.884
6.402
27.990
1.00
35.51
A


ATOM
1064
O
LYS
A
214
26.663
5.674
27.374
1.00
37.49
A


ATOM
1065
N
HIS
A
215
25.272
7.440
27.430
1.00
36.90
A


ATOM
1066
CA
HIS
A
215
25.492
7.828
26.034
1.00
38.06
A


ATOM
1067
CB
HIS
A
215
24.161
8.240
25.403
1.00
39.15
A


ATOM
1068
CG
HIS
A
215
24.256
8.634
23.961
1.00
42.53
A


ATOM
1069
CD2
HIS
A
215
24.657
9.789
23.377
1.00
42.04
A


ATOM
1070
ND1
HIS
A
215
23.861
7.802
22.934
1.00
44.41
A


ATOM
1071
CE1
HIS
A
215
24.008
8.429
21.780
1.00
44.09
A


ATOM
1072
NE2
HIS
A
215
24.490
9.638
22.021
1.00
45.63
A


ATOM
1073
C
HIS
A
215
26.439
9.033
26.062
1.00
39.69
A


ATOM
1074
O
HIS
A
215
26.408
9.807
27.016
1.00
40.24
A


ATOM
1075
N
ASN
A
216
27.263
9.187
25.025
1.00
40.51
A


ATOM
1076
CA
ASN
A
216
28.204
10.306
24.935
1.00
42.83
A


ATOM
1077
CB
ASN
A
216
28.959
10.291
23.596
1.00
42.89
A


ATOM
1078
CG
ASN
A
216
30.045
9.233
23.535
1.00
42.90
A


ATOM
1079
OD1
ASN
A
216
30.499
8.722
24.556
1.00
44.23
A


ATOM
1080
ND2
ASN
A
216
30.484
8.914
22.323
1.00
43.07
A


ATOM
1081
C
ASN
A
216
27.538
11.675
25.080
1.00
42.65
A


ATOM
1082
O
ASN
A
216
28.179
12.632
25.519
1.00
44.85
A


ATOM
1083
N
ASN
A
217
26.270
11.784
24.701
1.00
42.59
A


ATOM
1084
CA
ASN
A
217
25.567
13.067
24.782
1.00
43.43
A


ATOM
1085
CB
ASN
A
217
24.719
13.297
23.523
1.00
45.33
A


ATOM
1086
CG
ASN
A
217
25.558
13.388
22.257
1.00
46.89
A


ATOM
1087
OD1
ASN
A
217
26.711
13.826
22.291
1.00
49.15
A


ATOM
1088
ND2
ASN
A
217
24.976
12.986
21.131
1.00
47.14
A


ATOM
1089
C
ASN
A
217
24.679
13.267
26.003
1.00
43.78
A


ATOM
1090
O
ASN
A
217
23.786
14.110
25.980
1.00
43.62
A


ATOM
1091
N
ASP
A
218
24.915
12.513
27.073
1.00
43.35
A


ATOM
1092
CA
ASP
A
218
24.086
12.662
28.263
1.00
43.44
A


ATOM
1093
CB
ASP
A
218
22.721
11.989
28.032
1.00
45.80
A


ATOM
1094
CG
ASP
A
218
21.613
12.575
28.907
1.00
47.88
A


ATOM
1095
OD1
ASP
A
218
21.920
13.368
29.823
1.00
48.42
A


ATOM
1096
OD2
ASP
A
218
20.430
12.237
28.679
1.00
46.04
A


ATOM
1097
C
ASP
A
218
24.777
12.033
29.465
1.00
41.31
A


ATOM
1098
O
ASP
A
218
25.815
11.385
29.335
1.00
41.97
A


ATOM
1099
N
THR
A
219
24.211
12.244
30.644
1.00
41.11
A


ATOM
1100
CA
THR
A
219
24.771
11.658
31.847
1.00
40.99
A


ATOM
1101
CB
THR
A
219
24.389
12.487
33.081
1.00
42.30
A


ATOM
1102
OG1
THR
A
219
22.980
12.744
33.058
1.00
43.10
A


ATOM
1103
CG2
THR
A
219
25.150
13.836
33.078
1.00
44.68
A


ATOM
1104
C
THR
A
219
24.188
10.243
31.960
1.00
40.58
A


ATOM
1105
O
THR
A
219
23.305
9.868
31.185
1.00
40.58
A


ATOM
1106
N
GLN
A
220
24.689
9.463
32.908
1.00
40.36
A


ATOM
1107
CA
GLN
A
220
24.211
8.092
33.102
1.00
39.83
A


ATOM
1108
CB
GLN
A
220
25.248
7.299
33.900
1.00
40.67
A


ATOM
1109
CG
GLN
A
220
24.877
5.857
34.246
1.00
41.43
A


ATOM
1110
CD
GLN
A
220
26.102
5.047
34.647
1.00
42.56
A


ATOM
1111
OE1
GLN
A
220
26.056
4.239
35.578
1.00
44.52
A


ATOM
1112
NE2
GLN
A
220
27.204
5.252
33.935
1.00
41.54
A


ATOM
1113
C
GLN
A
220
22.858
8.062
33.806
1.00
38.08
A


ATOM
1114
O
GLN
A
220
22.641
8.747
34.812
1.00
35.10
A


ATOM
1115
N
HIS
A
221
21.947
7.246
33.275
1.00
36.61
A


ATOM
1116
CA
HIS
A
221
20.613
7.117
33.833
1.00
34.35
A


ATOM
1117
CB
HIS
A
221
19.587
7.755
32.888
1.00
37.28
A


ATOM
1118
CG
HIS
A
221
19.687
9.249
32.799
1.00
39.74
A


ATOM
1119
CD2
HIS
A
221
20.550
10.047
32.127
1.00
38.57
A


ATOM
1120
ND1
HIS
A
221
18.830
10.095
33.471
1.00
41.09
A


ATOM
1121
CE1
HIS
A
221
19.162
11.350
33.218
1.00
39.34
A


ATOM
1122
NE2
HIS
A
221
20.201
11.348
32.405
1.00
40.70
A


ATOM
1123
C
HIS
A
221
20.260
5.638
34.044
1.00
35.15
A


ATOM
1124
O
HIS
A
221
20.942
4.742
33.522
1.00
30.35
A


ATOM
1125
N
ILE
A
222
19.211
5.416
34.829
1.00
34.92
A


ATOM
1126
CA
ILE
A
222
18.706
4.071
35.128
1.00
35.77
A


ATOM
1127
CB
ILE
A
222
18.851
3.715
36.638
1.00
34.59
A


ATOM
1128
CG2
ILE
A
222
18.362
2.274
36.894
1.00
36.24
A


ATOM
1129
CG1
ILE
A
222
20.304
3.849
37.068
1.00
33.15
A


ATOM
1130
CD1
ILE
A
222
20.521
3.735
38.560
1.00
33.87
A


ATOM
1131
C
ILE
A
222
17.218
3.989
34.774
1.00
34.51
A


ATOM
1132
O
ILE
A
222
16.432
4.867
35.146
1.00
35.84
A


ATOM
1133
N
TRP
A
223
16.848
2.945
34.029
1.00
34.41
A


ATOM
1134
CA
TRP
A
223
15.462
2.679
33.643
1.00
33.72
A


ATOM
1135
CB
TRP
A
223
15.391
2.404
32.130
1.00
34.35
A


ATOM
1136
CG
TRP
A
223
14.032
2.052
31.545
1.00
34.80
A


ATOM
1137
CD2
TRP
A
223
13.444
0.743
31.460
1.00
33.67
A


ATOM
1138
CE2
TRP
A
223
12.243
0.868
30.726
1.00
34.03
A


ATOM
1139
CE3
TRP
A
223
13.825
−0.526
31.926
1.00
35.44
A


ATOM
1140
CD1
TRP
A
223
13.173
2.897
30.890
1.00
33.40
A


ATOM
1141
NE1
TRP
A
223
12.096
2.190
30.393
1.00
34.12
A


ATOM
1142
CZ2
TRP
A
223
11.414
−0.234
30.448
1.00
33.49
A


ATOM
1143
CZ3
TRP
A
223
12.992
−1.627
31.645
1.00
32.64
A


ATOM
1144
CH2
TRP
A
223
11.813
−1.468
30.914
1.00
31.20
A


ATOM
1145
C
TRP
A
223
15.150
1.406
34.438
1.00
34.21
A


ATOM
1146
O
TRP
A
223
16.015
0.537
34.559
1.00
34.33
A


ATOM
1147
N
GLU
A
224
13.956
1.306
35.008
1.00
35.10
A


ATOM
1148
CA
GLU
A
224
13.592
0.114
35.780
1.00
36.56
A


ATOM
1149
CB
GLU
A
224
13.952
0.308
37.263
1.00
38.53
A


ATOM
1150
CG
GLU
A
224
13.255
1.465
37.945
1.00
40.52
A


ATOM
1151
CD
GLU
A
224
11.948
1.052
38.592
1.00
45.21
A


ATOM
1152
OE1
GLU
A
224
11.152
1.951
38.931
1.00
45.71
A


ATOM
1153
OE2
GLU
A
224
11.723
−0.169
38.776
1.00
44.23
A


ATOM
1154
C
GLU
A
224
12.106
−0.196
35.608
1.00
35.60
A


ATOM
1155
O
GLU
A
224
11.263
0.708
35.554
1.00
35.06
A


ATOM
1156
N
SER
A
225
11.773
−1.481
35.507
1.00
33.72
A


ATOM
1157
CA
SER
A
225
10.390
−1.846
35.300
1.00
31.46
A


ATOM
1158
CB
SER
A
225
10.022
−1.636
33.830
1.00
33.63
A


ATOM
1159
OG
SER
A
225
8.750
−2.203
33.541
1.00
34.39
A


ATOM
1160
C
SER
A
225
10.074
−3.290
35.665
1.00
33.06
A


ATOM
1161
O
SER
A
225
10.934
−4.163
35.538
1.00
29.51
A


ATOM
1162
N
ASP
A
226
8.845
−3.515
36.116
1.00
34.95
A


ATOM
1163
CA
ASP
A
226
8.368
−4.858
36.454
1.00
36.21
A


ATOM
1164
CB
ASP
A
226
7.769
−4.909
37.869
1.00
37.64
A


ATOM
1165
CG
ASP
A
226
6.467
−4.129
37.994
1.00
38.79
A


ATOM
1166
OD1
ASP
A
226
5.726
−4.376
38.967
1.00
40.27
A


ATOM
1167
OD2
ASP
A
226
6.188
−3.265
37.137
1.00
38.30
A


ATOM
1168
C
ASP
A
226
7.301
−5.206
35.418
1.00
35.98
A


ATOM
1169
O
ASP
A
226
6.510
−6.133
35.603
1.00
36.56
A


ATOM
1170
N
SER
A
227
7.318
−4.437
34.324
1.00
32.04
A


ATOM
1171
CA
SER
A
227
6.410
−4.535
33.181
1.00
32.39
A


ATOM
1172
CB
SER
A
227
6.159
−6.007
32.791
1.00
33.69
A


ATOM
1173
OG
SER
A
227
5.115
−6.572
33.557
1.00
35.74
A


ATOM
1174
C
SER
A
227
5.066
−3.832
33.402
1.00
29.43
A


ATOM
1175
O
SER
A
227
4.309
−3.629
32.450
1.00
33.38
A


ATOM
1176
N
ASN
A
228
4.772
−3.453
34.641
1.00
28.29
A


ATOM
1177
CA
ASN
A
228
3.503
−2.793
34.955
1.00
29.31
A


ATOM
1178
CB
ASN
A
228
3.029
−3.265
36.324
1.00
31.61
A


ATOM
1179
CG
ASN
A
228
2.704
−4.764
36.340
1.00
35.15
A


ATOM
1180
OD1
ASN
A
228
3.127
−5.498
37.241
1.00
36.20
A


ATOM
1181
ND2
ASN
A
228
1.950
−5.211
35.344
1.00
30.27
A


ATOM
1182
C
ASN
A
228
3.588
−1.253
34.920
1.00
31.26
A


ATOM
1183
O
ASN
A
228
2.601
−0.565
35.170
1.00
31.12
A


ATOM
1184
N
ALA
A
229
4.773
−0.749
34.601
1.00
33.22
A


ATOM
1185
CA
ALA
A
229
5.059
0.690
34.505
1.00
33.19
A


ATOM
1186
CB
ALA
A
229
4.513
1.428
35.741
1.00
34.56
A


ATOM
1187
C
ALA
A
229
6.575
0.761
34.478
1.00
34.91
A


ATOM
1188
O
ALA
A
229
7.230
−0.270
34.609
1.00
33.58
A


ATOM
1189
N
PHE
A
230
7.149
1.954
34.313
1.00
32.04
A


ATOM
1190
CA
PHE
A
230
8.596
2.068
34.327
1.00
33.83
A


ATOM
1191
CB
PHE
A
230
9.186
1.764
32.936
1.00
35.52
A


ATOM
1192
CG
PHE
A
230
8.957
2.842
31.905
1.00
33.95
A


ATOM
1193
CD1
PHE
A
230
9.880
3.882
31.752
1.00
35.30
A


ATOM
1194
CD2
PHE
A
230
7.863
2.781
31.046
1.00
34.06
A


ATOM
1195
CE1
PHE
A
230
9.714
4.835
30.750
1.00
35.01
A


ATOM
1196
CE2
PHE
A
230
7.683
3.729
30.038
1.00
38.15
A


ATOM
1197
CZ
PHE
A
230
8.620
4.767
29.887
1.00
34.58
A


ATOM
1198
C
PHE
A
230
9.013
3.459
34.805
1.00
35.37
A


ATOM
1199
O
PHE
A
230
8.218
4.392
34.766
1.00
36.39
A


ATOM
1200
N
SER
A
231
10.236
3.575
35.301
1.00
36.27
A


ATOM
1201
CA
SER
A
231
10.728
4.870
35.765
1.00
37.81
A


ATOM
1202
CB
SER
A
231
10.650
4.976
37.295
1.00
37.18
A


ATOM
1203
OG
SER
A
231
11.683
4.254
37.931
1.00
43.77
A


ATOM
1204
C
SER
A
231
12.154
5.089
35.298
1.00
39.01
A


ATOM
1205
O
SER
A
231
12.888
4.139
34.996
1.00
35.80
A


ATOM
1206
N
VAL
A
232
12.543
6.356
35.219
1.00
37.11
A


ATOM
1207
CA
VAL
A
232
13.887
6.694
34.813
1.00
38.94
A


ATOM
1208
CB
VAL
A
232
13.939
7.228
33.362
1.00
40.57
A


ATOM
1209
CG1
VAL
A
232
13.119
8.494
33.233
1.00
44.75
A


ATOM
1210
CG2
VAL
A
232
15.382
7.481
32.964
1.00
42.03
A


ATOM
1211
C
VAL
A
232
14.385
7.755
35.777
1.00
38.34
A


ATOM
1212
O
VAL
A
232
13.663
8.696
36.094
1.00
36.03
A


ATOM
1213
N
ILE
A
233
15.598
7.557
36.270
1.00
38.95
A


ATOM
1214
CA
ILE
A
233
16.213
8.478
37.216
1.00
41.07
A


ATOM
1215
CB
ILE
A
233
16.231
7.891
38.650
1.00
41.10
A


ATOM
1216
CG2
ILE
A
233
14.824
7.535
39.093
1.00
44.79
A


ATOM
1217
CG1
ILE
A
233
17.123
6.641
38.694
1.00
43.25
A


ATOM
1218
CD1
ILE
A
233
17.451
6.156
40.100
1.00
44.38
A


ATOM
1219
C
ILE
A
233
17.658
8.702
36.808
1.00
40.36
A


ATOM
1220
O
ILE
A
233
18.263
7.838
36.181
1.00
37.92
A


ATOM
1221
N
ALA
A
234
18.212
9.866
37.144
1.00
40.64
A


ATOM
1222
CA
ALA
A
234
19.614
10.109
36.833
1.00
40.47
A


ATOM
1223
CB
ALA
A
234
19.992
11.559
37.151
1.00
39.23
A


ATOM
1224
C
ALA
A
234
20.341
9.150
37.774
1.00
39.79
A


ATOM
1225
O
ALA
A
234
19.917
8.975
38.917
1.00
41.32
A


ATOM
1226
N
ASP
A
235
21.414
8.520
37.311
1.00
39.90
A


ATOM
1227
CA
ASP
A
235
22.153
7.591
38.158
1.00
41.21
A


ATOM
1228
CB
ASP
A
235
23.121
6.745
37.322
1.00
39.33
A


ATOM
1229
CG
ASP
A
235
23.640
5.537
38.075
1.00
40.56
A


ATOM
1230
OD1
ASP
A
235
23.753
5.614
39.312
1.00
42.34
A


ATOM
1231
OD2
ASP
A
235
23.947
4.511
37.432
1.00
38.58
A


ATOM
1232
C
ASP
A
235
22.949
8.382
39.202
1.00
43.09
A


ATOM
1233
O
ASP
A
235
23.775
9.228
38.855
1.00
41.26
A


ATOM
1234
N
PRO
A
236
22.701
8.117
40.494
1.00
44.50
A


ATOM
1235
CD
PRO
A
236
21.702
7.176
41.044
1.00
43.52
A


ATOM
1236
CA
PRO
A
236
23.412
8.817
41.569
1.00
45.36
A


ATOM
1237
CB
PRO
A
236
22.626
8.411
42.818
1.00
45.58
A


ATOM
1238
CG
PRO
A
236
22.159
7.016
42.473
1.00
44.95
A


ATOM
1239
C
PRO
A
236
24.888
8.431
41.653
1.00
47.00
A


ATOM
1240
O
PRO
A
236
25.691
9.154
42.248
1.00
47.95
A


ATOM
1241
N
ARG
A
237
25.248
7.299
41.051
1.00
46.03
A


ATOM
1242
CA
ARG
A
237
26.631
6.835
41.069
1.00
47.36
A


ATOM
1243
CB
ARG
A
237
26.692
5.332
40.755
1.00
46.32
A


ATOM
1244
CG
ARG
A
237
26.028
4.434
41.799
1.00
47.54
A


ATOM
1245
CD
ARG
A
237
25.908
2.994
41.305
1.00
47.06
A


ATOM
1246
NE
ARG
A
237
25.047
2.902
40.128
1.00
46.65
A


ATOM
1247
CZ
ARG
A
237
24.705
1.763
39.529
1.00
47.42
A


ATOM
1248
NH1
ARG
A
237
25.152
0.598
39.995
1.00
45.44
A


ATOM
1249
NH2
ARG
A
237
23.911
1.792
38.464
1.00
43.05
A


ATOM
1250
C
ARG
A
237
27.495
7.597
40.060
1.00
47.83
A


ATOM
1251
O
ARG
A
237
28.704
7.383
39.984
1.00
48.93
A


ATOM
1252
N
GLY
A
238
26.873
8.483
39.290
1.00
48.46
A


ATOM
1253
CA
GLY
A
238
27.610
9.231
38.288
1.00
49.54
A


ATOM
1254
C
GLY
A
238
27.913
8.370
37.076
1.00
50.88
A


ATOM
1255
O
GLY
A
238
27.231
7.372
36.826
1.00
50.89
A


ATOM
1256
N
ASN
A
239
28.941
8.750
36.323
1.00
51.50
A


ATOM
1257
CA
ASN
A
239
29.335
8.015
35.121
1.00
52.78
A


ATOM
1258
CB
ASN
A
239
29.999
8.975
34.122
1.00
55.72
A


ATOM
1259
CG
ASN
A
239
30.638
8.252
32.951
1.00
59.41
A


ATOM
1260
OD1
ASN
A
239
30.160
7.203
32.518
1.00
60.19
A


ATOM
1261
ND2
ASN
A
239
31.720
8.819
32.422
1.00
61.11
A


ATOM
1262
C
ASN
A
239
30.275
6.859
35.444
1.00
51.17
A


ATOM
1263
O
ASN
A
239
31.491
7.035
35.486
1.00
52.67
A


ATOM
1264
N
THR
A
240
29.712
5.674
35.659
1.00
47.62
A


ATOM
1265
CA
THR
A
240
30.511
4.504
35.998
1.00
45.60
A


ATOM
1266
CB
THR
A
240
29.843
3.690
37.130
1.00
46.23
A


ATOM
1267
OG1
THR
A
240
28.571
3.197
36.685
1.00
43.74
A


ATOM
1268
CG2
THR
A
240
29.635
4.566
38.360
1.00
44.58
A


ATOM
1269
C
THR
A
240
30.754
3.572
34.816
1.00
45.28
A


ATOM
1270
O
THR
A
240
31.700
2.780
34.820
1.00
44.39
A


ATOM
1271
N
LEU
A
241
29.896
3.660
33.808
1.00
43.37
A


ATOM
1272
CA
LEU
A
241
30.025
2.819
32.629
1.00
43.66
A


ATOM
1273
CB
LEU
A
241
28.658
2.663
31.952
1.00
42.59
A


ATOM
1274
CG
LEU
A
241
27.577
1.918
32.740
1.00
42.51
A


ATOM
1275
CD1
LEU
A
241
26.249
2.021
32.011
1.00
40.99
A


ATOM
1276
CD2
LEU
A
241
27.987
0.456
32.911
1.00
41.69
A


ATOM
1277
C
LEU
A
241
31.034
3.353
31.611
1.00
42.94
A


ATOM
1278
O
LEU
A
241
31.632
2.574
30.867
1.00
43.16
A


ATOM
1279
N
GLY
A
242
31.219
4.672
31.590
1.00
42.04
A


ATOM
1280
CA
GLY
A
242
32.127
5.288
30.634
1.00
41.74
A


ATOM
1281
C
GLY
A
242
31.287
5.553
29.395
1.00
41.22
A


ATOM
1282
O
GLY
A
242
31.096
6.697
28.970
1.00
40.56
A


ATOM
1283
N
ARG
A
243
30.775
4.463
28.833
1.00
39.01
A


ATOM
1284
CA
ARG
A
243
29.895
4.469
27.674
1.00
37.25
A


ATOM
1285
CB
ARG
A
243
30.630
4.830
26.385
1.00
36.87
A


ATOM
1286
CG
ARG
A
243
29.684
4.953
25.194
1.00
36.89
A


ATOM
1287
CD
ARG
A
243
30.380
4.784
23.848
1.00
37.66
A


ATOM
1288
NE
ARG
A
243
31.083
3.509
23.744
1.00
36.50
A


ATOM
1289
CZ
ARG
A
243
32.393
3.356
23.901
1.00
39.23
A


ATOM
1290
NH1
ARG
A
243
33.167
4.409
24.162
1.00
38.79
A


ATOM
1291
NH2
ARG
A
243
32.931
2.142
23.809
1.00
39.28
A


ATOM
1292
C
ARG
A
243
29.358
3.046
27.543
1.00
37.57
A


ATOM
1293
O
ARG
A
243
30.110
2.073
27.652
1.00
36.86
A


ATOM
1294
N
GLY
A
244
28.062
2.916
27.309
1.00
37.87
A


ATOM
1295
CA
GLY
A
244
27.518
1.579
27.174
1.00
39.10
A


ATOM
1296
C
GLY
A
244
26.229
1.379
27.932
1.00
37.78
A


ATOM
1297
O
GLY
A
244
25.591
2.338
28.363
1.00
36.73
A


ATOM
1298
N
THR
A
245
25.866
0.115
28.122
1.00
37.45
A


ATOM
1299
CA
THR
A
245
24.618
−0.229
28.776
1.00
35.71
A


ATOM
1300
CB
THR
A
245
23.491
−0.395
27.728
1.00
38.09
A


ATOM
1301
OG1
THR
A
245
23.310
0.834
27.015
1.00
38.31
A


ATOM
1302
CG2
THR
A
245
22.180
−0.808
28.400
1.00
37.67
A


ATOM
1303
C
THR
A
245
24.720
−1.547
29.521
1.00
36.13
A


ATOM
1304
O
THR
A
245
25.351
−2.488
29.040
1.00
35.73
A


ATOM
1305
N
THR
A
246
24.106
−1.593
30.698
1.00
34.43
A


ATOM
1306
CA
THR
A
246
24.053
−2.804
31.492
1.00
35.99
A


ATOM
1307
CB
THR
A
246
24.645
−2.630
32.925
1.00
36.89
A


ATOM
1308
OG1
THR
A
246
26.055
−2.369
32.856
1.00
36.25
A


ATOM
1309
CG2
THR
A
246
24.408
−3.901
33.742
1.00
39.20
A


ATOM
1310
C
THR
A
246
22.578
−3.163
31.647
1.00
35.24
A


ATOM
1311
O
THR
A
246
21.762
−2.335
32.078
1.00
34.48
A


ATOM
1312
N
ILE
A
247
22.233
−4.388
31.265
1.00
34.48
A


ATOM
1313
CA
ILE
A
247
20.868
−4.878
31.421
1.00
33.45
A


ATOM
1314
CB
ILE
A
247
20.415
−5.765
30.224
1.00
33.63
A


ATOM
1315
CG2
ILE
A
247
18.964
−6.191
30.421
1.00
35.01
A


ATOM
1316
CG1
ILE
A
247
20.597
−5.030
28.883
1.00
32.43
A


ATOM
1317
CD1
ILE
A
247
19.686
−3.815
28.676
1.00
34.07
A


ATOM
1318
C
ILE
A
247
20.973
−5.777
32.652
1.00
34.92
A


ATOM
1319
O
ILE
A
247
21.689
−6.782
32.624
1.00
33.23
A


ATOM
1320
N
THR
A
248
20.316
−5.408
33.744
1.00
36.35
A


ATOM
1321
CA
THR
A
248
20.376
−6.245
34.938
1.00
36.95
A


ATOM
1322
CB
THR
A
248
20.753
−5.452
36.214
1.00
39.00
A


ATOM
1323
OG1
THR
A
248
19.665
−4.602
36.596
1.00
47.01
A


ATOM
1324
CG2
THR
A
248
21.974
−4.611
35.977
1.00
32.77
A


ATOM
1325
C
THR
A
248
19.006
−6.856
35.139
1.00
37.36
A


ATOM
1326
O
THR
A
248
17.991
−6.149
35.161
1.00
35.94
A


ATOM
1327
N
LEU
A
249
18.985
−8.178
35.261
1.00
35.99
A


ATOM
1328
CA
LEU
A
249
17.739
−8.910
35.457
1.00
36.92
A


ATOM
1329
CB
LEU
A
249
17.634
−10.069
34.455
1.00
35.98
A


ATOM
1330
CG
LEU
A
249
17.708
−9.770
32.949
1.00
34.21
A


ATOM
1331
CD1
LEU
A
249
17.275
−11.012
32.175
1.00
37.00
A


ATOM
1332
CD2
LEU
A
249
16.796
−8.605
32.595
1.00
36.30
A


ATOM
1333
C
LEU
A
249
17.647
−9.479
36.866
1.00
35.98
A


ATOM
1334
O
LEU
A
249
18.568
−10.154
37.336
1.00
36.96
A


ATOM
1335
N
VAL
A
250
16.541
−9.185
37.537
1.00
36.15
A


ATOM
1336
CA
VAL
A
250
16.274
−9.708
38.871
1.00
36.88
A


ATOM
1337
CB
VAL
A
250
15.448
−8.701
39.691
1.00
37.61
A


ATOM
1338
CG1
VAL
A
250
15.135
−9.274
41.075
1.00
39.94
A


ATOM
1339
CG2
VAL
A
250
16.228
−7.395
39.820
1.00
36.44
A


ATOM
1340
C
VAL
A
250
15.449
−10.959
38.557
1.00
36.98
A


ATOM
1341
O
VAL
A
250
14.244
−10.883
38.351
1.00
35.45
A


ATOM
1342
N
LEU
A
251
16.120
−12.107
38.511
1.00
38.82
A


ATOM
1343
CA
LEU
A
251
15.478
−13.366
38.138
1.00
39.06
A


ATOM
1344
CB
LEU
A
251
16.537
−14.470
37.987
1.00
40.36
A


ATOM
1345
CG
LEU
A
251
17.640
−14.374
36.924
1.00
40.29
A


ATOM
1346
CD1
LEU
A
251
17.025
−14.052
35.554
1.00
40.30
A


ATOM
1347
CD2
LEU
A
251
18.643
−13.310
37.322
1.00
45.74
A


ATOM
1348
C
LEU
A
251
14.338
−13.918
38.992
1.00
40.19
A


ATOM
1349
O
LEU
A
251
14.344
−13.823
40.219
1.00
38.35
A


ATOM
1350
N
LYS
A
252
13.368
−14.517
38.308
1.00
40.87
A


ATOM
1351
CA
LYS
A
252
12.226
−15.139
38.959
1.00
44.52
A


ATOM
1352
CB
LYS
A
252
11.185
−15.568
37.922
1.00
43.89
A


ATOM
1353
CG
LYS
A
252
10.500
−14.435
37.163
1.00
44.89
A


ATOM
1354
CD
LYS
A
252
9.645
−15.002
36.032
1.00
43.46
A


ATOM
1355
CE
LYS
A
252
8.860
−13.935
35.282
1.00
44.72
A


ATOM
1356
NZ
LYS
A
252
7.824
−13.287
36.132
1.00
42.45
A


ATOM
1357
C
LYS
A
252
12.768
−16.384
39.658
1.00
47.30
A


ATOM
1358
O
LYS
A
252
13.802
−16.931
39.264
1.00
45.15
A


ATOM
1359
N
GLU
A
253
12.068
−16.834
40.690
1.00
50.33
A


ATOM
1360
CA
GLU
A
253
12.499
−18.015
41.422
1.00
53.47
A


ATOM
1361
CB
GLU
A
253
11.426
−18.423
42.428
1.00
56.08
A


ATOM
1362
CG
GLU
A
253
11.834
−19.568
43.327
1.00
59.63
A


ATOM
1363
CD
GLU
A
253
10.674
−20.099
44.135
1.00
62.42
A


ATOM
1364
OE1
GLU
A
253
9.756
−20.701
43.531
1.00
63.41
A


ATOM
1365
OE2
GLU
A
253
10.676
−19.907
45.371
1.00
64.55
A


ATOM
1366
C
GLU
A
253
12.780
−19.185
40.477
1.00
53.70
A


ATOM
1367
O
GLU
A
253
13.838
−19.813
40.546
1.00
53.56
A


ATOM
1368
N
GLU
A
254
11.833
−19.462
39.588
1.00
55.23
A


ATOM
1369
CA
GLU
A
254
11.957
−20.568
38.640
1.00
56.74
A


ATOM
1370
CB
GLU
A
254
10.630
−20.774
37.905
1.00
59.86
A


ATOM
1371
CG
GLU
A
254
9.445
−21.073
38.811
1.00
64.81
A


ATOM
1372
CD
GLU
A
254
8.165
−21.334
38.035
1.00
67.53
A


ATOM
1373
OE1
GLU
A
254
7.114
−21.563
38.676
1.00
69.99
A


ATOM
1374
OE2
GLU
A
254
8.209
−21.311
36.783
1.00
69.08
A


ATOM
1375
C
GLU
A
254
13.065
−20.396
37.607
1.00
55.58
A


ATOM
1376
O
GLU
A
254
13.296
−21.290
36.791
1.00
55.55
A


ATOM
1377
N
ALA
A
255
13.745
−19.254
37.639
1.00
54.30
A


ATOM
1378
CA
ALA
A
255
14.814
−18.970
36.682
1.00
51.41
A


ATOM
1379
CB
ALA
A
255
14.560
−17.611
36.016
1.00
51.65
A


ATOM
1380
C
ALA
A
255
16.203
−18.990
37.311
1.00
50.31
A


ATOM
1381
O
ALA
A
255
17.177
−18.585
36.682
1.00
49.04
A


ATOM
1382
N
SER
A
256
16.292
−19.466
38.550
1.00
50.77
A


ATOM
1383
CA
SER
A
256
17.564
−19.531
39.266
1.00
51.61
A


ATOM
1384
CB
SER
A
256
17.366
−20.225
40.622
1.00
53.80
A


ATOM
1385
OG
SER
A
256
16.683
−21.464
40.485
1.00
56.72
A


ATOM
1386
C
SER
A
256
18.689
−20.218
38.486
1.00
52.07
A


ATOM
1387
O
SER
A
256
19.867
−19.906
38.679
1.00
50.98
A


ATOM
1388
N
ASP
A
257
18.329
−21.145
37.602
1.00
52.71
A


ATOM
1389
CA
ASP
A
257
19.321
−21.859
36.799
1.00
54.07
A


ATOM
1390
CB
ASP
A
257
18.635
−22.869
35.883
1.00
58.47
A


ATOM
1391
CG
ASP
A
257
18.605
−24.255
36.476
1.00
62.55
A


ATOM
1392
OD1
ASP
A
257
19.699
−24.786
36.774
1.00
65.44
A


ATOM
1393
OD2
ASP
A
257
17.498
−24.811
36.645
1.00
65.65
A


ATOM
1394
C
ASP
A
257
20.194
−20.944
35.949
1.00
53.58
A


ATOM
1395
O
ASP
A
257
21.346
−21.270
35.659
1.00
52.34
A


ATOM
1396
N
TYR
A
258
19.650
−19.805
35.537
1.00
51.92
A


ATOM
1397
CA
TYR
A
258
20.422
−18.879
34.720
1.00
52.25
A


ATOM
1398
CB
TYR
A
258
19.498
−17.829
34.095
1.00
50.79
A


ATOM
1399
CG
TYR
A
258
18.547
−18.422
33.084
1.00
50.49
A


ATOM
1400
CD1
TYR
A
258
17.204
−18.629
33.394
1.00
49.41
A


ATOM
1401
CE1
TYR
A
258
16.344
−19.230
32.485
1.00
50.81
A


ATOM
1402
CD2
TYR
A
258
19.005
−18.828
31.828
1.00
51.47
A


ATOM
1403
CE2
TYR
A
258
18.153
−19.430
30.912
1.00
51.69
A


ATOM
1404
CZ
TYR
A
258
16.827
−19.629
31.246
1.00
52.00
A


ATOM
1405
OH
TYR
A
258
15.988
−20.238
30.344
1.00
52.08
A


ATOM
1406
C
TYR
A
258
21.547
−18.219
35.515
1.00
51.93
A


ATOM
1407
O
TYR
A
258
22.375
−17.505
34.957
1.00
53.70
A


ATOM
1408
N
LEU
A
259
21.578
−18.476
36.817
1.00
52.47
A


ATOM
1409
CA
LEU
A
259
22.612
−17.927
37.688
1.00
52.53
A


ATOM
1410
CB
LEU
A
259
22.080
−17.774
39.115
1.00
52.35
A


ATOM
1411
CG
LEU
A
259
20.974
−16.743
39.338
1.00
54.26
A


ATOM
1412
CD1
LEU
A
259
20.439
−16.870
40.757
1.00
53.62
A


ATOM
1413
CD2
LEU
A
259
21.522
−15.341
39.088
1.00
52.70
A


ATOM
1414
C
LEU
A
259
23.834
−18.843
37.703
1.00
52.92
A


ATOM
1415
O
LEU
A
259
24.902
−18.459
38.181
1.00
52.22
A


ATOM
1416
N
GLU
A
260
23.661
−20.054
37.181
1.00
53.68
A


ATOM
1417
CA
GLU
A
260
24.739
−21.039
37.133
1.00
55.47
A


ATOM
1418
CB
GLU
A
260
24.158
−22.450
36.975
1.00
56.20
A


ATOM
1419
CG
GLU
A
260
23.290
−22.910
38.133
1.00
57.13
A


ATOM
1420
CD
GLU
A
260
24.044
−22.947
39.453
1.00
59.37
A


ATOM
1421
OE1
GLU
A
260
25.098
−23.615
39.521
1.00
59.76
A


ATOM
1422
OE2
GLU
A
260
23.580
−22.311
40.426
1.00
59.26
A


ATOM
1423
C
GLU
A
260
25.696
−20.746
35.978
1.00
56.29
A


ATOM
1424
O
GLU
A
260
25.265
−20.451
34.861
1.00
55.70
A


ATOM
1425
N
LEU
A
261
26.994
−20.838
36.254
1.00
57.25
A


ATOM
1426
CA
LEU
A
261
28.018
−20.571
35.252
1.00
58.21
A


ATOM
1427
CB
LEU
A
261
29.407
−20.595
35.896
1.00
58.81
A


ATOM
1428
CG
LEU
A
261
29.791
−19.375
36.737
1.00
59.50
A


ATOM
1429
CD1
LEU
A
261
31.140
−19.604
37.404
1.00
59.71
A


ATOM
1430
CD2
LEU
A
261
29.833
−18.145
35.846
1.00
60.31
A


ATOM
1431
C
LEU
A
261
28.004
−21.508
34.052
1.00
58.27
A


ATOM
1432
O
LEU
A
261
28.117
−21.057
32.915
1.00
59.29
A


ATOM
1433
N
ASP
A
262
27.875
−22.808
34.289
1.00
58.80
A


ATOM
1434
CA
ASP
A
262
27.868
−23.751
33.175
1.00
59.21
A


ATOM
1435
CB
ASP
A
262
27.896
−25.193
33.692
1.00
61.69
A


ATOM
1436
CG
ASP
A
262
26.714
−25.517
34.573
1.00
64.11
A


ATOM
1437
OD1
ASP
A
262
26.513
−24.812
35.585
1.00
65.99
A


ATOM
1438
OD2
ASP
A
262
25.987
−26.482
34.255
1.00
67.06
A


ATOM
1439
C
ASP
A
262
26.650
−23.525
32.286
1.00
58.16
A


ATOM
1440
O
ASP
A
262
26.699
−23.748
31.078
1.00
57.22
A


ATOM
1441
N
THR
A
263
25.559
−23.065
32.886
1.00
57.75
A


ATOM
1442
CA
THR
A
263
24.342
−22.804
32.130
1.00
57.17
A


ATOM
1443
CB
THR
A
263
23.146
−22.570
33.069
1.00
58.24
A


ATOM
1444
OG1
THR
A
263
22.972
−23.715
33.917
1.00
59.79
A


ATOM
1445
CG2
THR
A
263
21.874
−22.342
32.259
1.00
58.17
A


ATOM
1446
C
THR
A
263
24.514
−21.567
31.248
1.00
56.27
A


ATOM
1447
O
THR
A
263
24.284
−21.618
30.040
1.00
54.38
A


ATOM
1448
N
ILE
A
264
24.932
−20.464
31.864
1.00
55.73
A


ATOM
1449
CA
ILE
A
264
25.125
−19.208
31.148
1.00
56.53
A


ATOM
1450
CB
ILE
A
264
25.454
−18.053
32.138
1.00
57.51
A


ATOM
1451
CG2
ILE
A
264
26.717
−18.368
32.915
1.00
57.95
A


ATOM
1452
CG1
ILE
A
264
25.593
−16.728
31.383
1.00
58.53
A


ATOM
1453
CD1
ILE
A
264
24.274
−16.173
30.871
1.00
61.05
A


ATOM
1454
C
ILE
A
264
26.213
−19.296
30.075
1.00
56.05
A


ATOM
1455
O
ILE
A
264
26.019
−18.831
28.955
1.00
54.57
A


ATOM
1456
N
LYS
A
265
27.352
−19.899
30.406
1.00
57.12
A


ATOM
1457
CA
LYS
A
265
28.435
−20.020
29.436
1.00
57.54
A


ATOM
1458
CB
LYS
A
265
29.651
−20.711
30.060
1.00
58.26
A


ATOM
1459
CG
LYS
A
265
30.401
−19.831
31.051
1.00
59.29
A


ATOM
1460
CD
LYS
A
265
31.778
−20.388
31.414
1.00
60.73
A


ATOM
1461
CE
LYS
A
265
31.702
−21.629
32.294
1.00
62.73
A


ATOM
1462
NZ
LYS
A
265
31.164
−22.829
31.592
1.00
63.52
A


ATOM
1463
C
LYS
A
265
28.011
−20.758
28.172
1.00
57.48
A


ATOM
1464
O
LYS
A
265
28.331
−20.327
27.064
1.00
58.37
A


ATOM
1465
N
ASN
A
266
27.283
−21.859
28.335
1.00
56.86
A


ATOM
1466
CA
ASN
A
266
26.824
−22.637
27.189
1.00
56.53
A


ATOM
1467
CB
ASN
A
266
26.093
−23.899
27.661
1.00
59.04
A


ATOM
1468
CG
ASN
A
266
26.207
−25.050
26.671
1.00
62.82
A


ATOM
1469
OD1
ASN
A
266
27.238
−25.728
26.601
1.00
64.55
A


ATOM
1470
ND2
ASN
A
266
25.151
−25.271
25.894
1.00
63.03
A


ATOM
1471
C
ASN
A
266
25.885
−21.793
26.325
1.00
55.35
A


ATOM
1472
O
ASN
A
266
25.987
−21.794
25.099
1.00
55.31
A


ATOM
1473
N
LEU
A
267
24.974
−21.064
26.967
1.00
53.28
A


ATOM
1474
CA
LEU
A
267
24.024
−20.226
26.241
1.00
51.71
A


ATOM
1475
CB
LEU
A
267
22.939
−19.702
27.186
1.00
51.22
A


ATOM
1476
CG
LEU
A
267
22.005
−20.747
27.801
1.00
50.93
A


ATOM
1477
CD1
LEU
A
267
21.016
−20.060
28.732
1.00
50.55
A


ATOM
1478
CD2
LEU
A
267
21.274
−21.502
26.701
1.00
49.71
A


ATOM
1479
C
LEU
A
267
24.712
−19.055
25.553
1.00
50.17
A


ATOM
1480
O
LEU
A
267
24.414
−18.744
24.402
1.00
49.76
A


ATOM
1481
N
VAL
A
268
25.627
−18.405
26.264
1.00
49.61
A


ATOM
1482
CA
VAL
A
268
26.357
−17.278
25.700
1.00
50.51
A


ATOM
1483
CB
VAL
A
268
27.350
−16.676
26.719
1.00
50.05
A


ATOM
1484
CG1
VAL
A
268
28.276
−15.695
26.021
1.00
48.90
A


ATOM
1485
CG2
VAL
A
268
26.588
−15.970
27.839
1.00
49.12
A


ATOM
1486
C
VAL
A
268
27.139
−17.736
24.475
1.00
52.34
A


ATOM
1487
O
VAL
A
268
27.222
−17.023
23.477
1.00
51.12
A


ATOM
1488
N
LYS
A
269
27.715
−18.932
24.559
1.00
54.85
A


ATOM
1489
CA
LYS
A
269
28.485
−19.475
23.452
1.00
56.05
A


ATOM
1490
CB
LYS
A
269
29.190
−20.768
23.884
1.00
58.31
A


ATOM
1491
CG
LYS
A
269
30.220
−21.273
22.884
1.00
60.99
A


ATOM
1492
CD
LYS
A
269
31.043
−22.433
23.445
1.00
62.07
A


ATOM
1493
CE
LYS
A
269
32.103
−22.893
22.445
1.00
63.31
A


ATOM
1494
NZ
LYS
A
269
32.919
−24.033
22.962
1.00
63.67
A


ATOM
1495
C
LYS
A
269
27.573
−19.741
22.258
1.00
56.18
A


ATOM
1496
O
LYS
A
269
27.891
−19.366
21.131
1.00
57.66
A


ATOM
1497
N
ALA
A
270
26.429
−20.368
22.508
1.00
55.65
A


ATOM
1498
CA
ALA
A
270
25.489
−20.690
21.439
1.00
56.06
A


ATOM
1499
CB
ALA
A
270
24.380
−21.587
21.978
1.00
55.79
A


ATOM
1500
C
ALA
A
270
24.873
−19.472
20.761
1.00
56.08
A


ATOM
1501
O
ALA
A
270
24.352
−19.574
19.649
1.00
56.07
A


ATOM
1502
N
TYR
A
271
24.939
−18.318
21.416
1.00
56.18
A


ATOM
1503
CA
TYR
A
271
24.343
−17.108
20.864
1.00
56.34
A


ATOM
1504
CB
TYR
A
271
23.297
−16.571
21.845
1.00
56.37
A


ATOM
1505
CG
TYR
A
271
22.141
−17.506
22.145
1.00
55.02
A


ATOM
1506
CD1
TYR
A
271
21.598
−17.567
23.429
1.00
54.14
A


ATOM
1507
CE1
TYR
A
271
20.500
−18.362
23.708
1.00
55.32
A


ATOM
1508
CD2
TYR
A
271
21.547
−18.278
21.140
1.00
55.39
A


ATOM
1509
CE2
TYR
A
271
20.438
−19.080
21.412
1.00
54.95
A


ATOM
1510
CZ
TYR
A
271
19.920
−19.113
22.700
1.00
55.50
A


ATOM
1511
OH
TYR
A
271
18.809
−19.878
22.991
1.00
56.77
A


ATOM
1512
C
TYR
A
271
25.324
−15.981
20.524
1.00
57.27
A


ATOM
1513
O
TYR
A
271
24.896
−14.873
20.206
1.00
56.68
A


ATOM
1514
N
SER
A
272
26.627
−16.247
20.572
1.00
57.63
A


ATOM
1515
CA
SER
A
272
27.601
−15.194
20.286
1.00
59.42
A


ATOM
1516
CB
SER
A
272
28.397
−14.873
21.557
1.00
59.53
A


ATOM
1517
OG
SER
A
272
29.120
−16.006
22.011
1.00
60.07
A


ATOM
1518
C
SER
A
272
28.575
−15.471
19.138
1.00
60.05
A


ATOM
1519
O
SER
A
272
29.495
−14.684
18.897
1.00
59.38
A


ATOM
1520
N
ALA
A
273
28.363
−16.570
18.423
1.00
61.51
A


ATOM
1521
CA
ALA
A
273
29.244
−16.943
17.319
1.00
62.88
A


ATOM
1522
CB
ALA
A
273
28.935
−18.375
16.875
1.00
63.23
A


ATOM
1523
C
ALA
A
273
29.200
−15.997
16.117
1.00
63.61
A


ATOM
1524
O
ALA
A
273
30.034
−16.101
15.219
1.00
63.89
A


ATOM
1525
N
PHE
A
274
28.238
−15.078
16.093
1.00
65.13
A


ATOM
1526
CA
PHE
A
274
28.127
−14.130
14.986
1.00
66.18
A


ATOM
1527
CB
PHE
A
274
26.722
−14.166
14.384
1.00
68.29
A


ATOM
1528
CG
PHE
A
274
26.462
−15.365
13.518
1.00
70.72
A


ATOM
1529
CD1
PHE
A
274
26.325
−16.633
14.078
1.00
71.78
A


ATOM
1530
CD2
PHE
A
274
26.345
−15.221
12.136
1.00
72.16
A


ATOM
1531
CE1
PHE
A
274
26.071
−17.748
13.274
1.00
72.69
A


ATOM
1532
CE2
PHE
A
274
26.092
−16.324
11.317
1.00
73.51
A


ATOM
1533
CZ
PHE
A
274
25.953
−17.594
11.888
1.00
73.64
A


ATOM
1534
C
PHE
A
274
28.467
−12.688
15.359
1.00
66.07
A


ATOM
1535
O
PHE
A
274
28.710
−11.858
14.482
1.00
67.24
A


ATOM
1536
N
ALA
A
275
28.479
−12.385
16.652
1.00
65.00
A


ATOM
1537
CA
ALA
A
275
28.792
−11.032
17.100
1.00
64.39
A


ATOM
1538
CB
ALA
A
275
28.647
−10.935
18.619
1.00
63.46
A


ATOM
1539
C
ALA
A
275
30.212
−10.672
16.681
1.00
63.05
A


ATOM
1540
O
ALA
A
275
31.141
−11.443
16.903
1.00
63.35
A


ATOM
1541
N
ASN
A
276
30.374
−9.500
16.076
1.00
62.19
A


ATOM
1542
CA
ASN
A
276
31.687
−9.050
15.623
1.00
60.41
A


ATOM
1543
CB
ASN
A
276
31.538
−8.114
14.422
1.00
63.49
A


ATOM
1544
CG
ASN
A
276
31.060
−8.837
13.178
1.00
65.72
A


ATOM
1545
OD1
ASN
A
276
31.730
−9.739
12.677
1.00
68.63
A


ATOM
1546
ND2
ASN
A
276
29.896
−8.443
12.671
1.00
66.67
A


ATOM
1547
C
ASN
A
276
32.478
−8.357
16.725
1.00
58.37
A


ATOM
1548
O
ASN
A
276
33.331
−7.506
16.456
1.00
57.93
A


ATOM
1549
N
PHE
A
277
32.190
−8.737
17.965
1.00
54.60
A


ATOM
1550
CA
PHE
A
277
32.862
−8.179
19.131
1.00
51.97
A


ATOM
1551
CB
PHE
A
277
31.989
−7.088
19.753
1.00
51.91
A


ATOM
1552
CG
PHE
A
277
31.656
−5.967
18.808
1.00
51.47
A


ATOM
1553
CD1
PHE
A
277
32.551
−4.916
18.611
1.00
52.08
A


ATOM
1554
CD2
PHE
A
277
30.458
−5.976
18.099
1.00
51.35
A


ATOM
1555
CE1
PHE
A
277
32.256
−3.885
17.718
1.00
51.57
A


ATOM
1556
CE2
PHE
A
277
30.149
−4.955
17.203
1.00
52.12
A


ATOM
1557
CZ
PHE
A
277
31.051
−3.905
17.010
1.00
51.46
A


ATOM
1558
C
PHE
A
277
33.071
−9.315
20.131
1.00
50.36
A


ATOM
1559
O
PHE
A
277
32.320
−10.286
20.139
1.00
46.88
A


ATOM
1560
N
PRO
A
278
34.100
−9.216
20.980
1.00
50.28
A


ATOM
1561
CD
PRO
A
278
35.153
−8.187
21.072
1.00
50.22
A


ATOM
1562
CA
PRO
A
278
34.325
−10.291
21.954
1.00
50.16
A


ATOM
1563
CB
PRO
A
278
35.724
−9.989
22.470
1.00
49.80
A


ATOM
1564
CG
PRO
A
278
35.768
−8.479
22.431
1.00
50.62
A


ATOM
1565
C
PRO
A
278
33.283
−10.298
23.079
1.00
50.37
A


ATOM
1566
O
PRO
A
278
32.877
−9.236
23.565
1.00
50.51
A


ATOM
1567
N
ILE
A
279
32.839
−11.489
23.476
1.00
50.25
A


ATOM
1568
CA
ILE
A
279
31.871
−11.609
24.567
1.00
51.04
A


ATOM
1569
CB
ILE
A
279
30.549
−12.285
24.125
1.00
51.79
A


ATOM
1570
CG2
ILE
A
279
29.604
−12.405
25.320
1.00
52.28
A


ATOM
1571
CG1
ILE
A
279
29.859
−11.457
23.037
1.00
53.44
A


ATOM
1572
CD1
ILE
A
279
30.363
−11.729
21.632
1.00
56.53
A


ATOM
1573
C
ILE
A
279
32.484
−12.433
25.694
1.00
50.74
A


ATOM
1574
O
ILE
A
279
32.937
−13.561
25.481
1.00
51.89
A


ATOM
1575
N
TYR
A
280
32.482
−11.864
26.894
1.00
49.59
A


ATOM
1576
CA
TYR
A
280
33.058
−12.504
28.070
1.00
50.16
A


ATOM
1577
CB
TYR
A
280
34.070
−11.557
28.717
1.00
50.09
A


ATOM
1578
CG
TYR
A
280
35.165
−11.095
27.792
1.00
51.60
A


ATOM
1579
CD1
TYR
A
280
36.252
−11.920
27.501
1.00
51.31
A


ATOM
1580
CE1
TYR
A
280
37.264
−11.500
26.647
1.00
52.44
A


ATOM
1581
CD2
TYR
A
280
35.116
−9.832
27.201
1.00
51.35
A


ATOM
1582
CE2
TYR
A
280
36.122
−9.402
26.344
1.00
52.57
A


ATOM
1583
CZ
TYR
A
280
37.193
−10.242
26.073
1.00
52.17
A


ATOM
1584
OH
TYR
A
280
38.190
−9.828
25.223
1.00
53.86
A


ATOM
1585
C
TYR
A
280
32.024
−12.885
29.124
1.00
50.79
A


ATOM
1586
O
TYR
A
280
30.915
−12.353
29.144
1.00
49.68
A


ATOM
1587
N
VAL
A
281
32.416
−13.799
30.009
1.00
50.64
A


ATOM
1588
CA
VAL
A
281
31.571
−14.244
31.111
1.00
50.82
A


ATOM
1589
CB
VAL
A
281
30.900
−15.607
30.808
1.00
50.44
A


ATOM
1590
CG1
VAL
A
281
29.885
−15.459
29.691
1.00
50.15
A


ATOM
1591
CG2
VAL
A
281
31.951
−16.629
30.428
1.00
52.25
A


ATOM
1592
C
VAL
A
281
32.449
−14.376
32.359
1.00
51.99
A


ATOM
1593
O
VAL
A
281
33.454
−15.088
32.346
1.00
52.81
A


ATOM
1594
N
TRP
A
282
32.081
−13.669
33.424
1.00
52.59
A


ATOM
1595
CA
TRP
A
282
32.832
−13.705
34.677
1.00
54.91
A


ATOM
1596
CB
TRP
A
282
32.166
−12.781
35.696
1.00
54.67
A


ATOM
1597
CG
TRP
A
282
33.000
−12.444
36.899
1.00
55.63
A


ATOM
1598
CD2
TRP
A
282
34.314
−11.867
36.905
1.00
55.87
A


ATOM
1599
CE2
TRP
A
282
34.670
−11.650
38.253
1.00
56.26
A


ATOM
1600
CE3
TRP
A
282
35.223
−11.509
35.900
1.00
56.93
A


ATOM
1601
CD1
TRP
A
282
32.628
−12.556
38.210
1.00
55.67
A


ATOM
1602
NE1
TRP
A
282
33.624
−12.079
39.028
1.00
55.84
A


ATOM
1603
CZ2
TRP
A
282
35.901
−11.089
38.624
1.00
56.57
A


ATOM
1604
CZ3
TRP
A
282
36.448
−10.950
36.269
1.00
56.51
A


ATOM
1605
CH2
TRP
A
282
36.772
−10.746
37.619
1.00
56.85
A


ATOM
1606
C
TRP
A
282
32.798
−15.149
35.171
1.00
56.74
A


ATOM
1607
O
TRP
A
282
31.789
−15.598
35.708
1.00
56.36
A


ATOM
1608
N
SER
A
283
33.901
−15.870
34.983
1.00
59.27
A


ATOM
1609
CA
SER
A
283
33.979
−17.276
35.375
1.00
61.20
A


ATOM
1610
CB
SER
A
283
34.242
−18.133
34.137
1.00
61.51
A


ATOM
1611
OG
SER
A
283
33.307
−17.840
33.113
1.00
64.73
A


ATOM
1612
C
SER
A
283
35.039
−17.588
36.429
1.00
62.33
A


ATOM
1613
O
SER
A
283
35.887
−16.756
36.749
1.00
62.30
A


ATOM
1614
N
SER
A
284
34.983
−18.809
36.955
1.00
64.03
A


ATOM
1615
CA
SER
A
284
35.923
−19.257
37.976
1.00
65.62
A


ATOM
1616
CB
SER
A
284
35.234
−19.292
39.341
1.00
65.36
A


ATOM
1617
OG
SER
A
284
34.134
−20.184
39.324
1.00
65.82
A


ATOM
1618
C
SER
A
284
36.469
−20.643
37.640
1.00
66.68
A


ATOM
1619
O
SER
A
284
35.886
−21.375
36.835
1.00
66.59
A


ATOM
1620
N
LYS
A
285
37.587
−21.004
38.264
1.00
67.92
A


ATOM
1621
CA
LYS
A
285
38.208
−22.301
38.016
1.00
69.26
A


ATOM
1622
CB
LYS
A
285
39.173
−22.186
36.831
1.00
70.51
A


ATOM
1623
CG
LYS
A
285
39.780
−23.502
36.359
1.00
72.05
A


ATOM
1624
CD
LYS
A
285
40.592
−23.286
35.084
1.00
73.12
A


ATOM
1625
CE
LYS
A
285
41.195
−24.584
34.563
1.00
74.70
A


ATOM
1626
NZ
LYS
A
285
41.976
−24.375
33.307
1.00
74.12
A


ATOM
1627
C
LYS
A
285
38.950
−22.810
39.249
1.00
69.18
A


ATOM
1628
O
LYS
A
285
38.934
−22.084
40.265
1.00
69.24
A


ATOM
1629
CB
VAL
A
330
41.691
−18.076
39.440
1.00
65.70
A


ATOM
1630
CG1
VAL
A
330
42.175
−16.694
38.995
1.00
65.89
A


ATOM
1631
CG2
VAL
A
330
42.767
−18.794
40.241
1.00
66.10
A


ATOM
1632
C
VAL
A
330
39.284
−17.407
39.400
1.00
65.01
A


ATOM
1633
O
VAL
A
330
38.717
−18.144
38.596
1.00
65.36
A


ATOM
1634
N
VAL
A
330
40.012
−19.256
40.874
1.00
65.49
A


ATOM
1635
CA
VAL
A
330
40.408
−17.941
40.289
1.00
65.73
A


ATOM
1636
N
TRP
A
331
38.973
−16.123
39.553
1.00
65.05
A


ATOM
1637
CA
TRP
A
331
37.914
−15.471
38.782
1.00
65.10
A


ATOM
1638
CB
TRP
A
331
36.972
−14.715
39.727
1.00
67.00
A


ATOM
1639
CG
TRP
A
331
35.754
−15.482
40.159
1.00
69.49
A


ATOM
1640
CD2
TRP
A
331
35.509
−16.054
41.450
1.00
70.82
A


ATOM
1641
CE2
TRP
A
331
34.228
−16.648
41.409
1.00
71.28
A


ATOM
1642
CE3
TRP
A
331
36.246
−16.120
42.640
1.00
71.31
A


ATOM
1643
CD1
TRP
A
331
34.644
−15.750
39.406
1.00
70.76
A


ATOM
1644
NE1
TRP
A
331
33.723
−16.448
40.151
1.00
71.62
A


ATOM
1645
CZ2
TRP
A
331
33.668
−17.302
42.516
1.00
71.95
A


ATOM
1646
CZ3
TRP
A
331
35.689
−16.771
43.742
1.00
72.37
A


ATOM
1647
CH2
TRP
A
331
34.413
−17.351
43.670
1.00
72.81
A


ATOM
1648
C
TRP
A
331
38.466
−14.493
37.742
1.00
64.34
A


ATOM
1649
O
TRP
A
331
39.305
−13.647
38.058
1.00
62.86
A


ATOM
1650
N
ASP
A
332
37.983
−14.609
36.505
1.00
63.43
A


ATOM
1651
CA
ASP
A
332
38.419
−13.724
35.425
1.00
63.19
A


ATOM
1652
CB
ASP
A
332
39.826
−14.117
34.955
1.00
64.46
A


ATOM
1653
CG
ASP
A
332
40.680
−12.913
34.585
1.00
66.04
A


ATOM
1654
OD1
ASP
A
332
40.245
−12.092
33.748
1.00
66.86
A


ATOM
1655
OD2
ASP
A
332
41.795
−12.789
35.134
1.00
67.88
A


ATOM
1656
C
ASP
A
332
37.450
−13.784
34.238
1.00
61.65
A


ATOM
1657
O
ASP
A
332
36.635
−14.703
34.133
1.00
60.08
A


ATOM
1658
N
TRP
A
333
37.536
−12.793
33.353
1.00
59.80
A


ATOM
1659
CA
TRP
A
333
36.686
−12.759
32.169
1.00
58.58
A


ATOM
1660
CB
TRP
A
333
36.805
−11.410
31.452
1.00
56.51
A


ATOM
1661
CG
TRP
A
333
36.236
−10.257
32.216
1.00
54.90
A


ATOM
1662
CD2
TRP
A
333
34.864
−10.056
32.574
1.00
53.44
A


ATOM
1663
CE2
TRP
A
333
34.788
−8.833
33.276
1.00
54.00
A


ATOM
1664
CE3
TRP
A
333
33.690
−10.792
32.370
1.00
53.96
A


ATOM
1665
CD1
TRP
A
333
36.920
−9.179
32.704
1.00
54.32
A


ATOM
1666
NE1
TRP
A
333
36.057
−8.319
33.341
1.00
53.95
A


ATOM
1667
CZ2
TRP
A
333
33.577
−8.326
33.776
1.00
52.51
A


ATOM
1668
CZ3
TRP
A
333
32.485
−10.286
32.868
1.00
53.62
A


ATOM
1669
CH2
TRP
A
333
32.442
−9.067
33.561
1.00
52.56
A


ATOM
1670
C
TRP
A
333
37.119
−13.873
31.221
1.00
58.90
A


ATOM
1671
O
TRP
A
333
38.286
−13.952
30.832
1.00
58.73
A


ATOM
1672
N
GLU
A
334
36.178
−14.738
30.856
1.00
58.96
A


ATOM
1673
CA
GLU
A
334
36.473
−15.839
29.951
1.00
59.53
A


ATOM
1674
CB
GLU
A
334
35.928
−17.149
30.516
1.00
60.54
A


ATOM
1675
CG
GLU
A
334
36.514
−18.382
29.859
1.00
62.48
A


ATOM
1676
CD
GLU
A
334
35.800
−19.652
30.265
1.00
64.73
A


ATOM
1677
OE1
GLU
A
334
35.527
−19.825
31.470
1.00
65.42
A


ATOM
1678
OE2
GLU
A
334
35.515
−20.481
29.376
1.00
65.95
A


ATOM
1679
C
GLU
A
334
35.847
−15.581
28.584
1.00
59.18
A


ATOM
1680
O
GLU
A
334
34.631
−15.440
28.466
1.00
58.69
A


ATOM
1681
N
LEU
A
335
36.679
−15.523
27.552
1.00
58.58
A


ATOM
1682
CA
LEU
A
335
36.190
−15.282
26.200
1.00
59.22
A


ATOM
1683
CB
LEU
A
335
37.372
−15.091
25.248
1.00
59.10
A


ATOM
1684
CG
LEU
A
335
37.052
−14.785
23.786
1.00
59.02
A


ATOM
1685
CD1
LEU
A
335
36.417
−13.406
23.679
1.00
57.75
A


ATOM
1686
CD2
LEU
A
335
38.336
−14.850
22.968
1.00
59.37
A


ATOM
1687
C
LEU
A
335
35.321
−16.440
25.713
1.00
59.48
A


ATOM
1688
O
LEU
A
335
35.745
−17.596
25.727
1.00
59.46
A


ATOM
1689
N
MET
A
336
34.102
−16.122
25.290
1.00
59.17
A


ATOM
1690
CA
MET
A
336
33.169
−17.123
24.787
1.00
60.97
A


ATOM
1691
CB
MET
A
336
31.767
−16.862
25.349
1.00
61.75
A


ATOM
1692
CG
MET
A
336
31.675
−16.920
26.870
1.00
62.57
A


ATOM
1693
SD
MET
A
336
32.033
−18.560
27.531
1.00
64.96
A


ATOM
1694
CE
MET
A
336
33.730
−18.383
27.925
1.00
64.61
A


ATOM
1695
C
MET
A
336
33.152
−17.018
23.264
1.00
61.64
A


ATOM
1696
O
MET
A
336
32.490
−17.796
22.578
1.00
61.35
A


ATOM
1697
N
ASN
A
337
33.906
−16.037
22.772
1.00
62.88
A


ATOM
1698
CA
ASN
A
337
34.059
−15.710
21.355
1.00
64.29
A


ATOM
1699
CB
ASN
A
337
33.962
−16.952
20.467
1.00
66.06
A


ATOM
1700
CG
ASN
A
337
34.470
−16.689
19.063
1.00
67.89
A


ATOM
1701
OD1
ASN
A
337
35.553
−16.125
18.888
1.00
69.70
A


ATOM
1702
ND2
ASN
A
337
33.699
−17.096
18.056
1.00
68.64
A


ATOM
1703
C
ASN
A
337
33.031
−14.675
20.909
1.00
63.60
A


ATOM
1704
O
ASN
A
337
32.161
−15.008
20.078
1.00
63.24
A


ATOM
1705
OXT
ASN
A
337
33.111
−13.536
21.409
1.00
63.10
A


ATOM
1706
OH2
WAT
W
1
24.847
0.476
24.771
1.00
32.16
W


ATOM
1707
OH2
WAT
W
2
14.818
−5.124
21.757
1.00
33.62
W


ATOM
1708
OH2
WAT
W
3
8.952
−0.499
21.618
1.00
36.76
W


ATOM
1709
OH2
WAT
W
4
30.911
−29.736
33.235
1.00
79.70
W


ATOM
1710
OH2
WAT
W
5
2.130
−7.313
33.720
1.00
38.37
W


ATOM
1711
OH2
WAT
W
6
28.011
−7.152
33.287
1.00
42.07
W


ATOM
1712
OH2
WAT
W
7
8.579
−18.872
35.549
1.00
63.18
W


ATOM
1713
OH2
WAT
W
8
2.341
0.845
32.049
1.00
34.97
W


ATOM
1714
OH2
WAT
W
9
21.534
−5.717
25.523
1.00
37.67
W


ATOM
1715
OH2
WAT
W
10
18.884
−3.010
18.470
1.00
47.48
W


ATOM
1716
OH2
WAT
W
11
23.026
8.416
28.820
1.00
46.39
W


ATOM
1717
OH2
WAT
W
12
21.101
5.291
21.278
1.00
45.73
W


ATOM
1718
OH2
WAT
W
13
24.002
−5.027
27.071
1.00
36.22
W


ATOM
1719
OH2
WAT
W
14
3.071
7.977
31.329
1.00
40.54
W


ATOM
1720
OH2
WAT
W
15
27.441
7.441
22.808
1.00
33.79
W


ATOM
1721
OH2
WAT
W
16
2.583
7.339
37.179
1.00
65.71
W


ATOM
1722
OH2
WAT
W
17
18.873
−6.771
17.897
1.00
44.39
W


ATOM
1723
OH2
WAT
W
18
2.483
−1.807
31.770
1.00
32.18
W


ATOM
1724
OH2
WAT
W
19
7.866
−10.916
34.603
1.00
41.35
W


ATOM
1725
OH2
WAT
W
20
32.781
7.383
24.625
1.00
44.35
W


ATOM
1726
OH2
WAT
W
21
7.711
−20.828
22.884
1.00
41.13
W


ATOM
1727
OH2
WAT
W
22
21.547
12.590
16.946
1.00
70.66
W


ATOM
1728
OH2
WAT
W
23
18.791
−5.014
25.314
1.00
30.54
W


ATOM
1729
C5*
NEC
N
1
14.630
−1.760
21.594
1.00
42.82
N


ATOM
1730
O5*
NEC
N
1
15.095
−2.737
22.487
1.00
39.14
N


ATOM
1731
N5*
NEC
N
1
13.379
−1.892
20.912
1.00
47.41
N


ATOM
1732
C51
NEC
N
1
12.204
−2.356
21.609
1.00
47.38
N


ATOM
1733
C52
NEC
N
1
11.143
−2.661
20.558
1.00
47.12
N


ATOM
1734
C4*
NEC
N
1
15.422
−0.530
21.323
1.00
40.52
N


ATOM
1735
O4*
NEC
N
1
15.959
0.043
22.481
1.00
38.34
N


ATOM
1736
C3*
NEC
N
1
16.575
−0.698
20.373
1.00
40.65
N


ATOM
1737
O3*
NEC
N
1
16.181
−0.610
19.032
1.00
40.85
N


ATOM
1738
C2*
NEC
N
1
17.553
0.382
20.858
1.00
41.27
N


ATOM
1739
O2*
NEC
N
1
17.221
1.611
20.223
1.00
41.96
N


ATOM
1740
C1*
NEC
N
1
17.345
0.390
22.405
1.00
37.25
N


ATOM
1741
N9
NEC
N
1
18.267
−0.595
23.039
1.00
37.54
N


ATOM
1742
C8
NEC
N
1
17.971
−1.829
23.616
1.00
35.13
N


ATOM
1743
N7
NEC
N
1
19.036
−2.426
24.091
1.00
34.96
N


ATOM
1744
C5
NEC
N
1
20.093
−1.540
23.825
1.00
35.95
N


ATOM
1745
C6
NEC
N
1
21.496
−1.571
24.098
1.00
35.57
N


ATOM
1746
N6
NEC
N
1
22.092
−2.599
24.726
1.00
36.19
N


ATOM
1747
N1
NEC
N
1
22.270
−0.493
23.697
1.00
37.88
N


ATOM
1748
C2
NEC
N
1
21.683
0.546
23.065
1.00
34.37
N


ATOM
1749
N3
NEC
N
1
20.394
0.693
22.754
1.00
34.33
N


ATOM
1750
C4
NEC
N
1
19.632
−0.399
23.165
1.00
36.84
N
















TABLE 2





ATOMIC STRUCTURE COORDINATE DATA OBTAINED FROM X-RAY


DIFFRACTION FROM THE LIGAND BINDNG POCKET OF FORM 1 OF THE


LIGAND BINDING DOMAIN OF GRP94 (RESIDUES 48-316 OF SEQ ID NO: 6)


IN COMPLEX WITH NECA


























ATOM
106
N
MET
A
85
2.451
−5.703
22.762
1.00
36.19
A


ATOM
107
CA
MET
A
85
3.652
−6.287
22.182
1.00
36.75
A


ATOM
108
CB
MET
A
85
4.609
−5.184
21.693
1.00
38.82
A


ATOM
109
CG
MET
A
85
5.974
−5.703
21.210
1.00
41.23
A


ATOM
110
SD
MET
A
85
6.976
−4.519
20.233
1.00
43.17
A


ATOM
111
CE
MET
A
85
7.035
−3.128
21.369
1.00
45.81
A


ATOM
112
C
MET
A
85
4.374
−7.186
23.177
1.00
36.50
A


ATOM
113
O
MET
A
85
4.917
−8.229
22.797
1.00
37.23
A


ATOM
258
N
GLU
A
103
18.584
−12.439
22.390
1.00
35.80
A


ATOM
259
CA
GLU
A
103
18.172
−11.084
22.033
1.00
36.77
A


ATOM
260
CB
GLU
A
103
16.667
−10.927
22.244
1.00
38.94
A


ATOM
261
CG
GLU
A
103
15.845
−11.797
21.293
1.00
44.14
A


ATOM
262
CD
GLU
A
103
16.277
−11.613
19.843
1.00
48.10
A


ATOM
263
OE1
GLU
A
103
16.200
−10.471
19.344
1.00
47.73
A


ATOM
264
OE2
GLU
A
103
16.706
−12.603
19.205
1.00
48.75
A


ATOM
265
C
GLU
A
103
18.933
−9.987
22.764
1.00
37.18
A


ATOM
266
O
GLU
A
103
19.260
−8.956
22.167
1.00
38.35
A


ATOM
267
N
LEU
A
104
19.213
−10.181
24.052
1.00
36.53
A


ATOM
268
CA
LEU
A
104
19.967
−9.168
24.782
1.00
34.96
A


ATOM
269
CB
LEU
A
104
20.051
−9.514
26.266
1.00
36.16
A


ATOM
270
CG
LEU
A
104
18.724
−9.575
27.021
1.00
34.61
A


ATOM
271
CD1
LEU
A
104
18.994
−9.853
28.490
1.00
36.29
A


ATOM
272
CD2
LEU
A
104
17.971
−8.254
26.855
1.00
37.01
A


ATOM
273
C
LEU
A
104
21.369
−9.081
24.183
1.00
36.79
A


ATOM
274
O
LEU
A
104
21.956
−7.995
24.098
1.00
35.24
A


ATOM
289
N
ASN
A
107
21.038
−7.279
20.880
1.00
35.94
A


ATOM
290
CA
ASN
A
107
20.841
−5.846
21.110
1.00
38.70
A


ATOM
291
CB
ASN
A
107
19.829
−5.623
22.241
1.00
37.51
A


ATOM
292
CG
ASN
A
107
18.406
−5.947
21.826
1.00
36.75
A


ATOM
293
OD1
ASN
A
107
17.509
−6.028
22.667
1.00
38.88
A


ATOM
294
ND2
ASN
A
107
18.188
−6.120
20.531
1.00
32.53
A


ATOM
295
C
ASN
A
107
22.168
−5.161
21.457
1.00
38.73
A


ATOM
296
O
ASN
A
107
22.425
−4.029
21.030
1.00
38.05
A


ATOM
297
N
ALA
A
108
23.010
−5.854
22.224
1.00
37.25
A


ATOM
298
CA
ALA
A
108
24.306
−5.319
22.616
1.00
37.11
A


ATOM
299
CB
ALA
A
108
24.944
−6.218
23.689
1.00
35.32
A


ATOM
300
C
ALA
A
108
25.208
−5.220
21.384
1.00
38.35
A


ATOM
301
O
ALA
A
108
25.949
−4.248
21.219
1.00
36.53
A


ATOM
308
N
ASP
A
110
24.249
−4.776
18.322
1.00
37.55
A


ATOM
309
CA
ASP
A
110
23.765
−3.645
17.523
1.00
38.97
A


ATOM
310
CB
ASP
A
110
22.236
−3.578
17.545
1.00
39.76
A


ATOM
311
CG
ASP
A
110
21.580
−4.651
16.686
1.00
42.87
A


ATOM
312
OD1
ASP
A
110
20.344
−4.769
16.743
1.00
41.85
A


ATOM
313
OD2
ASP
A
110
22.290
−5.373
15.956
1.00
46.31
A


ATOM
314
C
ASP
A
110
24.327
−2.326
18.060
1.00
36.57
A


ATOM
315
O
ASP
A
110
24.750
−1.469
17.284
1.00
37.95
A


ATOM
316
N
ALA
A
111
24.315
−2.173
19.385
1.00
36.03
A


ATOM
317
CA
ALA
A
111
24.827
−0.969
20.047
1.00
35.99
A


ATOM
318
CB
ALA
A
111
24.577
−1.047
21.560
1.00
34.35
A


ATOM
319
C
ALA
A
111
26.315
−0.808
19.779
1.00
36.64
A


ATOM
320
O
ALA
A
111
26.801
0.310
19.586
1.00
34.89
A


ATOM
579
N
VAL
A
147
24.143
−8.039
31.844
1.00
35.57
A


ATOM
580
CA
VAL
A
147
24.662
−8.102
30.478
1.00
36.33
A


ATOM
581
CB
VAL
A
147
23.599
−8.553
29.478
1.00
35.23
A


ATOM
582
CG1
VAL
A
147
24.188
−8.592
28.082
1.00
37.63
A


ATOM
583
CG2
VAL
A
147
23.096
−9.941
29.858
1.00
37.09
A


ATOM
584
C
VAL
A
147
25.087
−6.669
30.160
1.00
36.34
A


ATOM
585
O
VAL
A
147
24.250
−5.778
29.980
1.00
34.90
A


ATOM
593
N
ASP
A
149
27.425
−3.938
27.840
1.00
37.49
A


ATOM
594
CA
ASP
A
149
28.112
−3.694
26.593
1.00
37.69
A


ATOM
595
CB
ASP
A
149
27.166
−3.865
25.388
1.00
38.06
A


ATOM
596
CG
ASP
A
149
26.100
−2.779
25.299
1.00
39.30
A


ATOM
597
OD1
ASP
A
149
26.449
−1.599
25.056
1.00
39.14
A


ATOM
598
OD2
ASP
A
149
24.905
−3.107
25.471
1.00
37.40
A


ATOM
599
C
ASP
A
149
28.687
−2.286
26.637
1.00
37.48
A


ATOM
600
O
ASP
A
149
28.236
−1.442
27.412
1.00
37.39
A


ATOM
612
N
VAL
A
152
26.921
2.139
21.990
1.00
38.14
A


ATOM
613
CA
VAL
A
152
26.539
3.547
21.916
1.00
36.85
A


ATOM
614
CB
VAL
A
152
25.663
3.800
20.660
1.00
36.97
A


ATOM
615
CG1
VAL
A
152
24.359
3.030
20.784
1.00
34.47
A


ATOM
616
CG2
VAL
A
152
25.398
5.312
20.473
1.00
36.38
A


ATOM
617
C
VAL
A
152
25.797
4.071
23.150
1.00
38.24
A


ATOM
618
O
VAL
A
152
25.862
5.264
23.457
1.00
37.41
A


ATOM
619
N
GLY
A
153
25.090
3.188
23.854
1.00
35.89
A


ATOM
620
CA
GLY
A
153
24.344
3.613
25.023
1.00
33.35
A


ATOM
621
C
GLY
A
153
23.074
4.371
24.641
1.00
33.66
A


ATOM
622
O
GLY
A
153
22.769
4.519
23.458
1.00
33.60
A


ATOM
623
N
MET
A
154
22.339
4.844
25.645
1.00
33.73
A


ATOM
624
CA
MET
A
154
21.098
5.583
25.435
1.00
35.02
A


ATOM
625
CB
MET
A
154
19.881
4.687
25.713
1.00
35.45
A


ATOM
626
CG
MET
A
154
19.672
3.579
24.711
1.00
36.62
A


ATOM
627
SD
MET
A
154
18.279
2.497
25.176
1.00
39.95
A


ATOM
628
CE
MET
A
154
19.165
1.378
26.258
1.00
38.04
A


ATOM
629
C
MET
A
154
20.991
6.802
26.353
1.00
34.74
A


ATOM
630
O
MET
A
154
21.330
6.734
27.532
1.00
34.79
A


ATOM
654
N
GLU
A
158
16.061
8.597
25.142
1.00
35.85
A


ATOM
655
CA
GLU
A
158
16.003
7.314
24.438
1.00
38.21
A


ATOM
656
CB
GLU
A
158
17.372
6.982
23.852
1.00
37.42
A


ATOM
657
CG
GLU
A
158
17.828
8.013
22.815
1.00
42.18
A


ATOM
658
CD
GLU
A
158
19.127
7.649
22.141
1.00
41.21
A


ATOM
659
OE1
GLU
A
158
20.131
7.435
22.843
1.00
42.08
A


ATOM
660
OE2
GLU
A
158
19.153
7.587
20.897
1.00
47.22
A


ATOM
661
C
GLU
A
158
15.511
6.174
25.346
1.00
37.73
A


ATOM
662
O
GLU
A
158
14.792
5.283
24.895
1.00
38.81
A


ATOM
678
N
ALA
A
161
11.730
6.786
25.413
1.00
34.61
A


ATOM
679
CA
ALA
A
161
11.091
6.516
24.128
1.00
35.52
A


ATOM
680
CB
ALA
A
161
11.512
7.573
23.110
1.00
35.95
A


ATOM
681
C
ALA
A
161
11.304
5.129
23.523
1.00
34.49
A


ATOM
682
O
ALA
A
161
10.349
4.476
23.115
1.00
36.23
A


ATOM
683
N
ASN
A
162
12.555
4.698
23.457
1.00
34.89
A


ATOM
684
CA
ASN
A
162
12.898
3.422
22.839
1.00
36.90
A


ATOM
685
CB
ASN
A
162
14.414
3.331
22.691
1.00
37.47
A


ATOM
686
CG
ASN
A
162
14.961
4.415
21.785
1.00
42.03
A


ATOM
687
OD1
ASN
A
162
14.383
5.505
21.688
1.00
43.34
A


ATOM
688
ND2
ASN
A
162
16.075
4.134
21.125
1.00
40.97
A


ATOM
689
C
ASN
A
162
12.354
2.196
23.551
1.00
37.70
A


ATOM
690
O
ASN
A
162
12.039
1.186
22.908
1.00
37.59
A


ATOM
691
N
LEU
A
163
12.232
2.277
24.869
1.00
37.09
A


ATOM
692
CA
LEU
A
163
11.722
1.135
25.624
1.00
38.10
A


ATOM
693
CB
LEU
A
163
12.587
0.906
26.864
1.00
35.47
A


ATOM
694
CG
LEU
A
163
14.093
0.725
26.645
1.00
34.53
A


ATOM
695
CD1
LEU
A
163
14.792
0.602
28.003
1.00
34.16
A


ATOM
696
CD2
LEU
A
163
14.361
−0.506
25.770
1.00
37.46
A


ATOM
697
C
LEU
A
163
10.262
1.297
26.035
1.00
38.71
A


ATOM
698
O
LEU
A
163
9.541
0.309
26.209
1.00
38.36
A


ATOM
718
N
ALA
A
167
8.904
3.353
19.167
1.00
53.09
A


ATOM
719
CA
ALA
A
167
9.809
4.375
18.658
1.00
55.59
A


ATOM
720
CB
ALA
A
167
11.139
4.277
19.400
1.00
55.40
A


ATOM
721
C
ALA
A
167
10.062
4.404
17.155
1.00
57.83
A


ATOM
722
O
ALA
A
167
10.185
5.483
16.575
1.00
57.75
A


ATOM
723
N
LYS
A
168
10.148
3.237
16.522
1.00
61.19
A


ATOM
724
CA
LYS
A
168
10.418
3.186
15.085
1.00
63.46
A


ATOM
725
CB
LYS
A
168
11.717
2.421
14.825
1.00
64.87
A


ATOM
726
CG
LYS
A
168
12.978
3.188
15.198
1.00
67.57
A


ATOM
727
CD
LYS
A
168
13.103
4.488
14.403
1.00
68.54
A


ATOM
728
CE
LYS
A
168
13.123
4.234
12.898
1.00
69.62
A


ATOM
729
NZ
LYS
A
168
14.290
3.409
12.479
1.00
70.51
A


ATOM
730
C
LYS
A
168
9.318
2.600
14.213
1.00
64.19
A


ATOM
731
O
LYS
A
168
8.276
2.159
14.701
1.00
64.53
A


ATOM
742
N
THR
A
171
9.191
−1.575
14.421
1.00
59.30
A


ATOM
743
CA
THR
A
171
8.412
−2.158
15.505
1.00
59.38
A


ATOM
744
CB
THR
A
171
8.724
−1.453
16.850
1.00
58.69
A


ATOM
745
OG1
THR
A
171
7.737
−1.810
17.824
1.00
60.23
A


ATOM
746
CG2
THR
A
171
8.750
0.041
16.675
1.00
61.04
A


ATOM
747
C
THR
A
171
6.911
−2.127
15.221
1.00
58.48
A


ATOM
748
O
THR
A
171
6.161
−2.973
15.707
1.00
58.04
A


ATOM
924
N
GLY
A
196
14.281
−6.880
15.898
1.00
43.45
A


ATOM
925
CA
GLY
A
196
14.991
−6.496
17.105
1.00
43.03
A


ATOM
926
C
GLY
A
196
14.139
−6.021
18.269
1.00
41.78
A


ATOM
927
O
GLY
A
196
14.488
−5.043
18.935
1.00
39.12
A


ATOM
928
N
VAL
A
197
13.032
−6.717
18.529
1.00
39.22
A


ATOM
929
CA
VAL
A
197
12.139
−6.353
19.629
1.00
37.80
A


ATOM
930
CB
VAL
A
197
10.659
−6.282
19.155
1.00
37.78
A


ATOM
931
CG1
VAL
A
197
10.498
−5.196
18.101
1.00
39.35
A


ATOM
932
CG2
VAL
A
197
10.212
−7.633
18.592
1.00
39.35
A


ATOM
933
C
VAL
A
197
12.228
−7.321
20.821
1.00
36.08
A


ATOM
934
O
VAL
A
197
11.379
−7.292
21.708
1.00
33.97
A


ATOM
935
N
GLY
A
198
13.264
−8.159
20.838
1.00
35.72
A


ATOM
936
CA
GLY
A
198
13.445
−9.133
21.910
1.00
34.04
A


ATOM
937
C
GLY
A
198
13.722
−8.639
23.324
1.00
35.00
A


ATOM
938
O
GLY
A
198
13.688
−9.427
24.269
1.00
32.54
A


ATOM
939
N
PHE
A
199
14.009
−7.349
23.500
1.00
34.83
A


ATOM
940
CA
PHE
A
199
14.267
−6.841
24.841
1.00
33.95
A


ATOM
941
CB
PHE
A
199
14.400
−5.308
24.804
1.00
35.31
A


ATOM
942
CG
PHE
A
199
14.375
−4.663
26.159
1.00
35.09
A


ATOM
943
CD1
PHE
A
199
15.521
−4.614
26.946
1.00
34.35
A


ATOM
944
CD2
PHE
A
199
13.183
−4.130
26.660
1.00
35.32
A


ATOM
945
CE1
PHE
A
199
15.490
−4.044
28.225
1.00
34.68
A


ATOM
946
CE2
PHE
A
199
13.131
−3.561
27.929
1.00
34.37
A


ATOM
947
CZ
PHE
A
199
14.295
−3.518
28.720
1.00
35.36
A


ATOM
948
C
PHE
A
199
13.165
−7.250
25.829
1.00
33.31
A


ATOM
949
O
PHE
A
199
13.449
−7.652
26.961
1.00
31.85
A


ATOM
950
N
TYR
A
200
11.913
−7.178
25.395
1.00
33.78
A


ATOM
951
CA
TYR
A
200
10.799
−7.515
26.270
1.00
35.65
A


ATOM
952
CB
TYR
A
200
9.479
−7.090
25.621
1.00
35.72
A


ATOM
953
CG
TYR
A
200
9.432
−5.610
25.293
1.00
36.99
A


ATOM
954
CD1
TYR
A
200
9.404
−4.645
26.304
1.00
35.32
A


ATOM
955
CE1
TYR
A
200
9.413
−3.267
25.990
1.00
37.74
A


ATOM
956
CD2
TYR
A
200
9.465
−5.177
23.971
1.00
38.74
A


ATOM
957
CE2
TYR
A
200
9.474
−3.826
23.654
1.00
40.33
A


ATOM
958
CZ
TYR
A
200
9.449
−2.877
24.665
1.00
38.60
A


ATOM
959
OH
TYR
A
200
9.463
−1.542
24.326
1.00
43.22
A


ATOM
960
C
TYR
A
200
10.716
−8.982
26.707
1.00
35.83
A


ATOM
961
O
TYR
A
200
10.067
−9.284
27.712
1.00
32.96
A


ATOM
1036
N
VAL
A
211
17.875
−1.148
33.430
1.00
34.49
A


ATOM
1037
CA
VAL
A
211
18.791
−0.793
32.366
1.00
32.75
A


ATOM
1038
CB
VAL
A
211
18.038
−0.519
31.036
1.00
33.29
A


ATOM
1039
CG1
VAL
A
211
19.032
−0.186
29.942
1.00
32.50
A


ATOM
1040
CG2
VAL
A
211
17.211
−1.759
30.608
1.00
32.87
A


ATOM
1041
C
VAL
A
211
19.558
0.456
32.786
1.00
34.30
A


ATOM
1042
O
VAL
A
211
18.958
1.504
33.015
1.00
31.53
A


ATOM
1133
N
TRP
A
223
16.848
2.945
34.029
1.00
34.41
A


ATOM
1134
CA
TRP
A
223
15.462
2.679
33.643
1.00
33.72
A


ATOM
1135
CB
TRP
A
223
15.391
2.404
32.130
1.00
34.35
A


ATOM
1136
CG
TRP
A
223
14.032
2.052
31.545
1.00
34.80
A


ATOM
1137
CD2
TRP
A
223
13.444
0.743
31.460
1.00
33.67
A


ATOM
1138
CE2
TRP
A
223
12.243
0.868
30.726
1.00
34.03
A


ATOM
1139
CE3
TRP
A
223
13.825
−0.526
31.926
1.00
35.44
A


ATOM
1140
CD1
TRP
A
223
13.173
2.897
30.890
1.00
33.40
A


ATOM
1141
NE1
TRP
A
223
12.096
2.190
30.393
1.00
34.12
A


ATOM
1142
CZ2
TRP
A
223
11.414
−0.234
30.448
1.00
33.49
A


ATOM
1143
CZ3
TRP
A
223
12.992
−1.627
31.645
1.00
32.64
A


ATOM
1144
CH2
TRP
A
223
11.813
−1.468
30.914
1.00
31.20
A


ATOM
1145
C
TRP
A
223
15.150
1.406
34.438
1.00
34.21
A


ATOM
1146
O
TRP
A
223
16.015
0.537
34.559
1.00
34.33
A


ATOM
1298
N
THR
A
245
25.866
0.115
28.122
1.00
37.45
A


ATOM
1299
CA
THR
A
245
24.618
−0.229
28.776
1.00
35.71
A


ATOM
1300
CB
THR
A
245
23.491
−0.395
27.728
1.00
38.09
A


ATOM
1301
OG1
THR
A
245
23.310
0.834
27.015
1.00
38.31
A


ATOM
1302
CG2
THR
A
245
22.180
−0.808
28.400
1.00
37.67
A


ATOM
1303
C
THR
A
245
24.720
−1.547
29.521
1.00
36.13
A


ATOM
1304
O
THR
A
245
25.351
−2.488
29.040
1.00
35.73
A


ATOM
1312
N
ILE
A
247
22.233
−4.388
31.265
1.00
34.48
A


ATOM
1313
CA
ILE
A
247
20.868
−4.878
31.421
1.00
33.45
A


ATOM
1314
CB
ILE
A
247
20.415
−5.765
30.224
1.00
33.63
A


ATOM
1315
CG2
ILE
A
247
18.964
−6.191
30.421
1.00
35.01
A


ATOM
1316
CG1
ILE
A
247
20.597
−5.030
28.883
1.00
32.43
A


ATOM
1317
CD1
ILE
A
247
19.686
−3.815
28.676
1.00
34.07
A


ATOM
1318
C
ILE
A
247
20.973
−5.777
32.652
1.00
34.92
A


ATOM
1319
O
ILE
A
247
21.689
−6.782
32.624
1.00
33.23
A


ATOM
1706
OH2
WAT
W
1
24.847
0.476
24.771
1.00
32.16
W


ATOM
1718
OH2
WAT
W
13
24.002
−5.027
27.071
1.00
36.22
W


ATOM
1722
OH2
WAT
W
17
18.873
−6.771
17.897
1.00
44.39
W


ATOM
1707
OH2
WAT
W
2
14.818
−5.124
21.757
1.00
33.62
W


ATOM
1708
OH2
WAT
W
3
8.952
−0.499
21.618
1.00
36.76
W


ATOM
1714
OH2
WAT
W
9
21.534
−5.717
25.523
1.00
37.67
W


ATOM
1715
OH2
WAT
W
10
18.884
−3.010
18.470
1.00
47.48
W


ATOM
1717
OH2
WAT
W
12
21.101
5.291
21.278
1.00
45.73
W


ATOM
1728
OH2
WAT
W
23
18.791
−5.014
25.314
1.00
30.54
W


ATOM
1729
C5*
NEC
N
1
14.630
−1.760
21.594
1.00
42.82
N


ATOM
1730
O5*
NEC
N
1
15.095
−2.737
22.487
1.00
39.14
N


ATOM
1731
N5*
NEC
N
1
13.379
−1.892
20.912
1.00
47.41
N


ATOM
1732
C51
NEC
N
1
12.204
−2.356
21.609
1.00
47.38
N


ATOM
1733
C52
NEC
N
1
11.143
−2.661
20.558
1.00
47.12
N


ATOM
1734
C4*
NEC
N
1
15.422
−0.530
21.323
1.00
40.52
N


ATOM
1735
O4*
NEC
N
1
15.959
0.043
22.481
1.00
38.34
N


ATOM
1736
C3*
NEC
N
1
16.575
−0.698
20.373
1.00
40.65
N


ATOM
1737
O3*
NEC
N
1
16.181
−0.610
19.032
1.00
40.85
N


ATOM
1738
C2*
NEC
N
1
17.553
0.382
20.858
1.00
41.27
N


ATOM
1739
O2*
NEC
N
1
17.221
1.611
20.223
1.00
41.96
N


ATOM
1740
C1*
NEC
N
1
17.345
0.390
22.405
1.00
37.25
N


ATOM
1741
N9
NEC
N
1
18.267
−0.595
23.039
1.00
37.54
N


ATOM
1742
C8
NEC
N
1
17.971
−1.829
23.616
1.00
35.13
N


ATOM
1743
N7
NEC
N
1
19.036
−2.426
24.091
1.00
34.96
N


ATOM
1744
C5
NEC
N
1
20.093
−1.540
23.825
1.00
35.95
N


ATOM
1745
C6
NEC
N
1
21.496
−1.571
24.098
1.00
35.57
N


ATOM
1746
N6
NEC
N
1
22.092
−2.599
24.726
1.00
36.19
N


ATOM
1747
N1
NEC
N
1
22.270
−0.493
23.697
1.00
37.88
N


ATOM
1748
C2
NEC
N
1
21.683
0.546
23.065
1.00
34.37
N


ATOM
1749
N3
NEC
N
1
20.394
0.693
22.754
1.00
34.33
N


ATOM
1750
C4
NEC
N
1
19.632
−0.399
23.165
1.00
36.84
N
















TABLE 3





ATOMIC STRUCTURE COORDINATE DATA OBTAINED FROM X-RAY


DIFFRACTION FROM FORM 2 OF THE LIGAND BINDING DOMAIN OF GRP94


(RESIDUES 48-316 OF SEQ ID NO: 6) IN COMPLEX WITH NECA


























ATOM
1
CB
LYS
A
72
33.507
−16.296
72.107
1.00
69.73
A


ATOM
2
CG
LYS
A
72
33.483
−15.288
73.248
1.00
70.11
A


ATOM
3
CD
LYS
A
72
33.524
−15.987
74.598
1.00
71.49
A


ATOM
4
CE
LYS
A
72
33.552
−14.988
75.745
1.00
72.79
A


ATOM
5
NZ
LYS
A
72
34.793
−14.165
75.741
1.00
72.91
A


ATOM
6
C
LYS
A
72
31.931
−15.062
70.609
1.00
67.84
A


ATOM
7
O
LYS
A
72
30.984
−15.730
70.200
1.00
68.80
A


ATOM
8
N
LYS
A
72
34.390
−14.667
70.454
1.00
69.61
A


ATOM
9
CA
LYS
A
72
33.324
−15.679
70.715
1.00
69.35
A


ATOM
10
N
SER
A
73
31.820
−13.789
70.987
1.00
65.64
A


ATOM
11
CA
SER
A
73
30.555
−13.056
70.944
1.00
63.60
A


ATOM
12
CB
SER
A
73
30.007
−12.863
72.361
1.00
64.12
A


ATOM
13
OG
SER
A
73
28.825
−12.079
72.353
1.00
63.95
A


ATOM
14
C
SER
A
73
30.779
−11.692
70.293
1.00
61.79
A


ATOM
15
O
SER
A
73
31.712
−10.975
70.662
1.00
63.42
A


ATOM
16
N
GLU
A
74
29.927
−11.331
69.335
1.00
57.06
A


ATOM
17
CA
GLU
A
74
30.072
−10.054
68.642
1.00
53.07
A


ATOM
18
CB
GLU
A
74
30.972
−10.244
67.417
1.00
53.91
A


ATOM
19
CG
GLU
A
74
31.283
−8.971
66.657
1.00
55.81
A


ATOM
20
CD
GLU
A
74
32.438
−9.142
65.689
1.00
57.46
A


ATOM
21
OE1
GLU
A
74
32.445
−10.135
64.931
1.00
56.10
A


ATOM
22
OE2
GLU
A
74
33.338
−8.276
65.684
1.00
59.85
A


ATOM
23
C
GLU
A
74
28.732
−9.431
68.231
1.00
50.14
A


ATOM
24
O
GLU
A
74
27.853
−10.111
67.694
1.00
48.22
A


ATOM
25
N
ALA
A
75
28.591
−8.132
68.489
1.00
45.60
A


ATOM
26
CA
ALA
A
75
27.369
−7.395
68.175
1.00
43.37
A


ATOM
27
CB
ALA
A
75
27.066
−6.390
69.287
1.00
43.20
A


ATOM
28
C
ALA
A
75
27.453
−6.673
66.834
1.00
41.49
A


ATOM
29
O
ALA
A
75
28.530
−6.237
66.413
1.00
40.95
A


ATOM
30
N
PHE
A
76
26.311
−6.544
66.167
1.00
37.00
A


ATOM
31
CA
PHE
A
76
26.254
−5.881
64.868
1.00
36.00
A


ATOM
32
CB
PHE
A
76
26.259
−6.904
63.721
1.00
34.46
A


ATOM
33
CG
PHE
A
76
27.463
−7.804
63.685
1.00
32.78
A


ATOM
34
CD1
PHE
A
76
27.530
−8.932
64.494
1.00
33.06
A


ATOM
35
CD2
PHE
A
76
28.515
−7.539
62.810
1.00
33.42
A


ATOM
36
CE1
PHE
A
76
28.631
−9.792
64.432
1.00
34.56
A


ATOM
37
CE2
PHE
A
76
29.624
−8.392
62.739
1.00
34.73
A


ATOM
38
CZ
PHE
A
76
29.681
−9.519
63.549
1.00
32.36
A


ATOM
39
C
PHE
A
76
24.989
−5.046
64.695
1.00
36.16
A


ATOM
40
O
PHE
A
76
23.945
−5.337
65.282
1.00
35.60
A


ATOM
41
N
ALA
A
77
25.089
−4.015
63.867
1.00
34.33
A


ATOM
42
CA
ALA
A
77
23.936
−3.188
63.553
1.00
33.00
A


ATOM
43
CB
ALA
A
77
24.306
−1.715
63.580
1.00
34.28
A


ATOM
44
C
ALA
A
77
23.555
−3.609
62.138
1.00
32.33
A


ATOM
45
O
ALA
A
77
24.427
−3.973
61.337
1.00
28.99
A


ATOM
46
N
PHE
A
78
22.261
−3.590
61.837
1.00
29.93
A


ATOM
47
CA
PHE
A
78
21.802
−3.948
60.503
1.00
28.73
A


ATOM
48
CB
PHE
A
78
20.275
−4.111
60.465
1.00
26.59
A


ATOM
49
CG
PHE
A
78
19.768
−5.389
61.083
1.00
24.26
A


ATOM
50
CD1
PHE
A
78
19.297
−5.409
62.398
1.00
23.07
A


ATOM
51
CD2
PHE
A
78
19.725
−6.564
60.341
1.00
23.49
A


ATOM
52
CE1
PHE
A
78
18.788
−6.577
62.957
1.00
23.95
A


ATOM
53
CE2
PHE
A
78
19.217
−7.738
60.888
1.00
20.39
A


ATOM
54
CZ
PHE
A
78
18.746
−7.746
62.201
1.00
21.62
A


ATOM
55
C
PHE
A
78
22.183
−2.843
59.522
1.00
30.30
A


ATOM
56
O
PHE
A
78
22.275
−1.670
59.893
1.00
29.90
A


ATOM
57
N
GLN
A
79
22.402
−3.218
58.268
1.00
30.62
A


ATOM
58
CA
GLN
A
79
22.717
−2.236
57.240
1.00
31.01
A


ATOM
59
CB
GLN
A
79
23.051
−2.941
55.925
1.00
29.52
A


ATOM
60
CG
GLN
A
79
23.557
−2.026
54.825
1.00
33.63
A


ATOM
61
CD
GLN
A
79
23.726
−2.756
53.499
1.00
36.38
A


ATOM
62
OE1
GLN
A
79
23.884
−3.975
53.468
1.00
34.09
A


ATOM
63
NE2
GLN
A
79
23.702
−2.006
52.397
1.00
37.63
A


ATOM
64
C
GLN
A
79
21.436
−1.407
57.082
1.00
30.67
A


ATOM
65
O
GLN
A
79
20.330
−1.942
57.201
1.00
28.06
A


ATOM
66
N
ALA
A
80
21.582
−0.113
56.816
1.00
29.05
A


ATOM
67
CA
ALA
A
80
20.426
0.769
56.664
1.00
30.86
A


ATOM
68
CB
ALA
A
80
20.873
2.122
56.098
1.00
29.60
A


ATOM
69
C
ALA
A
80
19.318
0.176
55.785
1.00
32.06
A


ATOM
70
O
ALA
A
80
18.152
0.131
56.188
1.00
33.23
A


ATOM
71
N
GLU
A
81
19.685
−0.275
54.588
1.00
31.42
A


ATOM
72
CA
GLU
A
81
18.728
−0.849
53.651
1.00
31.65
A


ATOM
73
CB
GLU
A
81
19.421
−1.265
52.352
1.00
33.71
A


ATOM
74
CG
GLU
A
81
19.909
−0.117
51.489
1.00
39.87
A


ATOM
75
CD
GLU
A
81
21.190
0.516
52.003
1.00
44.19
A


ATOM
76
OE1
GLU
A
81
21.693
1.447
51.334
1.00
46.79
A


ATOM
77
OE2
GLU
A
81
21.693
0.089
53.069
1.00
46.52
A


ATOM
78
C
GLU
A
81
17.978
−2.054
54.203
1.00
30.64
A


ATOM
79
O
GLU
A
81
16.822
−2.287
53.841
1.00
28.44
A


ATOM
80
N
VAL
A
82
18.634
−2.838
55.053
1.00
26.89
A


ATOM
81
CA
VAL
A
82
17.971
−4.001
55.615
1.00
27.07
A


ATOM
82
CB
VAL
A
82
18.968
−4.957
56.299
1.00
25.85
A


ATOM
83
CG1
VAL
A
82
18.209
−6.070
57.020
1.00
21.09
A


ATOM
84
CG2
VAL
A
82
19.919
−5.551
55.250
1.00
24.54
A


ATOM
85
C
VAL
A
82
16.910
−3.545
56.614
1.00
27.82
A


ATOM
86
O
VAL
A
82
15.856
−4.166
56.729
1.00
26.27
A


ATOM
87
N
ASN
A
83
17.188
−2.463
57.337
1.00
25.48
A


ATOM
88
CA
ASN
A
83
16.206
−1.938
58.278
1.00
26.67
A


ATOM
89
CB
ASN
A
83
16.738
−0.705
59.019
1.00
24.92
A


ATOM
90
CG
ASN
A
83
17.707
−1.056
60.135
1.00
28.46
A


ATOM
91
OD1
ASN
A
83
17.559
−2.080
60.813
1.00
25.85
A


ATOM
92
ND2
ASN
A
83
18.695
−0.191
60.347
1.00
26.16
A


ATOM
93
C
ASN
A
83
14.968
−1.536
57.471
1.00
29.03
A


ATOM
94
O
ASN
A
83
13.835
−1.833
57.861
1.00
28.84
A


ATOM
95
N
ARG
A
84
15.196
−0.857
56.348
1.00
29.73
A


ATOM
96
CA
ARG
A
84
14.104
−0.413
55.487
1.00
32.14
A


ATOM
97
CB
ARG
A
84
14.631
0.475
54.351
1.00
35.80
A


ATOM
98
CG
ARG
A
84
15.480
1.633
54.839
1.00
40.52
A


ATOM
99
CD
ARG
A
84
15.714
2.721
53.787
1.00
42.66
A


ATOM
100
NE
ARG
A
84
16.572
3.762
54.354
1.00
42.69
A


ATOM
101
CZ
ARG
A
84
17.871
3.890
54.101
1.00
42.60
A


ATOM
102
NH1
ARG
A
84
18.480
3.053
53.267
1.00
39.66
A


ATOM
103
NH2
ARG
A
84
18.573
4.828
54.724
1.00
43.08
A


ATOM
104
C
ARG
A
84
13.389
−1.621
54.905
1.00
32.38
A


ATOM
105
O
ARG
A
84
12.159
−1.684
54.902
1.00
33.16
A


ATOM
106
N
MET
A
85
14.162
−2.584
54.413
1.00
31.59
A


ATOM
107
CA
MET
A
85
13.576
−3.789
53.847
1.00
30.94
A


ATOM
108
CB
MET
A
85
14.675
−4.744
53.361
1.00
33.35
A


ATOM
109
CG
MET
A
85
14.176
−6.141
52.996
1.00
34.44
A


ATOM
110
SD
MET
A
85
15.307
−7.026
51.900
1.00
36.89
A


ATOM
111
CE
MET
A
85
16.747
−7.176
52.979
1.00
35.72
A


ATOM
112
C
MET
A
85
12.695
−4.482
54.882
1.00
29.99
A


ATOM
113
O
MET
A
85
11.614
−4.965
54.559
1.00
30.02
A


ATOM
114
N
MET
A
86
13.156
−4.529
56.126
1.00
28.78
A


ATOM
115
CA
MET
A
86
12.375
−5.169
57.174
1.00
29.00
A


ATOM
116
CB
MET
A
86
13.120
−5.130
58.517
1.00
29.54
A


ATOM
117
CG
MET
A
86
14.276
−6.125
58.632
1.00
29.72
A


ATOM
118
SD
MET
A
86
14.755
−6.426
60.361
1.00
31.17
A


ATOM
119
CE
MET
A
86
16.134
−5.288
60.552
1.00
32.15
A


ATOM
120
C
MET
A
86
11.014
−4.486
57.309
1.00
29.93
A


ATOM
121
O
MET
A
86
9.984
−5.152
57.369
1.00
29.47
A


ATOM
122
N
ALA
A
87
11.014
−3.157
57.339
1.00
30.60
A


ATOM
123
CA
ALA
A
87
9.772
−2.394
57.467
1.00
29.51
A


ATOM
124
CB
ALA
A
87
10.092
−0.909
57.636
1.00
30.35
A


ATOM
125
C
ALA
A
87
8.824
−2.600
56.277
1.00
28.78
A


ATOM
126
O
ALA
A
87
7.612
−2.712
56.447
1.00
26.77
A


ATOM
127
N
LEU
A
88
9.375
−2.646
55.072
1.00
29.52
A


ATOM
128
CA
LEU
A
88
8.548
−2.843
53.888
1.00
31.42
A


ATOM
129
CB
LEU
A
88
9.395
−2.740
52.621
1.00
33.48
A


ATOM
130
CG
LEU
A
88
9.893
−1.342
52.258
1.00
35.63
A


ATOM
131
CD1
LEU
A
88
10.754
−1.430
51.005
1.00
35.88
A


ATOM
132
CD2
LEU
A
88
8.696
−0.412
52.040
1.00
36.34
A


ATOM
133
C
LEU
A
88
7.869
−4.206
53.936
1.00
31.94
A


ATOM
134
O
LEU
A
88
6.684
−4.338
53.637
1.00
30.13
A


ATOM
135
N
ILE
A
89
8.635
−5.222
54.318
1.00
31.82
A


ATOM
136
CA
ILE
A
89
8.111
−6.572
54.395
1.00
31.63
A


ATOM
137
CB
ILE
A
89
9.241
−7.577
54.669
1.00
32.58
A


ATOM
138
CG2
ILE
A
89
8.660
−8.945
54.995
1.00
32.00
A


ATOM
139
CG1
ILE
A
89
10.181
−7.628
53.458
1.00
32.05
A


ATOM
140
CD1
ILE
A
89
11.358
−8.573
53.632
1.00
32.05
A


ATOM
141
C
ILE
A
89
7.058
−6.690
55.479
1.00
32.50
A


ATOM
142
O
ILE
A
89
5.972
−7.226
55.250
1.00
32.33
A


ATOM
143
N
ILE
A
90
7.380
−6.183
56.662
1.00
32.87
A


ATOM
144
CA
ILE
A
90
6.459
−6.246
57.780
1.00
34.48
A


ATOM
145
CB
ILE
A
90
7.081
−5.577
59.028
1.00
34.93
A


ATOM
146
CG2
ILE
A
90
6.002
−5.258
60.056
1.00
34.14
A


ATOM
147
CG1
ILE
A
90
8.173
−6.493
59.596
1.00
36.87
A


ATOM
148
CD1
ILE
A
90
8.941
−5.920
60.780
1.00
37.62
A


ATOM
149
C
ILE
A
90
5.128
−5.588
57.432
1.00
36.73
A


ATOM
150
O
ILE
A
90
4.072
−6.211
57.541
1.00
36.03
A


ATOM
151
N
ASN
A
91
5.184
−4.336
56.998
1.00
37.77
A


ATOM
152
CA
ASN
A
91
3.980
−3.601
56.648
1.00
41.16
A


ATOM
153
CB
ASN
A
91
4.346
−2.148
56.335
1.00
44.99
A


ATOM
154
CG
ASN
A
91
4.612
−1.334
57.599
1.00
49.01
A


ATOM
155
OD1
ASN
A
91
3.678
−0.943
58.303
1.00
52.43
A


ATOM
156
ND2
ASN
A
91
5.886
−1.091
57.901
1.00
50.00
A


ATOM
157
C
ASN
A
91
3.200
−4.232
55.492
1.00
40.60
A


ATOM
158
O
ASN
A
91
1.976
−4.138
55.441
1.00
41.03
A


ATOM
159
N
SER
A
92
3.907
−4.892
54.580
1.00
39.25
A


ATOM
160
CA
SER
A
92
3.264
−5.536
53.444
1.00
37.85
A


ATOM
161
CB
SER
A
92
4.308
−5.936
52.402
1.00
40.00
A


ATOM
162
OG
SER
A
92
3.763
−6.868
51.480
1.00
44.69
A


ATOM
163
C
SER
A
92
2.451
−6.769
53.825
1.00
35.95
A


ATOM
164
O
SER
A
92
1.382
−7.007
53.262
1.00
37.27
A


ATOM
165
N
LEU
A
93
2.959
−7.553
54.771
1.00
33.26
A


ATOM
166
CA
LEU
A
93
2.291
−8.775
55.212
1.00
32.62
A


ATOM
167
CB
LEU
A
93
3.303
−9.924
55.299
1.00
31.56
A


ATOM
168
CG
LEU
A
93
4.171
−10.233
54.074
1.00
33.63
A


ATOM
169
CD1
LEU
A
93
5.176
−11.333
54.432
1.00
32.56
A


ATOM
170
CD2
LEU
A
93
3.296
−10.665
52.901
1.00
32.73
A


ATOM
171
C
LEU
A
93
1.635
−8.606
56.580
1.00
34.15
A


ATOM
172
O
LEU
A
93
1.292
−9.593
57.241
1.00
33.21
A


ATOM
173
N
TYR
A
94
1.458
−7.365
57.011
1.00
35.00
A


ATOM
174
CA
TYR
A
94
0.873
−7.124
58.320
1.00
38.40
A


ATOM
175
CB
TYR
A
94
0.692
−5.626
58.565
1.00
40.76
A


ATOM
176
CG
TYR
A
94
0.583
−5.308
60.030
1.00
45.83
A


ATOM
177
CD1
TYR
A
94
1.712
−5.354
60.851
1.00
48.25
A


ATOM
178
CE1
TYR
A
94
1.618
−5.121
62.220
1.00
50.09
A


ATOM
179
CD2
TYR
A
94
−0.649
−5.018
60.615
1.00
47.99
A


ATOM
180
CE2
TYR
A
94
−0.754
−4.782
61.986
1.00
50.05
A


ATOM
181
CZ
TYR
A
94
0.384
−4.837
62.780
1.00
50.62
A


ATOM
182
OH
TYR
A
94
0.293
−4.618
64.136
1.00
54.45
A


ATOM
183
C
TYR
A
94
−0.462
−7.834
58.552
1.00
38.49
A


ATOM
184
O
TYR
A
94
−0.748
−8.279
59.665
1.00
36.56
A


ATOM
185
N
LYS
A
95
−1.277
−7.946
57.507
1.00
38.51
A


ATOM
186
CA
LYS
A
95
−2.575
−8.594
57.648
1.00
39.01
A


ATOM
187
CB
LYS
A
95
−3.640
−7.790
56.904
1.00
41.67
A


ATOM
188
CG
LYS
A
95
−3.935
−6.434
57.534
1.00
44.54
A


ATOM
189
CD
LYS
A
95
−4.232
−6.572
59.024
1.00
46.17
A


ATOM
190
CE
LYS
A
95
−4.967
−5.356
59.573
1.00
48.95
A


ATOM
191
NZ
LYS
A
95
−4.210
−4.085
59.382
1.00
50.46
A


ATOM
192
C
LYS
A
95
−2.601
−10.047
57.190
1.00
38.34
A


ATOM
193
O
LYS
A
95
−3.671
−10.654
57.078
1.00
37.55
A


ATOM
194
N
ASN
A
96
−1.417
−10.596
56.934
1.00
36.93
A


ATOM
195
CA
ASN
A
96
−1.266
−11.981
56.499
1.00
36.51
A


ATOM
196
CB
ASN
A
96
−1.174
−12.052
54.971
1.00
39.38
A


ATOM
197
CG
ASN
A
96
−2.487
−11.701
54.291
1.00
41.91
A


ATOM
198
OD1
ASN
A
96
−3.414
−12.512
54.249
1.00
43.88
A


ATOM
199
ND2
ASN
A
96
−2.576
−10.485
53.769
1.00
43.40
A


ATOM
200
C
ASN
A
96
0.012
−12.523
57.130
1.00
36.61
A


ATOM
201
O
ASN
A
96
0.885
−13.057
56.447
1.00
34.41
A


ATOM
202
N
LYS
A
97
0.101
−12.378
58.447
1.00
35.10
A


ATOM
203
CA
LYS
A
97
1.261
−12.810
59.209
1.00
34.80
A


ATOM
204
CB
LYS
A
97
0.995
−12.605
60.705
1.00
33.16
A


ATOM
205
CG
LYS
A
97
0.770
−11.150
61.086
1.00
34.93
A


ATOM
206
CD
LYS
A
97
0.336
−11.005
62.542
1.00
40.66
A


ATOM
207
CE
LYS
A
97
0.437
−9.561
63.020
1.00
41.82
A


ATOM
208
NZ
LYS
A
97
−0.331
−8.616
62.171
1.00
43.56
A


ATOM
209
C
LYS
A
97
1.685
−14.248
58.955
1.00
35.24
A


ATOM
210
O
LYS
A
97
2.881
−14.539
58.874
1.00
35.69
A


ATOM
211
N
GLU
A
98
0.712
−15.144
58.818
1.00
35.04
A


ATOM
212
CA
GLU
A
98
1.013
−16.553
58.605
1.00
35.34
A


ATOM
213
CB
GLU
A
98
−0.281
−17.353
58.378
1.00
37.17
A


ATOM
214
CG
GLU
A
98
−1.069
−17.009
57.120
1.00
42.04
A


ATOM
215
CD
GLU
A
98
−2.062
−15.872
57.321
1.00
44.24
A


ATOM
216
OE1
GLU
A
98
−2.874
−15.632
56.399
1.00
47.71
A


ATOM
217
OE2
GLU
A
98
−2.037
−15.219
58.388
1.00
43.42
A


ATOM
218
C
GLU
A
98
2.009
−16.836
57.476
1.00
33.13
A


ATOM
219
O
GLU
A
98
2.654
−17.886
57.462
1.00
32.97
A


ATOM
220
N
ILE
A
99
2.144
−15.903
56.541
1.00
31.36
A


ATOM
221
CA
ILE
A
99
3.070
−16.083
55.428
1.00
32.00
A


ATOM
222
CB
ILE
A
99
2.947
−14.914
54.411
1.00
33.50
A


ATOM
223
CG2
ILE
A
99
4.033
−15.002
53.345
1.00
34.71
A


ATOM
224
CG1
ILE
A
99
1.571
−14.975
53.739
1.00
38.54
A


ATOM
225
CD1
ILE
A
99
1.392
−13.998
52.590
1.00
40.32
A


ATOM
226
C
ILE
A
99
4.514
−16.213
55.923
1.00
31.10
A


ATOM
227
O
ILE
A
99
5.409
−16.604
55.168
1.00
29.83
A


ATOM
228
N
PHE
A
100
4.743
−15.899
57.197
1.00
29.56
A


ATOM
229
CA
PHE
A
100
6.085
−16.011
57.746
1.00
28.31
A


ATOM
230
CB
PHE
A
100
6.141
−15.512
59.204
1.00
26.69
A


ATOM
231
CG
PHE
A
100
5.755
−16.553
60.228
1.00
27.65
A


ATOM
232
CD1
PHE
A
100
4.439
−16.670
60.664
1.00
27.08
A


ATOM
233
CD2
PHE
A
100
6.715
−17.430
60.744
1.00
28.13
A


ATOM
234
CE1
PHE
A
100
4.074
−17.650
61.605
1.00
28.84
A


ATOM
235
CE2
PHE
A
100
6.367
−18.413
61.681
1.00
28.66
A


ATOM
236
CZ
PHE
A
100
5.042
−18.522
62.112
1.00
29.66
A


ATOM
237
C
PHE
A
100
6.519
−17.476
57.684
1.00
27.86
A


ATOM
238
O
PHE
A
100
7.671
−17.782
57.375
1.00
26.69
A


ATOM
239
N
LEU
A
101
5.586
−18.378
57.967
1.00
26.61
A


ATOM
240
CA
LEU
A
101
5.894
−19.803
57.966
1.00
27.71
A


ATOM
241
CB
LEU
A
101
4.761
−20.594
58.630
1.00
28.29
A


ATOM
242
CG
LEU
A
101
5.048
−22.074
58.906
1.00
31.62
A


ATOM
243
CD1
LEU
A
101
6.310
−22.197
59.757
1.00
31.66
A


ATOM
244
CD2
LEU
A
101
3.863
−22.711
59.622
1.00
32.58
A


ATOM
245
C
LEU
A
101
6.141
−20.324
56.556
1.00
27.21
A


ATOM
246
O
LEU
A
101
6.978
−21.204
56.354
1.00
29.92
A


ATOM
247
N
ARG
A
102
5.414
−19.782
55.583
1.00
26.05
A


ATOM
248
CA
ARG
A
102
5.587
−20.185
54.192
1.00
27.16
A


ATOM
249
CB
ARG
A
102
4.637
−19.414
53.274
1.00
27.20
A


ATOM
250
CG
ARG
A
102
4.666
−19.895
51.824
1.00
28.59
A


ATOM
251
CD
ARG
A
102
3.882
−18.953
50.906
1.00
29.28
A


ATOM
252
NE
ARG
A
102
4.616
−17.720
50.638
1.00
32.58
A


ATOM
253
CZ
ARG
A
102
4.129
−16.686
49.956
1.00
36.08
A


ATOM
254
NH1
ARG
A
102
2.893
−16.728
49.473
1.00
33.94
A


ATOM
255
NH2
ARG
A
102
4.888
−15.618
49.734
1.00
33.41
A


ATOM
256
C
ARG
A
102
7.014
−19.886
53.755
1.00
26.97
A


ATOM
257
O
ARG
A
102
7.674
−20.724
53.139
1.00
27.51
A


ATOM
258
N
GLU
A
103
7.478
−18.680
54.077
1.00
26.40
A


ATOM
259
CA
GLU
A
103
8.820
−18.243
53.715
1.00
25.90
A


ATOM
260
CB
GLU
A
103
8.967
−16.740
53.973
1.00
27.03
A


ATOM
261
CG
GLU
A
103
8.084
−15.892
53.074
1.00
30.54
A


ATOM
262
CD
GLU
A
103
8.227
−16.277
51.610
1.00
34.94
A


ATOM
263
OE1
GLU
A
103
9.367
−16.271
51.096
1.00
36.36
A


ATOM
264
OE2
GLU
A
103
7.200
−16.592
50.971
1.00
36.06
A


ATOM
265
C
GLU
A
103
9.933
−19.006
54.424
1.00
27.65
A


ATOM
266
O
GLU
A
103
10.949
−19.329
53.811
1.00
28.59
A


ATOM
267
N
LEU
A
104
9.762
−19.289
55.714
1.00
27.46
A


ATOM
268
CA
LEU
A
104
10.789
−20.027
56.437
1.00
28.00
A


ATOM
269
CB
LEU
A
104
10.446
−20.109
57.929
1.00
28.75
A


ATOM
270
CG
LEU
A
104
10.344
−18.774
58.677
1.00
27.18
A


ATOM
271
CD1
LEU
A
104
10.066
−19.029
60.150
1.00
29.40
A


ATOM
272
CD2
LEU
A
104
11.640
−17.990
58.510
1.00
26.56
A


ATOM
273
C
LEU
A
104
10.920
−21.433
55.846
1.00
28.30
A


ATOM
274
O
LEU
A
104
12.021
−21.973
55.739
1.00
26.95
A


ATOM
275
N
ILE
A
105
9.792
−22.023
55.466
1.00
27.22
A


ATOM
276
CA
ILE
A
105
9.805
−23.355
54.874
1.00
29.99
A


ATOM
277
CB
ILE
A
105
8.365
−23.937
54.770
1.00
31.43
A


ATOM
278
CG2
ILE
A
105
8.328
−25.119
53.793
1.00
29.81
A


ATOM
279
CG1
ILE
A
105
7.901
−24.372
56.167
1.00
31.88
A


ATOM
280
CD1
ILE
A
105
6.493
−24.890
56.231
1.00
34.12
A


ATOM
281
C
ILE
A
105
10.458
−23.277
53.499
1.00
30.30
A


ATOM
282
O
ILE
A
105
11.252
−24.138
53.125
1.00
30.17
A


ATOM
283
N
SER
A
106
10.138
−22.220
52.762
1.00
29.33
A


ATOM
284
CA
SER
A
106
10.710
−22.007
51.438
1.00
30.77
A


ATOM
285
CB
SER
A
106
10.097
−20.754
50.810
1.00
31.84
A


ATOM
286
OG
SER
A
106
10.662
−20.496
49.542
1.00
39.34
A


ATOM
287
C
SER
A
106
12.237
−21.857
51.545
1.00
30.70
A


ATOM
288
O
SER
A
106
12.981
−22.458
50.767
1.00
29.18
A


ATOM
289
N
ASN
A
107
12.700
−21.060
52.509
1.00
28.35
A


ATOM
290
CA
ASN
A
107
14.139
−20.866
52.705
1.00
28.64
A


ATOM
291
CB
ASN
A
107
14.409
−19.875
53.846
1.00
27.62
A


ATOM
292
CG
ASN
A
107
14.083
−18.434
53.472
1.00
29.03
A


ATOM
293
OD1
ASN
A
107
14.153
−17.529
54.317
1.00
29.37
A


ATOM
294
ND2
ASN
A
107
13.737
−18.210
52.211
1.00
23.85
A


ATOM
295
C
ASN
A
107
14.791
−22.212
53.044
1.00
28.48
A


ATOM
296
O
ASN
A
107
15.881
−22.530
52.563
1.00
27.33
A


ATOM
297
N
ALA
A
108
14.116
−22.995
53.881
1.00
27.12
A


ATOM
298
CA
ALA
A
108
14.628
−24.301
54.278
1.00
29.05
A


ATOM
299
CB
ALA
A
108
13.704
−24.934
55.303
1.00
26.23
A


ATOM
300
C
ALA
A
108
14.738
−25.194
53.039
1.00
29.49
A


ATOM
301
O
ALA
A
108
15.712
−25.930
52.874
1.00
29.41
A


ATOM
302
N
SER
A
109
13.725
−25.120
52.179
1.00
29.12
A


ATOM
303
CA
SER
A
109
13.696
−25.895
50.945
1.00
30.36
A


ATOM
304
CB
SER
A
109
12.400
−25.612
50.182
1.00
30.10
A


ATOM
305
OG
SER
A
109
12.408
−26.258
48.926
1.00
34.79
A


ATOM
306
C
SER
A
109
14.891
−25.535
50.066
1.00
31.21
A


ATOM
307
O
SER
A
109
15.545
−26.417
49.504
1.00
33.48
A


ATOM
308
N
ASP
A
110
15.172
−24.239
49.947
1.00
29.99
A


ATOM
309
CA
ASP
A
110
16.296
−23.776
49.136
1.00
30.22
A


ATOM
310
CB
ASP
A
110
16.341
−22.242
49.099
1.00
31.27
A


ATOM
311
CG
ASP
A
110
15.163
−21.635
48.345
1.00
32.67
A


ATOM
312
OD1
ASP
A
110
15.010
−20.398
48.382
1.00
30.92
A


ATOM
313
OD2
ASP
A
110
14.391
−22.388
47.713
1.00
35.49
A


ATOM
314
C
ASP
A
110
17.617
−24.311
49.682
1.00
29.51
A


ATOM
315
O
ASP
A
110
18.492
−24.720
48.915
1.00
27.25
A


ATOM
316
N
ALA
A
111
17.757
−24.302
51.007
1.00
26.85
A


ATOM
317
CA
ALA
A
111
18.969
−24.791
51.661
1.00
26.33
A


ATOM
318
CB
ALA
A
111
18.901
−24.520
53.163
1.00
25.25
A


ATOM
319
C
ALA
A
111
19.151
−26.286
51.406
1.00
27.62
A


ATOM
320
O
ALA
A
111
20.273
−26.768
51.235
1.00
27.32
A


ATOM
321
N
LEU
A
112
18.043
−27.020
51.385
1.00
26.45
A


ATOM
322
CA
LEU
A
112
18.101
−28.447
51.126
1.00
29.74
A


ATOM
323
CB
LEU
A
112
16.775
−29.116
51.514
1.00
28.30
A


ATOM
324
CG
LEU
A
112
16.596
−29.288
53.033
1.00
30.20
A


ATOM
325
CD1
LEU
A
112
15.130
−29.470
53.366
1.00
30.47
A


ATOM
326
CD2
LEU
A
112
17.425
−30.474
53.528
1.00
28.57
A


ATOM
327
C
LEU
A
112
18.442
−28.706
49.655
1.00
30.58
A


ATOM
328
O
LEU
A
112
19.209
−29.618
49.352
1.00
28.99
A


ATOM
329
N
ASP
A
113
17.893
−27.905
48.745
1.00
30.56
A


ATOM
330
CA
ASP
A
113
18.207
−28.090
47.327
1.00
32.58
A


ATOM
331
CB
ASP
A
113
17.479
−27.079
46.427
1.00
31.43
A


ATOM
332
CG
ASP
A
113
15.995
−27.366
46.278
1.00
31.87
A


ATOM
333
OD1
ASP
A
113
15.590
−28.544
46.286
1.00
32.19
A


ATOM
334
OD2
ASP
A
113
15.229
−26.396
46.119
1.00
34.18
A


ATOM
335
C
ASP
A
113
19.707
−27.871
47.159
1.00
32.53
A


ATOM
336
O
ASP
A
113
20.382
−28.626
46.458
1.00
31.55
A


ATOM
337
N
LYS
A
114
20.221
−26.831
47.814
1.00
32.96
A


ATOM
338
CA
LYS
A
114
21.636
−26.504
47.720
1.00
32.84
A


ATOM
339
CB
LYS
A
114
21.956
−25.259
48.547
1.00
37.08
A


ATOM
340
CG
LYS
A
114
21.351
−23.983
47.983
1.00
43.46
A


ATOM
341
CD
LYS
A
114
21.838
−22.746
48.731
1.00
50.51
A


ATOM
342
CE
LYS
A
114
21.249
−21.466
48.137
1.00
52.99
A


ATOM
343
NZ
LYS
A
114
21.695
−20.256
48.891
1.00
54.73
A


ATOM
344
C
LYS
A
114
22.556
−27.646
48.130
1.00
32.02
A


ATOM
345
O
LYS
A
114
23.448
−28.024
47.374
1.00
32.06
A


ATOM
346
N
ILE
A
115
22.358
−28.197
49.323
1.00
30.95
A


ATOM
347
CA
ILE
A
115
23.216
−29.290
49.751
1.00
30.36
A


ATOM
348
CB
ILE
A
115
23.006
−29.648
51.253
1.00
28.05
A


ATOM
349
CG2
ILE
A
115
21.703
−30.404
51.451
1.00
29.39
A


ATOM
350
CG1
ILE
A
115
24.171
−30.515
51.739
1.00
30.04
A


ATOM
351
CD1
ILE
A
115
25.540
−29.849
51.611
1.00
29.38
A


ATOM
352
C
ILE
A
115
22.984
−30.517
48.861
1.00
29.19
A


ATOM
353
O
ILE
A
115
23.897
−31.291
48.625
1.00
28.85
A


ATOM
354
N
ARG
A
116
21.769
−30.687
48.355
1.00
31.07
A


ATOM
355
CA
ARG
A
116
21.495
−31.815
47.474
1.00
35.04
A


ATOM
356
CB
ARG
A
116
20.004
−31.900
47.124
1.00
37.53
A


ATOM
357
CG
ARG
A
116
19.712
−32.923
46.022
1.00
43.83
A


ATOM
358
CD
ARG
A
116
18.230
−33.072
45.711
1.00
46.53
A


ATOM
359
NE
ARG
A
116
17.525
−33.854
46.724
1.00
50.14
A


ATOM
360
CZ
ARG
A
116
16.234
−34.165
46.666
0.00
49.23
A


ATOM
361
NH1
ARG
A
116
15.497
−33.759
45.642
0.00
49.63
A


ATOM
362
NH2
ARG
A
116
15.679
−34.886
47.631
0.00
49.63
A


ATOM
363
C
ARG
A
116
22.319
−31.656
46.190
1.00
35.93
A


ATOM
364
O
ARG
A
116
22.931
−32.608
45.720
1.00
35.09
A


ATOM
365
N
LEU
A
117
22.342
−30.448
45.634
1.00
35.02
A


ATOM
366
CA
LEU
A
117
23.109
−30.192
44.416
1.00
35.33
A


ATOM
367
CB
LEU
A
117
22.848
−28.769
43.898
1.00
35.62
A


ATOM
368
CG
LEU
A
117
21.416
−28.401
43.480
1.00
38.17
A


ATOM
369
CD1
LEU
A
117
21.396
−26.984
42.917
1.00
38.15
A


ATOM
370
CD2
LEU
A
117
20.906
−29.380
42.437
1.00
39.74
A


ATOM
371
C
LEU
A
117
24.604
−30.372
44.688
1.00
35.09
A


ATOM
372
O
LEU
A
117
25.348
−30.862
43.838
1.00
32.38
A


ATOM
373
N
ILE
A
118
25.036
−29.976
45.880
1.00
33.96
A


ATOM
374
CA
ILE
A
118
26.438
−30.102
46.258
1.00
35.20
A


ATOM
375
CB
ILE
A
118
26.719
−29.402
47.606
1.00
35.56
A


ATOM
376
CG2
ILE
A
118
28.155
−29.686
48.058
1.00
33.74
A


ATOM
377
CG1
ILE
A
118
26.481
−27.898
47.463
1.00
36.15
A


ATOM
378
CD1
ILE
A
118
26.556
−27.127
48.782
1.00
37.01
A


ATOM
379
C
ILE
A
118
26.856
−31.566
46.373
1.00
35.27
A


ATOM
380
O
ILE
A
118
28.006
−31.910
46.087
1.00
34.03
A


ATOM
381
N
SER
A
119
25.922
−32.422
46.784
1.00
35.20
A


ATOM
382
CA
SER
A
119
26.219
−33.842
46.939
1.00
36.19
A


ATOM
383
CB
SER
A
119
25.043
−34.581
47.599
1.00
34.53
A


ATOM
384
OG
SER
A
119
23.933
−34.700
46.727
1.00
33.75
A


ATOM
385
C
SER
A
119
26.532
−34.454
45.578
1.00
37.53
A


ATOM
386
O
SER
A
119
27.127
−35.529
45.494
1.00
36.37
A


ATOM
387
N
LEU
A
120
26.132
−33.761
44.515
1.00
38.69
A


ATOM
388
CA
LEU
A
120
26.396
−34.233
43.157
1.00
41.79
A


ATOM
389
CB
LEU
A
120
25.583
−33.436
42.127
1.00
40.22
A


ATOM
390
CG
LEU
A
120
24.063
−33.612
42.106
1.00
42.57
A


ATOM
391
CD1
LEU
A
120
23.472
−32.764
40.985
1.00
42.67
A


ATOM
392
CD2
LEU
A
120
23.710
−35.078
41.903
1.00
42.57
A


ATOM
393
C
LEU
A
120
27.879
−34.096
42.824
1.00
42.04
A


ATOM
394
O
LEU
A
120
28.428
−34.900
42.074
1.00
44.04
A


ATOM
395
N
THR
A
121
28.524
−33.079
43.388
1.00
43.08
A


ATOM
396
CA
THR
A
121
29.937
−32.834
43.127
1.00
44.56
A


ATOM
397
CB
THR
A
121
30.185
−31.371
42.751
1.00
44.06
A


ATOM
398
OG1
THR
A
121
29.882
−30.534
43.875
1.00
44.67
A


ATOM
399
CG2
THR
A
121
29.313
−30.969
41.574
1.00
43.68
A


ATOM
400
C
THR
A
121
30.841
−33.150
44.309
1.00
46.28
A


ATOM
401
O
THR
A
121
32.061
−33.211
44.162
1.00
45.91
A


ATOM
402
N
ASP
A
122
30.244
−33.338
45.480
1.00
48.08
A


ATOM
403
CA
ASP
A
122
31.011
−33.630
46.683
1.00
49.23
A


ATOM
404
CB
ASP
A
122
30.869
−32.480
47.681
1.00
51.96
A


ATOM
405
CG
ASP
A
122
31.901
−32.535
48.787
1.00
54.29
A


ATOM
406
OD1
ASP
A
122
32.357
−33.650
49.122
1.00
56.04
A


ATOM
407
OD2
ASP
A
122
32.246
−31.462
49.330
1.00
55.62
A


ATOM
408
C
ASP
A
122
30.494
−34.921
47.305
1.00
48.93
A


ATOM
409
O
ASP
A
122
29.392
−34.954
47.851
1.00
47.16
A


ATOM
410
N
ALA
A
123
31.299
−35.977
47.219
1.00
49.14
A


ATOM
411
CA
ALA
A
123
30.930
−37.288
47.752
1.00
49.93
A


ATOM
412
CB
ALA
A
123
32.026
−38.305
47.431
1.00
50.62
A


ATOM
413
C
ALA
A
123
30.644
−37.295
49.253
1.00
50.38
A


ATOM
414
O
ALA
A
123
29.828
−38.086
49.726
1.00
52.10
A


ATOM
415
N
ASN
A
124
31.308
−36.423
50.004
1.00
50.62
A


ATOM
416
CA
ASN
A
124
31.099
−36.372
51.451
1.00
51.30
A


ATOM
417
CB
ASN
A
124
32.447
−36.354
52.179
1.00
53.30
A


ATOM
418
CG
ASN
A
124
33.070
−37.733
52.283
1.00
55.50
A


ATOM
419
OD1
ASN
A
124
33.395
−38.357
51.275
1.00
56.73
A


ATOM
420
ND2
ASN
A
124
33.234
−38.218
53.511
1.00
55.50
A


ATOM
421
C
ASN
A
124
30.253
−35.197
51.936
1.00
49.31
A


ATOM
422
O
ASN
A
124
30.179
−34.937
53.134
1.00
48.17
A


ATOM
423
N
ALA
A
125
29.605
−34.498
51.012
1.00
47.50
A


ATOM
424
CA
ALA
A
125
28.786
−33.351
51.373
1.00
46.05
A


ATOM
425
CB
ALA
A
125
28.023
−32.857
50.152
1.00
43.99
A


ATOM
426
C
ALA
A
125
27.811
−33.626
52.521
1.00
44.71
A


ATOM
427
O
ALA
A
125
27.620
−32.780
53.393
1.00
43.86
A


ATOM
428
N
LEU
A
126
27.209
−34.812
52.525
1.00
44.79
A


ATOM
429
CA
LEU
A
126
26.224
−35.177
53.542
1.00
45.70
A


ATOM
430
CB
LEU
A
126
25.107
−35.990
52.883
1.00
45.39
A


ATOM
431
CG
LEU
A
126
24.417
−35.278
51.717
1.00
46.52
A


ATOM
432
CD1
LEU
A
126
23.404
−36.197
51.061
1.00
46.62
A


ATOM
433
CD2
LEU
A
126
23.742
−34.014
52.233
1.00
47.81
A


ATOM
434
C
LEU
A
126
26.747
−35.932
54.768
1.00
45.28
A


ATOM
435
O
LEU
A
126
25.965
−36.341
55.628
1.00
45.72
A


ATOM
436
N
ALA
A
127
28.062
−36.096
54.852
1.00
45.95
A


ATOM
437
CA
ALA
A
127
28.698
−36.816
55.953
1.00
46.85
A


ATOM
438
CB
ALA
A
127
30.211
−36.789
55.771
1.00
46.17
A


ATOM
439
C
ALA
A
127
28.344
−36.320
57.356
1.00
48.69
A


ATOM
440
O
ALA
A
127
28.477
−37.067
58.327
1.00
49.08
A


ATOM
441
N
GLY
A
128
27.897
−35.071
57.467
1.00
48.63
A


ATOM
442
CA
GLY
A
128
27.562
−34.521
58.771
1.00
48.99
A


ATOM
443
C
GLY
A
128
26.153
−34.823
59.244
1.00
49.66
A


ATOM
444
O
GLY
A
128
25.841
−34.662
60.422
1.00
50.11
A


ATOM
445
N
ASN
A
129
25.303
−35.265
58.326
1.00
49.94
A


ATOM
446
CA
ASN
A
129
23.920
−35.587
58.642
1.00
50.71
A


ATOM
447
CB
ASN
A
129
23.193
−34.323
59.121
1.00
50.81
A


ATOM
448
CG
ASN
A
129
21.807
−34.609
59.672
1.00
50.32
A


ATOM
449
OD1
ASN
A
129
21.224
−33.775
60.366
1.00
50.86
A


ATOM
450
ND2
ASN
A
129
21.269
−35.781
59.357
1.00
49.42
A


ATOM
451
C
ASN
A
129
23.290
−36.133
57.364
1.00
52.23
A


ATOM
452
O
ASN
A
129
22.938
−35.378
56.460
1.00
52.87
A


ATOM
453
N
GLU
A
130
23.157
−37.453
57.303
1.00
53.42
A


ATOM
454
CA
GLU
A
130
22.608
−38.138
56.137
1.00
53.84
A


ATOM
455
CB
GLU
A
130
22.679
−39.650
56.354
1.00
57.50
A


ATOM
456
CG
GLU
A
130
24.058
−40.162
56.758
1.00
62.46
A


ATOM
457
CD
GLU
A
130
25.093
−40.029
55.653
1.00
65.01
A


ATOM
458
OE1
GLU
A
130
26.273
−40.351
55.910
1.00
66.73
A


ATOM
459
OE2
GLU
A
130
24.731
−39.611
54.531
1.00
66.00
A


ATOM
460
C
GLU
A
130
21.179
−37.761
55.757
1.00
52.27
A


ATOM
461
O
GLU
A
130
20.741
−38.039
54.641
1.00
53.28
A


ATOM
462
N
ALA
A
131
20.450
−37.128
56.668
1.00
49.49
A


ATOM
463
CA
ALA
A
131
19.065
−36.754
56.383
1.00
46.55
A


ATOM
464
CB
ALA
A
131
18.272
−36.690
57.688
1.00
47.39
A


ATOM
465
C
ALA
A
131
18.899
−35.441
55.615
1.00
43.78
A


ATOM
466
O
ALA
A
131
19.746
−34.550
55.685
1.00
44.02
A


ATOM
467
N
LEU
A
132
17.793
−35.347
54.881
1.00
40.82
A


ATOM
468
CA
LEU
A
132
17.431
−34.165
54.100
1.00
39.49
A


ATOM
469
CB
LEU
A
132
17.586
−34.438
52.602
1.00
38.45
A


ATOM
470
CG
LEU
A
132
19.012
−34.686
52.111
1.00
39.04
A


ATOM
471
CD1
LEU
A
132
18.997
−34.935
50.606
1.00
40.56
A


ATOM
472
CD2
LEU
A
132
19.880
−33.480
52.446
1.00
37.22
A


ATOM
473
C
LEU
A
132
15.966
−33.894
54.424
1.00
39.45
A


ATOM
474
O
LEU
A
132
15.069
−34.289
53.673
1.00
38.76
A


ATOM
475
N
THR
A
133
15.728
−33.211
55.541
1.00
37.29
A


ATOM
476
CA
THR
A
133
14.369
−32.944
55.990
1.00
34.35
A


ATOM
477
CB
THR
A
133
13.972
−33.932
57.096
1.00
35.57
A


ATOM
478
OG1
THR
A
133
14.756
−33.654
58.265
1.00
34.87
A


ATOM
479
CG2
THR
A
133
14.229
−35.378
56.663
1.00
36.95
A


ATOM
480
C
THR
A
133
14.141
−31.554
56.578
1.00
33.92
A


ATOM
481
O
THR
A
133
15.079
−30.800
56.842
1.00
33.54
A


ATOM
482
N
VAL
A
134
12.864
−31.246
56.782
1.00
35.11
A


ATOM
483
CA
VAL
A
134
12.423
−30.007
57.404
1.00
35.04
A


ATOM
484
CB
VAL
A
134
11.559
−29.154
56.456
1.00
35.32
A


ATOM
485
CG1
VAL
A
134
11.063
−27.920
57.188
1.00
34.33
A


ATOM
486
CG2
VAL
A
134
12.370
−28.745
55.238
1.00
33.86
A


ATOM
487
C
VAL
A
134
11.558
−30.462
58.583
1.00
36.04
A


ATOM
488
O
VAL
A
134
10.492
−31.053
58.385
1.00
35.21
A


ATOM
489
N
LYS
A
135
12.030
−30.211
59.801
1.00
36.08
A


ATOM
490
CA
LYS
A
135
11.301
−30.608
61.006
1.00
36.29
A


ATOM
491
CB
LYS
A
135
12.157
−31.558
61.846
1.00
36.25
A


ATOM
492
CG
LYS
A
135
12.346
−32.934
61.220
1.00
39.42
A


ATOM
493
CD
LYS
A
135
13.328
−33.785
62.020
1.00
40.39
A


ATOM
494
CE
LYS
A
135
13.349
−35.230
61.522
1.00
40.38
A


ATOM
495
NZ
LYS
A
135
14.392
−36.039
62.230
1.00
39.55
A


ATOM
496
C
LYS
A
135
10.886
−29.403
61.847
1.00
37.12
A


ATOM
497
O
LYS
A
135
11.712
−28.564
62.214
1.00
36.87
A


ATOM
498
N
ILE
A
136
9.598
−29.337
62.157
1.00
37.18
A


ATOM
499
CA
ILE
A
136
9.042
−28.237
62.929
1.00
37.49
A


ATOM
500
CB
ILE
A
136
7.940
−27.524
62.116
1.00
37.75
A


ATOM
501
CG2
ILE
A
136
7.233
−26.486
62.983
1.00
37.38
A


ATOM
502
CG1
ILE
A
136
8.552
−26.896
60.863
1.00
39.62
A


ATOM
503
CD1
ILE
A
136
7.542
−26.337
59.893
1.00
41.02
A


ATOM
504
C
ILE
A
136
8.439
−28.686
64.257
1.00
38.72
A


ATOM
505
O
ILE
A
136
7.727
−29.688
64.312
1.00
36.70
A


ATOM
506
N
LYS
A
137
8.734
−27.955
65.328
1.00
38.86
A


ATOM
507
CA
LYS
A
137
8.153
−28.284
66.620
1.00
42.04
A


ATOM
508
CB
LYS
A
137
9.033
−29.267
67.416
1.00
45.10
A


ATOM
509
CG
LYS
A
137
10.511
−28.939
67.570
1.00
47.27
A


ATOM
510
CD
LYS
A
137
11.177
−30.056
68.373
1.00
49.53
A


ATOM
511
CE
LYS
A
137
12.693
−29.933
68.422
1.00
51.98
A


ATOM
512
NZ
LYS
A
137
13.150
−28.727
69.162
1.00
53.51
A


ATOM
513
C
LYS
A
137
7.806
−27.068
67.472
1.00
42.49
A


ATOM
514
O
LYS
A
137
8.510
−26.056
67.482
1.00
40.52
A


ATOM
515
N
CYS
A
138
6.687
−27.183
68.172
1.00
42.67
A


ATOM
516
CA
CYS
A
138
6.197
−26.120
69.029
1.00
44.44
A


ATOM
517
CB
CYS
A
138
4.678
−26.009
68.904
1.00
43.70
A


ATOM
518
SG
CYS
A
138
4.082
−25.900
67.213
1.00
45.30
A


ATOM
519
C
CYS
A
138
6.552
−26.414
70.474
1.00
43.99
A


ATOM
520
O
CYS
A
138
6.560
−27.565
70.901
1.00
43.91
A


ATOM
521
N
ASP
A
139
6.855
−25.367
71.222
1.00
44.81
A


ATOM
522
CA
ASP
A
139
7.173
−25.519
72.628
1.00
46.03
A


ATOM
523
CB
ASP
A
139
8.653
−25.252
72.885
1.00
47.30
A


ATOM
524
CG
ASP
A
139
9.105
−25.785
74.225
1.00
49.47
A


ATOM
525
OD1
ASP
A
139
8.436
−25.483
75.236
1.00
50.01
A


ATOM
526
OD2
ASP
A
139
10.123
−26.508
74.264
1.00
49.55
A


ATOM
527
C
ASP
A
139
6.319
−24.498
73.363
1.00
46.25
A


ATOM
528
O
ASP
A
139
6.811
−23.446
73.783
1.00
45.28
A


ATOM
529
N
ALA
A
140
5.033
−24.813
73.488
1.00
45.65
A


ATOM
530
CA
ALA
A
140
4.074
−23.936
74.145
1.00
48.13
A


ATOM
531
CB
ALA
A
140
2.738
−24.662
74.321
1.00
48.04
A


ATOM
532
C
ALA
A
140
4.591
−23.460
75.492
1.00
48.45
A


ATOM
533
O
ALA
A
140
4.490
−22.282
75.824
1.00
49.34
A


ATOM
534
N
GLU
A
141
5.155
−24.382
76.260
1.00
50.21
A


ATOM
535
CA
GLU
A
141
5.676
−24.057
77.578
1.00
51.71
A


ATOM
536
CB
GLU
A
141
6.163
−25.333
78.273
1.00
54.89
A


ATOM
537
CG
GLU
A
141
5.019
−26.268
78.643
1.00
59.36
A


ATOM
538
CD
GLU
A
141
5.485
−27.577
79.249
1.00
63.14
A


ATOM
539
OE1
GLU
A
141
6.137
−28.371
78.536
1.00
65.16
A


ATOM
540
OE2
GLU
A
141
5.195
−27.812
80.441
1.00
65.13
A


ATOM
541
C
GLU
A
141
6.779
−23.005
77.545
1.00
50.76
A


ATOM
542
O
GLU
A
141
6.847
−22.158
78.434
1.00
52.28
A


ATOM
543
N
ALA
A
142
7.633
−23.044
76.526
1.00
46.94
A


ATOM
544
CA
ALA
A
142
8.708
−22.062
76.424
1.00
43.37
A


ATOM
545
CB
ALA
A
142
10.010
−22.745
76.021
1.00
44.14
A


ATOM
546
C
ALA
A
142
8.373
−20.942
75.438
1.00
41.63
A


ATOM
547
O
ALA
A
142
9.180
−20.040
75.220
1.00
42.01
A


ATOM
548
N
ALA
A
143
7.179
−20.997
74.853
1.00
38.59
A


ATOM
549
CA
ALA
A
143
6.749
−19.983
73.893
1.00
36.98
A


ATOM
550
CB
ALA
A
143
6.676
−18.621
74.578
1.00
37.79
A


ATOM
551
C
ALA
A
143
7.683
−19.901
72.683
1.00
35.42
A


ATOM
552
O
ALA
A
143
7.976
−18.815
72.190
1.00
35.88
A


ATOM
553
N
LEU
A
144
8.144
−21.048
72.201
1.00
34.76
A


ATOM
554
CA
LEU
A
144
9.048
−21.074
71.056
1.00
34.83
A


ATOM
555
CB
LEU
A
144
10.415
−21.623
71.475
1.00
35.13
A


ATOM
556
CG
LEU
A
144
11.174
−20.954
72.622
1.00
36.82
A


ATOM
557
CD1
LEU
A
144
12.423
−21.779
72.951
1.00
35.69
A


ATOM
558
CD2
LEU
A
144
11.548
−19.525
72.233
1.00
35.89
A


ATOM
559
C
LEU
A
144
8.529
−21.925
69.902
1.00
34.02
A


ATOM
560
O
LEU
A
144
7.810
−22.901
70.107
1.00
33.48
A


ATOM
561
N
LEU
A
145
8.899
−21.536
68.687
1.00
34.45
A


ATOM
562
CA
LEU
A
145
8.540
−22.279
67.486
1.00
33.67
A


ATOM
563
CB
LEU
A
145
7.616
−21.452
66.582
1.00
34.01
A


ATOM
564
CG
LEU
A
145
7.163
−22.113
65.272
1.00
37.05
A


ATOM
565
CD1
LEU
A
145
6.402
−23.404
65.587
1.00
38.01
A


ATOM
566
CD2
LEU
A
145
6.281
−21.159
64.478
1.00
36.91
A


ATOM
567
C
LEU
A
145
9.884
−22.533
66.800
1.00
33.48
A


ATOM
568
O
LEU
A
145
10.660
−21.600
66.587
1.00
33.22
A


ATOM
569
N
HIS
A
146
10.172
−23.794
66.487
1.00
33.54
A


ATOM
570
CA
HIS
A
146
11.436
−24.157
65.845
1.00
32.05
A


ATOM
571
CB
HIS
A
146
12.164
−25.239
66.652
1.00
32.73
A


ATOM
572
CG
HIS
A
146
12.458
−24.865
68.073
1.00
30.80
A


ATOM
573
CD2
HIS
A
146
11.916
−25.301
69.237
1.00
29.75
A


ATOM
574
ND1
HIS
A
146
13.464
−23.990
68.421
1.00
30.71
A


ATOM
575
CE1
HIS
A
146
13.533
−23.909
69.740
1.00
33.06
A


ATOM
576
NE2
HIS
A
146
12.605
−24.695
70.258
1.00
30.34
A


ATOM
577
C
HIS
A
146
11.204
−24.707
64.439
1.00
31.91
A


ATOM
578
O
HIS
A
146
10.315
−25.527
64.234
1.00
31.45
A


ATOM
579
N
VAL
A
147
12.008
−24.251
63.482
1.00
29.51
A


ATOM
580
CA
VAL
A
147
11.927
−24.717
62.097
1.00
28.66
A


ATOM
581
CB
VAL
A
147
11.459
−23.605
61.137
1.00
28.09
A


ATOM
582
CG1
VAL
A
147
11.479
−24.112
59.711
1.00
31.64
A


ATOM
583
CG2
VAL
A
147
10.066
−23.154
61.510
1.00
26.76
A


ATOM
584
C
VAL
A
147
13.349
−25.129
61.739
1.00
28.95
A


ATOM
585
O
VAL
A
147
14.211
−24.284
61.491
1.00
26.81
A


ATOM
586
N
THR
A
148
13.581
−26.438
61.730
1.00
29.47
A


ATOM
587
CA
THR
A
148
14.900
−26.988
61.462
1.00
30.68
A


ATOM
588
CB
THR
A
148
15.342
−27.931
62.612
1.00
33.73
A


ATOM
589
OG1
THR
A
148
15.276
−27.229
63.862
1.00
32.55
A


ATOM
590
CG2
THR
A
148
16.773
−28.439
62.376
1.00
30.72
A


ATOM
591
C
THR
A
148
14.992
−27.765
60.162
1.00
29.42
A


ATOM
592
O
THR
A
148
14.126
−28.576
59.836
1.00
29.39
A


ATOM
593
N
ASP
A
149
16.061
−27.506
59.424
1.00
29.96
A


ATOM
594
CA
ASP
A
149
16.304
−28.192
58.170
1.00
29.42
A


ATOM
595
CB
ASP
A
149
16.159
−27.225
56.985
1.00
28.33
A


ATOM
596
CG
ASP
A
149
17.258
−26.169
56.948
1.00
27.59
A


ATOM
597
OD1
ASP
A
149
18.431
−26.534
56.737
1.00
26.90
A


ATOM
598
OD2
ASP
A
149
16.950
−24.971
57.126
1.00
27.46
A


ATOM
599
C
ASP
A
149
17.720
−28.741
58.228
1.00
28.78
A


ATOM
600
O
ASP
A
149
18.544
−28.274
59.016
1.00
27.96
A


ATOM
601
N
THR
A
150
17.987
−29.745
57.404
1.00
29.10
A


ATOM
602
CA
THR
A
150
19.304
−30.354
57.331
1.00
29.75
A


ATOM
603
CB
THR
A
150
19.211
−31.901
57.458
1.00
30.54
A


ATOM
604
OG1
THR
A
150
18.268
−32.422
56.508
1.00
30.80
A


ATOM
605
CG2
THR
A
150
18.758
−32.282
58.870
1.00
30.06
A


ATOM
606
C
THR
A
150
19.886
−29.948
55.980
1.00
31.93
A


ATOM
607
O
THR
A
150
20.487
−30.755
55.262
1.00
31.90
A


ATOM
608
N
GLY
A
151
19.681
−28.673
55.651
1.00
31.73
A


ATOM
609
CA
GLY
A
151
20.166
−28.115
54.404
1.00
29.51
A


ATOM
610
C
GLY
A
151
21.633
−27.743
54.463
1.00
30.05
A


ATOM
611
O
GLY
A
151
22.372
−28.209
55.333
1.00
30.34
A


ATOM
612
N
VAL
A
152
22.059
−26.880
53.549
1.00
28.78
A


ATOM
613
CA
VAL
A
152
23.460
−26.488
53.490
1.00
26.43
A


ATOM
614
CB
VAL
A
152
23.722
−25.600
52.256
1.00
25.38
A


ATOM
615
CG1
VAL
A
152
23.041
−24.258
52.421
1.00
23.19
A


ATOM
616
CG2
VAL
A
152
25.222
−25.439
52.039
1.00
22.48
A


ATOM
617
C
VAL
A
152
23.978
−25.779
54.746
1.00
28.64
A


ATOM
618
O
VAL
A
152
25.161
−25.858
55.062
1.00
27.06
A


ATOM
619
N
GLY
A
153
23.095
−25.098
55.470
1.00
27.77
A


ATOM
620
CA
GLY
A
153
23.533
−24.394
56.660
1.00
26.50
A


ATOM
621
C
GLY
A
153
24.267
−23.115
56.293
1.00
29.07
A


ATOM
622
O
GLY
A
153
24.364
−22.763
55.113
1.00
29.40
A


ATOM
623
N
MET
A
154
24.785
−22.419
57.302
1.00
27.36
A


ATOM
624
CA
MET
A
154
25.507
−21.171
57.084
1.00
28.29
A


ATOM
625
CB
MET
A
154
24.598
−19.961
57.358
1.00
27.33
A


ATOM
626
CG
MET
A
154
23.533
−19.684
56.299
1.00
31.21
A


ATOM
627
SD
MET
A
154
22.490
−18.255
56.745
1.00
29.78
A


ATOM
628
CE
MET
A
154
21.274
−19.072
57.723
1.00
26.73
A


ATOM
629
C
MET
A
154
26.728
−21.054
57.980
1.00
26.97
A


ATOM
630
O
MET
A
154
26.691
−21.446
59.142
1.00
27.36
A


ATOM
631
N
THR
A
155
27.804
−20.504
57.433
1.00
27.79
A


ATOM
632
CA
THR
A
155
29.023
−20.293
58.196
1.00
29.31
A


ATOM
633
CB
THR
A
155
30.237
−20.064
57.285
1.00
29.15
A


ATOM
634
OG1
THR
A
155
30.090
−18.806
56.610
1.00
30.37
A


ATOM
635
CG2
THR
A
155
30.358
−21.193
56.254
1.00
27.57
A


ATOM
636
C
THR
A
155
28.809
−19.013
58.991
1.00
30.74
A


ATOM
637
O
THR
A
155
27.861
−18.264
58.737
1.00
30.95
A


ATOM
638
N
ARG
A
156
29.695
−18.752
59.943
1.00
31.93
A


ATOM
639
CA
ARG
A
156
29.582
−17.549
60.747
1.00
31.40
A


ATOM
640
CB
ARG
A
156
30.781
−17.429
61.679
1.00
33.12
A


ATOM
641
CG
ARG
A
156
30.804
−16.141
62.466
1.00
36.80
A


ATOM
642
CD
ARG
A
156
31.986
−16.079
63.416
1.00
37.87
A


ATOM
643
NE
ARG
A
156
32.005
−14.810
64.132
1.00
42.31
A


ATOM
644
CZ
ARG
A
156
32.198
−13.633
63.550
1.00
43.07
A


ATOM
645
NH1
ARG
A
156
32.396
−13.562
62.244
1.00
45.16
A


ATOM
646
NH2
ARG
A
156
32.178
−12.525
64.271
1.00
47.44
A


ATOM
647
C
ARG
A
156
29.491
−16.313
59.854
1.00
30.98
A


ATOM
648
O
ARG
A
156
28.628
−15.446
60.057
1.00
29.50
A


ATOM
649
N
ALA
A
157
30.376
−16.238
58.862
1.00
28.79
A


ATOM
650
CA
ALA
A
157
30.405
−15.094
57.949
1.00
29.13
A


ATOM
651
CB
ALA
A
157
31.579
−15.216
56.977
1.00
28.16
A


ATOM
652
C
ALA
A
157
29.107
−14.925
57.167
1.00
27.42
A


ATOM
653
O
ALA
A
157
28.700
−13.810
56.873
1.00
27.13
A


ATOM
654
N
GLU
A
158
28.467
−16.031
56.821
1.00
28.87
A


ATOM
655
CA
GLU
A
158
27.217
−15.973
56.076
1.00
30.24
A


ATOM
656
CB
GLU
A
158
26.904
−17.343
55.490
1.00
31.06
A


ATOM
657
CG
GLU
A
158
27.982
−17.833
54.535
1.00
36.51
A


ATOM
658
CD
GLU
A
158
27.600
−19.120
53.849
1.00
36.81
A


ATOM
659
OE1
GLU
A
158
27.342
−20.120
54.549
1.00
38.69
A


ATOM
660
OE2
GLU
A
158
27.555
−19.130
52.606
1.00
43.45
A


ATOM
661
C
GLU
A
158
26.061
−15.504
56.960
1.00
31.29
A


ATOM
662
O
GLU
A
158
25.158
−14.804
56.494
1.00
30.64
A


ATOM
663
N
LEU
A
159
26.087
−15.900
58.231
1.00
29.36
A


ATOM
664
CA
LEU
A
159
25.046
−15.494
59.165
1.00
31.34
A


ATOM
665
CB
LEU
A
159
25.297
−16.107
60.549
1.00
28.12
A


ATOM
666
CG
LEU
A
159
24.976
−17.599
60.666
1.00
28.89
A


ATOM
667
CD1
LEU
A
159
25.520
−18.155
61.967
1.00
27.34
A


ATOM
668
CD2
LEU
A
159
23.469
−17.804
60.576
1.00
24.98
A


ATOM
669
C
LEU
A
159
25.067
−13.977
59.249
1.00
31.03
A


ATOM
670
O
LEU
A
159
24.023
−13.321
59.223
1.00
32.32
A


ATOM
671
N
VAL
A
160
26.275
−13.432
59.326
1.00
30.83
A


ATOM
672
CA
VAL
A
160
26.480
−11.996
59.410
1.00
31.27
A


ATOM
673
CB
VAL
A
160
27.953
−11.667
59.732
1.00
30.71
A


ATOM
674
CG1
VAL
A
160
28.198
−10.155
59.581
1.00
29.76
A


ATOM
675
CG2
VAL
A
160
28.288
−12.126
61.145
1.00
28.92
A


ATOM
676
C
VAL
A
160
26.108
−11.271
58.121
1.00
32.45
A


ATOM
677
O
VAL
A
160
25.329
−10.317
58.137
1.00
34.57
A


ATOM
678
N
ALA
A
161
26.667
−11.727
57.007
1.00
30.48
A


ATOM
679
CA
ALA
A
161
26.420
−11.093
55.719
1.00
31.23
A


ATOM
680
CB
ALA
A
161
27.477
−11.555
54.705
1.00
30.35
A


ATOM
681
C
ALA
A
161
25.022
−11.292
55.131
1.00
30.55
A


ATOM
682
O
ALA
A
161
24.348
−10.322
54.785
1.00
29.64
A


ATOM
683
N
ASN
A
162
24.604
−12.548
55.012
1.00
30.70
A


ATOM
684
CA
ASN
A
162
23.310
−12.901
54.429
1.00
31.28
A


ATOM
685
CB
ASN
A
162
23.180
−14.423
54.343
1.00
30.39
A


ATOM
686
CG
ASN
A
162
24.206
−15.047
53.414
1.00
32.65
A


ATOM
687
OD1
ASN
A
162
25.338
−14.580
53.319
1.00
34.15
A


ATOM
688
ND2
ASN
A
162
23.818
−16.120
52.741
1.00
32.64
A


ATOM
689
C
ASN
A
162
22.085
−12.346
55.147
1.00
31.78
A


ATOM
690
O
ASN
A
162
21.085
−12.019
54.504
1.00
31.02
A


ATOM
691
N
LEU
A
163
22.154
−12.256
56.472
1.00
29.42
A


ATOM
692
CA
LEU
A
163
21.025
−11.757
57.250
1.00
30.07
A


ATOM
693
CB
LEU
A
163
20.817
−12.631
58.499
1.00
29.43
A


ATOM
694
CG
LEU
A
163
20.621
−14.146
58.302
1.00
29.68
A


ATOM
695
CD1
LEU
A
163
20.443
−14.820
59.659
1.00
27.61
A


ATOM
696
CD2
LEU
A
163
19.405
−14.409
57.411
1.00
28.58
A


ATOM
697
C
LEU
A
163
21.180
−10.298
57.674
1.00
29.99
A


ATOM
698
O
LEU
A
163
20.192
−9.591
57.845
1.00
27.85
A


ATOM
699
N
GLY
A
164
22.420
−9.844
57.828
1.00
29.36
A


ATOM
700
CA
GLY
A
164
22.650
−8.480
58.277
1.00
29.63
A


ATOM
701
C
GLY
A
164
22.848
−7.419
57.214
1.00
30.34
A


ATOM
702
O
GLY
A
164
22.791
−6.225
57.514
1.00
30.77
A


ATOM
703
N
THR
A
165
23.075
−7.838
55.974
1.00
28.12
A


ATOM
704
CA
THR
A
165
23.288
−6.888
54.893
1.00
29.13
A


ATOM
705
CB
THR
A
165
24.783
−6.771
54.521
1.00
29.76
A


ATOM
706
OG1
THR
A
165
25.119
−7.813
53.595
1.00
26.17
A


ATOM
707
CG2
THR
A
165
25.670
−6.910
55.764
1.00
28.83
A


ATOM
708
C
THR
A
165
22.582
−7.342
53.630
1.00
30.18
A


ATOM
709
O
THR
A
165
22.001
−8.423
53.573
1.00
27.64
A


ATOM
710
N
ILE
A
166
22.655
−6.500
52.611
1.00
34.77
A


ATOM
711
CA
ILE
A
166
22.080
−6.818
51.320
1.00
40.37
A


ATOM
712
CB
ILE
A
166
21.673
−5.543
50.571
1.00
42.39
A


ATOM
713
CG2
ILE
A
166
21.394
−5.855
49.093
1.00
42.55
A


ATOM
714
CG1
ILE
A
166
20.444
−4.935
51.258
1.00
43.15
A


ATOM
715
CD1
ILE
A
166
19.822
−3.800
50.491
1.00
48.78
A


ATOM
716
C
ILE
A
166
23.200
−7.541
50.587
1.00
44.58
A


ATOM
717
O
ILE
A
166
24.079
−6.920
49.986
1.00
45.27
A


ATOM
718
N
ALA
A
167
23.177
−8.865
50.675
1.00
48.19
A


ATOM
719
CA
ALA
A
167
24.197
−9.693
50.057
1.00
52.05
A


ATOM
720
CB
ALA
A
167
24.266
−11.040
50.776
1.00
50.60
A


ATOM
721
C
ALA
A
167
23.977
−9.903
48.563
1.00
54.42
A


ATOM
722
O
ALA
A
167
24.671
−9.311
47.737
1.00
55.92
A


ATOM
723
N
LYS
A
168
23.000
−10.737
48.222
1.00
57.35
A


ATOM
724
CA
LYS
A
168
22.700
−11.053
46.829
1.00
59.01
A


ATOM
725
CB
LYS
A
168
21.674
−12.188
46.782
1.00
61.02
A


ATOM
726
CG
LYS
A
168
22.122
−13.426
47.546
1.00
63.34
A


ATOM
727
CD
LYS
A
168
21.095
−14.549
47.503
1.00
65.33
A


ATOM
728
CE
LYS
A
168
21.582
−15.744
48.320
1.00
66.58
A


ATOM
729
NZ
LYS
A
168
20.634
−16.891
48.295
1.00
67.20
A


ATOM
730
C
LYS
A
168
22.205
−9.865
46.006
1.00
59.60
A


ATOM
731
O
LYS
A
168
21.591
−8.939
46.538
1.00
59.93
A


ATOM
732
N
SER
A
169
22.480
−9.902
44.703
1.00
58.96
A


ATOM
733
CA
SER
A
169
22.064
−8.838
43.788
1.00
58.85
A


ATOM
734
CB
SER
A
169
22.587
−9.110
42.373
1.00
61.11
A


ATOM
735
OG
SER
A
169
24.004
−9.199
42.343
1.00
67.08
A


ATOM
736
C
SER
A
169
20.544
−8.749
43.739
1.00
56.48
A


ATOM
737
O
SER
A
169
19.975
−7.671
43.564
1.00
54.95
A


ATOM
738
N
GLY
A
170
19.894
−9.895
43.890
1.00
54.51
A


ATOM
739
CA
GLY
A
170
18.446
−9.932
43.853
1.00
52.69
A


ATOM
740
C
GLY
A
170
17.779
−9.114
44.942
1.00
51.35
A


ATOM
741
O
GLY
A
170
16.696
−8.566
44.730
1.00
52.46
A


ATOM
742
N
THR
A
171
18.416
−9.025
46.108
1.00
48.81
A


ATOM
743
CA
THR
A
171
17.845
−8.272
47.218
1.00
46.14
A


ATOM
744
CB
THR
A
171
18.731
−8.350
48.471
1.00
44.42
A


ATOM
745
OG1
THR
A
171
18.952
−9.723
48.814
1.00
44.07
A


ATOM
746
CG2
THR
A
171
18.052
−7.648
49.641
1.00
42.55
A


ATOM
747
C
THR
A
171
17.658
−6.810
46.843
1.00
45.48
A


ATOM
748
O
THR
A
171
16.618
−6.214
47.128
1.00
43.21
A


ATOM
749
N
SER
A
172
18.670
−6.241
46.198
1.00
46.26
A


ATOM
750
CA
SER
A
172
18.620
−4.848
45.774
1.00
47.43
A


ATOM
751
CB
SER
A
172
19.960
−4.443
45.154
1.00
49.11
A


ATOM
752
OG
SER
A
172
19.969
−3.067
44.815
1.00
53.00
A


ATOM
753
C
SER
A
172
17.484
−4.640
44.765
1.00
47.57
A


ATOM
754
O
SER
A
172
16.801
−3.614
44.784
1.00
46.65
A


ATOM
755
N
ALA
A
173
17.284
−5.620
43.887
1.00
47.00
A


ATOM
756
CA
ALA
A
173
16.225
−5.538
42.890
1.00
47.42
A


ATOM
757
CB
ALA
A
173
16.335
−6.704
41.909
1.00
46.58
A


ATOM
758
C
ALA
A
173
14.877
−5.574
43.610
1.00
46.92
A


ATOM
759
O
ALA
A
173
13.989
−4.765
43.330
1.00
46.07
A


ATOM
760
N
PHE
A
174
14.732
−6.514
44.542
1.00
46.28
A


ATOM
761
CA
PHE
A
174
13.494
−6.635
45.300
1.00
46.39
A


ATOM
762
CB
PHE
A
174
13.621
−7.703
46.391
1.00
44.34
A


ATOM
763
CG
PHE
A
174
12.585
−7.580
47.473
1.00
40.11
A


ATOM
764
CD1
PHE
A
174
12.841
−6.828
48.615
1.00
37.31
A


ATOM
765
CD2
PHE
A
174
11.333
−8.171
47.326
1.00
39.00
A


ATOM
766
CE1
PHE
A
174
11.864
−6.664
49.596
1.00
39.89
A


ATOM
767
CE2
PHE
A
174
10.345
−8.014
48.300
1.00
37.97
A


ATOM
768
CZ
PHE
A
174
10.608
−7.260
49.437
1.00
39.56
A


ATOM
769
C
PHE
A
174
13.092
−5.307
45.939
1.00
47.35
A


ATOM
770
O
PHE
A
174
11.918
−4.938
45.928
1.00
47.86
A


ATOM
771
N
LEU
A
175
14.062
−4.593
46.500
1.00
47.40
A


ATOM
772
CA
LEU
A
175
13.773
−3.311
47.132
1.00
48.97
A


ATOM
773
CB
LEU
A
175
15.045
−2.712
47.733
1.00
49.01
A


ATOM
774
CG
LEU
A
175
15.582
−3.390
48.994
1.00
50.83
A


ATOM
775
CD1
LEU
A
175
16.863
−2.695
49.445
1.00
50.32
A


ATOM
776
CD2
LEU
A
175
14.528
−3.326
50.094
1.00
52.00
A


ATOM
777
C
LEU
A
175
13.157
−2.321
46.149
1.00
50.06
A


ATOM
778
O
LEU
A
175
12.259
−1.557
46.508
1.00
49.84
A


ATOM
779
N
ASN
A
176
13.644
−2.331
44.912
1.00
50.71
A


ATOM
780
CA
ASN
A
176
13.127
−1.436
43.888
1.00
50.81
A


ATOM
781
CB
ASN
A
176
14.076
−1.391
42.690
1.00
53.31
A


ATOM
782
CG
ASN
A
176
15.414
−0.770
43.031
1.00
55.99
A


ATOM
783
OD1
ASN
A
176
15.482
0.363
43.515
1.00
57.64
A


ATOM
784
ND2
ASN
A
176
16.490
−1.506
42.778
1.00
57.47
A


ATOM
785
C
ASN
A
176
11.749
−1.891
43.426
1.00
50.13
A


ATOM
786
O
ASN
A
176
10.809
−1.097
43.374
1.00
50.94
A


ATOM
787
N
ALA
A
177
11.630
−3.171
43.091
1.00
48.58
A


ATOM
788
CA
ALA
A
177
10.361
−3.713
42.631
1.00
46.44
A


ATOM
789
CB
ALA
A
177
10.533
−5.166
42.211
1.00
45.93
A


ATOM
790
C
ALA
A
177
9.313
−3.600
43.729
1.00
46.54
A


ATOM
791
O
ALA
A
177
8.149
−3.305
43.458
1.00
44.04
A


ATOM
792
N
MET
A
178
9.734
−3.823
44.970
1.00
46.54
A


ATOM
793
CA
MET
A
178
8.825
−3.747
46.108
1.00
47.79
A


ATOM
794
CB
MET
A
178
9.555
−4.116
47.401
1.00
44.99
A


ATOM
795
CG
MET
A
178
8.658
−4.159
48.622
1.00
43.88
A


ATOM
796
SD
MET
A
178
7.324
−5.342
48.411
1.00
41.19
A


ATOM
797
CE
MET
A
178
6.603
−5.337
50.037
1.00
42.69
A


ATOM
798
C
MET
A
178
8.230
−2.353
46.241
1.00
49.94
A


ATOM
799
O
MET
A
178
7.016
−2.196
46.384
1.00
49.65
A


ATOM
800
N
THR
A
179
9.089
−1.341
46.181
1.00
52.61
A


ATOM
801
CA
THR
A
179
8.653
0.046
46.300
1.00
55.88
A


ATOM
802
CB
THR
A
179
9.864
0.998
46.418
1.00
56.76
A


ATOM
803
OG1
THR
A
179
10.748
0.786
45.311
1.00
57.84
A


ATOM
804
CG2
THR
A
179
10.616
0.746
47.720
1.00
56.79
A


ATOM
805
C
THR
A
179
7.789
0.491
45.121
1.00
57.35
A


ATOM
806
O
THR
A
179
6.826
1.237
45.299
1.00
58.41
A


ATOM
807
N
GLU
A
180
8.131
0.038
43.919
1.00
58.61
A


ATOM
808
CA
GLU
A
180
7.370
0.402
42.729
1.00
60.56
A


ATOM
809
CB
GLU
A
180
8.141
0.029
41.461
1.00
61.60
A


ATOM
810
CG
GLU
A
180
9.338
0.920
41.176
1.00
65.95
A


ATOM
811
CD
GLU
A
180
9.931
0.676
39.801
1.00
68.00
A


ATOM
812
OE1
GLU
A
180
10.894
1.383
39.430
1.00
69.00
A


ATOM
813
OE2
GLU
A
180
9.431
−0.223
39.089
1.00
70.25
A


ATOM
814
C
GLU
A
180
5.994
−0.255
42.696
1.00
61.73
A


ATOM
815
O
GLU
A
180
5.068
0.261
42.071
1.00
62.24
A


ATOM
816
N
ALA
A
181
5.863
−1.396
43.362
1.00
61.63
A


ATOM
817
CA
ALA
A
181
4.591
−2.105
43.400
1.00
61.69
A


ATOM
818
CB
ALA
A
181
4.829
−3.603
43.531
1.00
60.74
A


ATOM
819
C
ALA
A
181
3.762
−1.606
44.574
1.00
62.57
A


ATOM
820
O
ALA
A
181
2.583
−1.284
44.425
1.00
61.72
A


ATOM
821
N
GLN
A
182
4.399
−1.538
45.739
1.00
64.18
A


ATOM
822
CA
GLN
A
182
3.749
−1.100
46.970
1.00
65.69
A


ATOM
823
CB
GLN
A
182
4.733
−1.233
48.136
1.00
65.51
A


ATOM
824
CG
GLN
A
182
4.129
−1.023
49.506
1.00
66.97
A


ATOM
825
CD
GLN
A
182
4.866
−1.793
50.585
1.00
67.65
A


ATOM
826
OE1
GLN
A
182
4.906
−3.021
50.564
1.00
68.71
A


ATOM
827
NE2
GLN
A
182
5.453
−1.075
51.536
1.00
69.42
A


ATOM
828
C
GLN
A
182
3.241
0.333
46.852
1.00
66.94
A


ATOM
829
O
GLN
A
182
2.449
0.796
47.675
1.00
66.83
A


ATOM
830
N
GLU
A
183
3.695
1.024
45.813
1.00
68.18
A


ATOM
831
CA
GLU
A
183
3.289
2.400
45.565
1.00
69.69
A


ATOM
832
CB
GLU
A
183
4.479
3.209
45.038
1.00
70.89
A


ATOM
833
CG
GLU
A
183
4.120
4.592
44.522
1.00
73.65
A


ATOM
834
CD
GLU
A
183
5.333
5.370
44.044
1.00
75.88
A


ATOM
835
OE1
GLU
A
183
5.148
6.458
43.453
1.00
76.43
A


ATOM
836
OE2
GLU
A
183
6.470
4.896
44.264
1.00
76.85
A


ATOM
837
C
GLU
A
183
2.147
2.445
44.554
1.00
69.18
A


ATOM
838
O
GLU
A
183
1.225
3.252
44.680
1.00
69.16
A


ATOM
839
N
ASP
A
184
2.212
1.567
43.557
1.00
68.85
A


ATOM
840
CA
ASP
A
184
1.194
1.506
42.514
1.00
68.71
A


ATOM
841
CB
ASP
A
184
1.838
1.106
41.186
1.00
70.49
A


ATOM
842
CG
ASP
A
184
2.748
2.182
40.637
1.00
73.35
A


ATOM
843
OD1
ASP
A
184
2.278
3.327
40.471
1.00
75.84
A


ATOM
844
OD2
ASP
A
184
3.931
1.888
40.367
1.00
75.42
A


ATOM
845
C
ASP
A
184
0.031
0.563
42.830
1.00
67.37
A


ATOM
846
O
ASP
A
184
−0.901
0.432
42.036
1.00
67.70
A


ATOM
847
N
GLY
A
185
0.089
−0.097
43.983
1.00
65.71
A


ATOM
848
CA
GLY
A
185
−0.986
−0.994
44.373
1.00
63.02
A


ATOM
849
C
GLY
A
185
−0.868
−2.456
43.975
1.00
60.09
A


ATOM
850
O
GLY
A
185
−1.689
−3.274
44.396
1.00
60.36
A


ATOM
851
N
GLN
A
186
0.136
−2.793
43.169
1.00
56.39
A


ATOM
852
CA
GLN
A
186
0.335
−4.175
42.730
1.00
53.38
A


ATOM
853
CB
GLN
A
186
1.484
−4.234
41.724
1.00
54.26
A


ATOM
854
CG
GLN
A
186
1.215
−3.431
40.458
1.00
57.47
A


ATOM
855
CD
GLN
A
186
2.435
−3.317
39.566
1.00
58.77
A


ATOM
856
OE1
GLN
A
186
3.010
−4.322
39.147
1.00
60.77
A


ATOM
857
NE2
GLN
A
186
2.838
−2.086
39.272
1.00
58.40
A


ATOM
858
C
GLN
A
186
0.623
−5.093
43.922
1.00
49.70
A


ATOM
859
O
GLN
A
186
1.187
−4.654
44.921
1.00
49.64
A


ATOM
860
N
SER
A
187
0.229
−6.361
43.818
1.00
44.90
A


ATOM
861
CA
SER
A
187
0.443
−7.315
44.908
1.00
41.67
A


ATOM
862
CB
SER
A
187
−0.162
−8.672
44.557
1.00
39.85
A


ATOM
863
OG
SER
A
187
−0.242
−9.489
45.712
1.00
40.74
A


ATOM
864
C
SER
A
187
1.933
−7.468
45.215
1.00
38.72
A


ATOM
865
O
SER
A
187
2.762
−7.488
44.311
1.00
38.04
A


ATOM
866
N
THR
A
188
2.260
−7.587
46.497
1.00
37.99
A


ATOM
867
CA
THR
A
188
3.651
−7.688
46.926
1.00
38.34
A


ATOM
868
CB
THR
A
188
3.994
−6.564
47.917
1.00
37.82
A


ATOM
869
OG1
THR
A
188
3.044
−6.582
48.988
1.00
37.92
A


ATOM
870
CG2
THR
A
188
3.969
−5.209
47.230
1.00
35.82
A


ATOM
871
C
THR
A
188
4.102
−8.996
47.572
1.00
36.79
A


ATOM
872
O
THR
A
188
5.296
−9.275
47.605
1.00
38.88
A


ATOM
873
N
SER
A
189
3.177
−9.790
48.096
1.00
35.49
A


ATOM
874
CA
SER
A
189
3.581
−11.041
48.733
1.00
34.95
A


ATOM
875
CB
SER
A
189
2.362
−11.813
49.269
1.00
35.44
A


ATOM
876
OG
SER
A
189
1.528
−12.301
48.232
1.00
35.99
A


ATOM
877
C
SER
A
189
4.370
−11.910
47.755
1.00
35.51
A


ATOM
878
O
SER
A
189
5.301
−12.615
48.155
1.00
34.15
A


ATOM
879
N
ALA
A
190
4.014
−11.838
46.473
1.00
33.02
A


ATOM
880
CA
ALA
A
190
4.700
−12.624
45.453
1.00
33.42
A


ATOM
881
CB
ALA
A
190
3.964
−12.531
44.120
1.00
35.18
A


ATOM
882
C
ALA
A
190
6.146
−12.176
45.282
1.00
33.93
A


ATOM
883
O
ALA
A
190
7.020
−13.001
45.039
1.00
33.38
A


ATOM
884
N
LEU
A
191
6.393
−10.874
45.407
1.00
33.98
A


ATOM
885
CA
LEU
A
191
7.744
−10.339
45.271
1.00
35.42
A


ATOM
886
CB
LEU
A
191
7.742
−8.809
45.367
1.00
35.78
A


ATOM
887
CG
LEU
A
191
7.137
−8.018
44.208
1.00
38.28
A


ATOM
888
CD1
LEU
A
191
7.349
−6.517
44.448
1.00
36.74
A


ATOM
889
CD2
LEU
A
191
7.798
−8.443
42.906
1.00
37.70
A


ATOM
890
C
LEU
A
191
8.641
−10.902
46.366
1.00
35.52
A


ATOM
891
O
LEU
A
191
9.766
−11.342
46.100
1.00
35.56
A


ATOM
892
N
ILE
A
192
8.133
−10.880
47.595
1.00
34.69
A


ATOM
893
CA
ILE
A
192
8.871
−11.387
48.743
1.00
33.73
A


ATOM
894
CB
ILE
A
192
8.020
−11.254
50.034
1.00
33.89
A


ATOM
895
CG2
ILE
A
192
8.722
−11.924
51.208
1.00
31.29
A


ATOM
896
CG1
ILE
A
192
7.789
−9.767
50.332
1.00
32.32
A


ATOM
897
CD1
ILE
A
192
6.801
−9.494
51.435
1.00
33.13
A


ATOM
898
C
ILE
A
192
9.260
−12.843
48.495
1.00
33.70
A


ATOM
899
O
ILE
A
192
10.419
−13.210
48.639
1.00
32.55
A


ATOM
900
N
GLY
A
193
8.289
−13.666
48.109
1.00
34.34
A


ATOM
901
CA
GLY
A
193
8.585
−15.058
47.827
1.00
33.70
A


ATOM
902
C
GLY
A
193
9.557
−15.196
46.664
1.00
36.77
A


ATOM
903
O
GLY
A
193
10.534
−15.948
46.750
1.00
36.23
A


ATOM
904
N
GLN
A
194
9.301
−14.462
45.580
1.00
36.72
A


ATOM
905
CA
GLN
A
194
10.150
−14.511
44.390
1.00
37.60
A


ATOM
906
CB
GLN
A
194
9.680
−13.498
43.341
1.00
39.57
A


ATOM
907
CG
GLN
A
194
8.397
−13.862
42.616
1.00
43.85
A


ATOM
908
CD
GLN
A
194
8.021
−12.841
41.548
1.00
46.63
A


ATOM
909
OE1
GLN
A
194
6.938
−12.909
40.961
1.00
49.46
A


ATOM
910
NE2
GLN
A
194
8.919
−11.891
41.290
1.00
45.31
A


ATOM
911
C
GLN
A
194
11.619
−14.238
44.684
1.00
37.50
A


ATOM
912
O
GLN
A
194
12.499
−14.964
44.225
1.00
37.05
A


ATOM
913
N
PHE
A
195
11.878
−13.174
45.432
1.00
36.67
A


ATOM
914
CA
PHE
A
195
13.242
−12.801
45.765
1.00
37.67
A


ATOM
915
CB
PHE
A
195
13.341
−11.276
45.816
1.00
38.15
A


ATOM
916
CG
PHE
A
195
13.253
−10.637
44.461
1.00
41.48
A


ATOM
917
CD1
PHE
A
195
14.399
−10.435
43.698
1.00
42.51
A


ATOM
918
CD2
PHE
A
195
12.016
−10.301
43.915
1.00
43.12
A


ATOM
919
CE1
PHE
A
195
14.316
−9.909
42.406
1.00
45.11
A


ATOM
920
CE2
PHE
A
195
11.916
−9.776
42.626
1.00
44.12
A


ATOM
921
CZ
PHE
A
195
13.069
−9.579
41.867
1.00
45.02
A


ATOM
922
C
PHE
A
195
13.754
−13.444
47.055
1.00
35.33
A


ATOM
923
O
PHE
A
195
14.855
−13.154
47.507
1.00
34.49
A


ATOM
924
N
GLY
A
196
12.948
−14.338
47.622
1.00
33.40
A


ATOM
925
CA
GLY
A
196
13.330
−15.035
48.838
1.00
33.29
A


ATOM
926
C
GLY
A
196
13.854
−14.188
49.985
1.00
32.03
A


ATOM
927
O
GLY
A
196
14.838
−14.557
50.626
1.00
30.59
A


ATOM
928
N
VAL
A
197
13.192
−13.067
50.259
1.00
29.74
A


ATOM
929
CA
VAL
A
197
13.604
−12.185
51.348
1.00
28.13
A


ATOM
930
CB
VAL
A
197
13.693
−10.712
50.872
1.00
28.77
A


ATOM
931
CG1
VAL
A
197
14.739
−10.579
49.771
1.00
30.35
A


ATOM
932
CG2
VAL
A
197
12.336
−10.244
50.372
1.00
29.75
A


ATOM
933
C
VAL
A
197
12.604
−12.265
52.501
1.00
27.80
A


ATOM
934
O
VAL
A
197
12.524
−11.358
53.327
1.00
26.49
A


ATOM
935
N
GLY
A
198
11.863
−13.367
52.557
1.00
25.15
A


ATOM
936
CA
GLY
A
198
10.848
−13.546
53.582
1.00
24.56
A


ATOM
937
C
GLY
A
198
11.297
−13.804
55.013
1.00
25.45
A


ATOM
938
O
GLY
A
198
10.482
−13.725
55.931
1.00
25.76
A


ATOM
939
N
PHE
A
199
12.575
−14.110
55.216
1.00
25.68
A


ATOM
940
CA
PHE
A
199
13.088
−14.369
56.561
1.00
23.94
A


ATOM
941
CB
PHE
A
199
14.619
−14.477
56.534
1.00
22.33
A


ATOM
942
CG
PHE
A
199
15.250
−14.453
57.898
1.00
23.33
A


ATOM
943
CD1
PHE
A
199
15.252
−15.590
58.702
1.00
22.87
A


ATOM
944
CD2
PHE
A
199
15.821
−13.277
58.393
1.00
24.46
A


ATOM
945
CE1
PHE
A
199
15.816
−15.560
59.982
1.00
24.30
A


ATOM
946
CE2
PHE
A
199
16.385
−13.240
59.670
1.00
26.24
A


ATOM
947
CZ
PHE
A
199
16.381
−14.390
60.465
1.00
24.40
A


ATOM
948
C
PHE
A
199
12.681
−13.279
57.559
1.00
25.64
A


ATOM
949
O
PHE
A
199
12.303
−13.567
58.697
1.00
27.18
A


ATOM
950
N
TYR
A
200
12.750
−12.026
57.127
1.00
25.95
A


ATOM
951
CA
TYR
A
200
12.410
−10.908
57.994
1.00
25.91
A


ATOM
952
CB
TYR
A
200
12.806
−9.598
57.313
1.00
25.62
A


ATOM
953
CG
TYR
A
200
14.279
−9.534
56.957
1.00
26.90
A


ATOM
954
CD1
TYR
A
200
15.263
−9.518
57.948
1.00
24.43
A


ATOM
955
CE1
TYR
A
200
16.624
−9.494
57.614
1.00
27.21
A


ATOM
956
CD2
TYR
A
200
14.689
−9.524
55.627
1.00
27.49
A


ATOM
957
CE2
TYR
A
200
16.034
−9.500
55.287
1.00
27.42
A


ATOM
958
CZ
TYR
A
200
16.997
−9.485
56.278
1.00
28.42
A


ATOM
959
OH
TYR
A
200
18.326
−9.462
55.916
1.00
28.80
A


ATOM
960
C
TYR
A
200
10.938
−10.845
58.432
1.00
26.48
A


ATOM
961
O
TYR
A
200
10.624
−10.227
59.455
1.00
24.30
A


ATOM
962
N
SER
A
201
10.039
−11.488
57.687
1.00
24.40
A


ATOM
963
CA
SER
A
201
8.624
−11.449
58.065
1.00
25.63
A


ATOM
964
CB
SER
A
201
7.736
−12.040
56.961
1.00
23.58
A


ATOM
965
OG
SER
A
201
7.904
−13.439
56.842
1.00
24.58
A


ATOM
966
C
SER
A
201
8.395
−12.193
59.379
1.00
25.70
A


ATOM
967
O
SER
A
201
7.312
−12.129
59.963
1.00
26.74
A


ATOM
968
N
ALA
A
202
9.423
−12.898
59.841
1.00
25.08
A


ATOM
969
CA
ALA
A
202
9.332
−13.625
61.094
1.00
24.60
A


ATOM
970
CB
ALA
A
202
10.618
−14.416
61.347
1.00
25.15
A


ATOM
971
C
ALA
A
202
9.089
−12.640
62.236
1.00
24.58
A


ATOM
972
O
ALA
A
202
8.549
−13.009
63.277
1.00
25.71
A


ATOM
973
N
PHE
A
203
9.485
−11.386
62.040
1.00
25.13
A


ATOM
974
CA
PHE
A
203
9.298
−10.372
63.077
1.00
26.24
A


ATOM
975
CB
PHE
A
203
10.179
−9.153
62.794
1.00
27.79
A


ATOM
976
CG
PHE
A
203
11.633
−9.376
63.141
1.00
29.56
A


ATOM
977
CD1
PHE
A
203
12.014
−9.630
64.460
1.00
27.71
A


ATOM
978
CD2
PHE
A
203
12.609
−9.375
62.154
1.00
27.89
A


ATOM
979
CE1
PHE
A
203
13.348
−9.884
64.789
1.00
27.78
A


ATOM
980
CE2
PHE
A
203
13.947
−9.626
62.471
1.00
31.39
A


ATOM
981
CZ
PHE
A
203
14.317
−9.882
63.793
1.00
29.74
A


ATOM
982
C
PHE
A
203
7.842
−9.963
63.270
1.00
27.04
A


ATOM
983
O
PHE
A
203
7.502
−9.297
64.248
1.00
26.24
A


ATOM
984
N
LEU
A
204
6.975
−10.380
62.350
1.00
27.91
A


ATOM
985
CA
LEU
A
204
5.551
−10.078
62.479
1.00
26.99
A


ATOM
986
CB
LEU
A
204
4.797
−10.467
61.201
1.00
28.39
A


ATOM
987
CG
LEU
A
204
4.955
−9.565
59.972
1.00
28.86
A


ATOM
988
CD1
LEU
A
204
4.344
−10.225
58.766
1.00
30.50
A


ATOM
989
CD2
LEU
A
204
4.289
−8.219
60.229
1.00
30.89
A


ATOM
990
C
LEU
A
204
4.983
−10.864
63.662
1.00
27.43
A


ATOM
991
O
LEU
A
204
4.069
−10.402
64.341
1.00
27.90
A


ATOM
992
N
VAL
A
205
5.530
−12.055
63.908
1.00
26.39
A


ATOM
993
CA
VAL
A
205
5.052
−12.889
65.005
1.00
26.90
A


ATOM
994
CB
VAL
A
205
4.616
−14.283
64.497
1.00
28.55
A


ATOM
995
CG1
VAL
A
205
3.486
−14.143
63.483
1.00
25.90
A


ATOM
996
CG2
VAL
A
205
5.814
−15.014
63.884
1.00
27.28
A


ATOM
997
C
VAL
A
205
6.059
−13.115
66.135
1.00
27.16
A


ATOM
998
O
VAL
A
205
5.712
−13.667
67.175
1.00
26.02
A


ATOM
999
N
ALA
A
206
7.301
−12.689
65.936
1.00
26.54
A


ATOM
1000
CA
ALA
A
206
8.327
−12.913
66.942
1.00
28.79
A


ATOM
1001
CB
ALA
A
206
9.396
−13.874
66.384
1.00
27.34
A


ATOM
1002
C
ALA
A
206
9.003
−11.663
67.483
1.00
28.08
A


ATOM
1003
O
ALA
A
206
9.336
−10.744
66.732
1.00
30.28
A


ATOM
1004
N
ASP
A
207
9.202
−11.645
68.798
1.00
27.34
A


ATOM
1005
CA
ASP
A
207
9.888
−10.543
69.459
1.00
27.28
A


ATOM
1006
CB
ASP
A
207
9.498
−10.497
70.935
1.00
29.37
A


ATOM
1007
CG
ASP
A
207
8.166
−9.819
71.154
1.00
29.88
A


ATOM
1008
OD1
ASP
A
207
7.426
−10.251
72.060
1.00
33.62
A


ATOM
1009
OD2
ASP
A
207
7.872
−8.851
70.415
1.00
28.84
A


ATOM
1010
C
ASP
A
207
11.391
−10.781
69.325
1.00
27.50
A


ATOM
1011
O
ASP
A
207
12.191
−9.848
69.383
1.00
28.52
A


ATOM
1012
N
LYS
A
208
11.759
−12.045
69.130
1.00
24.98
A


ATOM
1013
CA
LYS
A
208
13.158
−12.434
68.976
1.00
29.19
A


ATOM
1014
CB
LYS
A
208
13.733
−12.875
70.326
1.00
30.51
A


ATOM
1015
CG
LYS
A
208
15.046
−13.627
70.216
1.00
34.73
A


ATOM
1016
CD
LYS
A
208
16.243
−12.747
70.521
1.00
37.07
A


ATOM
1017
CE
LYS
A
208
16.357
−12.473
72.009
1.00
35.69
A


ATOM
1018
NZ
LYS
A
208
17.669
−11.850
72.336
1.00
38.56
A


ATOM
1019
C
LYS
A
208
13.312
−13.574
67.970
1.00
27.58
A


ATOM
1020
O
LYS
A
208
12.540
−14.526
67.979
1.00
26.32
A


ATOM
1021
N
VAL
A
209
14.312
−13.465
67.104
1.00
25.41
A


ATOM
1022
CA
VAL
A
209
14.575
−14.501
66.114
1.00
25.21
A


ATOM
1023
CB
VAL
A
209
14.447
−13.963
64.665
1.00
24.58
A


ATOM
1024
CG1
VAL
A
209
14.799
−15.070
63.660
1.00
24.08
A


ATOM
1025
CG2
VAL
A
209
13.016
−13.471
64.419
1.00
24.90
A


ATOM
1026
C
VAL
A
209
15.988
−15.024
66.339
1.00
27.03
A


ATOM
1027
O
VAL
A
209
16.948
−14.256
66.400
1.00
25.92
A


ATOM
1028
N
ILE
A
210
16.101
−16.337
66.480
1.00
24.68
A


ATOM
1029
CA
ILE
A
210
17.381
−16.966
66.694
1.00
26.11
A


ATOM
1030
CB
ILE
A
210
17.398
−17.700
68.048
1.00
27.89
A


ATOM
1031
CG2
ILE
A
210
18.797
−18.221
68.337
1.00
26.45
A


ATOM
1032
CG1
ILE
A
210
16.940
−16.740
69.155
1.00
28.54
A


ATOM
1033
CD1
ILE
A
210
16.938
−17.347
70.523
1.00
32.12
A


ATOM
1034
C
ILE
A
210
17.653
−17.960
65.563
1.00
26.52
A


ATOM
1035
O
ILE
A
210
16.774
−18.736
65.167
1.00
24.24
A


ATOM
1036
N
VAL
A
211
18.872
−17.925
65.037
1.00
25.00
A


ATOM
1037
CA
VAL
A
211
19.247
−18.825
63.961
1.00
25.48
A


ATOM
1038
CB
VAL
A
211
19.536
−18.051
62.641
1.00
26.62
A


ATOM
1039
CG1
VAL
A
211
19.905
−19.030
61.539
1.00
24.54
A


ATOM
1040
CG2
VAL
A
211
18.309
−17.223
62.225
1.00
25.83
A


ATOM
1041
C
VAL
A
211
20.488
−19.626
64.350
1.00
25.39
A


ATOM
1042
O
VAL
A
211
21.578
−19.075
64.492
1.00
25.48
A


ATOM
1043
N
THR
A
212
20.312
−20.926
64.551
1.00
25.49
A


ATOM
1044
CA
THR
A
212
21.444
−21.777
64.883
1.00
25.56
A


ATOM
1045
CB
THR
A
212
21.089
−22.788
65.983
1.00
28.29
A


ATOM
1046
OG1
THR
A
212
20.580
−22.082
67.123
1.00
26.38
A


ATOM
1047
CG2
THR
A
212
22.337
−23.577
66.401
1.00
26.88
A


ATOM
1048
C
THR
A
212
21.778
−22.487
63.579
1.00
25.09
A


ATOM
1049
O
THR
A
212
20.896
−23.037
62.921
1.00
23.68
A


ATOM
1050
N
SER
A
213
23.045
−22.470
63.187
1.00
25.58
A


ATOM
1051
CA
SER
A
213
23.394
−23.079
61.916
1.00
27.51
A


ATOM
1052
CB
SER
A
213
23.306
−22.005
60.819
1.00
24.34
A


ATOM
1053
OG
SER
A
213
23.316
−22.565
59.521
1.00
25.66
A


ATOM
1054
C
SER
A
213
24.766
−23.736
61.892
1.00
29.07
A


ATOM
1055
O
SER
A
213
25.724
−23.223
62.473
1.00
30.35
A


ATOM
1056
N
LYS
A
214
24.838
−24.872
61.202
1.00
29.96
A


ATOM
1057
CA
LYS
A
214
26.072
−25.637
61.044
1.00
29.56
A


ATOM
1058
CB
LYS
A
214
26.002
−26.959
61.817
1.00
30.30
A


ATOM
1059
CG
LYS
A
214
27.229
−27.867
61.623
1.00
31.55
A


ATOM
1060
CD
LYS
A
214
28.509
−27.189
62.101
1.00
31.08
A


ATOM
1061
CE
LYS
A
214
29.749
−28.087
61.948
1.00
32.51
A


ATOM
1062
NZ
LYS
A
214
30.169
−28.275
60.520
1.00
31.25
A


ATOM
1063
C
LYS
A
214
26.281
−25.929
59.561
1.00
28.85
A


ATOM
1064
O
LYS
A
214
25.490
−26.632
58.933
1.00
28.51
A


ATOM
1065
N
HIS
A
215
27.345
−25.356
59.014
1.00
31.48
A


ATOM
1066
CA
HIS
A
215
27.719
−25.525
57.614
1.00
32.33
A


ATOM
1067
CB
HIS
A
215
27.997
−24.150
57.005
1.00
32.68
A


ATOM
1068
CG
HIS
A
215
28.395
−24.185
55.564
1.00
35.07
A


ATOM
1069
CD2
HIS
A
215
29.575
−24.486
54.971
1.00
35.69
A


ATOM
1070
ND1
HIS
A
215
27.526
−23.855
54.546
1.00
37.05
A


ATOM
1071
CE1
HIS
A
215
28.154
−23.949
53.386
1.00
36.12
A


ATOM
1072
NE2
HIS
A
215
29.398
−24.330
53.616
1.00
38.37
A


ATOM
1073
C
HIS
A
215
28.996
−26.377
57.606
1.00
33.45
A


ATOM
1074
O
HIS
A
215
29.836
−26.238
58.496
1.00
34.03
A


ATOM
1075
N
ASN
A
216
29.140
−27.249
56.609
1.00
34.16
A


ATOM
1076
CA
ASN
A
216
30.313
−28.117
56.505
1.00
34.54
A


ATOM
1077
CB
ASN
A
216
30.309
−28.901
55.180
1.00
33.51
A


ATOM
1078
CG
ASN
A
216
29.247
−29.968
55.137
1.00
31.18
A


ATOM
1079
OD1
ASN
A
216
28.825
−30.469
56.170
1.00
34.13
A


ATOM
1080
ND2
ASN
A
216
28.816
−30.336
53.932
1.00
32.44
A


ATOM
1081
C
ASN
A
216
31.656
−27.408
56.617
1.00
34.35
A


ATOM
1082
O
ASN
A
216
32.633
−28.006
57.054
1.00
35.40
A


ATOM
1083
N
ASN
A
217
31.713
−26.142
56.230
1.00
34.75
A


ATOM
1084
CA
ASN
A
217
32.975
−25.411
56.264
1.00
34.72
A


ATOM
1085
CB
ASN
A
217
33.090
−24.520
55.026
1.00
38.42
A


ATOM
1086
CG
ASN
A
217
33.241
−25.319
53.738
1.00
41.13
A


ATOM
1087
OD1
ASN
A
217
33.142
−24.770
52.636
1.00
43.67
A


ATOM
1088
ND2
ASN
A
217
33.489
−26.621
53.871
1.00
40.39
A


ATOM
1089
C
ASN
A
217
33.200
−24.569
57.506
1.00
35.80
A


ATOM
1090
O
ASN
A
217
34.084
−23.714
57.523
1.00
36.93
A


ATOM
1091
N
ASP
A
218
32.415
−24.802
58.551
1.00
35.43
A


ATOM
1092
CA
ASP
A
218
32.575
−24.024
59.771
1.00
35.16
A


ATOM
1093
CB
ASP
A
218
31.906
−22.654
59.606
1.00
36.23
A


ATOM
1094
CG
ASP
A
218
32.519
−21.595
60.497
1.00
37.78
A


ATOM
1095
OD1
ASP
A
218
33.285
−21.958
61.412
1.00
38.97
A


ATOM
1096
OD2
ASP
A
218
32.235
−20.398
60.286
1.00
37.63
A


ATOM
1097
C
ASP
A
218
31.963
−24.749
60.958
1.00
34.91
A


ATOM
1098
O
ASP
A
218
31.336
−25.794
60.802
1.00
36.71
A


ATOM
1099
N
THR
A
219
32.158
−24.198
62.149
1.00
34.34
A


ATOM
1100
CA
THR
A
219
31.590
−24.791
63.350
1.00
35.03
A


ATOM
1101
CB
THR
A
219
32.438
−24.459
64.588
1.00
36.33
A


ATOM
1102
OG1
THR
A
219
32.620
−23.041
64.671
1.00
37.48
A


ATOM
1103
CG2
THR
A
219
33.809
−25.140
64.493
1.00
36.36
A


ATOM
1104
C
THR
A
219
30.190
−24.207
63.517
1.00
34.77
A


ATOM
1105
O
THR
A
219
29.787
−23.325
62.753
1.00
34.10
A


ATOM
1106
N
GLN
A
220
29.455
−24.689
64.513
1.00
33.81
A


ATOM
1107
CA
GLN
A
220
28.095
−24.220
64.749
1.00
33.03
A


ATOM
1108
CB
GLN
A
220
27.346
−25.215
65.630
1.00
34.22
A


ATOM
1109
CG
GLN
A
220
25.861
−24.923
65.771
1.00
32.44
A


ATOM
1110
CD
GLN
A
220
25.081
−26.155
66.160
1.00
33.58
A


ATOM
1111
OE1
GLN
A
220
25.181
−27.195
65.505
1.00
35.72
A


ATOM
1112
NE2
GLN
A
220
24.291
−26.050
67.222
1.00
32.58
A


ATOM
1113
C
GLN
A
220
28.042
−22.835
65.378
1.00
33.09
A


ATOM
1114
O
GLN
A
220
28.769
−22.539
66.326
1.00
33.09
A


ATOM
1115
N
HIS
A
221
27.169
−21.989
64.843
1.00
32.12
A


ATOM
1116
CA
HIS
A
221
27.025
−20.632
65.350
1.00
31.17
A


ATOM
1117
CB
HIS
A
221
27.652
−19.636
64.368
1.00
31.00
A


ATOM
1118
CG
HIS
A
221
29.145
−19.735
64.281
1.00
34.24
A


ATOM
1119
CD2
HIS
A
221
29.943
−20.545
63.548
1.00
32.61
A


ATOM
1120
ND1
HIS
A
221
29.990
−18.953
65.041
1.00
36.07
A


ATOM
1121
CE1
HIS
A
221
31.244
−19.277
64.778
1.00
34.47
A


ATOM
1122
NE2
HIS
A
221
31.243
−20.240
63.876
1.00
35.07
A


ATOM
1123
C
HIS
A
221
25.572
−20.263
65.608
1.00
30.98
A


ATOM
1124
O
HIS
A
221
24.650
−20.929
65.129
1.00
27.49
A


ATOM
1125
N
ILE
A
222
25.385
−19.202
66.386
1.00
28.25
A


ATOM
1126
CA
ILE
A
222
24.063
−18.713
66.729
1.00
28.60
A


ATOM
1127
CB
ILE
A
222
23.772
−18.904
68.227
1.00
28.80
A


ATOM
1128
CG2
ILE
A
222
22.354
−18.429
68.543
1.00
27.64
A


ATOM
1129
CG1
ILE
A
222
23.946
−20.377
68.601
1.00
27.19
A


ATOM
1130
CD1
ILE
A
222
23.890
−20.640
70.104
1.00
28.34
A


ATOM
1131
C
ILE
A
222
23.939
−17.229
66.405
1.00
27.81
A


ATOM
1132
O
ILE
A
222
24.779
−16.424
66.801
1.00
27.24
A


ATOM
1133
N
TRP
A
223
22.893
−16.892
65.656
1.00
28.88
A


ATOM
1134
CA
TRP
A
223
22.590
−15.516
65.269
1.00
26.45
A


ATOM
1135
CB
TRP
A
223
22.313
−15.456
63.761
1.00
26.37
A


ATOM
1136
CG
TRP
A
223
21.935
−14.104
63.195
1.00
24.42
A


ATOM
1137
CD2
TRP
A
223
20.611
−13.551
63.091
1.00
23.57
A


ATOM
1138
CE2
TRP
A
223
20.722
−12.323
62.396
1.00
24.40
A


ATOM
1139
CE3
TRP
A
223
19.342
−13.976
63.512
1.00
24.61
A


ATOM
1140
CD1
TRP
A
223
22.768
−13.209
62.590
1.00
22.60
A


ATOM
1141
NE1
TRP
A
223
22.048
−12.139
62.103
1.00
24.95
A


ATOM
1142
CZ2
TRP
A
223
19.611
−11.512
62.112
1.00
22.27
A


ATOM
1143
CZ3
TRP
A
223
18.233
−13.168
63.226
1.00
23.64
A


ATOM
1144
CH2
TRP
A
223
18.380
−11.952
62.532
1.00
21.24
A


ATOM
1145
C
TRP
A
223
21.320
−15.211
66.055
1.00
26.45
A


ATOM
1146
O
TRP
A
223
20.431
−16.054
66.145
1.00
25.73
A


ATOM
1147
N
GLU
A
224
21.237
−14.028
66.645
1.00
27.69
A


ATOM
1148
CA
GLU
A
224
20.047
−13.670
67.410
1.00
28.57
A


ATOM
1149
CB
GLU
A
224
20.236
−14.049
68.882
1.00
28.25
A


ATOM
1150
CG
GLU
A
224
21.394
−13.352
69.561
1.00
32.38
A


ATOM
1151
CD
GLU
A
224
20.996
−12.040
70.203
1.00
35.31
A


ATOM
1152
OE1
GLU
A
224
21.908
−11.266
70.560
1.00
37.89
A


ATOM
1153
OE2
GLU
A
224
19.779
−11.787
70.362
1.00
34.10
A


ATOM
1154
C
GLU
A
224
19.755
−12.181
67.254
1.00
26.84
A


ATOM
1155
O
GLU
A
224
20.670
−11.365
67.201
1.00
26.00
A


ATOM
1156
N
SER
A
225
18.476
−11.832
67.179
1.00
26.97
A


ATOM
1157
CA
SER
A
225
18.089
−10.436
66.985
1.00
27.37
A


ATOM
1158
CB
SER
A
225
18.284
−10.084
65.504
1.00
27.31
A


ATOM
1159
OG
SER
A
225
17.842
−8.778
65.204
1.00
27.10
A


ATOM
1160
C
SER
A
225
16.643
−10.136
67.383
1.00
27.99
A


ATOM
1161
O
SER
A
225
15.772
−10.997
67.291
1.00
27.02
A


ATOM
1162
N
ASP
A
226
16.401
−8.908
67.830
1.00
29.13
A


ATOM
1163
CA
ASP
A
226
15.054
−8.466
68.185
1.00
28.16
A


ATOM
1164
CB
ASP
A
226
15.027
−7.811
69.568
1.00
28.71
A


ATOM
1165
CG
ASP
A
226
15.886
−6.565
69.641
1.00
29.58
A


ATOM
1166
OD1
ASP
A
226
15.726
−5.803
70.610
1.00
33.74
A


ATOM
1167
OD2
ASP
A
226
16.727
−6.346
68.742
1.00
30.11
A


ATOM
1168
C
ASP
A
226
14.692
−7.434
67.131
1.00
28.91
A


ATOM
1169
O
ASP
A
226
13.724
−6.689
67.276
1.00
29.07
A


ATOM
1170
N
SER
A
227
15.515
−7.406
66.082
1.00
26.17
A


ATOM
1171
CA
SER
A
227
15.399
−6.510
64.930
1.00
27.87
A


ATOM
1172
CB
SER
A
227
13.933
−6.314
64.516
1.00
26.81
A


ATOM
1173
OG
SER
A
227
13.322
−5.256
65.234
1.00
30.30
A


ATOM
1174
C
SER
A
227
16.059
−5.137
65.132
1.00
25.84
A


ATOM
1175
O
SER
A
227
16.197
−4.371
64.182
1.00
25.78
A


ATOM
1176
N
ASN
A
228
16.474
−4.834
66.357
1.00
25.68
A


ATOM
1177
CA
ASN
A
228
17.106
−3.544
66.654
1.00
26.22
A


ATOM
1178
CB
ASN
A
228
16.692
−3.079
68.048
1.00
26.07
A


ATOM
1179
CG
ASN
A
228
15.209
−2.802
68.142
1.00
30.49
A


ATOM
1180
OD1
ASN
A
228
14.535
−3.270
69.061
1.00
34.03
A


ATOM
1181
ND2
ASN
A
228
14.692
−2.034
67.191
1.00
22.67
A


ATOM
1182
C
ASN
A
228
18.628
−3.615
66.567
1.00
26.12
A


ATOM
1183
O
ASN
A
228
19.322
−2.605
66.713
1.00
25.83
A


ATOM
1184
N
ALA
A
229
19.119
−4.827
66.333
1.00
24.72
A


ATOM
1185
CA
ALA
A
229
20.538
−5.140
66.203
1.00
25.83
A


ATOM
1186
CB
ALA
A
229
21.324
−4.626
67.425
1.00
24.69
A


ATOM
1187
C
ALA
A
229
20.568
−6.661
66.173
1.00
26.02
A


ATOM
1188
O
ALA
A
229
19.521
−7.300
66.287
1.00
24.68
A


ATOM
1189
N
PHE
A
230
21.753
−7.240
66.013
1.00
25.02
A


ATOM
1190
CA
PHE
A
230
21.883
−8.689
66.023
1.00
28.61
A


ATOM
1191
CB
PHE
A
230
21.562
−9.285
64.639
1.00
27.93
A


ATOM
1192
CG
PHE
A
230
22.631
−9.067
63.595
1.00
26.10
A


ATOM
1193
CD1
PHE
A
230
23.700
−9.953
63.478
1.00
25.19
A


ATOM
1194
CD2
PHE
A
230
22.537
−8.007
62.693
1.00
24.79
A


ATOM
1195
CE1
PHE
A
230
24.653
−9.793
62.478
1.00
23.58
A


ATOM
1196
CE2
PHE
A
230
23.488
−7.836
61.688
1.00
24.57
A


ATOM
1197
CZ
PHE
A
230
24.550
−8.736
61.579
1.00
24.47
A


ATOM
1198
C
PHE
A
230
23.287
−9.061
66.459
1.00
28.38
A


ATOM
1199
O
PHE
A
230
24.203
−8.254
66.361
1.00
30.46
A


ATOM
1200
N
SER
A
231
23.451
−10.272
66.974
1.00
30.93
A


ATOM
1201
CA
SER
A
231
24.768
−10.725
67.396
1.00
31.70
A


ATOM
1202
CB
SER
A
231
24.914
−10.627
68.916
1.00
30.54
A


ATOM
1203
OG
SER
A
231
24.132
−11.611
69.558
1.00
34.51
A


ATOM
1204
C
SER
A
231
24.996
−12.161
66.949
1.00
30.94
A


ATOM
1205
O
SER
A
231
24.051
−12.893
66.645
1.00
28.43
A


ATOM
1206
N
VAL
A
232
26.262
−12.553
66.897
1.00
31.28
A


ATOM
1207
CA
VAL
A
232
26.628
−13.902
66.500
1.00
34.01
A


ATOM
1208
CB
VAL
A
232
27.173
−13.944
65.053
1.00
34.38
A


ATOM
1209
CG1
VAL
A
232
28.317
−12.965
64.899
1.00
38.67
A


ATOM
1210
CG2
VAL
A
232
27.648
−15.350
64.718
1.00
35.03
A


ATOM
1211
C
VAL
A
232
27.696
−14.438
67.436
1.00
33.64
A


ATOM
1212
O
VAL
A
232
28.686
−13.768
67.722
1.00
34.60
A


ATOM
1213
N
ILE
A
233
27.482
−15.651
67.919
1.00
34.01
A


ATOM
1214
CA
ILE
A
233
28.429
−16.282
68.816
1.00
32.94
A


ATOM
1215
CB
ILE
A
233
27.894
−16.340
70.256
1.00
34.86
A


ATOM
1216
CG2
ILE
A
233
27.550
−14.943
70.750
1.00
36.09
A


ATOM
1217
CG1
ILE
A
233
26.662
−17.251
70.307
1.00
33.82
A


ATOM
1218
CD1
ILE
A
233
26.219
−17.606
71.713
1.00
35.91
A


ATOM
1219
C
ILE
A
233
28.638
−17.717
68.367
1.00
32.97
A


ATOM
1220
O
ILE
A
233
27.757
−18.310
67.747
1.00
30.39
A


ATOM
1221
N
ALA
A
234
29.806
−18.270
68.670
1.00
32.69
A


ATOM
1222
CA
ALA
A
234
30.061
−19.660
68.338
1.00
33.35
A


ATOM
1223
CB
ALA
A
234
31.508
−20.023
68.649
1.00
31.89
A


ATOM
1224
C
ALA
A
234
29.115
−20.373
69.297
1.00
32.93
A


ATOM
1225
O
ALA
A
234
28.931
−19.920
70.426
1.00
33.49
A


ATOM
1226
N
ASP
A
235
28.495
−21.458
68.852
1.00
33.92
A


ATOM
1227
CA
ASP
A
235
27.565
−22.195
69.703
1.00
35.07
A


ATOM
1228
CB
ASP
A
235
26.740
−23.172
68.862
1.00
32.85
A


ATOM
1229
CG
ASP
A
235
25.509
−23.673
69.589
1.00
34.03
A


ATOM
1230
OD1
ASP
A
235
25.567
−23.815
70.824
1.00
35.68
A


ATOM
1231
OD2
ASP
A
235
24.483
−23.938
68.926
1.00
34.84
A


ATOM
1232
C
ASP
A
235
28.345
−22.966
70.772
1.00
37.82
A


ATOM
1233
O
ASP
A
235
29.114
−23.878
70.457
1.00
36.64
A


ATOM
1234
N
PRO
A
236
28.154
−22.612
72.056
1.00
40.23
A


ATOM
1235
CD
PRO
A
236
27.189
−21.645
72.613
1.00
40.64
A


ATOM
1236
CA
PRO
A
236
28.872
−23.310
73.129
1.00
40.88
A


ATOM
1237
CB
PRO
A
236
28.469
−22.524
74.378
1.00
41.33
A


ATOM
1238
CG
PRO
A
236
27.065
−22.102
74.053
1.00
41.51
A


ATOM
1239
C
PRO
A
236
28.500
−24.789
73.223
1.00
41.29
A


ATOM
1240
O
PRO
A
236
29.231
−25.581
73.816
1.00
41.99
A


ATOM
1241
N
ARG
A
237
27.368
−25.157
72.628
1.00
40.37
A


ATOM
1242
CA
ARG
A
237
26.900
−26.542
72.653
1.00
39.76
A


ATOM
1243
CB
ARG
A
237
25.396
−26.597
72.373
1.00
39.62
A


ATOM
1244
CG
ARG
A
237
24.532
−25.892
73.393
1.00
38.78
A


ATOM
1245
CD
ARG
A
237
23.089
−25.803
72.912
1.00
39.41
A


ATOM
1246
NE
ARG
A
237
22.978
−24.977
71.713
1.00
35.90
A


ATOM
1247
CZ
ARG
A
237
21.829
−24.689
71.107
1.00
39.52
A


ATOM
1248
NH1
ARG
A
237
20.679
−25.162
71.589
1.00
34.04
A


ATOM
1249
NH2
ARG
A
237
21.827
−23.927
70.018
1.00
37.40
A


ATOM
1250
C
ARG
A
237
27.617
−27.417
71.624
1.00
41.21
A


ATOM
1251
O
ARG
A
237
27.326
−28.610
71.506
1.00
40.45
A


ATOM
1252
N
GLY
A
238
28.545
−26.821
70.881
1.00
41.59
A


ATOM
1253
CA
GLY
A
238
29.261
−27.563
69.860
1.00
42.55
A


ATOM
1254
C
GLY
A
238
28.378
−27.872
68.660
1.00
43.55
A


ATOM
1255
O
GLY
A
238
27.328
−27.250
68.463
1.00
41.70
A


ATOM
1256
N
ASN
A
239
28.801
−28.837
67.852
1.00
44.11
A


ATOM
1257
CA
ASN
A
239
28.041
−29.231
66.672
1.00
44.67
A


ATOM
1258
CB
ASN
A
239
28.978
−29.857
65.632
1.00
46.49
A


ATOM
1259
CG
ASN
A
239
28.238
−30.685
64.597
1.00
48.61
A


ATOM
1260
OD1
ASN
A
239
27.163
−30.304
64.128
1.00
50.81
A


ATOM
1261
ND2
ASN
A
239
28.817
−31.820
64.225
1.00
50.33
A


ATOM
1262
C
ASN
A
239
26.928
−30.211
67.026
1.00
43.82
A


ATOM
1263
O
ASN
A
239
27.159
−31.415
67.095
1.00
46.79
A


ATOM
1264
N
THR
A
240
25.720
−29.695
67.244
1.00
40.25
A


ATOM
1265
CA
THR
A
240
24.584
−30.546
67.588
1.00
37.66
A


ATOM
1266
CB
THR
A
240
23.759
−29.958
68.756
1.00
38.88
A


ATOM
1267
OG1
THR
A
240
23.201
−28.699
68.357
1.00
37.45
A


ATOM
1268
CG2
THR
A
240
24.632
−29.763
69.990
1.00
35.95
A


ATOM
1269
C
THR
A
240
23.635
−30.751
66.416
1.00
36.12
A


ATOM
1270
O
THR
A
240
22.773
−31.630
66.455
1.00
35.77
A


ATOM
1271
N
LEU
A
241
23.777
−29.933
65.380
1.00
35.46
A


ATOM
1272
CA
LEU
A
241
22.915
−30.057
64.211
1.00
34.67
A


ATOM
1273
CB
LEU
A
241
22.696
−28.680
63.560
1.00
32.39
A


ATOM
1274
CG
LEU
A
241
21.837
−27.691
64.357
1.00
32.41
A


ATOM
1275
CD1
LEU
A
241
21.794
−26.340
63.664
1.00
30.77
A


ATOM
1276
CD2
LEU
A
241
20.443
−28.254
64.513
1.00
31.49
A


ATOM
1277
C
LEU
A
241
23.467
−31.037
63.174
1.00
32.75
A


ATOM
1278
O
LEU
A
241
22.707
−31.628
62.414
1.00
32.61
A


ATOM
1279
N
GLY
A
242
24.781
−31.225
63.160
1.00
32.82
A


ATOM
1280
CA
GLY
A
242
25.384
−32.113
62.176
1.00
33.49
A


ATOM
1281
C
GLY
A
242
25.607
−31.244
60.954
1.00
32.75
A


ATOM
1282
O
GLY
A
242
26.743
−30.972
60.560
1.00
33.31
A


ATOM
1283
N
ARG
A
243
24.494
−30.800
60.374
1.00
32.49
A


ATOM
1284
CA
ARG
A
243
24.473
−29.903
59.226
1.00
31.35
A


ATOM
1285
CB
ARG
A
243
24.797
−30.621
57.912
1.00
31.73
A


ATOM
1286
CG
ARG
A
243
24.934
−29.641
56.738
1.00
32.57
A


ATOM
1287
CD
ARG
A
243
24.839
−30.310
55.370
1.00
30.58
A


ATOM
1288
NE
ARG
A
243
23.582
−31.031
55.212
1.00
30.37
A


ATOM
1289
CZ
ARG
A
243
23.442
−32.338
55.409
1.00
28.39
A


ATOM
1290
NH1
ARG
A
243
24.489
−33.077
55.763
1.00
29.52
A


ATOM
1291
NH2
ARG
A
243
22.254
−32.900
55.262
1.00
27.01
A


ATOM
1292
C
ARG
A
243
23.056
−29.347
59.131
1.00
31.98
A


ATOM
1293
O
ARG
A
243
22.081
−30.075
59.314
1.00
29.48
A


ATOM
1294
N
GLY
A
244
22.936
−28.057
58.848
1.00
31.79
A


ATOM
1295
CA
GLY
A
244
21.611
−27.490
58.735
1.00
31.73
A


ATOM
1296
C
GLY
A
244
21.385
−26.208
59.501
1.00
28.94
A


ATOM
1297
O
GLY
A
244
22.325
−25.545
59.938
1.00
28.30
A


ATOM
1298
N
THR
A
245
20.112
−25.882
59.693
1.00
28.92
A


ATOM
1299
CA
THR
A
245
19.748
−24.646
60.359
1.00
27.12
A


ATOM
1300
CB
THR
A
245
19.585
−23.514
59.317
1.00
29.13
A


ATOM
1301
OG1
THR
A
245
20.752
−23.457
58.484
1.00
28.18
A


ATOM
1302
CG2
THR
A
245
19.374
−22.161
60.008
1.00
29.18
A


ATOM
1303
C
THR
A
245
18.448
−24.751
61.129
1.00
28.44
A


ATOM
1304
O
THR
A
245
17.491
−25.396
60.683
1.00
29.55
A


ATOM
1305
N
THR
A
246
18.421
−24.116
62.294
1.00
27.00
A


ATOM
1306
CA
THR
A
246
17.220
−24.080
63.104
1.00
27.61
A


ATOM
1307
CB
THR
A
246
17.414
−24.659
64.529
1.00
29.89
A


ATOM
1308
OG1
THR
A
246
17.545
−26.084
64.469
1.00
30.34
A


ATOM
1309
CG2
THR
A
246
16.201
−24.307
65.407
1.00
30.40
A


ATOM
1310
C
THR
A
246
16.834
−22.621
63.276
1.00
26.97
A


ATOM
1311
O
THR
A
246
17.636
−21.802
63.741
1.00
27.17
A


ATOM
1312
N
ILE
A
247
15.615
−22.294
62.880
1.00
25.19
A


ATOM
1313
CA
ILE
A
247
15.124
−20.944
63.054
1.00
25.58
A


ATOM
1314
CB
ILE
A
247
14.233
−20.479
61.887
1.00
25.50
A


ATOM
1315
CG2
ILE
A
247
13.833
−19.021
62.114
1.00
25.21
A


ATOM
1316
CG1
ILE
A
247
14.947
−20.684
60.541
1.00
24.91
A


ATOM
1317
CD1
ILE
A
247
16.199
−19.854
60.345
1.00
24.45
A


ATOM
1318
C
ILE
A
247
14.247
−21.081
64.285
1.00
24.63
A


ATOM
1319
O
ILE
A
247
13.279
−21.838
64.276
1.00
25.24
A


ATOM
1320
N
THR
A
248
14.603
−20.377
65.349
1.00
25.95
A


ATOM
1321
CA
THR
A
248
13.830
−20.424
66.584
1.00
25.55
A


ATOM
1322
CB
THR
A
248
14.738
−20.727
67.801
1.00
26.69
A


ATOM
1323
OG1
THR
A
248
15.296
−22.043
67.673
1.00
28.48
A


ATOM
1324
CG2
THR
A
248
13.933
−20.640
69.104
1.00
26.02
A


ATOM
1325
C
THR
A
248
13.153
−19.075
66.784
1.00
26.59
A


ATOM
1326
O
THR
A
248
13.813
−18.036
66.806
1.00
26.71
A


ATOM
1327
N
LEU
A
249
11.835
−19.089
66.920
1.00
26.73
A


ATOM
1328
CA
LEU
A
249
11.103
−17.851
67.122
1.00
28.46
A


ATOM
1329
CB
LEU
A
249
9.958
−17.737
66.109
1.00
26.10
A


ATOM
1330
CG
LEU
A
249
10.287
−17.887
64.618
1.00
27.20
A


ATOM
1331
CD1
LEU
A
249
9.060
−17.498
63.811
1.00
28.67
A


ATOM
1332
CD2
LEU
A
249
11.458
−17.012
64.227
1.00
25.85
A


ATOM
1333
C
LEU
A
249
10.535
−17.745
68.534
1.00
28.32
A


ATOM
1334
O
LEU
A
249
9.859
−18.655
69.014
1.00
28.30
A


ATOM
1335
N
VAL
A
250
10.846
−16.643
69.208
1.00
28.75
A


ATOM
1336
CA
VAL
A
250
10.318
−16.388
70.542
1.00
29.14
A


ATOM
1337
CB
VAL
A
250
11.298
−15.549
71.387
1.00
29.73
A


ATOM
1338
CG1
VAL
A
250
10.701
−15.274
72.763
1.00
29.16
A


ATOM
1339
CG2
VAL
A
250
12.628
−16.282
71.515
1.00
27.56
A


ATOM
1340
C
VAL
A
250
9.057
−15.580
70.234
1.00
30.79
A


ATOM
1341
O
VAL
A
250
9.114
−14.369
70.015
1.00
30.24
A


ATOM
1342
N
LEU
A
251
7.924
−16.274
70.208
1.00
31.70
A


ATOM
1343
CA
LEU
A
251
6.639
−15.684
69.857
1.00
33.83
A


ATOM
1344
CB
LEU
A
251
5.596
−16.803
69.749
1.00
33.73
A


ATOM
1345
CG
LEU
A
251
5.914
−17.822
68.649
1.00
35.88
A


ATOM
1346
CD1
LEU
A
251
4.901
−18.952
68.679
1.00
34.78
A


ATOM
1347
CD2
LEU
A
251
5.916
−17.121
67.288
1.00
39.61
A


ATOM
1348
C
LEU
A
251
6.066
−14.539
70.693
1.00
34.58
A


ATOM
1349
O
LEU
A
251
6.113
−14.552
71.922
1.00
31.56
A


ATOM
1350
N
LYS
A
252
5.522
−13.547
69.995
1.00
35.31
A


ATOM
1351
CA
LYS
A
252
4.894
−12.414
70.652
1.00
37.92
A


ATOM
1352
CB
LYS
A
252
4.429
−11.371
69.633
1.00
36.52
A


ATOM
1353
CG
LYS
A
252
5.531
−10.673
68.857
1.00
37.81
A


ATOM
1354
CD
LYS
A
252
4.929
−9.718
67.829
1.00
35.73
A


ATOM
1355
CE
LYS
A
252
6.004
−8.907
67.129
1.00
35.52
A


ATOM
1356
NZ
LYS
A
252
6.722
−8.038
68.096
1.00
34.94
A


ATOM
1357
C
LYS
A
252
3.668
−12.975
71.353
1.00
40.30
A


ATOM
1358
O
LYS
A
252
3.095
−13.980
70.920
1.00
36.25
A


ATOM
1359
N
GLU
A
253
3.263
−12.319
72.431
1.00
43.28
A


ATOM
1360
CA
GLU
A
253
2.095
−12.746
73.188
1.00
46.90
A


ATOM
1361
CB
GLU
A
253
1.740
−11.669
74.215
1.00
50.77
A


ATOM
1362
CG
GLU
A
253
0.690
−12.076
75.232
1.00
57.75
A


ATOM
1363
CD
GLU
A
253
0.475
−11.008
76.291
1.00
60.96
A


ATOM
1364
OE1
GLU
A
253
1.456
−10.636
76.974
1.00
61.81
A


ATOM
1365
OE2
GLU
A
253
−0.674
−10.540
76.438
1.00
63.03
A


ATOM
1366
C
GLU
A
253
0.914
−12.980
72.245
1.00
45.72
A


ATOM
1367
O
GLU
A
253
0.253
−14.015
72.303
1.00
45.27
A


ATOM
1368
N
GLU
A
254
0.679
−12.018
71.360
1.00
45.23
A


ATOM
1369
CA
GLU
A
254
−0.422
−12.077
70.405
1.00
47.70
A


ATOM
1370
CB
GLU
A
254
−0.560
−10.729
69.686
1.00
50.07
A


ATOM
1371
CG
GLU
A
254
−0.180
−9.528
70.540
1.00
54.85
A


ATOM
1372
CD
GLU
A
254
1.324
−9.360
70.667
1.00
56.21
A


ATOM
1373
OE1
GLU
A
254
1.951
−8.923
69.680
1.00
56.48
A


ATOM
1374
OE2
GLU
A
254
1.879
−9.673
71.744
1.00
58.20
A


ATOM
1375
C
GLU
A
254
−0.299
−13.178
69.356
1.00
46.43
A


ATOM
1376
O
GLU
A
254
−1.191
−13.334
68.521
1.00
47.19
A


ATOM
1377
N
ALA
A
255
0.790
−13.942
69.392
1.00
44.24
A


ATOM
1378
CA
ALA
A
255
0.995
−15.002
68.405
1.00
40.23
A


ATOM
1379
CB
ALA
A
255
2.275
−14.724
67.618
1.00
39.06
A


ATOM
1380
C
ALA
A
255
1.043
−16.408
68.998
1.00
39.13
A


ATOM
1381
O
ALA
A
255
1.506
−17.347
68.341
1.00
36.42
A


ATOM
1382
N
SER
A
256
0.559
−16.559
70.229
1.00
39.57
A


ATOM
1383
CA
SER
A
256
0.566
−17.861
70.899
1.00
40.85
A


ATOM
1384
CB
SER
A
256
−0.085
−17.759
72.287
1.00
42.00
A


ATOM
1385
OG
SER
A
256
−1.441
−17.356
72.201
1.00
46.83
A


ATOM
1386
C
SER
A
256
−0.114
−18.967
70.093
1.00
40.89
A


ATOM
1387
O
SER
A
256
0.205
−20.143
70.257
1.00
40.95
A


ATOM
1388
N
ASP
A
257
−1.049
−18.592
69.227
1.00
41.73
A


ATOM
1389
CA
ASP
A
257
−1.747
−19.562
68.387
1.00
43.77
A


ATOM
1390
CB
ASP
A
257
−2.666
−18.845
67.400
1.00
47.93
A


ATOM
1391
CG
ASP
A
257
−4.125
−19.113
67.670
1.00
52.41
A


ATOM
1392
OD1
ASP
A
257
−4.491
−20.304
67.800
1.00
54.34
A


ATOM
1393
OD2
ASP
A
257
−4.901
−18.134
67.744
1.00
53.33
A


ATOM
1394
C
ASP
A
257
−0.773
−20.431
67.592
1.00
44.09
A


ATOM
1395
O
ASP
A
257
−1.062
−21.592
67.294
1.00
43.05
A


ATOM
1396
N
TYR
A
258
0.375
−19.860
67.239
1.00
42.61
A


ATOM
1397
CA
TYR
A
258
1.371
−20.593
66.473
1.00
43.55
A


ATOM
1398
CB
TYR
A
258
2.368
−19.621
65.837
1.00
40.49
A


ATOM
1399
CG
TYR
A
258
1.725
−18.682
64.840
1.00
37.93
A


ATOM
1400
CD1
TYR
A
258
1.442
−17.360
65.175
1.00
37.43
A


ATOM
1401
CE1
TYR
A
258
0.808
−16.507
64.271
1.00
38.22
A


ATOM
1402
CD2
TYR
A
258
1.359
−19.133
63.572
1.00
38.12
A


ATOM
1403
CE2
TYR
A
258
0.725
−18.293
62.660
1.00
39.12
A


ATOM
1404
CZ
TYR
A
258
0.451
−16.982
63.014
1.00
40.73
A


ATOM
1405
OH
TYR
A
258
−0.186
−16.154
62.111
1.00
43.50
A


ATOM
1406
C
TYR
A
258
2.093
−21.620
67.336
1.00
44.26
A


ATOM
1407
O
TYR
A
258
3.070
−22.231
66.910
1.00
45.55
A


ATOM
1408
N
LEU
A
259
1.605
−21.800
68.558
1.00
45.18
A


ATOM
1409
CA
LEU
A
259
2.178
−22.778
69.470
1.00
45.20
A


ATOM
1410
CB
LEU
A
259
2.267
−22.207
70.889
1.00
43.71
A


ATOM
1411
CG
LEU
A
259
3.271
−21.077
71.135
1.00
46.13
A


ATOM
1412
CD1
LEU
A
259
3.118
−20.553
72.556
1.00
43.85
A


ATOM
1413
CD2
LEU
A
259
4.688
−21.589
70.904
1.00
43.81
A


ATOM
1414
C
LEU
A
259
1.279
−24.013
69.461
1.00
45.63
A


ATOM
1415
O
LEU
A
259
1.681
−25.085
69.911
1.00
44.85
A


ATOM
1416
N
GLU
A
260
0.064
−23.852
68.938
1.00
46.11
A


ATOM
1417
CA
GLU
A
260
−0.905
−24.945
68.867
1.00
47.29
A


ATOM
1418
CB
GLU
A
260
−2.329
−24.389
68.752
1.00
48.35
A


ATOM
1419
CG
GLU
A
260
−2.821
−23.653
69.992
1.00
50.10
A


ATOM
1420
CD
GLU
A
260
−2.796
−24.524
71.240
1.00
50.21
A


ATOM
1421
OE1
GLU
A
260
−3.467
−25.576
71.260
1.00
52.40
A


ATOM
1422
OE2
GLU
A
260
−2.103
−24.155
72.207
1.00
51.35
A


ATOM
1423
C
GLU
A
260
−0.629
−25.890
67.695
1.00
48.12
A


ATOM
1424
O
GLU
A
260
−0.473
−25.454
66.552
1.00
47.97
A


ATOM
1425
N
LEU
A
261
−0.591
−27.186
67.994
1.00
48.15
A


ATOM
1426
CA
LEU
A
261
−0.318
−28.219
67.000
1.00
49.42
A


ATOM
1427
CB
LEU
A
261
−0.326
−29.604
67.657
1.00
50.55
A


ATOM
1428
CG
LEU
A
261
0.737
−29.856
68.726
1.00
52.10
A


ATOM
1429
CD1
LEU
A
261
0.611
−31.278
69.246
1.00
52.02
A


ATOM
1430
CD2
LEU
A
261
2.119
−29.621
68.136
1.00
53.44
A


ATOM
1431
C
LEU
A
261
−1.267
−28.230
65.812
1.00
48.59
A


ATOM
1432
O
LEU
A
261
−0.834
−28.409
64.673
1.00
48.28
A


ATOM
1433
N
ASP
A
262
−2.557
−28.055
66.064
1.00
47.88
A


ATOM
1434
CA
ASP
A
262
−3.513
−28.066
64.965
1.00
48.90
A


ATOM
1435
CB
ASP
A
262
−4.950
−28.077
65.500
1.00
51.04
A


ATOM
1436
CG
ASP
A
262
−5.233
−26.930
66.441
1.00
54.48
A


ATOM
1437
OD1
ASP
A
262
−4.535
−26.817
67.475
1.00
55.33
A


ATOM
1438
OD2
ASP
A
262
−6.157
−26.143
66.148
1.00
57.16
A


ATOM
1439
C
ASP
A
262
−3.284
−26.869
64.046
1.00
48.05
A


ATOM
1440
O
ASP
A
262
−3.399
−26.982
62.823
1.00
46.94
A


ATOM
1441
N
THR
A
263
−2.938
−25.727
64.632
1.00
46.96
A


ATOM
1442
CA
THR
A
263
−2.683
−24.531
63.837
1.00
46.87
A


ATOM
1443
CB
THR
A
263
−2.473
−23.291
64.731
1.00
47.54
A


ATOM
1444
OG1
THR
A
263
−3.632
−23.089
65.551
1.00
49.30
A


ATOM
1445
CG2
THR
A
263
−2.240
−22.047
63.872
1.00
45.04
A


ATOM
1446
C
THR
A
263
−1.437
−24.718
62.972
1.00
45.88
A


ATOM
1447
O
THR
A
263
−1.478
−24.523
61.758
1.00
44.59
A


ATOM
1448
N
ILE
A
264
−0.334
−25.109
63.604
1.00
45.39
A


ATOM
1449
CA
ILE
A
264
0.921
−25.302
62.890
1.00
45.12
A


ATOM
1450
CB
ILE
A
264
2.080
−25.670
63.867
1.00
45.80
A


ATOM
1451
CG2
ILE
A
264
1.827
−27.018
64.523
1.00
45.69
A


ATOM
1452
CG1
ILE
A
264
3.409
−25.705
63.111
1.00
45.29
A


ATOM
1453
CD1
ILE
A
264
3.836
−24.354
62.576
1.00
48.05
A


ATOM
1454
C
ILE
A
264
0.820
−26.359
61.789
1.00
45.27
A


ATOM
1455
O
ILE
A
264
1.221
−26.107
60.652
1.00
43.64
A


ATOM
1456
N
LYS
A
265
0.279
−27.532
62.113
1.00
45.81
A


ATOM
1457
CA
LYS
A
265
0.157
−28.595
61.115
1.00
46.73
A


ATOM
1458
CB
LYS
A
265
−0.501
−29.838
61.721
1.00
47.68
A


ATOM
1459
CG
LYS
A
265
0.400
−30.575
62.703
1.00
50.09
A


ATOM
1460
CD
LYS
A
265
−0.121
−31.964
63.043
1.00
53.24
A


ATOM
1461
CE
LYS
A
265
−1.399
−31.915
63.861
1.00
55.91
A


ATOM
1462
NZ
LYS
A
265
−1.849
−33.281
64.256
1.00
60.96
A


ATOM
1463
C
LYS
A
265
−0.622
−28.142
59.888
1.00
46.00
A


ATOM
1464
O
LYS
A
265
−0.225
−28.420
58.758
1.00
46.17
A


ATOM
1465
N
ASN
A
266
−1.721
−27.432
60.109
1.00
46.21
A


ATOM
1466
CA
ASN
A
266
−2.540
−26.948
59.007
1.00
47.31
A


ATOM
1467
CB
ASN
A
266
−3.770
−26.216
59.547
1.00
51.04
A


ATOM
1468
CG
ASN
A
266
−4.830
−25.993
58.484
1.00
56.45
A


ATOM
1469
OD1
ASN
A
266
−5.543
−26.922
58.094
1.00
59.75
A


ATOM
1470
ND2
ASN
A
266
−4.934
−24.758
58.002
1.00
58.02
A


ATOM
1471
C
ASN
A
266
−1.720
−26.001
58.132
1.00
46.83
A


ATOM
1472
O
ASN
A
266
−1.745
−26.096
56.904
1.00
46.99
A


ATOM
1473
N
LEU
A
267
−0.993
−25.087
58.770
1.00
45.41
A


ATOM
1474
CA
LEU
A
267
−0.166
−24.124
58.045
1.00
44.41
A


ATOM
1475
CB
LEU
A
267
0.408
−23.078
59.005
1.00
44.81
A


ATOM
1476
CG
LEU
A
267
−0.557
−22.100
59.675
1.00
44.89
A


ATOM
1477
CD1
LEU
A
267
0.220
−21.201
60.626
1.00
45.00
A


ATOM
1478
CD2
LEU
A
267
−1.275
−21.269
58.618
1.00
44.38
A


ATOM
1479
C
LEU
A
267
0.981
−24.797
57.302
1.00
42.78
A


ATOM
1480
O
LEU
A
267
1.238
−24.490
56.139
1.00
42.03
A


ATOM
1481
N
VAL
A
268
1.676
−25.703
57.983
1.00
41.81
A


ATOM
1482
CA
VAL
A
268
2.798
−26.418
57.383
1.00
42.20
A


ATOM
1483
CB
VAL
A
268
3.442
−27.400
58.385
1.00
41.34
A


ATOM
1484
CG1
VAL
A
268
4.397
−28.338
57.658
1.00
40.94
A


ATOM
1485
CG2
VAL
A
268
4.188
−26.628
59.460
1.00
39.92
A


ATOM
1486
C
VAL
A
268
2.348
−27.204
56.159
1.00
43.14
A


ATOM
1487
O
VAL
A
268
3.004
−27.187
55.119
1.00
41.73
A


ATOM
1488
N
LYS
A
269
1.220
−27.890
56.291
1.00
45.44
A


ATOM
1489
CA
LYS
A
269
0.686
−28.687
55.198
1.00
48.18
A


ATOM
1490
CB
LYS
A
269
−0.511
−29.505
55.689
1.00
50.56
A


ATOM
1491
CG
LYS
A
269
−0.984
−30.560
54.707
1.00
55.15
A


ATOM
1492
CD
LYS
A
269
−2.110
−31.396
55.305
1.00
58.30
A


ATOM
1493
CE
LYS
A
269
−2.590
−32.470
54.338
1.00
58.74
A


ATOM
1494
NZ
LYS
A
269
−3.722
−33.259
54.907
1.00
61.58
A


ATOM
1495
C
LYS
A
269
0.273
−27.803
54.023
1.00
47.87
A


ATOM
1496
O
LYS
A
269
0.407
−28.196
52.865
1.00
48.99
A


ATOM
1497
N
ALA
A
270
−0.216
−26.605
54.322
1.00
47.71
A


ATOM
1498
CA
ALA
A
270
−0.647
−25.682
53.278
1.00
47.22
A


ATOM
1499
CB
ALA
A
270
−1.518
−24.593
53.880
1.00
47.69
A


ATOM
1500
C
ALA
A
270
0.532
−25.049
52.547
1.00
47.81
A


ATOM
1501
O
ALA
A
270
0.407
−24.625
51.398
1.00
47.91
A


ATOM
1502
N
TYR
A
271
1.681
−24.998
53.210
1.00
46.43
A


ATOM
1503
CA
TYR
A
271
2.867
−24.384
52.624
1.00
46.82
A


ATOM
1504
CB
TYR
A
271
3.422
−23.340
53.600
1.00
45.84
A


ATOM
1505
CG
TYR
A
271
2.527
−22.136
53.817
1.00
44.83
A


ATOM
1506
CD1
TYR
A
271
2.503
−21.477
55.049
1.00
43.90
A


ATOM
1507
CE1
TYR
A
271
1.727
−20.341
55.244
1.00
44.66
A


ATOM
1508
CD2
TYR
A
271
1.742
−21.624
52.782
1.00
43.86
A


ATOM
1509
CE2
TYR
A
271
0.960
−20.485
52.965
1.00
44.32
A


ATOM
1510
CZ
TYR
A
271
0.958
−19.848
54.200
1.00
46.19
A


ATOM
1511
OH
TYR
A
271
0.192
−18.715
54.392
1.00
49.38
A


ATOM
1512
C
TYR
A
271
3.982
−25.364
52.251
1.00
46.30
A


ATOM
1513
O
TYR
A
271
5.105
−24.944
51.971
1.00
45.31
A


ATOM
1514
N
SER
A
272
3.682
−26.659
52.228
1.00
46.53
A


ATOM
1515
CA
SER
A
272
4.714
−27.642
51.920
1.00
48.82
A


ATOM
1516
CB
SER
A
272
5.038
−28.457
53.175
1.00
49.85
A


ATOM
1517
OG
SER
A
272
3.923
−29.228
53.591
1.00
51.28
A


ATOM
1518
C
SER
A
272
4.417
−28.601
50.773
1.00
49.33
A


ATOM
1519
O
SER
A
272
5.227
−29.481
50.477
1.00
48.33
A


ATOM
1520
N
ALA
A
273
3.270
−28.438
50.124
1.00
51.93
A


ATOM
1521
CA
ALA
A
273
2.907
−29.320
49.019
1.00
54.85
A


ATOM
1522
CB
ALA
A
273
1.490
−29.002
48.543
1.00
54.46
A


ATOM
1523
C
ALA
A
273
3.891
−29.221
47.854
1.00
56.23
A


ATOM
1524
O
ALA
A
273
3.863
−30.041
46.937
1.00
57.06
A


ATOM
1525
N
PHE
A
274
4.765
−28.219
47.896
1.00
58.75
A


ATOM
1526
CA
PHE
A
274
5.752
−28.010
46.836
1.00
60.64
A


ATOM
1527
CB
PHE
A
274
5.776
−26.536
46.421
1.00
63.61
A


ATOM
1528
CG
PHE
A
274
4.505
−26.058
45.773
1.00
66.68
A


ATOM
1529
CD1
PHE
A
274
4.319
−24.701
45.509
1.00
68.18
A


ATOM
1530
CD2
PHE
A
274
3.502
−26.955
45.413
1.00
67.97
A


ATOM
1531
CE1
PHE
A
274
3.155
−24.243
44.896
1.00
68.69
A


ATOM
1532
CE2
PHE
A
274
2.332
−26.509
44.798
1.00
68.98
A


ATOM
1533
CZ
PHE
A
274
2.159
−25.149
44.539
1.00
69.46
A


ATOM
1534
C
PHE
A
274
7.167
−28.430
47.229
1.00
60.36
A


ATOM
1535
O
PHE
A
274
7.988
−28.744
46.363
1.00
61.16
A


ATOM
1536
N
ALA
A
275
7.456
−28.421
48.526
1.00
58.26
A


ATOM
1537
CA
ALA
A
275
8.781
−28.795
49.008
1.00
56.97
A


ATOM
1538
CB
ALA
A
275
8.768
−28.934
50.533
1.00
55.69
A


ATOM
1539
C
ALA
A
275
9.220
−30.103
48.364
1.00
55.44
A


ATOM
1540
O
ALA
A
275
8.450
−31.060
48.301
1.00
56.27
A


ATOM
1541
N
ALA
A
276
10.454
−30.136
47.876
1.00
53.60
A


ATOM
1542
CA
ALA
A
276
10.986
−31.336
47.243
1.00
51.53
A


ATOM
1543
CB
ALA
A
276
12.007
−30.960
46.179
1.00
52.85
A


ATOM
1544
C
ALA
A
276
11.633
−32.205
48.309
1.00
49.72
A


ATOM
1545
O
ALA
A
276
12.531
−32.997
48.023
1.00
50.08
A


ATOM
1546
N
PHE
A
277
11.163
−32.042
49.543
1.00
47.45
A


ATOM
1547
CA
PHE
A
277
11.676
−32.785
50.687
1.00
43.13
A


ATOM
1548
CB
PHE
A
277
12.794
−31.986
51.358
1.00
42.71
A


ATOM
1549
CG
PHE
A
277
13.944
−31.678
50.448
1.00
41.75
A


ATOM
1550
CD1
PHE
A
277
14.932
−32.626
50.213
1.00
40.75
A


ATOM
1551
CD2
PHE
A
277
14.021
−30.449
49.798
1.00
39.70
A


ATOM
1552
CE1
PHE
A
277
15.981
−32.354
49.343
1.00
41.23
A


ATOM
1553
CE2
PHE
A
277
15.063
−30.169
48.925
1.00
39.82
A


ATOM
1554
CZ
PHE
A
277
16.047
−31.122
48.696
1.00
40.51
A


ATOM
1555
C
PHE
A
277
10.550
−33.032
51.693
1.00
41.66
A


ATOM
1556
O
PHE
A
277
9.546
−32.322
51.709
1.00
40.72
A


ATOM
1557
N
PRO
A
278
10.705
−34.049
52.548
1.00
40.54
A


ATOM
1558
CD
PRO
A
278
11.812
−35.020
52.623
1.00
39.57
A


ATOM
1559
CA
PRO
A
278
9.674
−34.352
53.544
1.00
39.44
A


ATOM
1560
CB
PRO
A
278
10.059
−35.748
54.005
1.00
38.76
A


ATOM
1561
CG
PRO
A
278
11.561
−35.691
53.958
1.00
39.95
A


ATOM
1562
C
PRO
A
278
9.670
−33.336
54.693
1.00
39.01
A


ATOM
1563
O
PRO
A
278
10.728
−32.960
55.203
1.00
36.83
A


ATOM
1564
N
ILE
A
279
8.481
−32.886
55.085
1.00
39.22
A


ATOM
1565
CA
ILE
A
279
8.351
−31.923
56.175
1.00
41.37
A


ATOM
1566
CB
ILE
A
279
7.673
−30.624
55.701
1.00
43.60
A


ATOM
1567
CG2
ILE
A
279
7.599
−29.627
56.852
1.00
43.19
A


ATOM
1568
CG1
ILE
A
279
8.475
−30.025
54.540
1.00
46.61
A


ATOM
1569
CD1
ILE
A
279
8.026
−28.640
54.110
1.00
51.71
A


ATOM
1570
C
ILE
A
279
7.549
−32.522
57.326
1.00
41.16
A


ATOM
1571
O
ILE
A
279
6.405
−32.952
57.150
1.00
41.86
A


ATOM
1572
N
TYR
A
280
8.160
−32.536
58.505
1.00
40.12
A


ATOM
1573
CA
TYR
A
280
7.540
−33.104
59.696
1.00
40.38
A


ATOM
1574
CB
TYR
A
280
8.478
−34.138
60.315
1.00
41.53
A


ATOM
1575
CG
TYR
A
280
8.905
−35.230
59.371
1.00
43.98
A


ATOM
1576
CD1
TYR
A
280
8.066
−36.309
59.097
1.00
45.26
A


ATOM
1577
CE1
TYR
A
280
8.455
−37.316
58.216
1.00
45.37
A


ATOM
1578
CD2
TYR
A
280
10.147
−35.181
58.738
1.00
44.40
A


ATOM
1579
CE2
TYR
A
280
10.544
−36.180
57.856
1.00
45.61
A


ATOM
1580
CZ
TYR
A
280
9.692
−37.244
57.600
1.00
46.06
A


ATOM
1581
OH
TYR
A
280
10.076
−38.231
56.724
1.00
47.42
A


ATOM
1582
C
TYR
A
280
7.192
−32.084
60.774
1.00
40.73
A


ATOM
1583
O
TYR
A
280
7.767
−30.995
60.837
1.00
36.63
A


ATOM
1584
N
VAL
A
281
6.246
−32.470
61.627
1.00
39.60
A


ATOM
1585
CA
VAL
A
281
5.817
−31.655
62.750
1.00
40.50
A


ATOM
1586
CB
VAL
A
281
4.407
−31.066
62.534
1.00
40.77
A


ATOM
1587
CG1
VAL
A
281
4.414
−30.113
61.360
1.00
40.09
A


ATOM
1588
CG2
VAL
A
281
3.410
−32.181
62.302
1.00
44.48
A


ATOM
1589
C
VAL
A
281
5.791
−32.579
63.966
1.00
42.22
A


ATOM
1590
O
VAL
A
281
5.274
−33.699
63.898
1.00
41.86
A


ATOM
1591
N
TRP
A
282
6.375
−32.121
65.066
1.00
42.62
A


ATOM
1592
CA
TRP
A
282
6.412
−32.907
66.290
1.00
45.45
A


ATOM
1593
CB
TRP
A
282
7.432
−32.306
67.256
1.00
46.54
A


ATOM
1594
CG
TRP
A
282
7.677
−33.120
68.480
1.00
49.77
A


ATOM
1595
CD2
TRP
A
282
8.306
−34.405
68.544
1.00
51.05
A


ATOM
1596
CE2
TRP
A
282
8.348
−34.781
69.905
1.00
51.72
A


ATOM
1597
CE3
TRP
A
282
8.841
−35.276
67.585
1.00
51.26
A


ATOM
1598
CD1
TRP
A
282
7.368
−32.780
69.763
1.00
50.66
A


ATOM
1599
NE1
TRP
A
282
7.768
−33.771
70.626
1.00
50.98
A


ATOM
1600
CZ2
TRP
A
282
8.904
−35.994
70.334
1.00
51.83
A


ATOM
1601
CZ3
TRP
A
282
9.395
−36.483
68.009
1.00
51.83
A


ATOM
1602
CH2
TRP
A
282
9.421
−36.829
69.374
1.00
52.82
A


ATOM
1603
C
TRP
A
282
5.016
−32.860
66.890
1.00
46.75
A


ATOM
1604
O
TRP
A
282
4.639
−31.875
67.518
1.00
46.99
A


ATOM
1605
N
SER
A
283
4.241
−33.920
66.681
1.00
49.65
A


ATOM
1606
CA
SER
A
283
2.876
−33.965
67.196
1.00
53.00
A


ATOM
1607
CB
SER
A
283
1.882
−34.042
66.036
1.00
54.08
A


ATOM
1608
OG
SER
A
283
2.123
−35.185
65.237
1.00
59.95
A


ATOM
1609
C
SER
A
283
2.612
−35.109
68.172
1.00
53.85
A


ATOM
1610
O
SER
A
283
3.429
−36.016
68.328
1.00
52.62
A


ATOM
1611
N
SER
A
284
1.454
−35.048
68.824
1.00
55.39
A


ATOM
1612
CA
SER
A
284
1.047
−36.053
69.802
1.00
57.73
A


ATOM
1613
CB
SER
A
284
1.028
−35.436
71.197
1.00
57.15
A


ATOM
1614
OG
SER
A
284
0.161
−34.317
71.228
1.00
57.89
A


ATOM
1615
C
SER
A
284
−0.340
−36.592
69.465
1.00
59.00
A


ATOM
1616
O
SER
A
284
−1.101
−35.953
68.739
1.00
58.48
A


ATOM
1617
N
LYS
A
285
−0.671
−37.762
70.002
1.00
61.19
A


ATOM
1618
CA
LYS
A
285
−1.970
−38.368
69.732
1.00
62.47
A


ATOM
1619
CB
LYS
A
285
−1.786
−39.800
69.222
1.00
62.13
A


ATOM
1620
CG
LYS
A
285
−3.076
−40.463
68.751
1.00
64.42
A


ATOM
1621
CD
LYS
A
285
−2.824
−41.894
68.297
1.00
65.81
A


ATOM
1622
CE
LYS
A
285
−4.090
−42.549
67.762
1.00
66.71
A


ATOM
1623
NZ
LYS
A
285
−3.839
−43.956
67.323
1.00
68.96
A


ATOM
1624
C
LYS
A
285
−2.907
−38.372
70.938
1.00
62.88
A


ATOM
1625
O
LYS
A
285
−3.984
−37.776
70.889
1.00
63.71
A


ATOM
1626
N
THR
A
286
−2.501
−39.040
72.014
1.00
61.19
A


ATOM
1627
CA
THR
A
286
−3.326
−39.127
73.221
1.00
62.47
A


ATOM
1628
CB
THR
A
286
−3.689
−37.724
73.773
1.00
62.13
A


ATOM
1629
OG1
THR
A
286
−2.527
−37.110
74.345
1.00
19.86
A


ATOM
1630
CG2
THR
A
286
−4.761
−37.835
74.848
1.00
19.86
A


ATOM
1631
C
THR
A
286
−4.628
−39.881
72.944
1.00
62.88
A


ATOM
1632
O
THR
A
286
−4.619
−41.008
72.445
1.00
63.71
A


ATOM
1633
N
LYS
A
328
−2.207
−41.457
78.617
1.00
67.81
A


ATOM
1634
CA
LYS
A
328
−1.228
−41.967
77.661
1.00
68.19
A


ATOM
1635
CB
LYS
A
328
−1.633
−43.360
77.175
1.00
70.00
A


ATOM
1636
CG
LYS
A
328
−1.698
−44.415
78.266
1.00
73.35
A


ATOM
1637
CD
LYS
A
328
−2.084
−45.771
77.687
1.00
75.20
A


ATOM
1638
CE
LYS
A
328
−2.144
−46.846
78.762
1.00
76.19
A


ATOM
1639
NZ
LYS
A
328
−2.470
−48.180
78.184
1.00
77.71
A


ATOM
1640
C
LYS
A
328
−1.094
−41.040
76.458
1.00
67.51
A


ATOM
1641
O
LYS
A
328
−2.084
−40.710
75.802
1.00
67.72
A


ATOM
1642
N
THR
A
329
0.136
−40.626
76.168
1.00
65.72
A


ATOM
1643
CA
THR
A
329
0.391
−39.740
75.039
1.00
64.09
A


ATOM
1644
CB
THR
A
329
0.655
−38.300
75.504
1.00
63.35
A


ATOM
1645
OG1
THR
A
329
−0.463
−37.831
76.268
1.00
62.53
A


ATOM
1646
CG2
THR
A
329
0.865
−37.390
74.302
1.00
62.46
A


ATOM
1647
C
THR
A
329
1.600
−40.207
74.241
1.00
63.16
A


ATOM
1648
O
THR
A
329
2.645
−40.524
74.811
1.00
62.83
A


ATOM
1649
N
VAL
A
330
1.455
−40.250
72.920
1.00
62.00
A


ATOM
1650
CA
VAL
A
330
2.553
−40.674
72.061
1.00
60.58
A


ATOM
1651
CB
VAL
A
330
2.150
−41.858
71.159
1.00
60.87
A


ATOM
1652
CG1
VAL
A
330
3.398
−42.483
70.549
1.00
60.39
A


ATOM
1653
CG2
VAL
A
330
1.366
−42.889
71.957
1.00
60.97
A


ATOM
1654
C
VAL
A
330
3.007
−39.522
71.173
1.00
59.36
A


ATOM
1655
O
VAL
A
330
2.220
−38.958
70.413
1.00
58.20
A


ATOM
1656
N
TRP
A
331
4.286
−39.180
71.279
1.00
59.04
A


ATOM
1657
CA
TRP
A
331
4.865
−38.095
70.497
1.00
58.50
A


ATOM
1658
CB
TRP
A
331
5.615
−37.132
71.420
1.00
60.31
A


ATOM
1659
CG
TRP
A
331
4.918
−35.828
71.649
1.00
62.59
A


ATOM
1660
CD2
TRP
A
331
4.313
−35.382
72.867
1.00
63.34
A


ATOM
1661
CE2
TRP
A
331
3.804
−34.085
72.629
1.00
64.50
A


ATOM
1662
CE3
TRP
A
331
4.152
−35.951
74.136
1.00
63.96
A


ATOM
1663
CD1
TRP
A
331
4.753
−34.815
70.746
1.00
64.47
A


ATOM
1664
NE1
TRP
A
331
4.086
−33.764
71.327
1.00
64.58
A


ATOM
1665
CZ2
TRP
A
331
3.145
−33.346
73.616
1.00
64.39
A


ATOM
1666
CZ3
TRP
A
331
3.497
−35.216
75.118
1.00
66.44
A


ATOM
1667
CH2
TRP
A
331
3.001
−33.925
74.851
1.00
66.38
A


ATOM
1668
C
TRP
A
331
5.821
−38.624
69.432
1.00
57.08
A


ATOM
1669
O
TRP
A
331
6.648
−39.496
69.701
1.00
55.66
A


ATOM
1670
N
ASP
A
332
5.701
−38.090
68.221
1.00
55.73
A


ATOM
1671
CA
ASP
A
332
6.569
−38.495
67.125
1.00
54.33
A


ATOM
1672
CB
ASP
A
332
6.197
−39.898
66.644
1.00
55.67
A


ATOM
1673
CG
ASP
A
332
7.412
−40.726
66.271
1.00
57.50
A


ATOM
1674
OD1
ASP
A
332
8.260
−40.236
65.496
1.00
57.91
A


ATOM
1675
OD2
ASP
A
332
7.517
−41.873
66.754
1.00
59.97
A


ATOM
1676
C
ASP
A
332
6.454
−37.510
65.962
1.00
52.80
A


ATOM
1677
O
ASP
A
332
5.546
−36.674
65.927
1.00
51.02
A


ATOM
1678
N
TRP
A
333
7.383
−37.609
65.017
1.00
50.33
A


ATOM
1679
CA
TRP
A
333
7.366
−36.739
63.852
1.00
48.17
A


ATOM
1680
CB
TRP
A
333
8.682
−36.842
63.083
1.00
45.68
A


ATOM
1681
CG
TRP
A
333
9.867
−36.381
63.852
1.00
43.61
A


ATOM
1682
CD2
TRP
A
333
10.177
−35.031
64.222
1.00
41.57
A


ATOM
1683
CE2
TRP
A
333
11.398
−35.063
64.931
1.00
42.05
A


ATOM
1684
CE3
TRP
A
333
9.542
−33.798
64.023
1.00
40.61
A


ATOM
1685
CD1
TRP
A
333
10.881
−37.155
64.339
1.00
42.81
A


ATOM
1686
NE1
TRP
A
333
11.805
−36.372
64.987
1.00
43.41
A


ATOM
1687
CZ2
TRP
A
333
12.001
−33.905
65.445
1.00
40.00
A


ATOM
1688
CZ3
TRP
A
333
10.144
−32.642
64.536
1.00
39.42
A


ATOM
1689
CH2
TRP
A
333
11.360
−32.709
65.237
1.00
37.87
A


ATOM
1690
C
TRP
A
333
6.226
−37.168
62.945
1.00
47.93
A


ATOM
1691
O
TRP
A
333
6.101
−38.344
62.610
1.00
48.69
A


ATOM
1692
N
GLU
A
334
5.386
−36.217
62.560
1.00
47.26
A


ATOM
1693
CA
GLU
A
334
4.273
−36.520
61.674
1.00
47.77
A


ATOM
1694
CB
GLU
A
334
2.963
−35.974
62.241
1.00
47.55
A


ATOM
1695
CG
GLU
A
334
1.729
−36.494
61.521
1.00
48.73
A


ATOM
1696
CD
GLU
A
334
0.459
−35.807
61.974
1.00
52.26
A


ATOM
1697
OE1
GLU
A
334
0.229
−35.728
63.198
1.00
51.80
A


ATOM
1698
OE2
GLU
A
334
−0.313
−35.348
61.105
1.00
54.38
A


ATOM
1699
C
GLU
A
334
4.545
−35.889
60.313
1.00
47.58
A


ATOM
1700
O
GLU
A
334
4.742
−34.678
60.206
1.00
46.83
A


ATOM
1701
N
LEU
A
335
4.565
−36.719
59.277
1.00
47.77
A


ATOM
1702
CA
LEU
A
335
4.817
−36.248
57.922
1.00
48.66
A


ATOM
1703
CB
LEU
A
335
5.043
−37.443
56.991
1.00
47.95
A


ATOM
1704
CG
LEU
A
335
5.398
−37.137
55.535
1.00
47.37
A


ATOM
1705
CD1
LEU
A
335
6.661
−36.288
55.479
1.00
45.63
A


ATOM
1706
CD2
LEU
A
335
5.593
−38.447
54.776
1.00
47.26
A


ATOM
1707
C
LEU
A
335
3.657
−35.403
57.405
1.00
48.95
A


ATOM
1708
O
LEU
A
335
2.520
−35.863
57.344
1.00
50.26
A


ATOM
1709
N
MET
A
336
3.952
−34.159
57.046
1.00
49.79
A


ATOM
1710
CA
MET
A
336
2.942
−33.249
56.523
1.00
50.82
A


ATOM
1711
CB
MET
A
336
3.183
−31.829
57.045
1.00
51.61
A


ATOM
1712
CG
MET
A
336
3.193
−31.704
58.565
1.00
51.96
A


ATOM
1713
SD
MET
A
336
1.620
−32.164
59.335
1.00
54.16
A


ATOM
1714
CE
MET
A
336
1.964
−33.821
59.759
1.00
52.24
A


ATOM
1715
C
MET
A
336
3.064
−33.270
55.004
1.00
51.69
A


ATOM
1716
O
MET
A
336
2.179
−32.808
54.288
1.00
50.87
A


ATOM
1717
N
ASN
A
337
4.181
−33.822
54.539
1.00
53.30
A


ATOM
1718
CA
ASN
A
337
4.500
−33.952
53.119
1.00
55.08
A


ATOM
1719
CB
ASN
A
337
3.257
−34.343
52.318
1.00
55.89
A


ATOM
1720
CG
ASN
A
337
3.582
−34.679
50.885
1.00
57.84
A


ATOM
1721
OD1
ASN
A
337
4.363
−35.590
50.617
1.00
59.85
A


ATOM
1722
ND2
ASN
A
337
2.988
−33.942
49.950
1.00
60.12
A


ATOM
1723
C
ASN
A
337
5.107
−32.686
52.529
1.00
52.82
A


ATOM
1724
O
ASN
A
337
6.305
−32.640
52.258
1.00
40.66
A


ATOM
1725
C5*
NEC
N
1
18.101
−14.744
53.515
1.00
32.09
N


ATOM
1726
O5*
NEC
N
1
16.931
−15.464
53.805
1.00
33.94
N


ATOM
1727
N5*
NEC
N
1
18.132
−13.321
53.673
1.00
34.59
N


ATOM
1728
C51
NEC
N
1
18.051
−12.476
52.504
1.00
39.19
N


ATOM
1729
C52
NEC
N
1
16.800
−11.615
52.601
1.00
38.66
N


ATOM
1730
C4*
NEC
N
1
19.356
−15.441
53.081
1.00
32.40
N


ATOM
1731
O4*
NEC
N
1
19.996
−16.008
54.191
1.00
31.40
N


ATOM
1732
C3*
NEC
N
1
19.175
−16.570
52.078
1.00
30.36
N


ATOM
1733
O3*
NEC
N
1
19.248
−16.137
50.739
1.00
31.36
N


ATOM
1734
C2*
NEC
N
1
20.257
−17.579
52.503
1.00
33.06
N


ATOM
1735
O2*
NEC
N
1
21.430
−17.328
51.832
1.00
30.18
N


ATOM
1736
C1*
NEC
N
1
20.339
−17.393
54.061
1.00
29.68
N


ATOM
1737
N9
NEC
N
1
19.362
−18.313
54.711
1.00
31.55
N


ATOM
1738
C8
NEC
N
1
18.167
−18.005
55.335
1.00
29.83
N


ATOM
1739
N7
NEC
N
1
17.556
−19.059
55.807
1.00
30.83
N


ATOM
1740
C5
NEC
N
1
18.399
−20.121
55.490
1.00
30.97
N


ATOM
1741
C6
NEC
N
1
18.336
−21.533
55.729
1.00
31.98
N


ATOM
1742
N6
NEC
N
1
17.315
−22.108
56.374
1.00
29.34
N


ATOM
1743
N1
NEC
N
1
19.367
−22.326
55.284
1.00
32.04
N


ATOM
1744
C2
NEC
N
1
20.402
−21.758
54.633
1.00
31.64
N


ATOM
1745
N3
NEC
N
1
20.574
−20.454
54.344
1.00
31.62
N


ATOM
1746
C4
NEC
N
1
19.524
−19.676
54.805
1.00
30.07
N


ATOM
1747
OH2
WAT
W
1
20.447
−24.737
56.287
1.00
24.64
W


ATOM
1748
OH2
WAT
W
2
14.568
−14.841
53.450
1.00
22.42
W


ATOM
1749
OH2
WAT
W
3
19.296
−8.909
53.216
1.00
34.41
W


ATOM
1750
OH2
WAT
W
4
−9.791
−31.932
65.071
1.00
60.00
W


ATOM
1751
OH2
WAT
W
5
12.411
−2.377
65.438
1.00
32.85
W


ATOM
1752
OH2
WAT
W
6
12.886
−28.142
64.918
1.00
28.26
W


ATOM
1753
OH2
WAT
W
8
20.659
−2.334
63.849
1.00
27.95
W


ATOM
1754
OH2
WAT
W
9
14.414
−21.453
57.222
1.00
33.19
W


ATOM
1755
OH2
WAT
W
10
16.630
−18.755
50.263
1.00
39.77
W


ATOM
1756
OH2
WAT
W
11
28.580
−23.175
60.300
1.00
31.58
W


ATOM
1757
OH2
WAT
W
12
25.144
−21.263
53.006
1.00
35.45
W


ATOM
1758
OH2
WAT
W
13
14.906
−23.981
58.677
1.00
26.25
W


ATOM
1759
OH2
WAT
W
14
27.806
−3.028
62.773
1.00
37.16
W


ATOM
1760
OH2
WAT
W
15
27.329
−27.459
54.451
1.00
24.48
W


ATOM
1761
OH2
WAT
W
17
13.107
−18.978
49.611
1.00
38.19
W


ATOM
1762
OH2
WAT
W
18
17.985
−2.488
63.260
1.00
28.53
W


ATOM
1763
OH2
WAT
W
19
9.101
−7.920
66.355
1.00
30.77
W


ATOM
1764
OH2
WAT
W
20
27.388
−32.822
56.057
1.00
39.96
W


ATOM
1765
OH2
WAT
W
21
−0.940
−7.731
54.653
1.00
44.25
W


ATOM
1766
OH2
WAT
W
22
32.699
−22.173
48.909
1.00
59.25
W


ATOM
1767
OH2
WAT
W
23
15.056
−18.808
56.978
1.00
22.64
W


ATOM
1768
OH2
WAT
W
24
17.946
−21.501
66.688
1.00
26.54
W


ATOM
1769
OH2
WAT
W
25
13.721
−2.135
62.627
1.00
39.41
W


ATOM
1770
OH2
WAT
W
26
12.394
−25.571
72.991
1.00
35.33
W


ATOM
1771
OH2
WAT
W
27
20.012
−29.619
61.103
1.00
25.85
W


ATOM
1772
OH2
WAT
W
28
17.936
−20.651
52.066
1.00
28.66
W


ATOM
1773
OH2
WAT
W
29
0.586
−12.872
44.960
1.00
34.56
W


ATOM
1774
OH2
WAT
W
30
15.573
−27.732
68.375
1.00
44.80
W


ATOM
1775
OH2
WAT
W
31
22.608
−19.886
52.600
1.00
34.40
W


ATOM
1776
OH2
WAT
W
32
1.893
−11.084
65.797
1.00
34.78
W


ATOM
1777
OH2
WAT
W
33
24.264
0.954
56.229
1.00
35.33
W


ATOM
1778
OH2
WAT
W
34
16.289
−35.704
59.755
1.00
42.89
W


ATOM
1779
OH2
WAT
W
36
11.669
−3.349
62.349
1.00
47.32
W


ATOM
1780
OH2
WAT
W
37
22.055
−1.433
66.332
1.00
37.36
W


ATOM
1781
OH2
WAT
W
38
28.886
−27.612
51.781
1.00
35.01
W


ATOM
1782
OH2
WAT
W
39
12.130
−16.233
51.524
1.00
28.89
W


ATOM
1783
OH2
WAT
W
40
28.128
−29.763
58.789
1.00
38.41
W


ATOM
1784
OH2
WAT
W
41
10.890
−4.366
67.600
1.00
39.52
W


ATOM
1785
OH2
WAT
W
42
2.443
−14.674
47.358
1.00
30.52
W


ATOM
1786
OH2
WAT
W
43
21.091
−12.400
51.627
1.00
38.41
W


ATOM
1787
OH2
WAT
W
44
31.286
−29.871
68.533
1.00
48.27
W


ATOM
1788
OH2
WAT
W
45
11.801
−3.011
69.667
1.00
31.67
W


ATOM
1789
OH2
WAT
W
46
18.867
−8.703
69.847
1.00
51.34
W


ATOM
1790
OH2
WAT
W
47
28.938
−32.732
61.259
1.00
42.94
W


ATOM
1791
OH2
WAT
W
48
7.647
−12.538
73.493
1.00
43.37
W


ATOM
1792
OH2
WAT
W
49
15.138
−31.436
60.017
1.00
34.37
W


ATOM
1793
OH2
WAT
W
51
14.648
−30.717
65.458
1.00
59.12
W


ATOM
1794
OH2
WAT
W
52
−0.388
−35.583
57.653
1.00
59.38
W


ATOM
1795
OH2
WAT
W
54
4.408
0.061
38.685
1.00
69.58
W


ATOM
1796
OH2
WAT
W
55
4.392
−39.653
59.577
1.00
49.80
W


ATOM
1797
OH2
WAT
W
56
10.651
−17.908
48.691
1.00
48.73
W


ATOM
1798
OH2
WAT
W
57
8.040
−18.417
49.106
1.00
52.79
W


ATOM
1799
OH2
WAT
W
59
15.761
−30.831
45.009
1.00
79.83
W


ATOM
1800
OH2
WAT
W
60
2.973
−8.078
63.778
1.00
41.27
W


ATOM
1801
OH2
WAT
W
61
25.327
−5.578
59.264
1.00
45.94
W


ATOM
1802
OH2
WAT
W
63
6.882
−22.869
51.273
1.00
42.05
W


ATOM
1803
OH2
WAT
W
64
35.748
−20.905
61.008
1.00
69.02
W


ATOM
1804
OH2
WAT
W
65
−4.257
−31.908
65.453
1.00
57.93
W


ATOM
1805
OH2
WAT
W
67
10.789
−6.923
67.951
1.00
40.87
W


ATOM
1806
OH2
WAT
W
68
30.679
−29.809
51.561
1.00
47.80
W


ATOM
1807
OH2
WAT
W
70
14.686
−36.386
52.025
1.00
58.07
W


ATOM
1808
OH2
WAT
W
71
23.286
−34.743
65.028
1.00
64.95
W


ATOM
1809
OH2
WAT
W
74
13.476
−24.792
47.142
1.00
44.00
W


ATOM
1810
OH2
WAT
W
75
27.424
−8.021
52.318
1.00
50.04
W


ATOM
1811
OH2
WAT
W
76
17.185
−11.984
47.384
1.00
68.65
W


ATOM
1812
OH2
WAT
W
77
5.622
−29.583
68.226
1.00
54.52
W


ATOM
1813
OH2
WAT
W
80
18.815
−24.842
68.793
1.00
40.43
W


ATOM
1814
OH2
WAT
W
81
21.343
5.380
55.616
1.00
33.74
W


ATOM
1815
OH2
WAT
W
82
16.590
−29.691
67.097
1.00
46.02
W


ATOM
1816
OH2
WAT
W
83
20.194
−21.601
51.021
1.00
36.86
W


ATOM
1817
OH2
WAT
W
85
14.715
−27.836
70.942
1.00
51.90
W


ATOM
1818
OH2
WAT
W
86
13.436
−29.582
72.049
1.00
50.86
W


ATOM
1819
OH2
WAT
W
87
20.259
−10.157
51.078
1.00
44.59
W


ATOM
1820
OH2
WAT
W
88
26.900
−0.804
66.233
1.00
39.07
W


ATOM
1821
OH2
WAT
W
89
4.820
−9.903
73.149
1.00
40.81
W


ATOM
1822
OH2
WAT
W
90
−0.281
−12.552
65.555
1.00
49.05
W


ATOM
1823
OH2
WAT
W
92
12.724
−5.568
71.586
1.00
53.51
W


ATOM
1824
OH2
WAT
W
93
7.826
−43.002
69.415
1.00
54.98
W


ATOM
1825
OH2
WAT
W
95
20.986
−12.197
43.875
1.00
63.86
W


ATOM
1826
OH2
WAT
W
96
32.091
−11.891
74.247
1.00
56.91
W


ATOM
1827
OH2
WAT
W
97
14.437
−24.757
74.268
1.00
57.27
W


ATOM
1828
OH2
WAT
W
98
30.503
−21.733
47.723
1.00
55.05
W


ATOM
1829
OH2
WAT
W
100
−2.534
−12.377
60.294
1.00
46.67
W


ATOM
1830
OH2
WAT
W
101
6.007
−40.679
73.453
1.00
60.10
W


ATOM
1831
OH2
WAT
W
103
9.741
−6.705
70.312
1.00
51.80
W


ATOM
1832
OH2
WAT
W
104
18.884
−23.389
46.288
1.00
42.13
W


ATOM
1833
OH2
WAT
W
105
8.278
−40.462
72.427
1.00
54.94
W


ATOM
1834
OH2
WAT
W
106
30.185
−18.890
72.770
1.00
50.94
W


ATOM
1835
OH2
WAT
W
110
31.599
−30.829
60.833
1.00
45.28
W


ATOM
1836
OH2
WAT
W
111
7.128
−4.100
40.767
1.00
44.90
W


ATOM
1837
OH2
WAT
W
112
5.094
−27.238
75.588
1.00
59.12
W


ATOM
1838
OH2
WAT
W
113
18.993
−31.333
62.938
1.00
38.54
W


ATOM
1839
OH2
WAT
W
117
27.880
−23.843
50.359
1.00
50.76
W


ATOM
1840
OH2
WAT
W
118
−1.684
−13.822
63.153
1.00
59.71
W


ATOM
1841
OH2
WAT
W
123
17.812
−27.177
71.950
1.00
60.66
W


ATOM
1842
OH2
WAT
W
125
2.093
−27.356
71.451
1.00
48.69
W


ATOM
1843
OH2
WAT
W
126
−3.442
−17.247
61.865
1.00
54.19
W


ATOM
1844
OH2
WAT
W
127
1.759
−10.946
45.001
1.00
41.89
W


ATOM
1845
OH2
WAT
W
128
−8.441
−42.749
77.832
1.00
70.82
W


ATOM
1846
OH2
WAT
W
129
7.800
−36.923
51.217
1.00
53.53
W


ATOM
1847
OH2
WAT
W
130
10.080
−40.723
68.230
1.00
53.30
W


ATOM
1848
OH2
WAT
W
131
26.285
1.358
67.018
1.00
48.30
W


ATOM
1849
OH2
WAT
W
132
22.963
−11.064
60.039
1.00
90.89
W


ATOM
1850
OH2
WAT
W
134
10.353
−4.541
64.097
1.00
56.18
W


ATOM
1851
OH2
WAT
W
137
3.597
−17.838
45.819
1.00
54.21
W


ATOM
1852
OH2
WAT
W
139
−3.897
−18.680
64.339
1.00
54.18
W


ATOM
1853
OH2
WAT
W
140
16.956
−20.939
44.880
1.00
69.00
W


ATOM
1854
OH2
WAT
W
142
14.738
−38.326
59.530
1.00
58.88
W


ATOM
1855
OH2
WAT
W
145
14.855
−23.001
76.133
1.00
43.96
W


ATOM
1856
OH2
WAT
W
147
20.215
−26.877
69.337
1.00
64.57
W


ATOM
1857
OH2
WAT
W
155
16.455
−32.064
62.139
1.00
47.73
W


ATOM
1858
OH2
WAT
W
156
18.383
−23.330
74.540
1.00
59.11
W


ATOM
1859
OH2
WAT
W
160
26.823
−3.191
54.242
1.00
60.75
W


ATOM
1860
OH2
WAT
W
161
34.156
−28.509
60.729
1.00
70.43
W


ATOM
1861
OH2
WAT
W
170
21.242
−28.298
75.598
1.00
51.15
W


ATOM
1862
OH2
WAT
W
171
23.362
4.612
56.588
1.00
46.95
W


ATOM
1863
OH2
WAT
W
172
−6.396
−44.988
78.159
1.00
54.39
W


ATOM
1864
OH2
WAT
W
173
18.594
−21.905
72.366
1.00
56.44
W


ATOM
1865
OH2
WAT
W
174
10.847
−2.441
73.845
1.00
45.58
W


ATOM
1866
OH2
WAT
W
180
12.073
−24.440
77.663
1.00
65.15
W


ATOM
1867
OH2
WAT
W
186
11.906
−40.592
66.069
1.00
71.20
W


ATOM
1868
OH2
WAT
W
194
25.784
−30.114
73.492
1.00
56.89
W


ATOM
1869
OH2
WAT
W
195
3.140
−16.054
72.571
1.00
59.02
W


ATOM
1870
OH2
WAT
W
196
4.981
−17.689
72.622
1.00
95.43
W


ATOM
1871
OH2
WAT
W
197
18.315
−22.761
69.952
1.00
56.52
W


ATOM
1872
OH2
WAT
W
198
−0.003
−0.358
39.802
1.00
51.25
W


ATOM
1873
OH2
WAT
W
199
8.744
−26.111
49.896
1.00
66.37
W


ATOM
1874
OH2
WAT
W
202
24.098
2.931
58.413
1.00
46.53
W


ATOM
1875
OH2
WAT
W
203
16.861
−34.165
43.335
1.00
81.19
W


ATOM
1876
OH2
WAT
W
204
19.382
−26.393
74.298
1.00
67.51
W


ATOM
1877
OH2
WAT
W
206
24.225
3.592
53.248
1.00
58.61
W


ATOM
1878
OH2
WAT
W
207
31.912
−18.264
54.777
1.00
56.00
W


ATOM
1879
OH2
WAT
W
208
11.384
−32.171
71.466
1.00
80.10
W


ATOM
1880
OH2
WAT
W
211
24.900
−11.183
43.677
1.00
52.88
W


ATOM
1881
OH2
WAT
W
212
16.768
−16.252
49.717
1.00
53.52
W


ATOM
1882
OH2
WAT
W
214
10.711
−26.840
77.276
1.00
64.18
W


ATOM
1883
OH2
WAT
W
215
28.159
−2.789
65.544
1.00
53.99
W


ATOM
1884
OH2
WAT
W
216
−5.027
−22.160
56.481
1.00
67.91
W


ATOM
1885
OH2
WAT
W
217
5.564
−10.768
41.516
1.00
62.02
W


ATOM
1886
OH2
WAT
W
218
34.758
−32.991
44.233
1.00
67.86
W


ATOM
1887
OH2
WAT
W
219
15.383
−35.199
64.512
1.00
57.58
W


ATOM
1888
OH2
WAT
W
220
11.042
−0.993
71.541
1.00
40.57
W


ATOM
1889
OH2
WAT
W
221
−1.373
−35.364
55.318
1.00
63.40
W


ATOM
1890
OH2
WAT
W
222
−1.414
−28.056
71.313
1.00
51.06
W


ATOM
1891
OH2
WAT
W
223
0.579
−15.614
44.883
1.00
57.51
W


ATOM
1892
OH2
WAT
W
224
20.213
−21.409
45.285
1.00
59.50
W


ATOM
1893
OH2
WAT
W
226
16.554
−23.850
44.983
1.00
53.48
W


ATOM
1894
OH2
WAT
W
227
31.301
−14.218
66.543
1.00
67.17
W


ATOM
1895
OH2
WAT
W
229
17.319
−12.439
44.761
1.00
62.52
W


ATOM
1896
OH2
WAT
W
230
33.266
−19.036
74.435
1.00
52.88
W


ATOM
1897
OH2
WAT
W
231
−4.526
−13.517
58.476
1.00
63.03
W


ATOM
1898
OH2
WAT
W
232
25.471
−17.826
51.113
1.00
50.66
W


ATOM
1899
OH2
WAT
W
233
30.569
−27.352
65.414
1.00
58.69
W


ATOM
1900
OH2
WAT
W
234
32.392
−27.907
51.765
1.00
56.34
W


ATOM
1901
OH2
WAT
W
235
18.193
−0.620
46.316
1.00
66.93
W


ATOM
1902
OH2
WAT
W
236
28.994
−9.891
51.100
1.00
57.32
W


ATOM
1903
OH2
WAT
W
237
14.361
−17.969
45.938
1.00
67.16
W


ATOM
1904
OH2
WAT
W
239
29.694
−37.643
43.980
1.00
59.17
W


ATOM
1905
OH2
WAT
W
240
9.220
−28.951
72.297
1.00
53.08
W


ATOM
1906
OH2
WAT
W
242
7.337
−31.202
73.280
1.00
80.10
W


ATOM
1907
OH2
WAT
W
243
11.834
−33.939
69.589
1.00
80.10
W


ATOM
1908
OH2
WAT
W
244
11.476
−0.583
66.761
1.00
80.10
W









It will be understood that various details of the invention can be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation—the invention being defined by the claims.

## Claims

1. A method of designing a potential modulator that selectively modulates the activity of a glucose regulated protein 94 kDa (GRP94) polypeptide, the method comprising:
(a) obtaining a crystalline form of a GRP94 ligand binding domain polypeptide complex, wherein said GRP94 polypeptide comprises amino acids 48-316 of SEQ ID NO: 6 complexed with N-ethylcarboxamidoadenosine (NECA) and forms in space group C2 with unit cell parameters of a=99-89.9 Å, b=89.614 Å, c=63.066 Å and β=90.132° or in space group C2221 with unit cell parameters of a=89.200 Å, b=99.180 Å, c=63.071 Å and β=90°;
(b) determining the three-dimensional structure of the crystalline form of the GRP94 ligand binding domain polypeptide from step (a);
(c) analyzing the determined three-dimensional GRP94 ligand binding domain structure by inputting and displaying on a computer the atomic coordinates according to Tables 1-3 to assess the binding pocket dimensions and designing a modulator therefrom; and
(d) synthesizing said modulator based on the three-dimensional structure of the crystalline form of the GRP94 ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of a GRP94 polypeptide is designed.

2. The method of claim 1, wherein the method further comprises contacting a GRP94 ligand binding domain polypeptide with the potential modulator; and assaying the GRP94 ligand binding domain polypeptide for binding of the potential modulator and change in activity of the GRP94 ligand binding domain polypeptide.

3. The method of claim 1, wherein the crystalline form is an orthorhombic form.

4. The method of claim 1, wherein the crystals are such that the three-dimensional structure of the crystallized GRP94 ligand binding domain polypeptide can be determined to a resolution of about 1.8 A or better.

5. A method for identifying a potential GRP94 modulator, the method comprising:
(a) providing atomic coordinates of a GRP94 ligand binding domain selected from the group consisting of Tables 1-3 to a computerized modeling system and displaying said computerized model on said computer;
(b) facilitating the identification of potential ligand binding pocket residues comprising amino acids selected from the group consisting of Met 85, Glu 103, Leu 104, Asn 107, Ala 108, ASP 110, Ala 111, Val 147, ASP 149, Val 152, Gly 153, Met 154, Glu 158, Lys 161, Asn 162, Leu 163, Ala 167, Lys 168, Thr 171, Gly 196, Val 197, Gly 198, Phe 199, Tyr 200, Val 211, Trp 223, Thr 245, Ile 247, and combinations thereof according to Tables 1-3; and
(c) modeling ligands that fit spatially into said identified binding pocket of the GRP94 ligand binding domain to thereby identify a potential GRP94 modulator.

6. The method of claim 5, wherein the method further comprises identifying in an assay for GRP94 -mediated activity a modeled ligand that increases or decreases the activity of the GRP94.

7. A method of identifying modulator that selectively modulates the activity of a GRP94 polypeptide compared to other Hsp90 polypeptides, the method comprising:
(a) providing atomic coordinates of a GRP94 ligand binding domain selected from the group consisting of Tables 1-3 to a computerized modeling system and displaying said computerized model on said computer;
(b) facilitating the identification of potential ligand binding pocket residues comprising amino acids selected from the group consisting of Met 85, Glu 103, Leu 104, Asn 107, Ala 108, ASP 110, Ala 111, Val 147, ASP 149, Val 152, Gly 153, Met 154, Glu 158, Lys 161, Asn 162, Leu 163, Ala 167, Lys 168, Thr 171, Gly 196, Val 197, Gly 198, Phe 199, Tyr 200, Val 211, Trp 223, Thr 245, Ile 247, and combinations thereof according to Tables 1-3; and
(c) modeling a ligand that fits selectively into said identified binding pocket of a GRP94 ligand binding domain wherein said ligand interacts selectively with conformationally constrained residues of said GRP94 ligand binding domain polypeptide that are conserved among Hsp90 subtypes but which are selective for GRP94 polypeptides, whereby a modulator that selectively modulates the activity of a GRP94 polypeptide compared to other Hsp90 polypeptides is identified.

8. The method of claim 7, wherein the method further comprises identifying in a biological assay for GRP94 activity a modeled ligand that selectively binds to GRP94 and increases or decreases the activity of said GRP94 .

9. The method of claim 7, wherein the atomic structure coordinates further comprises a ligand bound to the GRP94 ligand binding domain polypeptide.

10. The method of claim 9, wherein the atomic structure coordinates are the atomic structural coordinates shown in Tables 1-2.

11. The method of claim 9, wherein the ligand is an adenosine derivative.

12. The method of claim 11, wherein the adenosine derivative is NECA.

13. A method of designing a modulator of a GRP94 polypeptide, the method comprising:
(a) selecting a candidate GRP94 ligand;
(b) determining which amino acid or amino acids of a GRP94 polypeptide interact with said ligand by inputting and displaying on a computer a three-dimensional model of a crystallized a GRP94 ligand binding domain polypeptide according to Tables 1-3 and a three dimensional-model of said candidate ligand and identifying the amino acid interaction site(s);
(c) identifying in a biological assay for GRP94 activity the degree to which said candidate ligand modulates the activity of said GRP94 polypeptide;
(d) based upon the determined activity from step (c) and the determined amino acid:ligand interaction sites as determined in step (b), selecting a chemical modification of the candidate ligand to make a potential modified ligand which improves said modified ligands interaction and modulation activity of GRP94 polypeptides;
(e) synthesizing said modified ligand;
(f) contacting the modified ligand with a GRP94 polypeptide;
(g) identifying in a biological assay for GRP94 activity the degree to which the modified ligand modulates the biological activity of the GRP94 polypeptide; and
h) comparing the biological activities from step (c) and step (g) to identify the extent of activity modulation the modified ligand possesses, wherein those having a greater degree of activity modulation are identified and thereby designed.

14. The method of claim 13, wherein the method further comprises repeating steps (a) through (h), if the biological activity of the GRP94 polypeptide in the presence of the modified ligand varies from the biological activity of the GRP94 polypeptide in the presence of the unmodified ligand.

15. An assay method for identifying a compound that inhibits binding of a ligand to a GRP94 polypeptide, the assay method comprising:
(a) designing a test inhibitor compound according to any one of claim 1, 5, 7 or 13, wherein those candidate modulators that inhibit GRP94 activity are selected;
(b) incubating a GRP94 polypeptide with a known ligand in the presence of said test inhibitor compound;
(c) determining an amount of ligand that is bound to the GRP94 polypeptide, wherein decreased binding of ligand to the GRP94 protein in the presence of the test inhibitor compound relative to binding of ligand in the absence of the test inhibitor compound is indicative of inhibition; and
(d) identifying the test inhibitor as an inhibitor of ligand binding if decreased ligand binding is observed, whereby a compound that inhibits binding of a ligand to a GRP94 polypeptide is identified.