# Patent

## Title
Methods of screening for agents that modulate the interaction of RGS and Gαq and urinary incontinence

## Abstract

The present invention provides methods that are useful for the treatment or prevention of smooth muscle disorders such as urinary incontinence and compounds that are useful in such methods.

## Background
This application is entitled to and claims priority to U.S. Provisional Application Ser. No. 60/352,720, filed Jan. 28, 2002, which is hereby incorporated by reference in its entirety.


1. FIELD OF THE INVENTION
The present invention provides methods for treating and/or preventing conditions in smooth muscle such as urinary incontinence and compounds useful in such methods. In certain embodiments of the invention, the compounds are capable of modulating Gαq and RGS complex activity. In one aspect, the invention provides compounds and methods for identifying compounds that have agonizing effects on RGS and that affect the RGS/Gαq complex. The compounds are capable of altering G-protein coupled receptor protein pathway signals in vitro or in vivo.
2. BACKGROUND OF THE INVENTION
Urinary incontinence is a common condition that is a frequent cause of confinement to nursing homes among the elderly. It afflicts significant numbers among both men and women of all ages. Urinary incontinence is believed to currently affect over 12 million people in the United States alone, and to occur in between 15 and 30% of the population over the age of 60. In addition, studies show some degree of daily incontinence reported among as many as 17% of young, apparently healthy women.
Urinary incontinence is a manifestation of the failure to control the muscles of the bladder or urinary sphincter. Incontinence results when the pressure within the bladder is too great as a result of excessive force exerted by the bladder muscles, or when the sphincter muscles are too weak. Urinary incontinence can be a manifestation of other diseases such as Parkinsonism, multiple sclerosis, lesions of the central nervous system, or bladder infections. Interstitial cysts can result in instability of the bladder detusor muscles and a particularly unpleasant form of urge incontinence.
Current treatments for urinary incontinence rely on the control of G protein coupled receptors (GPCR) of the muscarinic class. These GPCR proteins are intracellular proteins that act as transducers of binding by extracellular ligands to cell surface G protein coupled receptors (“GPCRs”). Zhong and Neubig, 2001, Perspectives in Pharmacology 297:837-845, and the references cited therein, provide a review of GPCRs and their functions. Modulation of the signaling pathways downstream of the muscarinic GPCR's is responsible for proper muscle contraction within the bladder, and antagonists of these receptors have been utilized for this purpose, but have problems with specificity and side effects do to a lack of specificity.
The current standard of care is quite unsatisfactory. All of the current drugs now utilized to treat urinary incontinence suffer from polypharmacology and unwanted side effects. Safe and reliable methods and compounds are needed to improve the treatment and/or prevention of conditions involving defects in muscle contraction or the control of muscle contraction with one such example being urinary incontinence.
3. SUMMARY OF THE INVENTION
Accordingly, the present invention provides methods for the treatment of smooth muscle disorders such as urinary incontinence. The invention is based, in part, on the discovery that RGS/Gαq polypeptides complexes are involved in smooth muscle function and that this function can be modulated by agents that increase or decrease the affinity or activity of complex formation of these proteins. Accordingly, the present invention provides compounds that are capable of modulating the interaction of an RGS/Gαq protein complex, methods for identifying such compounds and methods of their use. The proper regulation of RGS/Gαq complexes downstream from GPCR signals is critical for conditions such as urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosis, muscle disorders, skeletal muscle disorders, cardiac muscle disorders, smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, smooth muscle spasms, muscle contraction disorders, and muscle relaxation disorders. As such the compounds of the invention can be used for the treatment of such disorders.
In one aspect, the present invention provides methods for the identification of compounds that can be used to treat and/or prevent disorders such as urinary incontinence. In particular, the present invention provides the use of any RGS polypeptide or any Gαq polypeptide to identify compounds that can bind to and/or modulate the activity of an RGS or a Gαq polypeptide or a complex of the polypeptides. The identification of such compounds can comprise assay methods described herein as well as those known to one of skill in the art. Such compounds can be useful, for example, for the treatment and/or prevention of urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosis, muscle disorders, skeletal muscle disorders, cardiac muscle disorders, preferably smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, preferably smooth muscle spasms, muscle contraction disorders and muscle relaxation disorders. The present invention also encompasses the use of such compounds for the treatment of such disorders.
In another aspect, the present invention provides mutant Gαq polypeptides. Typically, a mutant Gαq polypeptide of the invention comprises a mutation of a conserved methionine residue within its switch III region. The mutant Gαq polypeptides of the invention can be used to screen for compounds useful for the treatment of conditions such as urinary incontinence, or to counter-screen compounds for specificity with regards to compounds that increase or decrease the affinity of RGS binding to Gαq with a resultant effect on Gαq signaling and muscle contraction. In another embodiment the conserved methionine residue in the switch III region defines a compound interaction site for compounds that increases the affinity of Gαq for RGS protein. Compounds which interact with this site may antagonize or agonize Gαq activity and be useful for the treatment and/or prevention of urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosis, muscle disorders, skeletal muscle disorders, cardiac muscle disorders, preferably smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, preferably smooth muscle spasms, muscle contraction disorders and muscle relaxation disorders. The present invention also encompasses the use of such compounds for the treatment of such disorders.
In another aspect, the present invention provides mutant RGS polypeptides. A mutant RGS polypeptide of the invention comprises a DEP domain and a GGL domain, an RGS domain, and, typically, a mutation of a glutamine residue between the DEP domain and the GGL domain. Preferably, the mutant RGS polypeptide of the invention activates Gαq. Experiments here have shown that mutation of this amino acid to other non-conserved amino acids renders compounds I, II and III unable to affect the Gαq pathway. These experiments show convincingly that this amino acid residue defines a site critical for compound action and therefore is the likely compound binding site. The mutant RGS polypeptides of the invention can be used to screen for compounds useful for the treatment of conditions such as urinary incontinence. In another preferred embodiment residue 158 in eat-16 or the conserved motif in an RGS family member defines a binding site for compounds which affect its affinity for Gαq or other proteins, and compounds which bind such site that agonize RGS activity or antagonize Gαq signals or act as uncompetitive inhibitors of the complex such that Gαq is unable to recycle to the active state. Such compounds can be useful, for example, for the treatment and/or prevention of urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosis, muscle disorders, skeletal muscle disorders, cardiac muscle disorders, preferably smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, preferably smooth muscle spasms, muscle contraction disorders and muscle relaxation disorders. The present invention also encompasses the use of such compounds for the treatment of such disorders.
In another aspect, the invention provides computer-readable media embedded with the three-dimensional structural information obtained from the C. elegans RGS polypeptide eat-16, or portions or substrates thereof. This aspect of the invention is illustrated by way of working examples demonstrating the determination and analysis of the three-dimensional structure of eat-16. Such three-dimensional structural information typically includes the atomic structure coordinates of the polypeptide, or the atomic structure coordinates of a portion thereof such as, for example, an active or binding site, but may include other structural information, such as vector representations of the atomic structure coordinates, etc. The computer readable media of the invention may further comprise additional information that is useful for representing the three-dimensional structure, including, but not limited to, thermal parameters, chain identifiers, and connectivity information.
In yet another aspect, the invention provides computer-readable media embedded with the three-dimensional structural information obtained from the human RGS polypeptide RGS-7, or portions or substrates thereof. This aspect of the invention is illustrated by way of working examples demonstrating the determination and analysis of the three-dimensional structure of RGS-7. Such three-dimensional structural information typically includes the atomic structure coordinates of the polypeptide, or the atomic structure coordinates of a portion thereof such as, for example, an active or binding site, but may include other structural information, such as vector representations of the atomic structure coordinates, etc. The computer readable media of the invention may further comprise additional information that is useful for representing the three-dimensional structure, including, but not limited to, thermal parameters, chain identifiers, and connectivity information.
The atomic structure coordinates of the invention have a variety of uses. For example, the coordinates are useful for a variety of molecular modeling and computer-based screening applications to, for example, computationally design and identify compounds that bind the eat-16 or RGS-7 polypeptide or a portion or fragment of the eat-16 or RGS-7 polypeptide, such as domains therein or the active site. Such compounds may be used as lead compounds in pharmaceutical efforts to identify compounds that agonize or inhibit eat-16 or RGS-7 as a therapeutic approach toward the treatment of, e.g., urinary incontinence.
The present invention also comprises compounds identified by screening compounds or libraries of compounds against the RGS or Gαq polypeptides or complexes thereof. Any compound identified by the methods of the invention can be assayed to determine its efficacy of binding or their modulation of, for example, Gαq activity or RGS/Gαq affinity.
Compounds which modulate complexes containing RGS and/or Gαq polypeptides have utility in all diseases where hyperactivating or inactivating G-protein coupled receptors would be of therapeutic value, including urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosis, muscle disorders, skeletal muscle disorders, cardiac muscle disorders, smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, smooth muscle spasms, muscle contraction disorders, muscle relaxation disorders.



4. BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B provide an amino acid sequence alignment of RGS polypeptides and G polypeptides of the invention (RGS7 (SEQ ID NO.:7); EGL-10 (SEQ ID NO.:8); RGS11 SEQ ID NO.:9); EAT-16 SEQ ID NO.:10); RGS9 (SEQ ID NO.:11); EGL-30 (SEQ ID NO.:12); G(q) (SEQ ID NO.:13); G(i) (SEQ ID NO.:14); G(t) (SEQ ID NO.:15); G(o) (SEQ ID NO.:16); G(x) (SEQ ID NO.:17); G(s) (SEQ ID NO.:18); and G(13) (SEQ ID NO.:19));
FIGS. 2A-2D provide the sequences of exemplary RGS and G alpha q wild-type and mutant polypeptides of the invention (wt egl-30 ((SEQ ID NO.:20); mutant egl-30 (SEQ ID NO.:21); wt eat-16(SEQ ID NO.:22); and mutant eat-16(SEQ ID NO.:23).
FIG. 3A provides compound I;
FIG. 3B provides compound II;
FIG. 3C provides compound III;
FIG. 4 provides the effects of compounds I and III on smooth muscle cells;
FIG. 5 provides the results of a calcium assay illustrating that compound I specifically inhibits Gαq signalling;
FIG. 6 provides an immunoprecipitation blot indicating that compounds I and II increase the affinity of Gαq and RGS;
FIG. 7 provides a bioinformatics work flow diagram;
FIG. 8A provides a depiction of the PDE binding site in the Gα/RGS/PDE trimer;
FIG. 8B provides a depiction of compound II docked into the binding site in the Gα/RGS/PDE trimer, showing surface complementarity.
FIG. 9A provides a ribbon diagram of the eat-16 model;
FIG. 9B provides a “molecular elevation” plot view of the eat-16 model;
FIG. 9C provides the surface of the eat-16 model colored according to hydrophobic character;
FIG. 9D provides superposition of compound II, stick figure, onto the hydrophobic pocket, shown in white, showing that they are of similar size; and
FIG. 10 provides an illustration of a computer system for use in the present invention.
FIG. 11 provides the effects of compound III on the micturition pressure and bladder capacity of a normal rabbit: each bar represents the mean +/− SEM of between 3 and 5 individual preparations.
FIG. 12 provides the effects of compound III on initial bladder pressure (at 10% of bladder capacity) and compliance (resistance to stretch between 10% and 20% of capacity). Each bar represents the mean +/− SEM of 4-6 individual preparations.
FIG. 13 provides the effects of compound III on the frequency and amplitude of unstable bladder contractions induced by partial outlet obstruction. Each bar represents the mean +/− SEM of 4-6 individual preparations.



5. BRIEF DESCRIPTION OF THE TABLES
Table 1 provides a structure-based sequence alignment of eat-16 and rat RGS-4;
Table 2 provides a sequence alignment of human RGS-7 and rat RGS-4;
Table 3 provides the atomic structure coordinates of wild type eat-16; and
Table 4 provides the atomic structure coordinates of wild type RGS-7.
6. ABBREVIATIONS
The amino acid notations used herein for the twenty genetically encoded L-amino acids are conventional and are as follows:
















One-Letter
Three-Letter



Amino Acid
Symbol
Symbol









Alanine
A
Ala



Arginine
R
Arg



Asparagine
N
Asn



Aspartic acid
D
Asp



Cysteine
C
Cys



Glutamine
Q
Gln



Glutamic acid
E
Glu



Glycine
G
Gly



Histidine
H
His



Isoleucine
I
Ile



Leucine
L
Leu



Lysine
K
Lys



Methionine
M
Met



Phenylalanine
F
Phe



Proline
P
Pro



Serine
S
Ser



Threonine
T
Thr



Tryptophan
W
Trp



Tyrosine
Y
Tyr



Valine
V
Val










As used herein, unless specifically delineated otherwise, the three-letter amino acid abbreviations designate amino acids in the L-configuration. Amino acids in the D-configuration are preceded with a “D-.” For example, Arg designates L-arginine and D-Arg designates D-arginine. Likewise, the capital one-letter abbreviations refer to amino acids in the L-configuration. Lower-case one-letter abbreviations designate amino acids in the D-configuration. For example, “R” designates L-arginine and “r” designates D-arginine.
Unless noted otherwise, when polypeptide sequences are presented as a series of one-letter and/or three-letter abbreviations, the sequences are presented in the N→C direction, in accordance with common practice wherein “N” refers to the amino terminus of a polypeptide, and “C” refers to the carboxy terminus of a polypeptide.
7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is based, in part, on the inventors' discovery of the importance of Gαq polypeptides and RGS polypeptides in cellular pathways that have been linked to a variety of conditions such as urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosis, muscle disorders, skeletal muscle disorders, cardiac muscle disorders, smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, smooth muscle spasms, muscle contraction disorders, muscle relaxation disorders.
The present invention targets a protein complex of Gαq and RGS, which regulates aspects of signals downstream of GPCR signaling, and therefore allows for better specificity in controlling that signal due to tissue specific expression of RGS proteins. Ligand binding to GPCRs results in the activation of one or more heterotrimeric G-proteins. G-proteins comprise several families including Gi, Gq, Gs and G12. Heterotrimeric G-proteins in their resting states are complexes comprised of three subunits, known as Gα, Gβ and Gγ. In mammalian cells there are at least three classes of subunits, comprising 15 Gα, 5 Gβ and 7 Gγ genes. G-proteins have also been identified in invertebrate animals and some of these are capable of functionally substituting for their mammalian counterparts and are therefore equivalent.
The Gα subunits have intrinsic GTPase activity. This structure and associated mechanism are shared with the monomeric GTP-binding proteins of the ras superfamily. In the resting state, the complex contains bound GDP (Gα-GDP .beta..gamma.). Activation sets in motion a release of GDP and the subsequent binding of GTP resulting in the concurrent dissociation of the complex into two signaling complexes: Gα-GTP and .beta..gamma.. Signaling through Gα-GTP is terminated by GTP hydrolysis to GDP, and this reaction is catalyzed by regulator of G-protein signaling (“RGS”) proteins. The regulation of Gαq by RGS proteins has been shown to depend on the “RGS” catalytic domain of these proteins and in vitro many RGS domain containing members of the RGS family are interchangeable in this activity. Specifically RGS6 (Genbank Accession No. XM.sub.—027292; SEQ ID NO.:27), RGS7 (Genbank Accession No. XM.sub.—010645), RGS9 (Genbank Accession No. XM.sub.—032141), RGS11 (Genbank Accession No. XM.sub.—027524, Genbank Accession No. XM.sub.—027525), RGS 1 (Genbank Accession No. XM.sub.—001330), RGS2 (Genbank Accession No. XM.sub.—029884; SEQ ID NO.:26), RGS3 (Genbank Accession No. XM.sub.—005324), RGS4 (Genbank Accession No. XM.sub.—034023), RGS5 (Genbank Accession No. XM.sub.—002185), and RGS16 (Genbank Accession No. XM.sub.—046463) have been published previously to effect in vitro Gαq signaling in an essentially equivalent fashion. Upon catalysis, Gα-GDP is free to reassociate with βγ to reform the inactive, heterotrimeric complex.
The present invention provides compounds that modulate Gαq activity, RGS activity and/or activity of the protein complex of RGS and Gαq in vivo and/or in vitro. In particular, the present invention provides methods of using Gαq polypeptides, mutant Gαq polypeptides, RGS polypeptides and mutant RGS polypeptides to identify compounds that can be used to treat or prevent conditions such as urinary incontinence.
7.1 Methods of Identifying Compounds that Modulate the Activity of RGS and Gαq Complexes
RGS polypeptides of the invention, such as human RGS- and its conserved human homologs, can be used to identify compounds that selectively bind to and/or modulate the activity of the RGS polypeptide, a domain thereof or complex thereof with Gαq molecules. In addition, any of the mutant RGS polypeptides of the invention discussed below, can be used to identify a compound that selectively binds to and/or modulates the activity of the corresponding wild-type polypeptide, or domain thereof or complex with Gαq. Such compounds include, for example, compound I (FIG. 3A) and compound II (FIG. 3B) which are also described in co-pending U.S. Provisional Application No. 60/336,865, filed Nov. 2, 2001, the content of which is hereby incorporated by reference in its entirety.
As used in the present invention, the term modulation refers to a change in the activity of an active agent such as a polypeptide of the invention. The activity can be increased or decreased as measured by techniques described herein for detecting the activity of the active agent. The change in activity can be detected, for example, when the active agent is contacted with a compound of the invention. A modulator of an active agent refers to a substance, for example a compound of the invention, that is capable of altering the activity of the active agent.
In general, such methods comprise contacting a test compound with a polypeptide of the invention and assaying for the presence of the bound test compound or assaying for modulation of the activity of the polypeptide. The polypeptide can be, for example, expressed within a cell, and immunoprecipitated with an antibody according to standard protocols in the presence or absence of a radioactively or fluorescently labeled compound. The binding of the compound to the immunoprecipitated protein or protein complex can be measured by the degree of radiation or fluorescence that is precipitated with the protein. Controls from cells not transfected and expression the polypeptide or competition studies with non-radioactive compound can be utilized to prove the specificity of binding.
As used herein the term “selectively binds” refers to a compound (e.g., an antibody, a peptide, a lipid or a small organic molecule) that binds to a native polypeptide or to a chimeric polypeptide or a complex of polypeptides preferentially relative to other unrelated polypeptides. A compound selectively binds to a wild-type polypeptide or a mutant polypeptide of the invention if it has at least a 10%, preferably at least a 25%, at least a 50%, at least a 75%, at least a 90%, at least a 95%, or at least a 100% higher affinity and/or avidity for the native polypeptide or mutant polypeptide than an unrelated polypeptide.
The assay for the presence of the bound test compound can be any assay known to those of skill in the art to be useful for assaying binding to the wild-type polypeptide or mutant polypeptide and/or any assay known to those of skill in the art to be useful for assaying activation of the wild-type polypeptide or mutant polypeptide or protein complexes thereof. In particularly convenient embodiments of the invention, the presence of the test compound can be assayed by detecting the activity of a downstream effector of the wild-type polypeptide or mutant polypeptide such as phospholipase C (PLC) activity and/or mobilization of intracellular calcium. Examples of such assays are discussed in detail below. In addition direct binding of a labeled compound to a polypeptide or complex of a polypeptide can be detected by ligand binding assays known to one skilled in the art.
The assay for the presence of the bound test compound can be any assay known to those of skill in the art to be useful for assaying binding to the wild-type polypeptide or mutant polypeptide and/or any assay known to those of skill in the art to be useful for assaying activation of the wild-type polypeptide or mutant polypeptide or protein complexes thereof. In certain embodiments of the invention, the presence of the test compound can be assayed by detecting the activity of a downstream effector of a wild-type polypeptide or mutant polypeptide such as phospholipase C (PLC) activity and/or mobilization of intracellular calcium. Examples of such assays are discussed in detail herein. Optionally, a compound that binds a mutant polypeptide can then be tested against a corresponding wild-type polypeptide from the same species or from another species according to standard techniques.
In addition, direct binding of a labeled compound to a polypeptide or complex of a polypeptide is standard for ligand binding assays known to one skilled in the art. Direct binding of radioactive compounds to proteins or displacement of a radioligand by a subsequent higher affinity compound is often utilized as a method of screening for novel compounds by one skilled in the art.
For example, a Gαq polypeptide and an RGS polypeptide can be produced according to methods known to those of skill in the art, such as those discussed in detail below. The polypeptides can then be contacted with a candidate compound under suitable conditions. The polypeptides can then be assayed for complex formation according to standard techniques such as immunoprecipitation. In order to conduct assays, one or more of the components, such as the candidate compound, the Gαq polypeptide or the RGS polypeptide, can be suitably labeled for use in the assay for complex formation, as will be apparent to those of skill in the art. For instance, the candidate compound can be radiolabeled to facilitate radioassays. Also for instance, either or both polypeptides can be epitope-labeled to facilitate immunoprecipitation assays.
Such methods can be used to identify compounds that interact with and produce agonist or antagonistic activity on RGS/Gαq complexes and subsequent signals downstream of these complexes. Such perturbations of signaling are useful for the treatment and/or prevention of, for example, urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosismuscle disorders, skeletal muscle disorders, cardiac muscle disorders, preferably smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, preferably smooth muscle spasms, muscle contraction disorders and muscle relaxation disorders.
Fluorescence resonance energy transfer microscopy (FRET) can also be used to identify compounds that affect the affinity of RGS and G alpha proteins. FRET technology is capable of detecting the affinity of interactions of two proteins. Compounds which inhibit this signal or increase this signal indicating a stronger or weaker affinity of RGS and G alpha q for one another can be found utilizing this technology as a screen. Such techniques are described, for example, in PCT publication WO 9806737 and in Janetopoulous et al., 2001, Science 291:2408-2410, the contents of which are hereby incorporated by reference in their entireties. For example, an RGS polypeptide can be fused to CFP or YFP using methods and materials readily available to those of skill in the art. Other similar fusion proteins can be easily designed by one skilled in the arts of Polymerase chain reaction (PCR) and FRET technologies, and either N-terminal protein fusions or C terminal protein fusions utilizing other conserved RGS proteins could easily be utilized by one skilled in the arts and simply substituted into this screening assay.
In addition, BRET technology which is based on very similar principles could also be utilized; the use of BRET is extensively taught in WO 9966324, the content of which is hereby incorporated by reference in its entirety. In this case PCR of RGS family members, G alpha q or G beta 5 could be utilized to create expression vectors and tools suitable for BRET analysis.
Other assays can be used to detect G-protein activation (see, e.g., “Regulation of G Protein-Coupled Receptor Function and Expression” ed. Benovic, J. L. pp 119-132., 2000, Wiley-Liss, New York). Such assays include receptor-stimulated GTP Binding to Gα subunits. Since activation of GPCR results in GDP-GTP exchange in the Gα subunit, this exchange can be quantified and used as a direct measurement of receptor-G protein interaction. This typically involves the use of radiolabeled guanine nucleotide (35S-GTPγS or α32P-GTP) incubated with the receptor (either in cell-free membrane preparations or artificial lipid membranes). The amount of 35S-GTPγS incorporated can be used as a measure of the extent of G protein activation. Another assay for receptor mediated G-protein activation is measurement of the hydrolysis of GTP by Gα. The final outcome of Gα activation is hydrolysis of bound GTP to GDP by intrinsic GTPase activity. Using γ32P-GTP, the release of 32Pi upon GTP-GDP exchange can be used as an indication of G-protein activation.
Such methods can be used to identify compounds that are useful for the treatment and/or prevention of, for example, urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosismuscle disorders, skeletal muscle disorders, cardiac muscle disorders, preferably smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, preferably smooth muscle spasms, muscle contraction disorders and muscle relaxation disorders.
Furthermore, compounds that interact with and produce agonist or antagonistic activity on RGS/Gαq complexes and subsequent signals downstream of these complexes can be further tested in in vivo assays to assess their utility in the treatment and/or prevention of, for example, urinary incontinence, depression, anxiety, arrhythmia, cognition, psychosismuscle disorders, skeletal muscle disorders, cardiac muscle disorders, preferably smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, preferably smooth muscle spasms, muscle contraction disorders and muscle relaxation disorders. The in vivo assays can be any assay known to one of skill in the art to be effective in assessing a test compound's utility in treating such disorders known to one of skill in the art without limitation.
When testing a compound's effect on urinary incontinence, the in vivo assay can be an in situ assay that tests a compound's effect on bladder cystometry. In other embodiments, the in vivo assay can be an in situ assay that tests a compound's effect on bladder contractile response. In certain embodiments, the bladder can be a normal bladder. In other embodiments, the bladder can be a hypertrophied bladder. These assays can be used to identify compounds that decrease initial bladder pressure during filling, increase bladder compliance, and decrease the amplitude of unstable bladder contractions. A decrease in initial bladder pressure during filling, decreased resistance to stretch (increased compliance), and inhibition of the amplitude of unstable bladder contractions, would all be beneficial in the treatment of urinary incontinence, and in the treatment of bladder dysfunction secondary to benign prostatic hyperplasia (“BPH”).
7.2 Gαq Polypeptides
In the methods of identifying compounds of the invention, a Gαq polypeptide or a complex of a Gαq with a related RGS polypeptide can be used to identify compounds that are useful for the treatment of conditions such as urinary incontinence. Preferred Gαq polypeptides for use in methods to identify such compounds include human Gαq polypeptides such as those described in GI:12620875 and GI:1181671, the contents of which are hereby incorporated by reference in their entireties.
In certain embodiments of the invention, mutant Gαq polypeptides that display increased Gαq activity in vivo and/or in vitro or which define a binding site for compounds which affect the affinity of complex formation between Gαq and RGS proteins can be used to identify compounds of the invention. According to this aspect of the invention, the mutant Gαq polypeptide comprises a mutation of a conserved methionine residue in its switch III domain. Preferably, the methionine residue is mutated to a hydrophobic residue. More preferably, the methionine residue is mutated to an aliphatic residue. Most preferably, the methionine residue is mutated to isoleucine.
In preferred embodiments, the remainder of the amino acid sequence of the mutant Gαq polypeptide corresponds identically to the amino acid sequence of the wild-type Gαq polypeptide. However, in other embodiments of the invention, the remainder of the polypeptide can comprise additional mutations. The mutations can, for example, be conservative or non-conservative. For example, a mutated residue of the mutant polypeptide can belong to the same amino acid class or sub-class as the corresponding residue of the wild type Gαq polypeptide.
Preferably, the mutant Gαq polypeptide displays increased Gαq activity in vivo and/or in vitro. Gαq activity can be measured readily according to assays known to those of skill in the art. For example, Gαq activity can be measured by assaying the activity of downstream effectors of Gαq such as phospholipase C (PLC) activity or mobilization of intracellular calcium. Calcium mobilization can be observed by contacting cells comprising the mutant Gαq polypeptide with an appropriate calcium-sensitive dye. Changes in fluorescence of the dye indicate changes in intracellular calcium resulting from the activation of a Gαq —coupled GPCR. Such changes can be measured advantageously in whole cells in “real-time” (See, e.g., Berridge et al., Nature Reviews 2000 1:11-21). In another method, a mutant Gαq polypeptide can be expressed in Xenopus laevis oocytes followed by measurement of calcium activated chloride currents (see Weber, 1999, Biochim Biophys Acta 1421:213-233).
7.3 RGS Polypeptides
In the methods of the identifying compounds of the invention, an RGS polypeptide or a complex of an RGS polypeptide with a related Gαq polypeptide can be used to identify compounds useful for the treatment of conditions such as urinary incontinence. Preferred RGS polypeptides include the human RGS polypeptides RGS6, RGS7, RGS9, RGS11, RGS1, RGS2, RGS3, RGS4, RGS5, and RGS16.
RGS proteins are known to regulate agonist-evoked calcium oscillations in muscle which control proper bladder function. In particular, RGS proteins are thought to catalyze the hydrolysis of GTP by Gα thereby regulating the activity of Gα. Thus, by modulating these RGS proteins, it should be possible to control the strength and timing of bladder muscle contractions and therefore modulate diseases in the area of urinary incontinence, as well as cardiovascular and neuropathology. In the area of urinary incontinence it can reasonably be expected that compounds which act as agonists of bladder specific RGS protein activity will have therapeutic effects on the muscle spasms characteristic of incontinence disease.
The founding member of the regulator of G-protein signals is yeast SST-2, a protein identified in genetic screens for negative regulators of the pheromone response pathway in yeast. Genetic analysis in Caenorhabditis elegans (C. elegans) uncovered the SST-2 related gene, Egl-10, which was shown to negatively regulate C. elegans Gα0 involved in the control of egg-laying. RGS proteins have been found in all eukaryotic species for which searches have been performed, and are generally split into several different families based on protein structure. All of the RGS proteins share a common “RGS” core domain, and all are thought to affect the duration of active Gα by catalyzing GTP hydrolysis.
RGS proteins have been found in all eukaryotic species for which they have been searched, and are generally split into several different families based on protein structure. All of the RGS proteins share a common “RGS” core domain, and all are thought to affect the duration of active Gα by catalyzing GTP hydrolysis. These RGS domains in many cases are interchangeable in their activity. A subfamily of RGS proteins has been identified in which each member possesses a “DEP” (disheveled, Egl-10, pleckstrin) domain, and a “GGL” (G-protein γ subunit-like) domain, in addition to, their core RGS domain. The functions of these other domains is not clear although there is some evidence that they may control the cellular localization and protein-protein interactions of this class of RGS proteins. In C. elegans there are two RGS homologues that contain the DEP and GGL domains: egl-10 and eat-16; while in humans there are four that have been identified thus far: RGS 6, 7, 9, and 11. This family of RGS proteins is found predominantly in the nervous system, and the extent of their cellular roles is still being determined.
In one aspect, the present invention provides mutant RGS polypeptides that increase Gαq activity in vivo and/or in vitro. According to this aspect of the invention, the mutant RGS polypeptide comprises an RGS domain, a DEP domain, a GGL domain and a mutation of a glutamine residue between the DEP domain and the GGL domain. Preferably, the glutamine residue is mutated to a hydrophilic residue. More preferably, the methionine residue is mutated to a basic residue. Most preferably, the methionine residue is mutated to lysine. In preferred embodiments, the mutant RGS polypeptide is a mutant eat-16 polypeptide (see FIG. 2D) from C. elegans or a mutant human RGS-7.
In preferred embodiments, the remainder of the amino acid sequence of the mutant RGS polypeptide corresponds identically to the amino acid sequence of the wild-type RGS polypeptide. However, in other embodiments of the invention, the remainder of the polypeptide can comprise additional mutations. The mutations can be, for example, conservative or non-conservative. In certain embodiments, a mutated residue of the mutant polypeptide can belong to the same amino acid class or sub-class as the corresponding residue of the wild type RGS polypeptide.
Preferably, the mutant RGS polypeptide of the invention modulates, e.g. increases, Gαq activity in vivo or in vitro. Gαq activity can be measured readily according to assays known to those of skill in the art such as those described above. In certain embodiments, mutant C. elegans RGS polypeptides of the invention can be assayed for resistance to the egg-laying defects caused by compound I, II or III as described above.
7.4 Production of Polypeptides
The native and mutated polypeptides described herein may be chemically synthesized in whole or part using techniques, that are well-known in the art (see, e.g., Creighton, Proteins: Structures and Molecular Principles, 1983 W. H. Freeman & Co., NY.). Alternatively, methods that are well known to those skilled in the art can be used to construct expression vectors containing the native or mutated polypeptide coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.
A variety of host-expression vector systems may be utilized to express the polypeptide coding sequence. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the polypeptide coding sequence; or animal cell systems. The expression elements of these systems vary in their strength and specificities.
Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that causes mRNAs to be initiated at high frequency.
Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as the T7 promoter, pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies of the tyrosine kinase domain DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce eat-16 or egl-30. Identification of polypeptide expressing host cell clones may be done by several means, including but not limited to immunological reactivity with antibodies with specificity for the polypeptide, and the presence of host cell-associated polypeptide activity.
Expression of cDNA encoding the polypeptide may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes.
To determine cDNA sequence(s) that yields optimal amounts of polypeptide and/or levels of polypeptide activity, modified cDNA molecules are constructed. Host cells are transformed with the cDNA molecules and the levels of RNA and/or protein are measured.
Levels of polypeptide in host cells are quantitated by a variety of methods such as immunoaffinity and/or ligand affinity techniques, polypeptide-specific affinity beads or polypeptide-specific antibodies are used to isolate 35S-methionine labeled or unlabeled polypeptide protein. Labeled or unlabeled polypeptide is analyzed by SDS-PAGE. Unlabeled polypeptide is detected by Western blotting, ELISA or RIA employing polypeptide-specific antibodies.
Following expression of the polypeptide in a recombinant host cell, the polypeptide may be recovered to provide the polypeptide in active form. Several polypeptide purification procedures are available and suitable for use. Recombinant polypeptide may be purified from cell lysates or from conditioned culture media, by various combinations of, or individual application of, fractionation, or chromatography steps that are known in the art.
In addition, recombinant polypeptide can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full length nascent polypeptide or fragments thereof.
Alternatively, the polypeptide may be recovered from a host cell in an unfolded, inactive form, e.g., from inclusion bodies of bacteria. Polypeptides recovered in this form may be solublized using a denaturant, e.g., guanidinium hydrochloride, and then refolded into an active form using methods known to those skilled in the art, such as dialysis.
7.5 Further Manipulations of the RGS or Gαq Polypeptide Structures and Binding Molecules
Once an RGS or Gαq polypeptide-binding compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or chemical 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, polarity and charge as the original group. For selection of appropriate groups, any of several chemical models can be used, e.g., isolobal or isosteric analogies. Groups known to be bio-isosteres of one another are particularly preferred. One of skill in the art will understand that substitutions known in the art to alter conformation should be avoided. Such altered chemical compounds may then be analyzed for efficiency of binding to or modulation of the activity of an RGS polypeptide or a Gαq polypeptide, or a complex thereof, by the methods described in detail above.
7.6 Activity of Binding Molecules
The binding molecules described above can be synthesized according to synthetic techniques well known to those of skill in the art. The binding molecules preferably modulate the activity of the RGS or Gαq polypeptide and/or a molecule that interacts with the RGS or Gαq polypeptide. The activity of a binding molecule can be assayed easily by methods well known to those of skill in the art. For instance, an RGS or Gαq polypeptide or a cell comprising the RGS or Gαq polypeptide can be contacted with the binding molecule and then assayed for modulation of RGS activity or Gαq activity. Assays for RGS activity or Gαq activity are described in detail above.
Preferably, binding molecules may be identified by high throughput screening methods, according to which large libraries of ligands are screened against a particular target. A large library of ligands preferably contains more than 1,000 distinct ligands, more preferably contains more than 10,000 distinct ligands, even more preferably contains more than 100,000 distinct ligands and most preferably contains more than 1,000,000 distinct ligands. High throughput screening techniques typically employ robotically controlled assay systems, and take advantage of the latest improvements in miniaturization and automation. Samples are typically assayed on 96-well plates or microtiter plate arrays, and measurements may be taken in parallel. For an overview of high throughput screening techniques, see, for example, Razvi, E. S., “High-Throughput Screening—Where Are We Today?,” Drug & Market Development Publications, (June 1999), and Razvi, E. S., “Industry Trends in High-Throughput Screening,” Drug & Market Development Publications, (August 2000).
7.7 Therapeutic Methods
The present invention also provides methods of treating conditions such as urinary incontinence by administering a therapeutically effective amount of an agent of the invention or a compound identified in the methods described above.
The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant Gαq activity or smooth muscle finction. In particular, the compounds of the invention can be used to treat or prevent urinary incontinence, depression, anxiety, arrythmia, cognition, psychosismuscle disorders, skeletal muscle disorders, cardiac muscle disorders, smooth muscle disorders, muscle spasms, skeletal muscle spasms, cardiac muscle spasms, smooth muscle spasms, muscle contraction disorders, muscle relaxation disorders.
A compound of the invention can be administered in a composition comprising the compound to treat or prevent the disorder.
7.7.1 Compositions
The pharmaceutical compositions of the invention include compositions which comprise compounds of the invention. These compounds are also referred to herein as “active compounds” or “active agents.”
The compositions of the invention typically comprise an active agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
7.7.2 Effective Dosages
The agents of the invention, or compositions thereof, will generally be used in an amount effective to achieve the intended purpose. Of course, it is to be understood that the amount used will depend on the particular application.
For example, for use to treat urinary incontinence, a therapeutically effective amount of an agent, or composition thereof, is applied or administered to an animal or human in need thereof. By therapeutically effective amount is meant an amount of agent or composition that inhibits or ameliorates urinary incontinence in the subject. The actual therapeutically effective amount will depend on a particular application. An ordinarily skilled artisan will be able to determine therapeutically effective amounts of particular agents for particular applications without undue experimentation using, for example, the in vitro assays for the particular disease target known to those of skill in the art.
For use to treat or prevent diseases related to the function or abnormal expression of a Gαq polypeptide, the agents of the invention, or compositions thereof, are administered or applied in a therapeutically effective amount. By therapeutically effective amount is meant an amount effective to ameliorate the symptoms of, or ameliorate, treat or prevent diseases related to the function or abnormal expression of the Gαq polypeptide. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating agent concentration range that includes the I50 as determined in cell culture (i.e., the concentration of the agent that is lethal to 50% of a cell culture), the MIC, as determined in cell culture (i.e., the minimal inhibitory concentration for growth) or the I100 as determined in cell culture (i.e., the concentration of the agent that is lethal to 100% of a cell culture). Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
The amount of agent administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
The therapy may be repeated intermittently. The therapy may be provided alone or in combination with other drugs, such as for example other antiviral entities or other pharmaceutically effective entities.
7.7.3 Toxicity
Preferably, a therapeutically effective dose of the agents described herein will provide therapeutic benefit without causing substantial toxicity.
Toxicity of the agents described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. Agents which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the agents described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1, p.1).
7.7.4 Therapeutic Methods
In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression of a Gαq polypeptide or a molecule that interacts with a Gαq polypeptide, by administering to the subject a composition comprising an agent of the invention. In another aspect, the present invention provides a method of treating or preventing such a disease or condition by administering to the subject composition comprising a nucleic acid encoding a polypeptide of the invention. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
Another aspect of the invention pertains to methods of modulating expression or activity of a Gαq polypeptide or a molecule that interacts with a Gαq polypeptide. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the Gαq polypeptide or molecule that interacts with the Gαq polypeptide. An agent that modulates activity can be an agent as described herein, such as a compound of the invention. In one embodiment, the agent stimulates one or more of the biological activities of the Gαq polypeptide. Examples of such stimulatory agents include a compound of the invention. In another embodiment, the agent inhibits one or more of the biological activities of the Gαq polypeptide. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a Gαq polypeptide or a molecule that interacts with a Gαq polypeptide. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity. In another embodiment, the method involves administering a compound of the invention as therapy to compensate for reduced or aberrant expression or activity of the Gαq polypeptide or a molecule that interacts with the Gαq polypeptide.
7.8 Structure of C. elegans eat-16
The present invention provides, for the first time, the high-resolution predicted three-dimensional structures and atomic structure coordinates of eat-16. The specific methods used to obtain the structure coordinates are provided in the examples, infra. The atomic structure coordinates of eat-16, are listed in Table 3, infra.
The following abbreviations are used in Table 3:
“Residue” refers to amino acid by number.
“Atom Name” refers to the atom whose coordinates are provided.
“X coord”, “Y coord” and “Z coord” provide the Cartesian coordinates of the element.
Structure coordinates for eat-16 according to Table 3 may be modified by mathematical manipulation. Such manipulations include, but are not limited to, fractionalization of the raw structure coordinates, integer additions or subtractions to sets of the raw structure coordinates, inversion of the raw structure coordinates and any combination of the above.
Those having skill in the art will recognize that atomic structure coordinates are not without error. Thus, it is to be understood that any set of structure coordinates obtained for eat-16, that have a root mean square deviation (“r.m.s.d.”) of less than or equal to about 2.0 Å when superimposed, using backbone atoms (N, Cα, C and O), on the structure coordinates listed in Table 3 are considered to be identical with the structure coordinates listed in the Table when at least about 50% to 100% of the backbone atoms of eat-16 are included in the superposition.
7.9 Structure of RGS-7
The present invention provides, for the first time, the high-resolution three-dimensional structures and atomic structure coordinates of RGS-7. The specific methods used to obtain the structure coordinates are provided in the examples, infra. The atomic structure coordinates of RGS-7, are listed in Table 4, infra.
The following abbreviations are used in Table 4:
“Residue” refers to amino acid by number.
“Atom Name” refers to the atom whose coordinates are provided.
“X coord”, “Y coord” and “Z coord” provide the Cartesian coordinates of the element.
Structure coordinates for RGS-7 according to Table 4 may be modified by mathematical manipulation. Such manipulations include, but are not limited to, fractionalization of the raw structure coordinates, integer additions or subtractions to sets of the raw structure coordinates, inversion of the raw structure coordinates and any combination of the above.
Those having skill in the art will recognize that atomic structure coordinates are not without error. Thus, it is to be understood that any set of structure coordinates obtained for eat-16, that have a root mean square deviation (“r.m.s.d.”) of less than or equal to about 2.0 Å when superimposed, using backbone atoms (N, Cα, C and O), on the structure coordinates listed in Table 4 are considered to be identical with the structure coordinates listed in the Table when at least about 50% to 100% of the backbone atoms of eat-16 are included in the superposition.
7.9 Structure Coordinates
7.9.1 Obtaining Structure Coordinates
Structure coordinates of eat-16 and RGS-7 are provided in Table 3 and Table 4, respectively. Alternatively, structure coordinates of eat-16 or RGS-7 can be obtained using principles of homology modeling according to the description of the present invention. For instance, such homology models can be based on structures of related polypeptides known to those of skill in the art such as rat RGS-4 (Tesmer et al., 1997, Cell:251-61; Protein Data Bank structure 1AGR). Rat RGS-4 displays 33% primary sequence homology to eat-16 and 34% primary sequence identity to human RGS-7 (see Tables 1&2).
Programs are available to one of ordinary skill in the art for the purpose of homology modeling, often embedded within a larger molecular modeling package or suite of related programs. For examples of homology modeling tools, see:

    
    
        1. SEGMOD, part of LOOK (Levitt, 1992, J. Mol. Biol. 226: 507-533; Levitt, 1983, J. Mol. Biol. 170: 723-764; formerly available from the Molecular Applications Group, Palo Alto, Calif.)
        2. MoE: The Structure Prediction tool within the “Molecular Operating Environment,” (Chemical Computing Group Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, Quebec, Canada.
        3. Modeler (within the Quanta suite of programs, available from Accelrys, a subsidiary of Pharmacopeia, Inc.;
        4. COMPOSER (Blundell et al., see e.g., Protein Eng., 1:377-384, 1987; available as part of the Sybyl package, from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.; see www.tripos.com/software/composer.html)
    
    


7.9.2 Representations of Structure Coordinates
The atomic structure coordinates of eat-16 or RGS-7 (either of which are referred to herein as the “RGS polypeptide”) can be used in molecular modeling and design, as described more fully below. The present invention encompasses the structure coordinates and other information, e.g., amino acid sequence, connectivity tables, vector-based representations, temperature factors, etc., used to generate the three-dimensional structure of the RGS polypeptide for use in the software programs described herein and other software programs.
While Cartesian coordinates are important and convenient representations of the three-dimensional structure of a polypeptide, those of skill in the art will readily recognize that other representations of the structure are also useful. Therefore, the three-dimensional structure of a polypeptide, as discussed herein, includes not only the Cartesian coordinate representation, but also all alternative representations of the three-dimensional distribution of atoms. For example, atomic coordinates may be represented as a Z-matrix, wherein a first atom of the protein is chosen, a second atom is placed at a defined distance from the first atom, a third atom is placed at a defined distance from the second atom so that it makes a defined angle with the first atom. Each subsequent atom is placed at a defined distance from a previously placed atom with a specified angle with respect to a third atom, and at a specified torsion angle with respect to a fourth atom.
Atomic coordinates may also be represented as a Patterson function, wherein all interatomic vectors are drawn and are then placed with their tails at the origin. This representation is particularly useful for locating heavy atoms in a unit cell. In addition, atomic coordinates may be represented as a series of vectors having magnitude and direction and drawn from a chosen origin to each atom in the polypeptide structure. Furthermore, the positions of atoms in a three-dimensional structure may be represented as fractions of the unit cell (fractional coordinates), or in spherical polar coordinates.
Additional information, such as thermal parameters, which measure the motion of each atom in the structure, chain identifiers, which identify the particular chain of a multi-chain protein in which an atom is located, and connectivity information, which indicates to which atoms a particular atom is bonded, are also useful for representing a three-dimensional molecular structure.
7.9.3 Computational Implementation:
The structural coordinates of the proteins of the present invention are stored in electronic form on computer-readable medium for use with a computer. Additionally, methods of rational drug design and virtual screening that utilize the coordinates of the proteins of the present invention are preferably performed on one or more computers, as depicted in FIG. 10.
According to FIG. 10, a computer system 100 on which the molecular modeling methods of the present invention may be carried out, comprises:

    
    
        at least one central-processing unit 102 coupled via a bus 104 to working memory 106, a user interface 108, a network interface 110 and a machine-readable memory 107, for processing machine readable data; and
        a machine-readable memory 107 comprising a data storage material encoded with machine-readable data, wherein the data comprises the structural coordinates 134 of at least one of eat-16 and RGS-7 proteins;
        a working memory 106 for storing an operating system 112, optionally one or more molecular structure databases 114, one or more pharmacophores 116 derived from the structural coordinates 134, a graphical user interface 118 and instructions for processing machine-readable data comprising one or more molecular modeling programs 120 such as a deformation energy calculator 122, a homology modeling tool 124, a de novo design tool, 126, a “docking tool” 128, a database search engine 130, a 2D-3D structure converter 132 and a file format interconverter 134.
    
    


Computer system 100 may be any of the varieties of laptop or desktop personal computer, or workstation, or a networked or mainframe computer or super-computer, that would be available to one of skill in the art. For example, computer system 100 may be an IBM-compatible personal computer, a Silicon Graphics, Hewlett-Packard, Fujitsu, NEC, Sun or DEC workstation, or may be a Convex supercomputer. Computer system 100 may also support multiple processors, as, for example in a Silicon Graphics Origin system.
Operating system 112 may be any suitable variety that runs on any of computer systems 100. For example, in one embodiment, operating system 112 is selected from the UNIX family of operating systems, for example, Ultrix from DEC, AIX from IBM, or IRIX from Silicon Graphics. It may also be a LINUX operating system. In another embodiment, operating system 112 may be a VAX VMS system. In a preferred embodiment, operating system 112 is a Windows operating system such as Windows 3.1, Windows NT, Windows 95, Windows 98, Windows 2000, or Windows XP. In yet another embodiment, operating system 112 is a Macintosh operating system such as MacOS 7.5.x, MacOS 8.0, MacOS 8.1, MacOS 8.5, MacOS 8.6, MacOS 9.x and MaxOS X.
The graphical user interface (GUI) 118 is preferably used for displaying representations of structural coordinates 134, or variations thereof, in 3-dimensional form on user interface 108. GUI 118 also preferably permits the user to manipulate the display of the structure that corresponds to structural coordinates 134 in a number of ways, including, but not limited to: rotations in any of three orthogonal degrees of freedom; translations; zooming in on specific portions of the structure; coloring of the structure according to a property that varies amongst to different regions of the structure; displaying subsets of the atoms in the structure; coloring the structure by atom type; and displaying tertiary structure such as α-helices and β-sheets as solid objects. Structural coordinates 134 are also optionally copied into memory 106 to facilitate manipulations with one or more of the molecular modeling programs 120.
Network interface 110 may optionally be used to access one or more molecular structure databases stored in the memory of one or more other computers.
7.9.4 Data Storage Media
The invention encompasses machine readable media embedded with the three-dimensional structure of the model described herein, or with portions thereof. As used herein, “machine readable medium” or “computer readable medium” refers to any media that can be read and accessed directly by a computer or scanner. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage media and magnetic tape; optical storage media such as optical discs; CD-ROM, CD-R or CD-RW; electronic storage media such as RAM or ROM; and hybrids of these categories such as magnetic/optical storage media. Such media further include paper on which is recorded a representation of the atomic structure coordinates, e.g., Cartesian coordinates, that can be read by a scanning device and converted into a three-dimensional structure with optical character recognition (OCR) technology.
A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon the atomic structure coordinates of the invention or portions thereof and/or X-ray diffraction data. The choice of the data storage structure will generally be based on the means chosen to access the stored information.
In addition, a variety of data processor programs and formats can be used to store the sequence and structure information on a computer readable medium. Such formats include, but are not limited to, Protein Data Bank (“PDB”) format (Research Collaboratory for Structural Bioinformatics); Cambridge Crystallographic Data Centre format; Structure-data (“SD”) file format (MDL Information Systems, Inc.; Dalby et al., 1992, J. Chem. Inf. Comp. Sci. 32:244-255), and line-notation, e.g., as used in SMILES (Weininger, D., “SMILES, a Chemical Language and Information System. 1. Introduction to Methodology and Encoding Rules,” 1988, J. Chem. Inf. Comp. Sci. 28:31-36), and CHUCKLES (Siani, M. A., Weininger, D., Blaney, J., “CHUCKLES: a method for representing and searching peptide and peptoid sequences on both monomer and atomic levels,” J. Chem. Inf. Comp. Sci., 1994, 34:588-593).
Methods of converting between various formats read by different computer software will be readily apparent to those of skill in the art and programs for carrying out such conversions are widely available, either as stand-alone programs, e.g., BABEL (v. 1.06, Walters, P. & Stahl, M., ©1992, 1993, 1994) or integrated into other computer packages. All format representations of the polypeptide coordinates described herein, or portions thereof, are contemplated by the present invention. By providing a computer readable medium having stored thereon the atomic coordinates of the invention, one of skill in the art can routinely access the atomic coordinates of the invention, or portions thereof, and related information for use in modeling and design programs, described in detail below.
7.10 Uses of the Atomic Structure Coordinates
7.10.1 Molecular Modeling Methods In General
Structure information, typically in the form of the atomic structure coordinates, can be used in a variety of computational or computer-based methods to, for example, design, screen for and/or identify compounds that bind the RGS or Gαq polypeptide or a portion or fragment thereof, or to intelligently design mutants that have altered biological properties.
In one embodiment, the structure coordinates of the present invention are useful for identifying and/or designing compounds that bind the RGS or Gαq polypeptide as an approach towards developing new therapeutic agents.
In another embodiment, the structure is probed with a plurality of molecules to determine their ability to bind to the RGS or Gαq polypeptide at various sites. Such compounds can be used as targets or leads in medicinal chemistry efforts to identify modulators, for example, inhibitors of potential therapeutic importance.
In still another embodiment, compounds that can isomerize to short-lived reaction intermediates in the chemical reaction of an RGS or Gαq polypeptide-binding compound with an RGS or Gαq polypeptide can be developed. Thus, the time-dependent analysis of structural changes in the RGS or Gαq polypeptide during its interaction with other molecules is enabled. The reaction intermediates of an RGS or Gαq polypeptide can also be deduced from the reaction product in co-complex with the RGS or Gαq polypeptide. Such information is useful to design improved analogues of known RGS or Gαq polypeptide modulators, e.g., inhibitors or to design novel classes of modulators based on the reaction intermediates of RGS or Gαq polypeptide-inhibitor co-complexes. This provides a novel route for designing RGS or Gαq polypeptide modulators, e.g., inhibitors, with both high specificity and stability.
In yet another embodiment, the structure can be used to computationally screen small molecule databases for chemical entities or compounds that can bind in whole, or in part, to an RGS or Gαq polypeptide. In this screening, the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity or by estimated interaction energy. Meng et al., 1992, J. Comp. Chem., 13:505-524.
The computational methods of the present invention may be carried out with commercially available programs or with computer programs that are developed specially for the purpose and implemented on computer system 100. Commercially available programs comprise large integrated molecular modeling packages that contain at least two of the types of molecular modeling programs 120 shown in FIG. 10. Examples of such large integrated packages that are known to those skilled in the art include: Cerius2(available from Accelrys, a subsidiary of Pharmacopeia, Inc.), Molecular Operating Environment (available from, Chemical Computing Group Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, Quebec, Canada), Sybyl (available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.)and Quanta (available from Accelrys, a subsidiary of Pharmacopeia, Inc). Alternatively, the computational methods of the present invention may be achieved with one or more stand-alone programs that carries out one of the functions performed by molecular modeling programs 120. In particular certain aspects of the display and visualization of molecular structures may be accomplished by specialized tools, for example, GRASP (Nicholls, A.; Sharp, K.; and Honig, B., PROTEINS, Structure, Function and Genetics, 1991, Vol. 11 (No.4), pg. 281ff; available from Department of Biochemistry Rm 221, Columbia University Box 36, 630, W. 168th St., New York.
7.10.2 Computational Screening
In a preferred method, potential binding compounds may be obtained by rapid computational screening. Such a screening comprises testing a large number, which may be hundreds, or may preferably be thousands, or more preferably tens of thousands or even more preferably hundreds of thousands of molecules whose formulae are known. The molecules are obtained from one or more molecular structure databases, available in electronic form, for example, the “Available Chemicals Directory” (“ACD”, available from MDL Information Systems, Inc., 14600 Catalina Street, San Leandro, Calif.); the National Cancer Institute database (NCIDB; also available from MDL Information Systems, Inc., 14600 Catalina Street, San Leandro, Calif.); the “MDL Drug Data Report” (MDDR, available from MDL Information Systems, Inc., 14600 Catalina Street, San Leandro, Calif.); the Comprehensive Medicinal Chemistry Database (CMC, available from MDL Information Systems, Inc., 14600 Catalina Street, San Leandro, Calif.); and any proprietary database of compounds with known medicinal properties, as is found in a large or small pharmaceutical company. The molecules in such databases are preferably stored as a connection table with or without a 2D representation comprising coordinates in just 2 dimensions, say x and y, and more preferably stored as at least one set of 3D coordinates corresponding to an experimentally derived or computer-generated molecular conformation. If the molecules are only stored as a connection table or a 2D set of coordinates, then it can be necessary to generate a 3D structure for each molecule before proceeding. Programs for converting 2D molecular structures or molecule connection tables to 3D structures include Converter (available from Accelrys, a subsidiary of Pharmacopeia, Inc.) and CONCORD (A. Rusinko III, J. M. Skell, R. Balducci, C. M. McGarity, and R. S. Pearlman, “CONCORD, A Program for the Rapid Generation of High Quality Approximate 3-Dimensional Molecular PlStructures,” 1988 The University of Texas at Austin and Tripos Associates, available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.
As part of a computational screen, it is possible to “dock” 3D structures of molecules from a database into the active site of the protein in question, on a high throughput basis. Such a procedure can normally be subject to a number of user-defined parameters and thresholds according to desired speed of throughput and accuracy of result. Such parameters include the number of different starting positions from which to start a docking simulation and the number of energy calculations to carry out before rejecting or accepting a docked structure. Such parameters and their choices are familiar to one of skill in the art. Structures from the database can be selected for synthesis if their docked energy is below a certain threshold.
Alternatively, it is possible to carry out a “molecular similarity” search if a pharmacophore has been developed from the active site of the protein in question. A pharmacophore defines a set of contact sites on the surface of the active site, accompanied by the distances between them. A similarity search attempts to find molecules in a database that have at least one favorable 3D conformation whose structure overlaps favorably with the pharmacophore. For example, a pharmacophore may comprise a lipophilic pocket at a particular position, a hydrogen-bond acceptor site at another position and a hydrogen bond donor site at yet another specified position accompanied by distance ranges between them. A molecule that could potentially fit into the active site is one that can adopt a conformation in which a H-bond acceptor can reach the H-bond acceptor site on the pharmacophore, a H-bond donor can simultaneously reach the H-bond donor site of the pharmacophore and, for example, a group such as a phenyl ring can orient itself into the lipophilic pocket.
Even where a pharmacophore has not been developed, molecular similarity principles may be employed in a database searching regime. (See, for example, Johnson, M. A.; Maggiora, G. M., Eds. Concepts and Applications of Molecular Similarity, New York: John Wiley & Sons (1990)) In one embodiment, it is possible to search for molecules that have certain properties in common, for example, numbers of hydrogen bond donors or numbers of hydrogen bond acceptors, or overall hydrophobicity within a particular range of values. Alternatively, even where a pharmacophore is not known, similar molecules may be selected on the basis of optimizing an overlap criterion with the molecule of interest.
In searching a molecular structure database, a specialized database searching tool that permits searching molecular structures and sub-structures is typically employed. Examples of suitable database searching tools, known to one of skill in the art are: ISIS/Host and ISIS/Base (available from MDL Information Systems, Inc., 14600 Catalina Street, San Leandro, Calif.), Unity (available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.) or Catalyst (available from Accelrys, a subsidiary of Pharmacopeia, Inc.).
A molecular property of particular interest when assessing suitability of drug compounds is its hydrophobicity. An accepted and widespread measure of hydrophobicity is LogP, the Log.sub.10 of the octanol-water partition coefficient. Measured values of LogP are available for many compounds. Methods and programs for calculating LogP are also available, see for example: CLOGP (Hansch, C., and Leo, A.; available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.); and ACD/LogP DB (Advanced Chemistry Development Inc., 90 Adelaide Street West, Suite 702, Toronto, Ontario Canada)
7.10.2 Rational Design Considerations
The design of compounds that bind to or inhibit an RGS or Gαq polypeptide according to this invention generally involves consideration of two factors. First, the compound must be capable of physically and structurally associating with an RGS or Gαq polypeptide. This association can be covalent or non-covalent. For example, covalent interactions may be important for designing irreversible or suicide inhibitors of a protein. Non-covalent molecular interactions important in the association of an RGS or Gαq polypeptide with its substrate include hydrogen bonding, ionic interactions and van der Waals and hydrophobic interactions. Second, the compound must be able to assume a conformation that allows it to associate with an RGS or Gαq polypeptide. Although certain portions of the compound will not directly participate in this association with an RGS or Gαq polypeptide, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical group or compound in relation to all or a portion of the binding site, or the spacing between functional groups of a compound comprising several chemical groups that directly interact with an RGS or Gαq polypeptide.
The potential modulatory or binding effect of a chemical compound on an RGS or Gαq polypeptide may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association between it and an RGS or Gαq polypeptide, synthesis and testing of the compound is unnecessary. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to an RGS or Gαq polypeptide and inhibit its activity. In this manner, synthesis of ineffective compounds may be avoided. Visualization of molecular properties can be particularly important and may be aided by computer programs such as MOLCAD (Brickmann, J., and coworkers, see, for example, J. Comp.-Aid. Molec. Des., 7:503(1993); available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.).
7.10.3 Constructing Potential Molecules That Bind to RGS or Gαq Polypeptides
A modulatory or other binding compound of an RGS or Gαq polypeptide may be computationally evaluated and designed by means of a series of steps in which chemical groups or fragments are screened and selected for their ability to associate with the individual binding pockets or other areas of an RGS or Gαq polypeptide, for example, hydrophobic patches 1, 2 and/or 3 of an RGS polypeptide, as described hereinbelow. One skilled in the art may use one of several methods to screen chemical groups or fragments for their ability to associate with an RGS or Gαq polypeptide. This process may begin by visual inspection of, for example, the active site on the computer display based on the RGS or Gαq polypeptide coordinates. Selected fragments or chemical groups may then be positioned in a variety of orientations, or docked, within an individual binding pocket of an RGS or Gαq polypeptide as defined supra. Docking may be accomplished using software such as QUANTA (available from Accelrys, a subsidiary of Pharmacopeia, Inc.) and SYBYL, (available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.), followed by energy minimization and molecular dynamics simulations with molecular mechanics forcefields such as MM2 (see, e.g., Rev. Comp. Chem., 3, 81 (1991)), MM3 (Allinger, N. L., Bowen, J. P., and coworkers, University of Georgia; see, J. Comp. Chem., 17:429(1996); available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.), CHARMM and AMBER version 6 (Kollman, P. A., et al., School of Pharmacy, Department of Pharmaceutical Chemistry, University of California at San Francisco, ©2000), and Discover (available from Accelrys, a subsidiary of Pharmacopeia, Inc.).
Specialized computer programs may also assist in the process of selecting fragments or chemical groups. These include:
1. GRID (Goodford, (1985), J. Med. Chem., 28:849-857). GRID is available from Oxford University, Oxford, UK;
2. MCSS (Miranker & Karplus, (1991), Proteins: Structure, Function and Genetics 11:29-34). MCSS is available from Accelrys, a subsidiary of Pharmacopeia, Inc., as part of the Quanta package;
3. AUTODOCK (Goodsell & Olsen, (1990), Proteins: Structure, Function, and Genetics 8:195-202). AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; and
4. DOCK (Kuntz et al., (1982), J. Mol. Biol., 161:269-288). DOCK is available from University of California, San Francisco, Calif.
5. GOLD (Jones, et al., (1995), J. Mol. Biol., 245:43-53). GOLD is available from the Cambridge Crystallographic Data Center, 12 Union Road. Cambridge, U.K.;
6. GLIDE. GLIDE is available from Schrodinger, Inc.
7. ICM (Abagayan, et al.) ICM is available from MolSoft, L.L.C., 3366 North Torrey Pines Court, Suite 300, La Jolla, Calif. 92037.
Once suitable chemical groups or fragments have been selected, they can be assembled into a single compound or inhibitor. Assembly may proceed by visual inspection of the relationship of the fragments to each other in the three-dimensional image displayed on a computer display in relation to the structure coordinates of an RGS or Gαq polypeptide. This would be followed by manual model building using software such as QUANTA or SYBYL.
Useful programs to aid one of skill in the art in connecting the individual chemical groups or fragments include:
1. CAVEAT (Bartlett et al., 1989, “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules,” In Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. 78:182-196). CAVEAT is available from the University of California, Berkeley, Calif.;
2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Martin, (1992), J. Med. Chem., 35:2145-2154); and
3. HOOK (available from Accelrys, a subsidiary of Pharmacopeia, Inc., as part of the Quanta package).
Instead of proceeding to build a RGS or Gαq modulator in a step-wise fashion one fragment or chemical group at a time, as described above, RGS or Gαq binding compounds may be designed as a whole or de novo using either an empty active site or optionally including some portion(s) of a known inhibitor(s). These methods include:
1. LUDI (Bohm, (1992), J. Comp. Aid. Molec. Design 6:61-78). LUDI is available from Accelrys, a subsidiary of Pharmacopeia, Inc., as part of the Insight package, see www.accelrys.com/insight/ludi.html;
2. LEGEND (Nishibata & Itai, (1991), Tetrahedron 47:8985). LEGEND is available from Molecular Simulations, Burlington, Mass.; and
3. LeapFrog (available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.; www.tripos.com/software/leapfrog.html).
Other molecular modeling techniques may also be employed in accordance with this invention. See, e.g., Cohen et al., (1990), J. Med. Chem. 33:883-894. See also, Navia & Murcko, (1992), Current Opinions in Structural Biology 2:202-210.
7.10.4 Quantifying Potential Binding Molecules
Once a compound has been designed or selected by the above methods, the efficiency with which that compound may bind to RGS or Gαq polypeptide may be tested and optimized by computational evaluation. For example, a compound that has been designed or selected to function as a RGS or Gαq polypeptide activator (agonist) or inhibitor (antagonist) also preferably occupies a volume not overlapping the volume occupied by the active site residues when the native substrate is bound. An effective RGS or Gαq polypeptide activator or inhibitor must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., it must have a small deformation energy of binding). Thus, the most efficient RGS or Gαq polypeptide activators or inhibitors should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mol, preferably, not greater than 7 kcal/mol. RGS or Gαq polypeptide activators or inhibitors may interact with the protein 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 activator or inhibitor binds to the enzyme.
A compound selected or designed for binding to RGS or Gαq polypeptide may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target protein. Such non-complementary electrostatic interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the activator or inhibitor and the protein when the activator or inhibitor is bound to it 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 fall into approximately three levels of sophistication. The crudest level of approximation, molecular mechanics, is also the cheapest to compute and can most usefully be used to calculate deformation energies. Molecular mechanics programs find application for calculations on small organic molecules as well as polypeptides, nucleic acids, proteins and most other biomolecules. Examples of programs which have implemented molecular mechanics force fields include: AMBER, version 6 (Kollman, P. A., et al., School of Pharmacy, Department of Pharmaceutical Chemistry, University of California at San Francisco, ©2000); CHARMM (see B. R. Brooks, R. E. Bruccoleri, B. D. Olafson, D. J. States, S. Swaminathan, and M. Karplus, “CHARMM: A Program for Macromolecular Energy, Minimization, and Dynamics Calculations,” J. Comp. Chem., 4, 187-217, (1983); A. D. MacKerell, Jr., B. Brooks, C. L. Brooks, Ill, L. Nilsson, B. Roux, Y. Won, and M. Karplus, “CHARMM: The Energy Function and Its Parameterization with an Overview of the Program,” in The Encyclopedia of Computational Chemistry, 1, 271-277, P. v. R. Schleyer et al., editors, John Wiley & Sons: Chichester, (1998); QUANTA/CHARMm (available from Accelrys, a subsidiary of Pharmacopeia, Inc.); and Insight II/Discover (available from Accelrys, a subsidiary of Pharmacopeia, Inc.).
The next level of sophistication comprises the so-called “semi-empirical” methods, which are relatively inexpensive to compute and are most useful for calculating deformation energies of organic molecules. Examples of program packages that provide semi-empirical capability are MOPAC 2000 (Stewart, J. J. P., et al., available from Schrodinger, Inc., 1500 S.W. First Avenue, Suite 1180, Portland, Oreg.) and AMPAC (Holder, A., et al., available from Tripos, Inc., 1699 South Hanley Road, St. Louis, Mo.;
The highest level of sophistication is achieved by those programs that employ so-called ab initio quantum chemical methods and those from density functional theory, for example: Gaussian 98, Revision A.9, (available from Gaussian, Inc., Carnegie Office Park, Building 6, Suite 230, Carnegie, Pa.); and Q-Chem2.0 (“A high-performance ab initio electronic structure program,” J. Kong, et al., J. Comput. Chem., (2000) 21, 1532-1548; available from Four Triangle Lane, Suite 160, Export, Pa). These programs may be installed, for instance, on a computer workstation, as is well-known in the art. Other hardware systems and software packages will be known to those skilled in the art.
7.10.5 Further Manipulations of the RGS or Gαq Polypeptide Structures and Binding Molecules
Once an RGS or Gαq polypeptide-binding compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or chemical 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, polarity and charge as the original group. For selection of appropriate groups, any of several chemical models can be used, e.g., isolobal or isosteric analogies. Groups known to be bio-isosteres of one another are particularly preferred. One of skill in the art will understand that substitutions known in the art to alter conformation should be avoided. Such altered chemical compounds may then be analyzed for efficiency of binding to eat-16 by the same computer methods described in detail above.
The structure coordinates of wild-type eat-16 and its mutants will also facilitate the identification of related proteins or enzymes analogous to eat-16 in function, structure or both, thereby further leading to novel therapeutic modes for treating or preventing eat-16 mediated diseases.
Subsets of the atomic structure coordinates can be used in any of the above methods. Particularly useful subsets of the coordinates include, but are not limited to, coordinates of single domains, coordinates of residues lining an active site, coordinates of residues that participate in important protein-protein contacts at an interface, and Cα coordinates. For example, the coordinates of one domain of a protein that contains the active site may be used to design inhibitors that bind to that site, even though the protein is fully described by a larger set of atomic coordinates. Therefore, a set of atomic coordinates that define the entire polypeptide chain, although useful for many applications, do not necessarily need to be used for the methods described herein.
7.10.6 Activity of Binding Molecules
The binding molecules described above can be synthesized according to synthetic techniques well known to those of skill in the art. The binding molecules preferably modulate the activity of the RGS or Gαq polypeptide and/or a molecule that interacts with the RGS or Gαq polypeptide. The activity of a binding molecule can be assayed easily by methods well known to those of skill in the art. For instance, an RGS or Gαq polypeptide or a cell comprising the RGS or Gαq polypeptide can be contacted with the binding molecule and then assayed for modulation of RGS activity or Gαq activity. Assays for RGS activity or Gαq activity are described in detail above.
Preferably, binding molecules may be identified by high throughput screening methods, according to which large libraries of ligands are screened against a particular target. A large library of ligands preferably contains more than 1,000 distinct ligands, more preferably contains more than 10,000 distinct ligands, even more preferably contains more than 100,000 distinct ligands and most preferably contains more than 1,000,000 distinct ligands. High throughput screening techniques typically employ robotically controlled assay systems, and take advantage of the latest improvements in miniaturization and automation. Samples are typically assayed on 96-well plates or microtiter plate arrays, and measurements may be taken in parallel. For an overview of high throughput screening techniques, see, for example, Razvi, E. S., “High-Throughput Screening—Where Are We Today?,” Drug & Market Development Publications, (June 1999), and Razvi, E. S., “Industry Trends in High-Throughput Screening,” Drug & Market Development Publications, (August 2000).
8. EXAMPLES
8.1 Example 1
Compounds That Modulate Bladder Contractility
This example demonstrates the identification of Compound I (FIG. 3A), Compound II (FIG. 3B) and Compound III (FIG. 3C) via screening methods of the instant invention. Compounds I, II and III are capable of modulating bladder contractility.
Isolated Rat Bladder Strips
Tissue Preparation
Male rats (Harlan, Sprague Dawley, 250-350 g) were sacrificed by decapitation, the bladder removed and cleaned of connective tissue. Strips of bladder were then cut from the bladder body and each was placed in an organ bath, suspended between a fixed hook and a force transducer, containing oxygenated physiological buffer (composition in mM: NaCl 118.4, KCl 4.7, KH2PO4 1.2, MgSO4 1.3, CaCl2 1.8, glucose 10.1, NaHCO3 25, gassed with 95%O2/5%CO2) and maintained at 37° C. Force generated by the bladder strips was recorded and measured using an AcqKnowledge data acquisition system (AcqKnowledge for MP100WS, Biopac Systems Inc., Goleta, Calif.).
Experimental Design:
The bladder strips were primed by stimulating them with 10 μM carbachol to evoke a contraction. The strips were then washed multiple times with fresh physiological buffer and allowed to fully relax. Following a period of recovery, the strips were again challenged with 10 μM carbachol to produce a contraction; this contractile response served as the control response. The strips were again washed multiple times and allowed to fully relax (45 minutes). Test compound or vehicle was then added to each organ bath. Following a one hour incubation period, the strips were again stimulated with 10 μM carbachol and the contractile response measured.
Data Analysis
The percent inhibition of the carbachol response by the test compounds was calculated by comparing control and post-compound carbachol responses corrected for vehicle effects.










Results











Compound
Inhibition
N















Compound I (10 μM)
++
10



Compound III (3 μM)
+
6



Compound II (3 μM)
+
3







+ 20-40%



++ 40-60%






8.2 Example 2
Methods for Preparation of Compounds I, II and III
This example provides methods for synthesizing compounds I, II and III.
Compound I may be prepared by the general procedures described by J. L. Romine et al., in U.S. Pat. No. 5,869,509, issued Feb. 9, 1999, and, more specifically, in Example 140 of said U.S. patent. Compound III is identical to Formula XXVIII of J. L. Romine et al., U.S. Pat. No. 6,077,861, issued Jun. 20, 2000, and may be prepared by the general procedures described therein and, more specifically, in Example 141 of said U.S. patent. The contents of U.S. Pat. Nos. 5,869,509 and 6,077,861 are hereby incorporated by reference in their entireties.
Compound II was prepared according to the method depicted in Reaction Scheme 1.




Preparation of Intermediate 1

Step A: Preparation of Ethyl 2-(5-bromothien-2-yl)glyoxalate
A solution of AlCl3 (61 g, 455 mmol) in nitromethane (125 mL) was added dropwise to a stirring mixture of ethyloxalylchloride (50.8 ml, 455 mmol) and 2-bromothiophene (49.5 g, 304 mmol) at 0-5° C. The reaction was stirred for 1 hour at 0.5° C. following the addition step, then 3 hours at room temperature. The reaction was then poured into 1 L ice water and extracted with diethyl ether (3×250 mL). The combined diethyl ether layers were extracted with saturated aqueous NaHCO3 solution (250 mL), dried over Na2SO4, and concentrated under vacuum. The residue was Kugelrohr distilled to yield ethyl 2-(5-bromothien-2-yl)glyoxalate as a yellow solid (45.7 g, 57%). MS (MH+): 262.98; 1H NMR (300 MHz, CDCl3) d 7.90 (1H, d, J=4.1 Hz), 7.17 (1H, d, J=4.2 Hz), 4.42 (2H, q, J=7.2 Hz), 1.43 (3H, t, J=7.1 Hz); m.p. 63-66° C.
Step B: Preparation of 2-(5-bromothien-2-yl)glyoxalic Acid, Intermediate 1
Ethyl 2-(5-bromothien-2-yl)glyoxalate (45.6 g, 173 mmol) from Step A was dissolved in a mixture of 100 mL THF, 100 mL methanol, and 300 mL water, then treated with 10 N NaOH (26 ml, 260 mmol) at room temperature for 20 hours. Volatile solvents were removed by rotary evaporation. The residue was dissolved in 2 L water and extracted with diethyl ether (250 mL). The aqueous layer was acidified to pH 1 with 6 N HCl, and extracted with diethyl ether (3×300 mL). The combined diethyl ether extracts were dried over Na2SO4 and concentrated in vacuo to yield 2-(5-bromothien-2-yl)glyoxalic acid, Intermediate 1 (38.7 g, 95%). The product was then recrystallized from diethyl ether. m.p. 119-120° C.; MS (M−H) 235.1; 1H NMR (300 MHz, CDCl3) d 9.00 (1H, br s), 8.15 (1H, d, J=4.2 Hz), 7.16 (1H, d, J=4.2 Hz); 13C NMR (75 MHz, CDCl3) d 173.80, 159.58, 140.14, 137.17, 132.58, 130.48; IR (KBr) 3326, 3124, 1756, 1634, 1415, 1363, 1308, 1253cm−1;
Analytic composition for C6H3O3SBr:


















Calculated:
C, 30.66;
H, 1.29;
Br, 33.99.



Found:
C, 30.92;
H, 1.46;
Br, 33.94.










Preparation of Intermediate 2:

Step A: Preparation of 4-chloro-5-bromo-2-aminoanisole
Bromine (26.4 g) was added to a solution of 4-chloro-o-anisidine (23.55 g) in dichloromethane (400 mL) at room temperature. The resulting mixture was stirred for 10 hours and quenched with NaOH. The organic layer was washed with brine, dried over MgSO4, and concentrated. Purification by flash chromatography over silica gel (elution with 10% ethyl acetate in hexanes) gave 11.8 g (33%) of the desired 4-chloro-5-bromo-2-aminoanisole. 1H NMR (300 MHz, CDC13) d 6.94 (s, 1 H), 6.78 (s, 1 H), 3.83 (s, 3 H).
Step B: Preparation of (4-bromo-5-chloro-2-methoxyphenyl)hydrazine, Intermediate 2
A suspension of 4-chloro-5-bromo-2-aminoanisole (7.68 g) from Step A in 100 mL concentrated HCl was cooled to −10° C. With mechanical stirring, a solution of sodium nitrite (2.25 g) in 10 mL water was added slowly, maintaining the reaction temperature at −10° C. The tip of the addition pipette was kept below the surface of the reaction. The reaction was warmed to 0° C. and stirred for 1 hour. The reaction was then cooled to −35° C., and a solution of SnCl2—2H2O (15 g) in 17 mL concentrated HCl was added slowly, maintaining the reaction temperature at −35° C. The reaction was warmed to 0° C. and stirred for 1 hour. The solid product was filtered and washed with concentrated HCl and water. The solid was then stirred in a mixture of 200 mL ethyl acetate and 335 mL 3N NaOH for 1 hour. The organic layer was separated, dried over sodium sulfate, and concentrated to dryness under vacuum to yield 5.5 g (73%) of the desired product (4-bromo-5-chloro-2-methoxyphenyl)hydrazine, Intermediate 2, which was used in the next step without purification.
Preparation of Intermediate 3:
(4-Bromo-5-chloro-2-methoxyphenyl)hydrazine (Intermediate 2) (5.0 g) and 2-(5-bromothien-2-yl)glyoxylic acid (4.7 g) were refluxed in acetonitrile (15-25 ml/mmol starting material) for 30-60 minutes. The reaction was cooled to room temperature. Triethylamine (2.22 g) and diphenylphosphorylazide (6.05 g) were added, and the reaction was heated at reflux 3-18 hours. After cooling to room temperature, solids were filtered and washed with acetonitrile and ether to yield (75%) 2-(4-bromo-5-chloro-2-methoxyphenyl)-2,4-dihydro-5-(5-bromothien-2-yl)-3H-1,2,4-triazol-3-one (Intermediate 3).
1H NMR (300 MHz, DMSO-d6) δ12.60 (s, 1 H), 7.70 (s, 1 H), 7.59 (s, 1 H), 7.40 (d, J=4.0 Hz, 1 H), 7.34 (d, J=4.0 Hz, 1 H), 3.82 (s, 3 H) ppm;
13C NMR (75 MHz, DMSO-d6)—154.3, 152.8, 140.8, 131.4, 130.0, 129.9, 128.2, 125.2, 124.0, 122.7, 117.9, 114.5, 56.8 ppm; IR (KBr, cm−1) 3442, 1712; MS(ESI) 464 (M−1)+;
Anal composition for C13H9Br2ClN3O2S


















Calculated:
C, 33.54;
H, 1.73;
N, 9.03.



Found:
C, 33.95;
H, 1.92;
N, 9.04.










Preparation of 2-(4-bromo-5-chloro-2-hydroxyphenyl)-2,4-dihydro-5-(5-bromothien-2-yl)-3H-1,2,4-triazol-3-one (Compound II)
2-(4-Bromo-5-chloro-2-methoxyphenyl)-2,4-dihydro-5-(5-bromothien-2-yl)-3H-1,2,4-triazol-3-one (Intermediate 3) (1.395 g) was suspended in anhydrous dichloromethane (20-25 mL/mmol of Intermediate 3) under argon and cooled to −78° C. A 1 M solution of BBr3 (9 mL) in anhydrous dichloromethane was added via a dropping funnel over a 45 minute period. After the addition was complete, the reaction was warmed to room temperature and stirred for 5 hours. The reaction was quenched by the addition of 5-10 mL water. Volatile solvent was removed under vacuum and the crude product was filtered and washed with water. The solid was refluxed in a mixture of acetone and ethanol for 15 minutes. After cooling to room temperature, the purified product was filtered and washed with acetone and ethanol. The solid was dried under high vacuum to yield 1.1 g (81%) of the desired product, Compound II.
1H NMR (300 MHz, DMSO-d6)—12.6 (s, 1 H), 10.5 (s, 1 H), 7.63 (s, 1 H), 7.43 (d, J=4 Hz, 1 H), 7.34 (d, J=4 Hz, 1 H), 7.33 (s, 1 H) ppm;
13C NMR (75 MHz, DMSO-d6)—153.1, 152.5, 140.9, 131.4, 130.1, 129.2, 128.2, 124.3, 122.4, 121.7, 121.5, 114.5 ppm; IR (KBr, cm−1) 3224 (br), 1776; MS(ESI) 450(M−1)+;
Analytic composition for C12H6Br2ClN3O2S


















Calculated:
C, 31.92;
H, 1.34;
N, 9.31.



Found:
C, 32.34;
H, 1.46;
N, 9.24.










8.3 Example 3
Validation of Activity of Compounds I and II
This example demonstrates the activity of Compounds I, II and III in in vitro bladder function assays.
In Vitro Whole Bladder Model
Isolated Bladder Preparation
The model used for these experiments was a modified version of that previously described by Malkowicz et al., 1986, J. Urol. 136: 1324-1329. Briefly, a female rat (Harlan, Sprague Dawley, 250-350 g) was sacrificed by decapitation. The bladder was excised, cleaned of connective tissue and the ureters were tied. The bladder was emptied, catheterized at the urethral opening and mounted in a 50 ml organ bath containing physiological buffer (composition in mM: NaCl 118.4, KCl 4.7, KH2PO4 1.2, MgSO4 1.3, CaCl2 1.8, glucose 10.1, NaHCO3 25, gassed with 95%O2/5%CO2 and maintained at 37° C.). A three-way valve connected the bladder catheter to an infusion pump and to another three-way valve. The latter valve was connected to a pressure transducer and an “emptying tube”. The emptying tube was suspended approximately 10 cm above the pressure transducer and it was graduated to allow a direct reading of the volume of bladder emptying at the end of the infusion. The bladder was held at the same height as the pressure transducer.
Experimental Design
The bladder was infused with physiologfical buffer at a rate of 0.05 ml/min for 30 minutes. The pressure developed in the bladder during the infusion was recorded on-line using an AcqKnowledge data acquisition system (AcqKnowledge for MP100WS, Biopac Systems Inc., Goleta, Calif.). When the infusion was complete, the second three-way valve was opened allowing the bladder to empty (“spontaneous bladder emptying”). After spontaneous emptying, which produced only a partial emptying, 1 μM carbachol and then 10 μM carbachol was added to the bath to induce complete emptying. The volume of spontaneous emptying and emptying due to 1 μM and 10 μM carbachol were measured. This procedure was repeated multiple times in the same bladder. The second bladder filling served as a pre-drug or pre-vehicle baseline. Bladders were exposed to vehicle or drug for 1 hour prior to the third bladder filling.
Data Analysis
Bladder function was assessed by comparing the pressure developed in the bladders as a function of volume (statistical analysis compared pressure developed from 0.2 to 1.3 ml filling volume in 0.1 ml increments). Additionally, changes in spontaneous and carbachol-induced emptying volumes were also compared. Statistical analysis of the data was performed using a t-test and the significance of difference was determined by two-tailed P value of less than 0.05.
Results
Compound III (100 and 300 nM) significantly reduced the pressure developed within the bladder at any given infusion volume when compared to the pre-drug control filling (i.e. bladder compliance was improved). Vehicle treatment did not produce any significant changes.
8.4 Example 4
Identification of Pathway and Targets via Model Organisms
This example demonstrates that compounds I and II are capable of modulating the interaction of Gαq proteins and RGS proteins.
Methods
Phenotypic and Genetic Analysis
C. elegans strains were cultured and maintained according to standard procedures (Brenner, 1974; Hodgkin, 1988). All strains were assayed at 20° C. unless otherwise indicated.
Compound Treatment
Treatment of C. elegans with various compounds was conducted as follows:
compound was mixed with killed bacteria slurry (strain OP50, taken through multiple freeze-thaw cycles) to 2× desired final concentration. Adult wildtype (Bristol N2) or mutant hermaphrodites were collected in M9. Worms were mixed 1:1 with compound/bacteria mixture, and plated on peptone-free NGM plates. Plates were allowed to dry, and then placed at 20° C. until assayed.
For egg-laying assays, adults were treated overnight with compound. Approximately thirty animals were loaded onto agar pads made on glass slides and examined under Nomarski optics. Animals were scored as egg-laying defective if they contained>comma stage embryos.
EMS Mutagenesis/Screening
EMS mutagenesis was conducted according to standard procedures (Brenner, 1974). Briefly, Bristol N2 hermaphrodites of L4 stage were treated with 0.25% EMS (Sigma) in M9 for 4 hours at 20° C. Worms were washed 4× in M9, and plated onto seeded NGM plates (Po). Staged collections were taken of the F1 generation, and these were plated onto NGM plates at either 20° C. or 15° C. Staged collections of the F2 generation were plated onto NGM plates and allowed to grow until adulthood. These adults were then collected and treated with compound. After overnight treatment, animals that were not visibly egg-laying defective were isolated and re-tested for resistance to compound.
Mapping/Cloning Resistant Mutants
Mutant hermaphrodites were crossed to males of the polymorphic strain CB4856 (Hawaiian isolate). Recombinant homozygous mutants in the F2 generation were selected by visible phenotype and assayed for SNPs identified both through the Washington University SNP project and at Exelixis. Genotyping of SNP markers was performed using standard methods (Kwok, 2000). For eat-16(ep273) and egl-30(ep271), the genes were identified by sequence analysis of the corresponding genes in the mutant strains. Sequencing was performed according to manufacturer's instructions (Perkin Elmer Applied Biosystems). The eat-16(ep273) missense mutation changes a glutamine at position 158 to a lysine. egl-30(ep271) results in an amino acid substitution of an isoleucine at position 244 for methionine.
References

    Brenner, 1974, Genetics 77, 71-94.
    Hodgkin et al., 1988, The Nematode Caenorhabditis elegans (ed. W. B. Wood), Cold Spring
    Harbor, N.Y. Cold Spring Harbor Laboratory.
    Kwok, 2000, Pharmacogenomics 1, 95-100.


8.5 Example 5
Identification of C. elegans Mutants
This example demonstrates the identification of the biological targets of Compounds I and II in C. elegans. 
Compound I, causes neuromuscular defects in C. elegans, including pharyngeal pumping, body movement, and egg-laying. The C. elegans genome was surveyed for mutations that confer resistance to the egg-laying defect caused by compound. Mutations were identified in members of a conserved G-protein signaling pathway. Loss of function mutations in an RGS homolog, eat-16, conferred very strong resistance to compound effect. In addition, an activating mutation in a Gaq homolog, egl-30(ep271), was also identified. In C. elegans, RGS/eat-16 had been shown to negatively regulate the activity of Gαq/egl-30.
The mutation identified in Gαq/egl-30(ep271) was unique in its effect on compound response: other activating mutations in Gαq/egl-30 did not confer resistance (See below). The altered residue in egl-30(ep271) is absolutely conserved in all heterotrimeric Gα subunits that are regulated by RGS proteins. It is therefore possible to speculate that this mutation disrupts the ability of the RGS to regulate Gaq, mimicking a loss of RGS function. Experiments in yeast back this contention. Taken together, these results suggest that compound AG4A modulates the activity of the RGS or RGS/Gaq complex, resulting in decreased Gaq signaling.
Identification of the egl-30(M2441) Mutation:
The r4A-1 strain was identified in a screen for resistance to the BMS AG4A compound. The mutation causing resistance in this strain was mapped to a region containing a C. elegans Gaq homolog, egl-30. The phenotypes of known activating mutations in egl-30 resembles that observed in the r4A-1 strain. Sequencing of the egl-30 genomic region in the r4A-1 strain identified a G to A change in exon 6 of the coding sequence. This results in a methionine(M) to isoleucine(I) change at position 244 in the amino acid sequence of the protein. This residue is located in the Switch III region of the protein, which is known to be involved in many aspects of Gα function, including GTP hydrolysis, effector binding, and RGS binding. M244 is absolutely conserved in all Gαs that are regulated by RGS proteins. Interestingly, in those Gαs that are not regulated by RGS proteins, the analogous residue is a leucine (L). The conserved nature of this change suggest that this region of the protein may be involved in compound binding.
Identification of the eat-16(E158K) Mutation: Method Above
The r4A-3 strain was identified in an EMS mutagenensis screen for resistance to the BMS AG4A compound. The mutation causing resistance in this strain was mapped to a region containing a C. elegans RGS homolog, eat-16. The phenotypes of known loss of function mutations in eat-16 resembles that observed in the r4A-3 strain. Sequencing of the eat-16 genomic region in the r4A-3 strain identified a G to A change in exon 6 of the coding sequence. This results in a glutamine(E) to lysine(K) change at position 158 in the amino acid sequence of the protein. This region of the protein lies is in the N-terminus, separating the DEP domain from the GGL domain. To date, there is no known functional role for this region.
8.6 Example 6
Human Targets of Compounds I and II
This example demonstrates the identification of human homologues of the C. elegans polypeptides of Example 5. The human homologues are targets of Compounds I and II.
C. elegans EAT16 protein has been hypothesized to play a role in the mechanism of action of UI (Urge Incontinence) Compound I. Bioinformatics analysis was initiated to find the potential human homologue of C. elegans EAT16.
EAT16 is a 473 amino acid protein. Protein domain analysis using Hidden Markov Models (HMM) profiles revealed there are two domains in this protein. The domain information is as follows:

    DEP (Domain found in Dishevelled, EGL10 and Pleckstrin): Amino acids: 15-96
    RGS (Regulator of G-protein Signaling domain): Amino acids: 287-407


The EAT16 sequence was searched against the non-redundant protein database using the gapped BLAST program. The search identified the human RGS7 (Regulator of G-protein signaling) protein as the top hit. The other top hits include RGS6, RGS9 and RGS 11.
The RGS protein superfamily consists of six sub-families. RGS6, 7, 9 and 11 are related to each other and they have been shown to belong in a single subfamily by phylogenetic analysis (B. Zheng, et al. TIBS, 24:411-414, (1999)). The search results are consistent with the phylogenetic analysis. EAT16 is closer to the RGS 6,7,9 and 11 proteins when compared to other members of the RGS protein family. Among the sub-family members, EAT16 is closer to RGS7 when compared to RGS6, 9 and 11.
Comparison of EAT16 Against Various RGS Proteins:


    EAT 16 vs. RGS6: Identity=156/439 (35%), Similarity=244/439 (55%)
    
        RGS7: Identity=161/440 (36%), Similarity=255/440 (57%)
        RGS9: Identity=139/423 (32%), Similarity=235/423 (54%)
        RGS11: Identity=136/417 (32%), Similarity=237/417 (56%)
    
    


Domain analysis of RGS6 and RGS7 revealed that they have DEP and RGS domains consistent with EAT16. RGS6 has a G-gamma domain in addition to the above two domains. The profile score for the G-protein gamma subunit is not high. No conclusion can be derived based on this score. Nonetheless, from the absence of this domain and from sequence similarity, it appears as if EAT16 is closer to RGS7 than RGS6.
The next step is to take the human RGS6 and 7 proteins and search against all proteins present in C. elegans. This will detect which C. elegans proteins are closer to the human RGS6 and 7. Both RGS6 and 7 identified EGL10, another C. elegans protein, as the top hit. FIG. 7 illustrates the bioinformatics analysis workflow starting from EAT16.
EGL10 is a 558 amino acid protein. The sequence similarity between the RGS proteins and EGL10 is in two distinct regions.










Comparison of EGL10 against RGS6 and 7 proteins:
















EGL10:
Amino acids 19 to 256: 61% identity (76% similarity) with



RGS6



Amino acids 357 to 548: 41% identity (63% similarity) with



RGS6


EGL10:
Amino acids 19 to 226: 71% identity (84% similarity) with



RGS7



Amino acids 337 to 546: 41% identity (60% similarity) with



RGS7









The region between amino acid 225 to amino acid 350 of EGL10 does not match the RGS proteins. To determine how the matching regions correspond to the functional domains of EGL10, an analysis using Hidden Markov Model (HMM) profiles was performed. The results of the analysis are given below:
















EGL10:
Amino acids 37 to 118: DEP domain




Amino acids 329 to 395: G-protein gamma subunit




Amino acids 421 to 537: RGS domain










There is no functional domain present in the sequence region between amino acid 225 to amino acid 330. This region seems to be unique to EGL10. This suggests that EGL10 and EAT16 are both closer to the RGS6 and 7 proteins. However, EGL10 differs from these proteins in having a unique domain that does not correspond to RGS6, RGS7 or EAT 16. Also, this region of EGL10 is not similar to any of the proteins present in the complete protein database. FIG. 1A gives the alignment of various domains in EGL10, EAT16, RGS6 and RGS7 proteins.
Experimental analysis revealed that EGL10 is not involved in the mode of action of Compound I. The difference between EAT16 and EGL10 can be attributed to:

    1. Absence of an obvious G-protein gamma subunit domain in EAT16
    2. Unique region in EGL10 which does not have homology to any protein in the database.

There are two conclusions:

    1. The closest human protein to C. elegans EAT16 seems to be RGS7. The second best match, based on sequence similarity, is RGS6. Human RGS6 is highly related to RGS7 (73% identity and 82% similarity) and also aligns closely to EAT16. They also belong to the sub-family of RGS proteins and may have functional commonality with EAT 16.
    2. EGL10 has a striking sequence similarity with the N-terminus region of RGS 6 and 7. This fact suggests that EGL10 and EAT16 are closely related to the same human proteins RGS6 and RGS7. But they seem to play a different role in their response to Compound I. This difference can be attributed to the unique region in EGL10 and absence of an obvious G-protein gamma subunit domain in EAT16.


8.7 Example 7
RGS Domain mRNA Expression in Bladder Cells
This example demonstrates that the human RGS polypeptide is expressed in human bladder cells.
Total RNA from a primary smooth muscle cell line was isolated and hybridized to affymetrix gene chips. The results show that following RGS protein are expressed in Bladder and 293 cells. (Qiagen RNeasy midi-prep) for the HEK293 and smooth muscle cells. Confluent t175 cm flask each (approximately 10E7 cells). Qiashredders were utilized for the homogenization step, and RNA was eluted in 250 uL of RNAse-free water. mRNA was labeled and hybridized to Affymetrix chips to analyze gene expression levels. Analysis clearly showed differential specific expression of RGS proteins between HEK 293 human kidney cells and primary mouse bladder cell lines. In Primary bladder cells there was evidence for expression of RGS 1-7,9,10,20. In HEK 293 cells there was evidence for expression of RGS 1,2,7,9,10,16 and 19. The overlapping expressors are RGS 1,2,7,9, and 10.
8.8 Example 8
RGS Expression Across Affymetrix Chips Bladder Cells
This example demonstrates the expression patterns of the human RGS polypeptide.
Array Hybridization and Data Analysis
The cRNA preparation and array hybridization was performed according to the Affymetrix protocol (Affymetrix, CA). Briefly, cRNA was prepared from 10 mg of total RNA. The RNA was denatured at 70 C with T7-tagged oligo-dT primers and then reverse transcribed with Superscript II (GIBCO BRL) at 42 C for 1 hr. Second-strand cDNA was synthesized by adding DNA pol I, E. coli DNA ligase and RNase H, and incubation was carried out for 2 hrs at 16° C. After extracted once with phenol/chloroform, the synthesized cDNA was used for in vitro transcription with a BioArray High Yield RNA Transcript Labeling Kit (Enzo). Labeled cRNA was purified with RNeasy columns (Qiagen) and then fragmented (10 mg/per chip) before hybridization.
Human GenomeU95A arrays (HG-U95A, Affymetrix), containing  ̃12,000 full-length genes, were used for hybridization. The oligo array cartridges were prehybridized at 45 C for 10 min. The cRNA samples were added to cartridges and hybridization was performed for 16 hrs at 45° C. with 60 rpm rotation. After hybridization, the chips were washed and stained in a fluidics station using the antibody amplification protocol from Affymetrix. The chips were then scanned using a Hewlett-Packard GeneArray scanner. The data was analyzed using GeneChip software (Affymetrix). An intensity value and presence/absence (P/A) call was derived from hybridization signal for each gene to represent its expression level.












RGS
Intensity
Presence/Absence















Bladder Cell Line









1
9
P


2
53
P


3
79
P


4
166
P


5
8
P


6
18
P


7
50
P


9
250
A


10
250
P


11
47
A


12
16
A


14
84
A


16
46
A


19
19
A


20
32
P







HEK 293









1
9
P


2
14
P


3
10
A


4
8
A


5
18
A


6
28
A


7
28
P


9
387
A


10
127
P


12
30
A


14
97
A


16
59
P


19
29
P


20
2
A









Common RGS Proteins in Bladder and HEK: 1,2,7,10

8.9 Example 9
Compounds I and II Selectively Modulate the Gαq Signaling Pathway
This example demonstrates that the effects of Compounds I and II on Hek293 Cells and bladder smooth muscle cells stimulated by carbochol or histamine are consistent with and RGS/Gq mechanism.
Procedure for [Ca2+]i Measurements


    1. HEK 293 or Clonetics primary human bladder smooth muscle cells were plated at a concentration of 7e3 cells/well in Poly-L lysine coated 96 well plates, white with clear bottoms (Costar).
    2. The plates are allowed to attach overnight at 37° C. 5%CO2. The cells were rinsed 2× with Krebs-HEPES buffer and then loaded with 4 uM Fluo4 (Molecular Probes) for 60 minutes in the same buffer containing 1% (wt/vol) Pluronic F-127 and 0.25 mM sulfinpyrazone.
    3. After loading the cells were rinsed 3× with Krebs-HEPES buffer containing 0.5% (wt/vol) BSA (Sigma Chemical)
    4. Buffer alone or buffer containing test compounds, was injected sequentially into separate wells and the fluorescence intensity monitored in a Victor2 96 well plate fluorometer (PE-Wallac). Measurements were taken at 1-s intervals using an excitation wavelength of 485 nm and emissions filter of 535 nm.
    5. Five baseline measurements were taken at 1-s intervals prior to each injection (see FIGS. 4A and 4B).


8.10 Example 10
Compound I Targets Gαq and RGS Polypeptides
This example demonstrates that Compound I specifically targets Gαq signaling.
Calcium assays were run by standard protocols as described above and in Zhu et al., 1998, J. Biol Chem 273:133-142. It is known that calcium signals in response to carbachol have both a Gq and a Go/Gi signaling component. Pertussis toxin is a known inhibitor of Go/Gi signaling in cells. We asked the question if Compound I was inhibiting Gq or Go/Gi signals in Hek 293 kidney cells. We already were able to show that carbachol induced calcium influxes were blocked by Compound I, as well as Compounds II and III, the question was if this was a Gq specific effect. Go/Gi is known to block the Go/I but not Gq signals. If the compound blocked Go/Gi then we would expect the combination of pertussis toxin and Compound I would not be cumulative as they would both be inhibiting the same signal. If however Compound I were inhibiting Gq signal specifically, then we would expect the combination of pertussis toxin (hitting Go/Gi) and Gq. Our results (see FIG. 5A) show that the effect of Compound I as exemplified in this figure clearly shows the effects of Compound I and Pertussis toxin are cumulative, there by showing that the Compound I is specifically inhibiting Gq signaling and not Go/Gi.
8.11 Example 11
Compounds I, II and III Modulate the Interaction of Gαq Polypeptide and RGS Polypeptide
This example demonstrates that compounds I, II and III modulate the affinity of a Gαq polypeptide and an RGS polypeptide for one another.
Compounds I and III were discovered in assays of smooth muscle contraction. Specifically these compounds are capable of relaxing strips of bladder smooth muscle that have been induced to contract with the muscarinic agonist carbochol. The compounds were synthesized and retested for activity. The molecular targets of these compounds were not know but have been discovered utilizing a combination of model systems genetics and biochemistry.
C. elegans animals were treated with Compounds I and III and their effects were observed on the pumping of the animals pharynx, the contraction of the egg laying muscles and on the ability of the mobility muscles to function properly. The ability of these compounds to effect muscle contraction in human tissue strips is consistent with the effects on muscle seen in C. elegans. A mutagenesis and selection process was carried out to find C. elegans animals that were resistant to the muscle effects of the compounds and via this process genes involved in the compounds action were identified. In particular the Eat-16(RGS) and Egl-30 (Galpha q) proteins were implicated in the action of these compounds by virtue of specific amino-acid changes rendering the compound unable to effect its action on C. elegans muscle. Human orthologs for Eat-16 and Egl-30 were identified as RGS 6,7,9, and 11 (other RGS family members of which there are 23 are known to play similar roles and in may cases have interchangeable activities), and G alpha q respectively. Based on the information from the C. elegans experiments these human proteins are hypothesized to play a role in the function of Compounds I and III.
Compounds I and III were then tested in a human tissue culture cell line Human Kidney cell line Hek 293 for their ability to effect the function of human RGS and Gq signaling proteins via their role in Ca++ signaling. Compounds I and III were clearly able to effect the function of Gq and RGS in these assays, thereby validating their role in the action of Compounds I and III. The mammalian assays and C. elegans assays suggested that Compounds I and III were able to agonize the ability of RSG proteins to inactivate the signaling of G alpha q. This agonistic activity could be explain in several ways and these were tested. In particular we have shown biochemically that Compounds I and III increase the affinity of RGS7 with Gq in immunoprecipitation assays. This explains the biological activity of Compounds I and III, in that they increase the affinity of RGS-7 and Gq thereby increasing the ability of RGS-7 to inactivate Gq thereby agonizing G protein signals. In addition we have modeled the ability of Compounds I and III to bind RGS proteins or the combination of RGS-7 and Gq and have found likely binding pockets for these compounds.
GDP ALF(4)- is known to mimic the putative pentavalent transition state of Gq between active GTP bound and inactive GDP bound. GDP ALF4- also is known to increase the affinity of RGS proteins for Gq such that this interaction can be visualized by immunopreciptation assay. Here we are able to show that Compound III increase the affinity of RGS7 for Gq in a similar fashion to GDP Alf(4)-. This provides direct biochemical evidence that Compound III increase the affinity of RGS7 (and family members) to Gq and is completely consistent with its RGS7 agonist activity. Furthermore this ability proves a functional screening assay for additional compounds that act as agonists/antagonist or RGS related proteins and their binding to their respective G proteins.
Hek 293 cells were transfected with CMV-G alpha qHA, CMV-mycRGS-7 and CMV-Gbeta5. 48 hours later the cells were harvested and lysed. Antibody to the myc epitope was utilized to immunoprecipitate RGS-7 in the presence or absence of ALF4-. The precipitated proteins were split into two fractions and loaded onto gels and western blotted on to nitrocellulose. In FIG. 6, the left blot (1) was probed with anti-myc antibody to detect RGS-7. RGS-7 was immunoprecipitated by the anti-myc antibody in the presence and absence of ALF4-. The right blot shows the second fraction which was probed against the Hemagglutanin (HA) tag that was fused to G alpha q. A G alpha q protein band was detected to have associated with RGS-7 and precipitated in the presence of ALF4- (lanes A) but not in the absence of ALF4- (lane B). In the second experiment, RGS-7 was immuno-precipitated again with the Myc epitope to which it was fused and the presence of G alpha q was detected in the presence or absence of ALF4- or different concentrations of Compound III. As shown in 3) Lane (F) G alpha q was detected in the presence of ALF4- but also was present in the presence of Compound III (Lane F) this was true for multiple concentrations of Compound III (Lane H) and shows biochemically that Compound III and related compounds affect directly the affinity of RGS-7 and Gq. This increase in RGS-7 affinity would be predicted to be the same for other RGS7 family members such as RGS 6,9 and 11, as they have been shown previously to have similar affinities and effects on G alpha q. In addition this assay provides a method for screening for compounds that affect the interaction of RGS proteins and G alpha q.
In FIG. 5B, Compounds I, II and III affect the affinity of Gq and RGS proteins for one another. FIG. 5B is an immunoprecipitation blot of myc tagged RGS which has been probed with an anti hemagglutinin antibody to detect a hemagglutinin tagged Gαq. The negative control lane “Ctrl” shows (lane 10 from the left) that very little Gq is immunoprecipitated I the absence of Alf4-. Alf4- mimics the transition state of Gq and increases its affinity with RGS such that it can be detected “Alf4-” lane 10 from the left. Compound I, and Compound II alone, lane 4 and 6 from the left respectively, clearly increase the affinity of Gq for RGS enough to be visualized as compared to the Ctrl lane alone. The ability of Compounds I, II and III to increase the affinity of Gq and RGS is a novel mechanism by which one can Gαq activity and such compounds and screens for additional compounds with such activity are useful wherever one desires to antagonize g protein signaling that involves Gαq and RGS proteins.
Protocol for co-IP of Gq with RGS7 Protein


    Add 1 ug mouse IgG, 20 ul A/G agarose beads to lysate
    Rotate at 4° C. for 30 mins
    Spin
    Split the lysate into two tubes, add volume to 1 ml
    Add 4 ul of AlF4 mix to one of the tube
    Let sit on ice for 1 hour
    Add 4 ug Ab to supernate
    Rotate at 4° C. at least 1 hr
    Add 40 ul A/G plus agarose beads
    Rotate at 4° C. at lease 1 hr
    Spin, aspirate supe
    Wash three times
    Spin, aspirate super
    Boil
    Load on gel

Buffer Condition:

    20 mM Tris pH 7.4
    1 mM EDTA
    1 mM DTT
    100 mM NaCI
    5 mM MgCI2
    0.7%
    riton X-100
    1 mM PMSF
    leupeptin
    aprotinin
    50 uM AlCI3
    10 mM MgCI2
    5 mM NaF


8.12 Example 12
Determination of the Structure of eat-16
A model of the C-terminal domain of C. elegans protein eat-16 was developed based on sequence alignment with the homologous (33% identity with substantial additional conservation) protein rat RGS-4 (Genbank accession no. gi| 8394182) whose structure in complex with a G-protein is available in the Protein Data Bank structure as 1AGR (Tesmer et al., (1997), Cell, 89:251-61). Sequence alignments are shown in Table 1.







TABLE 1





Sequence Alignment of C. elegans eat-16 onto 1AGR chain E (Rat


RGS4)
















Rat RGS4
VSQEEVKKWA ESLENLINHE CGLAAFKAFL KSEYSEENID FWISCEEYKK


(SEQ ID


NO.: 1)


 


eat-16
.TEKRVKRWG LSVQELVKDP IGRQVLETFL ESEFSSENIR FWIAIQDLK.


(SEQ ID


NO.: 2)


 


Rat RGS4
IKSPSKLSPK AKKIYNEFIS VQATKEVNLD SCTREET... .SRNMLEPTI


(SEQ ID


NO.: 1)


 


eat-16
YAPNEQIYQK AERIREEFLA QGAPAQVNVD NRTLDQTLEC ISKAKDASQM


(SEQ ID


NO.: 2)


 


Rat RGS4
T.CFDEAQKK IFNLMEKDSY RRFLKSRFYL DLT


(SEQ ID


NO.: 1)


 


eat 16
RFAFYHSEEH VFTLMAKDSY PRFVRSQIYK AVL


(SEQ ID


NO.: 2)









The program LOOK was used for alignments and the model building module within LOOK, SEGMOD, was used to build the homology models (Levitt, (1992), J. Mol. Biol. 226: 507-533; Levitt, (1983), J. Mol. Biol. 170: 723-764).

Table 3, infra, provides the atomic structure coordinates of eat-16 built in this way.
Modeling Compounds in the eat-16 Active Site:
The surface of the resulting model of eat-16 was analyzed for pockets or depressions of a size that could accommodate the active compounds I and II, shown hereinbelow. (Surfaces were rendered and displayed using program GRASP (Nicholls, A.; Sharp, K.; and Honig, B., PROTEINS, Structure, Function and Genetics, (1991), Vol. 11 (No.4), pg. 281ff). A “molecular elevation” surface rendering was used to highlight pockets (FIG. 9B). The “molecular elevation” plot view of the eat-16 model accentuates pockets, which appear darker than the surrounding protein. A distinct, relatively large pocket can be seen on the middle left of the protein. As discussed hereinbelow, the region around the pocket is decidedly hydrophobic. FIG. 9A shows a ribbon diagram of the eat-16 model. The N-terminus is the upper terminus and the C terminus is the lower terminus. The pocket occurs on the C terminal face and is composed of residues from helices 1, 2, and 7.
Because both of the compounds I and II that were known to bind are substantially hydrophobic, the surface was further analyzed for hydrophobic characteristics, again using the program GRASP (Nicholls, 1991).




Two to three hydrophobic patches (identified as hydrophobic patches 1-3) of a size that could accommodate the two molecules (FIGS. 9C and 9D) were identified. In FIG. 9C, the surface of the eat-16 model has been shaded according to the hydrophobic character of the underlying amino acids: lighter denotes hydrophobic; darker, hydrophilic. The region around the pocket is decidedly hydrophobic. A tyrosine residue lies at the bottom of the pocket. FIG. 9D depicts a superposition of structure II, as a stick figure, onto the hydrophobic pocket, shown in white, showing that the molecule and the pocket are of similar size. The pocket and structure I are also of similar size.
One of the hydrophobic patches (hydrophobic patch 1) was coincident with a pocket of substantial size, as discussed above. Starting the numbering at the RGS domain of the eat-16 protein with sequence TEKRVKRWGLSVQ (SEQ ID NO:24), (TEKR being numbered 1, 2, 3, 4, respectively) this patch was comprised of residues including: Arg 4, Val 5, Lys 6, Arc 7, Trp 8, Leu 15, Ile 20, Gly 21, Val 24, Phe 121, Val 122, Ile 126, Tyr 127, Ala 129, Val 130. The corresponding residues in the human protein are (numbering in a similar way from sequence SQQRVKRWGRGMD (SEQ ID NO.:25)): Arg 4, Val 5, Lys 6, Arg 7, Trp 8, Ala 15, Val 20, Gly 21, Gln 24, Phe 116, Val 117, Ile 121, Tyr 122, Ala 124, Val 125.
Another hydrophobic patch (hydrophobic patch 2) was located on the reverse side of the eat-16 molecule. This patch is not associated with a well-defined “pocket” however, but it is of a size comparable to that of active molecules. Also, it is in a region of the RGS proteins that demonstrates conformational flexibility between the free and G-protein-bound forms and consequently is potentially a sensitive site for regulating the conformational change and hence activity. For the C. elegans eat-16 protein model, this patch was composed of residues (numbering in the same way as above): Leu 10, Ser 11, Val 12, Val 24, Val 122, Tyr 127, Ala 129, Val 130.
Additionally, another small pocket with hydrophobic characteristics (hydrophobic patch 3) was found in the other domain of the protein. It is composed of residues: Phe 102 (at the center of the pocket), and (surrounding the pocket) Lys 48, Leu 47, Arg 99, Tyr 103, His 104.
In general terms, active molecules are expected to be hydrophobic in character or to contain two or more substantially hydrophobic groups, wherein two hydrophobic portions of the molecules separated from one another by a group containing a hydrogen bonding moiety.
This information permits molecules that are potentially inhibitory of eat-16 to be identified in databases and through screening.
Applications to Docking and Structure Prediction from the Distorted Native Conformation: RGS Mechanism of Action eat16 and Human RGS7
None of the three binding sites is in the region found to be in direct contact with the G-I-alpha-1 protein in its complex with RGS4. It is known (from X-ray crystallographic and NMR structural determination studies) that RGS4 undergoes a substantial conformational change on binding to the G-protein (Moy, F. J.; Chanda, P. K.; Cockett, M. I.; Edris, W.; Jones, P. G.; Mason, K.; Semus, S.; Powers, R., Biochemistry, 39(24):7063-73, (2000)). “NMR Structure of Free RGS4 Reveals an Induced Conformational Change upon Binding G∝” Biochemistry, 39(24): 7063-7073 (2000); Tesmer, J. J., Berman, D. M., Gilman, A. G., Sprang, S. R., “Structure of RGS4 Bound to ALF4(−)-Activated G(I Alphal): Stabilization of the Transition State for GTP Hydrolysis,” Cell (Cambridge, Mass.) 89:251, (1997).
This results in a repacking of the N and C terminal regions of RGS4 which induces the formation of the binding pocket on RGS4 by which RGS4 binds to the G-protein. This binding pocket and the interactions that it can make with the G-protein are not fully realized in the free protein. The latter conformation could be considered to be an “active” conformation. The two principal binding pockets that we disclose on our RGS7 model are found in the corresponding N and C terminal regions of the model. It is proposed that compounds could activate the RGS proteins, and hence the GTP hydrolytic activity of the G-proteins, by stabilizing the active conformation in solution. It is expected that in solution, the predominant conformation of the RGS proteins would be the inactive form with a small probability that the active form would be transiently assumed. The compounds in question could bind to the pockets during those low probability events and could stabilize the active form and so effectively increase the concentration of the active conformer. This would effectively increase the probability of agonism of the GTPase activity of the G-protein.
Alternatively, the compounds could act directly on the RGS-G-protein complex after it is formed. By binding to the pockets, they could “lock” RGS into the already-assumed active conformation. Possibly this binding and locking could not only increase the percentage of RGS assuming an active conformation at any given time, but also—and possibly preferentially—increase the time that the two proteins stay in contact by preventing a separation of the proteins once a complex between them has been formed.
Additionally, the possibility that the compounds in question could exert a similar effect by binding at the some interfacial region between the RGS and G-protein or in a pocket formed when the two proteins complex should not be excluded.
Another possible mechanism of action involves potentiation of the GTPase activity through direct interaction with the G-alpha protein. It has been shown that the G-alpha effector cyclic GMP phosphodiesterase as well as the unrelated adenylyl cyclase proteins induce changes in the G-alpha conformation that potentiates its interaction with the RGS proteins (Slep, K. C., Kercher, M. A., He, W., Cowan, C. W., Wensel, T. G., Sigler, P. B., “Structural Determinants for Regulation of Phosphodiesterase by a G-Protein at 2.0 Å,” Nature, 409:1071, (2001); Tesmer, J. J. G., Dessauer, C. A., Sunahara, R. K., Murray, L. D., Johnson, R. A., Gilman, A. G., Sprang, S. R., “Molecular Basis for P-Site Inhibition of Adenylyl Cyclase,” Biochemistry, 39:14464 (2000)). This potentiation increases RGS binding and consequently GTPase activity. The compounds discussed could induce or stabilize the same or a similar conformational change in the G-alpha protein that would similarly potentiate and stabilize RGS binding to the G-protein.
8.13 Example 13
Determination of the Structure of RGS-7
Following the same protocol as for Example 13, a model of the C-terminal domain of human RGS-7 was developed based on sequence alignment with the homologous (34% identity with substantial additional conservation) protein rat RGS-4 whose structure in complex with a G-protein is available in the Protein Data Bank structure as 1AGR (Tesmer et al., (1997), Cell, 89: 251-61). The program LOOK was used for alignments and the model building module within LOOK, SEGMOD, was used to build the homology models (Levitt, (1992), J. Mol. Biol., 226: 507-533; Levitt, (1983), J. Mol. Biol., 170: 723-764). Sequence alignments are shown in Table 2.







TABLE 2





Sequence Alignment of RGS-7 onto 1AGR chain E (Rat RGS4)
















Rat RGS4
....V.SQEE VKKWAESLEN LINHECGLAA FKAFLKSEYS EENIDFWISC


(SEQ ID


NO.: 1)


 


Human RGS-7
GLVP.RGSHR VKRWGFGMDE ALKDPVGREQ FLKFLESEFS SENLRFWLAV


(SEQ ID


NO.: 4)


 


Rat RGS4
EEYKKIKSPS KLSPKAKKIY NEFISVQATK EVNLDSCTRE ETSRNMLEPT


(SEQ ID


NO.: 1)


 


Human RGS-7
EDLKK.RPIK EVPSRVQEIW QEFLAPGAPS AINLDSKSYD KTTQNVKEPG


(SEQ ID


NO.: 4)


 


Rat RGS4
ITCFDEAQKK IFNLMEKDSY RRFLKSRFYL DLT


(SEQ ID


NO.: 1)


 


Human RGS-7
RYTFEDAQEH IYKLMKSDSY PRFIRSSAYQ EL.


(SEQ ID


NO.: 4)









Table 4 provides the atomic structure coordinates of RGS-7.
Structures coordinates for RGS-7 according to Table 4 may be modified by mathematical manipulation. Such manipulations include, but are not limited to, fractionalization of the raw structure coordinates, integer additions or subtractions to sets of the raw structure coordinates, inversion of the raw structure coordinates and any combination of the above.
8.14 Example 14
RGS Modeling and Analysis: Gα/RGS/PDE Trimeric Complex: Binding Sites for UI Compounds on the Gα Protein at the PDE Binding Site.
The compounds I and II behave much like the effector, PDE, as well as adenylate cyclase. Both of these effectors bind at the same location of the G-protein chimera. The crystal structure of the heterotrimeric complex of the RGS domain of RGS-9, the γ-subunit of phosphodiesterase and the Gt/I1chimera α subunit [(rgs9)-(PDE γ)--(gt/i1α)-(GDP)-(α4-)-(Mg2+)] chimera of guanine nucleotide-binding protein G(t) α-1 subunit and guanine nucleotide-binding protein G(I), α-1 subunit (PDB entry 1FQJ; K. C. Slep, M. A. Kercher, W. He, C. W. Cowan, T. G. Wensel, P. B. Sigler, “Structural Determinants for Regulation of Phosphodiesterase by a G-protein at 2.0 A,” Nature, (2001) Vol. 409, page 1071) were examined for possible binding sites. The effector molecule, PDE, was removed from its complex with the G-protein. This resulted in a well-defined pocket. Such pockets are not necessarily typical of protein-protein interactions. This pocket had a hydrophobic face and so could be complementary to the hydrophobic ligands. It was composed of residues: Leu 201, Val 202, Ala 204, Glu 205, Glu 207, Glu 208, Arg 211, Phe 236, Asp 241, Glu 244, Glu 245, Lys 248, Lys 249, Ile 254.
The program ICM (the underlying methods of which are described in: Abagyan, R. A., and Totrov, M. M., “Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins.” J. Mol. Biol. 235: 983-1002, (1994); and Abagyan, R. A., Totrov, M. M., and Kuznetsov, D. N., “ICM—a new method for protein modeling and design,” J. Comp. Chem., 15: 488-506 (1994)) was used to investigate possible binding interactions of one of the molecules, II, with the pocket. It found favorable binding orientations of the molecule within the pocket, showing the characteristic surface complementarity seen in protein/ligand complexes and favorable hydrogen-bonding interactions. The ICM scoring function for protein/ligand interactions was comparable with that calculated for known protein/ligand complexes. See FIGS. 8 and 9.
8.15 Example 15
Effects of Compound III on Rabbit Bladder Cystometry and the Contractile Response to Acetylcholine Using an in situ Preparation: Normal Rabbit Bladder
The effects of compound III on bladder compliance were tested as follows. Each rabbit was sedated with Ketamine xylazine. A femoral artery catheter was placed for monitoring blood pressure and heart rate, a urethral catheter was placed for performing cystometry and monitoring bladder pressure, and a second catheter was fed through the other femoral artery, through the descending aorta and to the level of the bladder base to allow drugs to be administered directly to the bladder.
Saline was infused into the bladder at a rate of 1.5 ml/min until a micturition contraction was recorded; bladder pressure was monitored continually. The bladder was emptied and 15 ml of saline placed in the bladder. Acetylcholine (3×10−5·M Ach) was administered and the pressure response measured and recorded. After 30 minutes, compound III (5 μM/kg) was given i.v. and the rabbit allowed to rest for 30 minutes. At the end of this period a second cystometry and Ach stimulation were performed. At this time, 10 μM/kg was given and after another 30 minute equilibration period, a third set of studies performed.
Compound III was dissolved in a vehicle of DMSO and propylene glycol. Preliminary studies demonstrated that vehicle alone had no effect on the cystometry or response to Ach. Additionally, there were no significant differences among three sets of studies on each control animal.
There was a progressive decrease in the initial intravesical pressure mediated by compound III but no change in the final pressure at the time of micturition. In addition, compound III significantly increased the volume at the time of the first micturition contraction (bladder capacity; FIG. 11). Compound III did not affect micturition pressure or the pressure response to Ach.
These results indicate that compound III increases bladder compliance thus allowing for a greater bladder capacity at micturition. Clinically, this would be beneficial for the treatment of several conditions characterized by low compliance and early micturition contractions (urgency, frequency, nocturia). Compound III had no negative effects on either the micturition pressure or the response to Ach.
8.16 Example 16
Effects of Compound III on Rabbit Bladder Cystometry and the Contractile Response to Acetylcholine Using an in situ Preparation: Hypertrophied Rabbit Bladder
In order to assess the effects of compound III on hypertrophied bladders, model hypertrophied bladders were constructed in rabbits by obstructing the proximal urethra bladder neck. Each rabbit was anesthetized with an intramuscular injection of 3.0 ml of a ketamine/xylazine mixture (25 mg/ml ketamine, 8 mg/ml xylazine), surgical anesthesia was maintained with intravenous infusion of nembutal (25 mg/ml). The animal was shaved, painted with povidone-iodine solution and draped in a sterile fashion. The urethra was catheterized using an 8Fr. feeding-tube. Through a midline incision, the bladder was exposed. The bladder neck was cleared of surrounding connective and fatty tissues. A 3-0 silk ligature was passed around the proximal urethra bladder neck area and tied loosely, such that a small hemostat could be placed through the ligature, along side the urethra, without tension. The abdominal wound was closed in two layers with 2-0 vicryl, using a running stitch on the inner layer and interrupted, vertical mattress stitches on the skin. Each rabbit was recovered under observation.
At 4 weeks following obstruction, the effect of compound III on bladder compliance, unstable bladder contractions, and response to acetylcholine (Ach) was determined using the in situ model (see Section 8.15, above). Bladder capacity of the obstructed bladders were very variable.
8.17 Example 17
Effects of Compound III on Hyperreflexia in Normal Rabbits
The effects of compound III on hyperreflexia in normal rabbits were tested as follows. Each rabbit was anesthetized with ketamine/xylazine as described above. A catheter was placed into the bladder for both filling the bladder and monitoring bladder pressure. Under anesthesia, penile ligature was placed around the base of the penis. 30 minutes later, the bladder was emptied, and filled in 5 ml steps at 15 minute intervals until hyperreflexia was initiated (usually at approximately 30 mls). After 15 minutes of stable contractions, BMS-1 was administered as given above, and the effect on the frequency and amplitude of hyperreflexia was determined.
Partial outlet induces unstable bladder contractions which are mediated primarily by myogenic mechanisms (contractions initiated within the bladder smooth muscle matrix, and they are not dependent upon spinal or supraspinal reflexes). However, the hyperreflexia induced by penile ligation is strictly neurogenic in origin, resulting from the stimulation of spinal reflex mechanisms. This is the key difference between unstable bladder contractions and hyperreflexia.
The administration of 5 or 10 μM/kg compound III had no effects on either blood pressure or heart rate. Partial outlet obstruction induced a significant increase in bladder weight. The cystometric studies demonstrated that compound III produced significant decrease in the initial pressure of the bladder during filling, and an increase in compliance. (decreased resistance to stretch) of the bladder during the initial stages of bladder filling (FIG. 12). Similar to what was observed in the normal bladders (see above), compound III had no effect on the sensitivity of the bladder to Ach stimulation, and had no significant effect on the maximal response to Ach. Partial outlet obstruction induced unstable bladder contractions in approximately 50% of rabbits. Compound III administration significantly reduced the amplitude of the unstable contractions without significantly affecting the frequency of contractions (FIG. 13).
The present invention is not to be limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those having skill in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall with in the scope of the appended claims. All references cited herein are hereby incorporated by reference in their entireties.







TABLE 3







REMARK 4 C. elegans Eat-16 protein modeled on Rat RGS-4 (E. chain from PDB


REMARK 4 entry 1AGR. See: Tesmer J. J., D. M. Berman, A. G. Gilman, S. R. Sprang


REMARK 4 Structure of RGS4 Bound to ALF4(−)-Activated G(I Alpha1):


REMARK 4 Stabilization of the Transition State for GTP Hydrolysis,


REMARK 4 Cell(Cambridge, Mass.) V. 89 251 1997)


REMARK 4


REMARK 4 WRGS COMPLIES WITH FORMAT V. 2.0, 22-NOV-2000


















ATOM
1
N
THR
E
1
6.560
26.957
66.192
1.00
0.03
N


ATOM
2
CA
THR
E
1
5.444
26.073
66.568
1.00
0.02
C


ATOM
3
CB
THR
E
1
4.108
26.670
66.137
1.00
0.10
C


ATOM
4
OG1
THR
E
1
4.071
26.710
64.717
1.00
0.10
O


ATOM
5
CG2
THR
E
1
3.924
28.088
66.666
1.00
0.22
C


ATOM
6
C
THR
E
1
5.610
24.713
65.910
1.00
0.03
C


ATOM
7
O
THR
E
1
6.279
24.596
64.877
1.00
0.03
O


ATOM
8
N
GLU
E
2
4.807
23.761
66.355
1.00
0.03
N


ATOM
9
CA
GLU
E
2
4.886
22.401
65.807
1.00
0.04
C


ATOM
10
CB
GLU
E
2
4.138
21.457
66.738
1.00
0.18
C


ATOM
11
CG
GLU
E
2
4.287
20.001
66.307
1.00
0.64
C


ATOM
12
CD
GLU
E
2
3.468
19.115
67.238
1.00
1.13
C


ATOM
13
OE1
GLU
E
2
3.055
19.627
68.269
1.00
1.52
O


ATOM
14
OE2
GLU
E
2
3.182
17.988
66.859
1.00
1.49
O


ATOM
15
C
GLU
E
2
4.285
22.320
64.405
1.00
0.02
C


ATOM
16
O
GLU
E
2
4.823
21.591
63.564
1.00
0.02
O


ATOM
17
N
LYS
E
3
3.430
23.276
64.074
1.00
0.04
N


ATOM
18
CA
LYS
E
3
2.861
23.346
62.728
1.00
0.03
C


ATOM
19
CB
LYS
E
3
1.663
24.280
62.773
1.00
0.30
C


ATOM
20
CG
LYS
E
3
0.689
23.894
63.882
1.00
1.06
C


ATOM
21
CD
LYS
E
3
−0.474
24.879
64.051
1.00
1.20
C


ATOM
22
CE
LYS
E
3
−0.103
26.179
64.777
1.00
1.19
C


ATOM
23
NZ
LYS
E
3
0.595
27.162
63.927
1.00
1.80
N


ATOM
24
C
LYS
E
3
3.874
23.914
61.737
1.00
0.02
C


ATOM
25
O
LYS
E
3
3.933
23.447
60.596
1.00
0.01
O


ATOM
26
N
ARG
E
4
4.840
24.654
62.257
1.00
0.02
N


ATOM
27
CA
ARG
E
4
5.876
25.261
61.425
1.00
0.04
C


ATOM
28
CB
ARG
E
4
6.431
26.460
62.190
1.00
0.20
C


ATOM
29
CG
ARG
E
4
7.525
27.195
61.428
1.00
0.81
C


ATOM
30
CD
ARG
E
4
7.013
27.807
60.130
1.00
1.64
C


ATOM
31
NE
ARG
E
4
6.013
28.849
60.400
1.00
2.18
N


ATOM
32
CZ
ARG
E
4
6.100
30.094
59.928
1.00
3.04
C


ATOM
33
NH1
ARG
E
4
5.113
30.962
60.157
1.00
3.95
N


ATOM
34
NH2
ARG
E
4
7.146
30.453
59.181
1.00
3.35
N


ATOM
35
C
ARG
E
4
6.968
24.236
61.154
1.00
0.01
C


ATOM
36
O
ARG
E
4
7.434
24.125
60.015
1.00
0.02
O


ATOM
37
N
VAL
E
5
7.088
23.294
62.078
1.00
0.03
N


ATOM
38
CA
VAL
E
5
8.008
22.170
61.905
1.00
0.00
C


ATOM
39
CB
VAL
E
5
8.192
21.486
63.256
1.00
0.02
C


ATOM
40
CG1
VAL
E
5
9.120
20.281
63.150
1.00
0.00
C


ATOM
41
CG2
VAL
E
5
8.715
22.469
64.297
1.00
0.01
C


ATOM
42
C
VAL
E
5
7.442
21.182
60.891
1.00
0.02
C


ATOM
43
O
VAL
E
5
8.151
20.811
59.946
1.00
0.02
O


ATOM
44
N
LYS
E
6
6.126
21.030
60.901
1.00
0.01
N


ATOM
45
CA
LYS
E
6
5.455
20.187
59.905
1.00
0.02
C


ATOM
46
CB
LYS
E
6
3.994
20.027
60.305
1.00
0.02
C


ATOM
47
CG
LYS
E
6
3.845
19.343
61.657
1.00
0.01
C


ATOM
48
CD
LYS
E
6
2.391
19.354
62.106
1.00
0.01
C


ATOM
49
CE
LYS
E
6
2.230
18.733
63.486
1.00
0.00
C


ATOM
50
NZ
LYS
E
6
0.831
18.811
63.937
1.00
0.03
N


ATOM
51
C
LYS
E
6
5.505
20.818
58.517
1.00
0.03
C


ATOM
52
O
LYS
E
6
5.808
20.117
57.544
1.00
0.02
O


ATOM
53
N
ARG
E
7
5.480
22.141
58.465
1.00
0.02
N


ATOM
54
CA
ARG
E
7
5.603
22.846
57.188
1.00
0.03
C


ATOM
55
CB
ARG
E
7
5.063
24.260
57.351
1.00
0.17
C


ATOM
56
CG
ARG
E
7
3.549
24.259
57.503
1.00
0.96
C


ATOM
57
CD
ARG
E
7
2.993
25.674
57.605
1.00
1.31
C


ATOM
58
NE
ARG
E
7
3.399
26.347
58.849
1.00
2.47
N


ATOM
59
CZ
ARG
E
7
2.505
26.763
59.750
1.00
3.62
C


ATOM
60
NH1
ARG
E
7
2.893
27.490
60.799
1.00
4.65
N


ATOM
61
NH2
ARG
E
7
1.205
26.545
59.544
1.00
4.04
N


ATOM
62
C
ARG
E
7
7.033
22.901
56.651
1.00
0.02
C


ATOM
63
O
ARG
E
7
7.194
23.058
55.438
1.00
0.02
O


ATOM
64
N
TRP
E
8
8.026
22.588
57.467
1.00
0.03
N


ATOM
65
CA
TRP
E
8
9.399
22.476
56.962
1.00
0.03
C


ATOM
66
CB
TRP
E
8
10.378
22.572
58.124
1.00
0.01
C


ATOM
67
CG
TRP
E
8
10.392
23.883
58.871
1.00
0.02
C


ATOM
68
CD1
TRP
E
8
9.965
25.108
58.415
1.00
0.01
C


ATOM
69
NE1
TRP
E
8
10.137
26.017
59.405
1.00
0.03
N


ATOM
70
CE2
TRP
E
8
10.676
25.450
60.500
1.00
0.02
C


ATOM
71
CZ2
TRP
E
8
11.016
25.948
61.749
1.00
0.02
C


ATOM
72
CH2
TRP
E
8
11.574
25.101
62.702
1.00
0.01
C


ATOM
73
CZ3
TRP
E
8
11.785
23.758
62.408
1.00
0.03
C


ATOM
74
CE3
TRP
E
8
11.437
23.248
61.164
1.00
0.03
C


ATOM
75
CD2
TRP
E
8
10.877
24.090
60.216
1.00
0.01
C


ATOM
76
C
TRP
E
8
9.608
21.113
56.323
1.00
0.02
C


ATOM
77
O
TRP
E
8
10.237
20.996
55.263
1.00
0.02
O


ATOM
78
N
GLY
E
9
8.904
20.134
56.867
1.00
0.00
N


ATOM
79
CA
GLY
E
9
8.974
18.760
56.364
1.00
0.00
C


ATOM
80
C
GLY
E
9
8.101
18.563
55.131
1.00
0.02
C


ATOM
81
O
GLY
E
9
8.337
17.643
54.339
1.00
0.02
O


ATOM
82
N
LEU
E
10
7.077
19.388
55.001
1.00
0.01
N


ATOM
83
CA
LEU
E
10
6.248
19.362
53.796
1.00
0.02
C


ATOM
84
CB
LEU
E
10
4.856
19.846
54.187
1.00
0.13
C


ATOM
85
CG
LEU
E
10
3.866
19.747
53.032
1.00
0.65
C


ATOM
86
CD1
LEU
E
10
3.722
18.302
52.564
1.00
0.72
C


ATOM
87
CD2
LEU
E
10
2.510
20.315
53.436
1.00
1.20
C


ATOM
88
C
LEU
E
10
6.821
20.275
52.708
1.00
0.01
C


ATOM
89
O
LEU
E
10
6.758
19.945
51.518
1.00
0.01
O


ATOM
90
N
SER
E
11
7.457
21.358
53.120
1.00
0.02
N


ATOM
91
CA
SER
E
11
8.028
22.300
52.161
1.00
0.01
C


ATOM
92
CB
SER
E
11
7.144
23.539
52.128
1.00
0.02
C


ATOM
93
OG
SER
E
11
7.853
24.556
51.434
1.00
0.02
O


ATOM
94
C
SER
E
11
9.447
22.714
52.519
1.00
0.00
C


ATOM
95
O
SER
E
11
9.660
23.475
53.469
1.00
0.03
O


ATOM
96
N
VAL
E
12
10.341
22.475
51.572
1.00
0.00
N


ATOM
97
CA
VAL
E
12
11.743
22.880
51.724
1.00
0.01
C


ATOM
98
CB
VAL
E
12
12.585
22.110
50.706
1.00
1.16
C


ATOM
99
CG1
VAL
E
12
11.993
22.174
49.301
1.00
0.74
C


ATOM
100
CG2
VAL
E
12
14.044
22.557
50.711
1.00
1.93
C


ATOM
101
C
VAL
E
12
11.923
24.388
51.537
1.00
0.02
C


ATOM
102
O
VAL
E
12
12.794
24.987
52.179
1.00
0.02
O


ATOM
103
N
GLN
E
13
10.958
25.024
50.894
1.00
0.02
N


ATOM
104
CA
GLN
E
13
10.994
26.476
50.786
1.00
0.02
C


ATOM
105
CB
GLN
E
13
10.105
26.913
49.633
1.00
0.14
C


ATOM
106
CG
GLN
E
13
10.336
28.382
49.309
1.00
1.08
C


ATOM
107
CD
GLN
E
13
9.438
28.805
48.155
1.00
1.65
C


ATOM
108
OE1
GLN
E
13
8.718
27.981
47.580
1.00
2.23
O


ATOM
109
NE2
GLN
E
13
9.488
30.087
47.840
1.00
2.39
N


ATOM
110
C
GLN
E
13
10.524
27.108
52.094
1.00
0.03
C


ATOM
111
O
GLN
E
13
11.194
28.020
52.588
1.00
0.02
O


ATOM
112
N
GLU
E
14
9.564
26.486
52.766
1.00
0.01
N


ATOM
113
CA
GLU
E
14
9.155
26.956
54.102
1.00
0.00
C


ATOM
114
CB
GLU
E
14
7.951
26.130
54.535
1.00
0.24
C


ATOM
115
CG
GLU
E
14
7.398
26.548
55.893
1.00
1.07
C


ATOM
116
CD
GLU
E
14
6.502
27.773
55.760
1.00
1.75
C


ATOM
117
OE1
GLU
E
14
6.127
28.305
56.795
1.00
2.82
O


ATOM
118
OE2
GLU
E
14
6.041
28.014
54.653
1.00
1.72
O


ATOM
119
C
GLU
E
14
10.264
26.741
55.128
1.00
0.02
C


ATOM
120
O
GLU
E
14
10.536
27.628
55.953
1.00
0.02
O


ATOM
121
N
LEU
E
15
10.984
25.646
54.942
1.00
0.03
N


ATOM
122
CA
LEU
E
15
12.159
25.316
55.736
1.00
0.01
C


ATOM
123
CB
LEU
E
15
12.690
24.003
55.167
1.00
0.02
C


ATOM
124
CG
LEU
E
15
13.951
23.484
55.838
1.00
0.02
C


ATOM
125
CD1
LEU
E
15
13.709
23.193
57.309
1.00
0.03
C


ATOM
126
CD2
LEU
E
15
14.431
22.225
55.129
1.00
0.02
C


ATOM
127
C
LEU
E
15
13.192
26.436
55.644
1.00
0.02
C


ATOM
128
O
LEU
E
15
13.333
27.171
56.631
1.00
0.01
O


ATOM
129
N
VAL
E
16
13.599
26.782
54.431
1.00
0.01
N


ATOM
130
CA
VAL
E
16
14.666
27.780
54.252
1.00
0.02
C


ATOM
131
CB
VAL
E
16
15.350
27.541
52.911
1.00
0.50
C


ATOM
132
CG1
VAL
E
16
15.970
26.152
52.867
1.00
1.11
C


ATOM
133
CG2
VAL
E
16
14.401
27.740
51.736
1.00
0.96
C


ATOM
134
C
VAL
E
16
14.233
29.250
54.344
1.00
0.02
C


ATOM
135
O
VAL
E
16
15.108
30.120
54.424
1.00
0.02
O


ATOM
136
N
LYS
E
17
12.941
29.542
54.381
1.00
0.02
N


ATOM
137
CA
LYS
E
17
12.521
30.933
54.592
1.00
0.03
C


ATOM
138
CB
LYS
E
17
11.174
31.187
53.926
1.00
0.24
C


ATOM
139
CG
LYS
E
17
11.244
31.121
52.403
1.00
0.57
C


ATOM
140
CD
LYS
E
17
9.925
31.494
51.711
1.00
1.24
C


ATOM
141
CE
LYS
E
17
8.809
30.449
51.826
1.00
1.82
C


ATOM
142
NZ
LYS
E
17
8.078
30.487
53.104
1.00
2.56
N


ATOM
143
C
LYS
E
17
12.391
31.234
56.077
1.00
0.02
C


ATOM
144
O
LYS
E
17
12.502
32.391
56.499
1.00
0.02
O


ATOM
145
N
ASP
E
18
12.186
30.195
56.865
1.00
0.01
N


ATOM
146
CA
ASP
E
18
12.153
30.366
58.314
1.00
0.00
C


ATOM
147
CB
ASP
E
18
11.276
29.251
58.876
1.00
0.63
C


ATOM
148
CG
ASP
E
18
11.136
29.340
60.392
1.00
1.37
C


ATOM
149
OD1
ASP
E
18
10.054
29.669
60.852
1.00
2.14
O


ATOM
150
OD2
ASP
E
18
12.087
28.957
61.065
1.00
1.84
O


ATOM
151
C
ASP
E
18
13.578
30.276
58.846
1.00
0.01
C


ATOM
152
O
ASP
E
18
14.262
29.283
58.587
1.00
0.03
O


ATOM
153
N
PRO
E
19
13.972
31.218
59.691
1.00
0.02
N


ATOM
154
CA
PRO
E
19
15.375
31.311
60.121
1.00
0.02
C


ATOM
155
CB
PRO
E
19
15.460
32.601
60.877
1.00
0.30
C


ATOM
156
CG
PRO
E
19
14.080
33.230
60.967
1.00
0.17
C


ATOM
157
CD
PRO
E
19
13.149
32.321
60.187
1.00
0.15
C


ATOM
158
C
PRO
E
19
15.859
30.147
60.999
1.00
0.01
C


ATOM
159
O
PRO
E
19
17.040
29.783
60.936
1.00
0.02
O


ATOM
160
N
ILE
E
20
14.948
29.458
61.667
1.00
0.02
N


ATOM
161
CA
ILE
E
20
15.339
28.290
62.455
1.00
0.02
C


ATOM
162
CB
ILE
E
20
14.341
28.108
63.594
1.00
0.22
C


ATOM
163
CG2
ILE
E
20
14.664
26.851
64.394
1.00
0.17
C


ATOM
164
CG1
ILE
E
20
14.329
29.328
64.509
1.00
0.46
C


ATOM
165
CD1
ILE
E
20
15.673
29.522
65.204
1.00
1.43
C


ATOM
166
C
ILE
E
20
15.353
27.049
61.570
1.00
0.02
C


ATOM
167
O
ILE
E
20
16.357
26.331
61.553
1.00
0.02
O


ATOM
168
N
GLY
E
21
14.414
27.000
60.639
1.00
0.03
N


ATOM
169
CA
GLY
E
21
14.313
25.887
59.686
1.00
0.01
C


ATOM
170
C
GLY
E
21
15.530
25.819
58.770
1.00
0.02
C


ATOM
171
O
GLY
E
21
16.228
24.797
58.732
1.00
0.03
O


ATOM
172
N
ARG
E
22
15.888
26.965
58.218
1.00
0.02
N


ATOM
173
CA
ARG
E
22
17.055
27.092
57.344
1.00
0.01
C


ATOM
174
CB
ARG
E
22
17.052
28.529
56.845
1.00
0.12
C


ATOM
175
CG
ARG
E
22
18.322
28.877
56.084
1.00
0.50
C


ATOM
176
CD
ARG
E
22
18.437
30.389
55.930
1.00
0.54
C


ATOM
177
NE
ARG
E
22
18.244
31.061
57.230
1.00
1.34
N


ATOM
178
CZ
ARG
E
22
19.208
31.289
58.129
1.00
2.11
C


ATOM
179
NH1
ARG
E
22
18.926
31.948
59.254
1.00
2.95
N


ATOM
180
NH2
ARG
E
22
20.461
30.892
57.894
1.00
2.30
N


ATOM
181
C
ARG
E
22
18.372
26.842
58.076
1.00
0.03
C


ATOM
182
O
ARG
E
22
19.231
26.128
57.547
1.00
0.03
O


ATOM
183
N
GLN
E
23
18.403
27.160
59.359
1.00
0.02
N


ATOM
184
CA
GLN
E
23
19.610
26.977
60.165
1.00
0.02
C


ATOM
185
CB
GLN
E
23
19.418
27.867
61.382
1.00
0.17
C


ATOM
186
CG
GLN
E
23
20.578
27.863
62.361
1.00
1.00
C


ATOM
187
CD
GLN
E
23
20.246
28.877
63.448
1.00
1.48
C


ATOM
188
OE1
GLN
E
23
20.905
28.945
64.492
1.00
1.82
O


ATOM
189
NE2
GLN
E
23
19.207
29.655
63.188
1.00
2.18
N


ATOM
190
C
GLN
E
23
19.800
25.519
60.590
1.00
0.03
C


ATOM
191
O
GLN
E
23
20.921
25.000
60.518
1.00
0.03
O


ATOM
192
N
VAL
E
24
18.696
24.807
60.744
1.00
0.02
N


ATOM
193
CA
VAL
E
24
18.753
23.384
61.086
1.00
0.01
C


ATOM
194
CB
VAL
E
24
17.429
23.013
61.750
1.00
0.31
C


ATOM
195
CG1
VAL
E
24
17.305
21.517
61.990
1.00
0.74
C


ATOM
196
CG2
VAL
E
24
17.254
23.767
63.063
1.00
0.32
C


ATOM
197
C
VAL
E
24
19.013
22.528
59.844
1.00
0.02
C


ATOM
198
O
VAL
E
24
19.812
21.583
59.910
1.00
0.02
O


ATOM
199
N
LEU
E
25
18.607
23.038
58.690
1.00
0.02
N


ATOM
200
CA
LEU
E
25
18.945
22.377
57.426
1.00
0.02
C


ATOM
201
CB
LEU
E
25
18.065
22.933
56.312
1.00
0.10
C


ATOM
202
CG
LEU
E
25
18.434
22.313
54.966
1.00
0.17
C


ATOM
203
CD1
LEU
E
25
18.163
20.812
54.953
1.00
0.27
C


ATOM
204
CD2
LEU
E
25
17.704
22.997
53.818
1.00
0.25
C


ATOM
205
C
LEU
E
25
20.404
22.636
57.070
1.00
0.02
C


ATOM
206
O
LEU
E
25
21.113
21.703
56.675
1.00
0.03
O


ATOM
207
N
GLU
E
26
20.902
23.786
57.494
1.00
0.02
N


ATOM
208
CA
GLU
E
26
22.306
24.130
57.290
1.00
0.02
C


ATOM
209
CB
GLU
E
26
22.468
25.618
57.581
1.00
0.32
C


ATOM
210
CG
GLU
E
26
23.906
26.084
57.398
1.00
1.01
C


ATOM
211
CD
GLU
E
26
24.015
27.567
57.724
1.00
0.99
C


ATOM
212
OE1
GLU
E
26
23.122
28.065
58.398
1.00
1.00
O


ATOM
213
OE2
GLU
E
26
24.918
28.199
57.193
1.00
1.50
O


ATOM
214
C
GLU
E
26
23.217
23.327
58.210
1.00
0.04
C


ATOM
215
O
GLU
E
26
24.298
22.927
57.770
1.00
0.02
O


ATOM
216
N
THR
E
27
22.693
22.868
59.336
1.00
0.04
N


ATOM
217
CA
THR
E
27
23.478
22.025
60.240
1.00
0.02
C


ATOM
218
CB
THR
E
27
22.875
22.144
61.633
1.00
0.14
C


ATOM
219
OG1
THR
E
27
22.944
23.514
62.006
1.00
0.18
O


ATOM
220
CG2
THR
E
27
23.656
21.332
62.660
1.00
0.21
C


ATOM
221
C
THR
E
27
23.492
20.567
59.782
1.00
0.01
C


ATOM
222
O
THR
E
27
24.547
19.920
59.833
1.00
0.03
O


ATOM
223
N
PHE
E
28
22.449
20.172
59.070
1.00
0.02
N


ATOM
224
CA
PHE
E
28
22.420
18.835
58.471
1.00
0.01
C


ATOM
225
CB
PHE
E
28
20.979
18.514
58.098
1.00
0.02
C


ATOM
226
CG
PHE
E
28
20.797
17.180
57.383
1.00
0.03
C


ATOM
227
CD1
PHE
E
28
20.912
15.991
58.092
1.00
0.02
C


ATOM
228
CE1
PHE
E
28
20.749
14.776
57.440
1.00
0.04
C


ATOM
229
CZ
PHE
E
28
20.469
14.751
56.081
1.00
0.02
C


ATOM
230
CE2
PHE
E
28
20.353
15.939
55.372
1.00
0.02
C


ATOM
231
CD2
PHE
E
28
20.519
17.154
56.023
1.00
0.02
C


ATOM
232
C
PHE
E
28
23.304
18.783
57.226
1.00
0.03
C


ATOM
233
O
PHE
E
28
24.035
17.805
57.018
1.00
0.01
O


ATOM
234
N
LEU
E
29
23.420
19.916
56.557
1.00
0.02
N


ATOM
235
CA
LEU
E
29
24.334
20.017
55.426
1.00
0.02
C


ATOM
236
CB
LEU
E
29
23.877
21.182
54.555
1.00
0.03
C


ATOM
237
CG
LEU
E
29
22.520
20.905
53.914
1.00
0.02
C


ATOM
238
CD1
LEU
E
29
21.938
22.149
53.262
1.00
0.02
C


ATOM
239
CD2
LEU
E
29
22.606
19.780
52.896
1.00
0.02
C


ATOM
240
C
LEU
E
29
25.777
20.215
55.895
1.00
0.03
C


ATOM
241
O
LEU
E
29
26.701
19.723
55.235
1.00
0.03
O


ATOM
242
N
GLU
E
30
25.961
20.697
57.113
1.00
0.02
N


ATOM
243
CA
GLU
E
30
27.306
20.810
57.696
1.00
0.03
C


ATOM
244
CB
GLU
E
30
27.236
21.650
58.968
1.00
0.21
C


ATOM
245
CG
GLU
E
30
27.142
23.140
58.668
1.00
0.56
C


ATOM
246
CD
GLU
E
30
26.505
23.876
59.842
1.00
1.50
C


ATOM
247
OE1
GLU
E
30
26.538
23.334
60.938
1.00
2.43
O


ATOM
248
OE2
GLU
E
30
25.819
24.859
59.583
1.00
2.00
O


ATOM
249
C
GLU
E
30
27.863
19.440
58.042
1.00
0.03
C


ATOM
250
O
GLU
E
30
28.972
19.115
57.600
1.00
0.03
O


ATOM
251
N
SER
E
31
26.989
18.565
58.516
1.00
0.02
N


ATOM
252
CA
SER
E
31
27.402
17.196
58.848
1.00
0.01
C


ATOM
253
CB
SER
E
31
26.420
16.610
59.858
1.00
0.02
C


ATOM
254
OG
SER
E
31
25.148
16.503
59.236
1.00
0.01
O


ATOM
255
C
SER
E
31
27.485
16.282
57.621
1.00
0.02
C


ATOM
256
O
SER
E
31
27.931
15.136
57.739
1.00
0.02
O


ATOM
257
N
GLU
E
32
27.061
16.773
56.467
1.00
0.02
N


ATOM
258
CA
GLU
E
32
27.256
16.041
55.213
1.00
0.02
C


ATOM
259
CB
GLU
E
32
25.992
16.151
54.379
1.00
0.02
C


ATOM
260
CG
GLU
E
32
24.802
15.458
55.014
1.00
0.01
C


ATOM
261
CD
GLU
E
32
23.586
15.747
54.148
1.00
0.01
C


ATOM
262
OE1
GLU
E
32
23.428
16.898
53.761
1.00
0.00
O


ATOM
263
OE2
GLU
E
32
22.845
14.818
53.870
1.00
0.02
O


ATOM
264
C
GLU
E
32
28.397
16.611
54.374
1.00
0.02
C


ATOM
265
O
GLU
E
32
28.695
16.056
53.309
1.00
0.03
O


ATOM
266
N
PHE
E
33
28.995
17.703
54.836
1.00
0.03
N


ATOM
267
CA
PHE
E
33
29.990
18.467
54.059
1.00
0.03
C


ATOM
268
CB
PHE
E
33
31.239
17.624
53.811
1.00
0.97
C


ATOM
269
CG
PHE
E
33
31.964
17.198
55.083
1.00
1.76
C


ATOM
270
CD1
PHE
E
33
32.616
18.148
55.860
1.00
2.00
C


ATOM
271
CE1
PHE
E
33
33.274
17.764
57.020
1.00
3.14
C


ATOM
272
CZ
PHE
E
33
33.281
16.431
57.406
1.00
3.86
C


ATOM
273
CE2
PHE
E
33
32.630
15.480
56.631
1.00
3.76
C


ATOM
274
CD2
PHE
E
33
31.972
15.863
55.470
1.00
2.85
C


ATOM
275
C
PHE
E
33
29.383
18.946
52.740
1.00
0.02
C


ATOM
276
O
PHE
E
33
29.960
18.798
51.658
1.00
0.01
O


ATOM
277
N
SER
E
34
28.215
19.549
52.876
1.00
0.02
N


ATOM
278
CA
SER
E
34
27.426
20.045
51.751
1.00
0.01
C


ATOM
279
CB
SER
E
34
26.397
18.984
51.387
1.00
0.02
C


ATOM
280
OG
SER
E
34
25.605
18.758
52.544
1.00
0.02
O


ATOM
281
C
SER
E
34
26.710
21.326
52.159
1.00
0.03
C


ATOM
282
O
SER
E
34
25.733
21.748
51.524
1.00
0.00
O


ATOM
283
N
SER
E
35
27.274
22.000
53.151
1.00
0.02
N


ATOM
284
CA
SER
E
35
26.649
23.209
53.705
1.00
0.03
C


ATOM
285
CB
SER
E
35
27.169
23.443
55.113
1.00
1.70
C


ATOM
286
OG
SER
E
35
26.361
24.453
55.708
1.00
2.24
O


ATOM
287
C
SER
E
35
26.938
24.439
52.862
1.00
0.02
C


ATOM
288
O
SER
E
35
26.203
25.431
52.939
1.00
0.02
O


ATOM
289
N
GLU
E
36
27.811
24.266
51.884
1.00
0.02
N


ATOM
290
CA
GLU
E
36
28.118
25.330
50.932
1.00
0.02
C


ATOM
291
CB
GLU
E
36
29.395
24.991
50.144
1.00
0.02
C


ATOM
292
CG
GLU
E
36
29.263
23.954
49.015
1.00
0.02
C


ATOM
293
CD
GLU
E
36
29.246
22.509
49.512
1.00
0.02
C


ATOM
294
OE1
GLU
E
36
29.607
22.335
50.672
1.00
0.02
O


ATOM
295
OE2
GLU
E
36
28.523
21.728
48.912
1.00
0.03
O


ATOM
296
C
GLU
E
36
26.955
25.587
49.972
1.00
0.01
C


ATOM
297
O
GLU
E
36
26.765
26.747
49.600
1.00
0.02
O


ATOM
298
N
ASN
E
37
26.028
24.646
49.848
1.00
0.01
N


ATOM
299
CA
ASN
E
37
24.880
24.862
48.972
1.00
0.02
C


ATOM
300
CB
ASN
E
37
24.277
23.505
48.619
1.00
0.02
C


ATOM
301
CG
ASN
E
37
25.309
22.604
47.940
1.00
0.02
C


ATOM
302
OD1
ASN
E
37
26.009
23.009
47.000
1.00
0.02
O


ATOM
303
ND2
ASN
E
37
25.415
21.391
48.454
1.00
0.02
N


ATOM
304
C
ASN
E
37
23.825
25.740
49.649
1.00
0.03
C


ATOM
305
O
ASN
E
37
23.328
26.683
49.018
1.00
0.00
O


ATOM
306
N
ILE
E
38
23.709
25.636
50.965
1.00
0.02
N


ATOM
307
CA
ILE
E
38
22.724
26.471
51.664
1.00
0.02
C


ATOM
308
CB
ILE
E
38
22.171
25.734
52.887
1.00
0.01
C


ATOM
309
CG2
ILE
E
38
23.272
25.333
53.859
1.00
0.02
C


ATOM
310
CG1
ILE
E
38
21.109
26.557
53.612
1.00
0.02
C


ATOM
311
CD1
ILE
E
38
19.898
26.823
52.724
1.00
0.00
C


ATOM
312
C
ILE
E
38
23.338
27.816
52.046
1.00
0.02
C


ATOM
313
O
ILE
E
38
22.642
28.835
51.979
1.00
0.02
O


ATOM
314
N
ARG
E
39
24.660
27.858
52.098
1.00
0.01
N


ATOM
315
CA
ARG
E
39
25.359
29.112
52.364
1.00
0.03
C


ATOM
316
CB
ARG
E
39
26.772
28.758
52.798
1.00
0.23
C


ATOM
317
CG
ARG
E
39
27.526
29.957
53.353
1.00
0.42
C


ATOM
318
CD
ARG
E
39
28.953
29.560
53.709
1.00
0.91
C


ATOM
319
NE
ARG
E
39
28.959
28.374
54.581
1.00
1.27
N


ATOM
320
CZ
ARG
E
39
29.656
27.270
54.304
1.00
1.63
C


ATOM
321
NH1
ARG
E
39
30.395
27.209
53.195
1.00
2.16
N


ATOM
322
NH2
ARG
E
39
29.611
26.226
55.134
1.00
1.77
N


ATOM
323
C
ARG
E
39
25.395
29.951
51.093
1.00
0.02
C


ATOM
324
O
ARG
E
39
25.187
31.170
51.144
1.00
0.03
O


ATOM
325
N
PHE
E
40
25.369
29.260
49.966
1.00
0.02
N


ATOM
326
CA
PHE
E
40
25.250
29.911
48.666
1.00
0.02
C


ATOM
327
CB
PHE
E
40
25.608
28.881
47.605
1.00
0.03
C


ATOM
328
CG
PHE
E
40
25.280
29.289
46.176
1.00
0.02
C


ATOM
329
CD1
PHE
E
40
26.094
30.188
45.502
1.00
0.02
C


ATOM
330
CE1
PHE
E
40
25.786
30.556
44.200
1.00
0.02
C


ATOM
331
CZ
PHE
E
40
24.666
30.026
43.574
1.00
0.01
C


ATOM
332
CE2
PHE
E
40
23.853
29.128
44.249
1.00
0.02
C


ATOM
333
CD2
PHE
E
40
24.160
28.759
45.550
1.00
0.02
C


ATOM
334
C
PHE
E
40
23.836
30.419
48.426
1.00
0.02
C


ATOM
335
O
PHE
E
40
23.688
31.550
47.956
1.00
0.01
O


ATOM
336
N
TRP
E
41
22.840
29.733
48.964
1.00
0.01
N


ATOM
337
CA
TRP
E
41
21.461
30.210
48.828
1.00
0.03
C


ATOM
338
CB
TRP
E
41
20.509
29.111
49.291
1.00
0.02
C


ATOM
339
CG
TRP
E
41
19.046
29.477
49.133
1.00
0.01
C


ATOM
340
CD1
TRP
E
41
18.278
29.305
48.004
1.00
0.02
C


ATOM
341
NE1
TRP
E
41
17.034
29.785
48.253
1.00
0.03
N


ATOM
342
CE2
TRP
E
41
16.938
30.264
49.507
1.00
0.02
C


ATOM
343
CZ2
TRP
E
41
15.907
30.875
50.205
1.00
0.02
C


ATOM
344
CH2
TRP
E
41
16.109
31.278
51.521
1.00
0.02
C


ATOM
345
CZ3
TRP
E
41
17.339
31.074
52.138
1.00
0.02
C


ATOM
346
CE3
TRP
E
41
18.379
30.470
51.443
1.00
0.01
C


ATOM
347
CD2
TRP
E
41
18.185
30.072
50.129
1.00
0.03
C


ATOM
348
C
TRP
E
41
21.240
31.472
49.660
1.00
0.02
C


ATOM
349
O
TRP
E
41
20.731
32.468
49.127
1.00
0.02
O


ATOM
350
N
ILE
E
42
21.882
31.528
50.817
1.00
0.02
N


ATOM
351
CA
ILE
E
42
21.806
32.720
51.668
1.00
0.03
C


ATOM
352
CB
ILE
E
42
22.416
32.370
53.019
1.00
0.03
C


ATOM
353
CG2
ILE
E
42
22.415
33.584
53.943
1.00
0.02
C


ATOM
354
CG1
ILE
E
42
21.670
31.214
53.670
1.00
0.03
C


ATOM
355
CD1
ILE
E
42
22.412
30.706
54.900
1.00
0.01
C


ATOM
356
C
ILE
E
42
22.579
33.887
51.055
1.00
0.03
C


ATOM
357
O
ILE
E
42
22.043
35.001
50.979
1.00
0.02
O


ATOM
358
N
ALA
E
43
23.665
33.574
50.367
1.00
0.01
N


ATOM
359
CA
ALA
E
43
24.456
34.609
49.700
1.00
0.02
C


ATOM
360
CB
ALA
E
43
25.820
34.028
49.349
1.00
0.05
C


ATOM
361
C
ALA
E
43
23.786
35.144
48.436
1.00
0.02
C


ATOM
362
O
ALA
E
43
23.872
36.352
48.194
1.00
0.01
O


ATOM
363
N
ILE
E
44
22.922
34.358
47.811
1.00
0.02
N


ATOM
364
CA
ILE
E
44
22.199
34.858
46.635
1.00
0.03
C


ATOM
365
CB
ILE
E
44
21.756
33.708
45.737
1.00
0.39
C


ATOM
366
CG2
ILE
E
44
21.088
34.254
44.481
1.00
0.92
C


ATOM
367
CG1
ILE
E
44
22.931
32.824
45.349
1.00
0.66
C


ATOM
368
CD1
ILE
E
44
24.047
33.626
44.694
1.00
1.09
C


ATOM
369
C
ILE
E
44
20.980
35.654
47.074
1.00
0.02
C


ATOM
370
O
ILE
E
44
20.687
36.704
46.487
1.00
0.02
O


ATOM
371
N
GLN
E
45
20.505
35.348
48.269
1.00
0.03
N


ATOM
372
CA
GLN
E
45
19.447
36.151
48.880
1.00
0.02
C


ATOM
373
CB
GLN
E
45
18.902
35.380
50.078
1.00
0.15
C


ATOM
374
CG
GLN
E
45
17.783
36.137
50.788
1.00
0.97
C


ATOM
375
CD
GLN
E
45
16.551
36.242
49.894
1.00
1.26
C


ATOM
376
OE1
GLN
E
45
15.850
35.247
49.676
1.00
1.64
O


ATOM
377
NE2
GLN
E
45
16.324
37.429
49.358
1.00
1.96
N


ATOM
378
C
GLN
E
45
19.990
37.506
49.343
1.00
0.02
C


ATOM
379
O
GLN
E
45
19.317
38.531
49.172
1.00
0.02
O


ATOM
380
N
ASP
E
46
21.274
37.531
49.676
1.00
0.03
N


ATOM
381
CA
ASP
E
46
21.975
38.768
50.048
1.00
0.02
C


ATOM
382
CB
ASP
E
46
23.133
38.407
50.977
1.00
0.08
C


ATOM
383
CG
ASP
E
46
22.660
37.666
52.226
1.00
0.16
C


ATOM
384
OD1
ASP
E
46
21.586
37.992
52.713
1.00
0.15
O


ATOM
385
OD2
ASP
E
46
23.445
36.880
52.743
1.00
0.31
O


ATOM
386
C
ASP
E
46
22.543
39.511
48.831
1.00
0.03
C


ATOM
387
O
ASP
E
46
23.039
40.636
48.969
1.00
0.02
O


ATOM
388
N
LEU
E
47
22.399
38.922
47.653
1.00
0.03
N


ATOM
389
CA
LEU
E
47
22.897
39.512
46.407
1.00
0.03
C


ATOM
390
CB
LEU
E
47
23.281
38.341
45.496
1.00
3.01
C


ATOM
391
CG
LEU
E
47
23.938
38.707
44.167
1.00
4.01
C


ATOM
392
CD1
LEU
E
47
22.944
38.979
43.043
1.00
5.12
C


ATOM
393
CD2
LEU
E
47
24.986
39.803
44.300
1.00
4.53
C


ATOM
394
C
LEU
E
47
21.828
40.406
45.781
1.00
0.02
C


ATOM
395
O
LEU
E
47
22.153
41.297
44.984
1.00
0.02
O


ATOM
396
N
LYS
E
48
20.614
40.322
46.307
1.00
0.03
N


ATOM
397
CA
LYS
E
48
19.497
41.155
45.838
1.00
0.01
C


ATOM
398
CB
LYS
E
48
18.220
40.366
46.090
1.00
0.02
C


ATOM
399
CG
LYS
E
48
18.389
38.937
45.588
1.00
0.03
C


ATOM
400
CD
LYS
E
48
17.142
38.095
45.817
1.00
0.01
C


ATOM
401
CE
LYS
E
48
17.394
36.643
45.428
1.00
0.02
C


ATOM
402
NZ
LYS
E
48
17.862
36.546
44.036
1.00
0.02
N


ATOM
403
C
LYS
E
48
19.445
42.499
46.578
1.00
0.02
C


ATOM
404
O
LYS
E
48
18.413
42.883
47.139
1.00
0.02
O


ATOM
405
N
TYR
E
49
20.559
43.210
46.526
1.00
0.03
N


ATOM
406
CA
TYR
E
49
20.752
44.462
47.255
1.00
0.03
C


ATOM
407
CB
TYR
E
49
22.227
44.490
47.653
1.00
1.91
C


ATOM
408
CG
TYR
E
49
22.580
45.320
48.883
1.00
2.68
C


ATOM
409
CD1
TYR
E
49
21.651
45.493
49.903
1.00
3.02
C


ATOM
410
CE1
TYR
E
49
21.981
46.250
51.020
1.00
4.05
C


ATOM
411
CZ
TYR
E
49
23.242
46.825
51.114
1.00
4.78
C


ATOM
412
OH
TYR
E
49
23.566
47.588
52.214
1.00
5.92
O


ATOM
413
CE2
TYR
E
49
24.173
46.641
50.102
1.00
4.52
C


ATOM
414
CD2
TYR
E
49
23.843
45.886
48.987
1.00
3.43
C


ATOM
415
C
TYR
E
49
20.388
45.657
46.367
1.00
0.03
C


ATOM
416
O
TYR
E
49
20.096
45.497
45.177
1.00
0.02
O


ATOM
417
N
ALA
E
50
20.283
46.822
46.980
1.00
0.02
N


ATOM
418
CA
ALA
E
50
19.981
48.048
46.228
1.00
0.02
C


ATOM
419
CB
ALA
E
50
19.160
49.000
47.099
1.00
0.58
C


ATOM
420
C
ALA
E
50
21.206
48.750
45.604
1.00
0.03
C


ATOM
421
O
ALA
E
50
21.187
48.908
44.377
1.00
0.03
O


ATOM
422
N
PRO
E
51
22.223
49.178
46.357
1.00
0.02
N


ATOM
423
CA
PRO
E
51
23.363
49.850
45.714
1.00
0.03
C


ATOM
424
CB
PRO
E
51
24.258
50.311
46.822
1.00
1.18
C


ATOM
425
CG
PRO
E
51
23.686
49.851
48.150
1.00
1.48
C


ATOM
426
CD
PRO
E
51
22.395
49.122
47.820
1.00
1.19
C


ATOM
427
C
PRO
E
51
24.109
48.916
44.771
1.00
0.03
C


ATOM
428
O
PRO
E
51
24.719
47.927
45.202
1.00
0.03
O


ATOM
429
N
ASN
E
52
24.278
49.388
43.548
1.00
0.01
N


ATOM
430
CA
ASN
E
52
24.831
48.559
42.466
1.00
0.02
C


ATOM
431
CB
ASN
E
52
24.460
49.206
41.136
1.00
0.19
C


ATOM
432
CG
ASN
E
52
22.972
49.553
41.103
1.00
0.21
C


ATOM
433
OD1
ASN
E
52
22.610
50.735
41.079
1.00
0.31
O


ATOM
434
ND2
ASN
E
52
22.136
48.529
41.099
1.00
0.27
N


ATOM
435
C
ASN
E
52
26.348
48.431
42.556
1.00
0.02
C


ATOM
436
O
ASN
E
52
26.910
47.401
42.162
1.00
0.02
O


ATOM
437
N
GLU
E
53
26.937
49.309
43.351
1.00
0.03
N


ATOM
438
CA
GLU
E
53
28.378
49.278
43.601
1.00
0.03
C


ATOM
439
CB
GLU
E
53
28.815
50.593
44.254
1.00
0.22
C


ATOM
440
CG
GLU
E
53
28.401
51.840
43.471
1.00
0.87
C


ATOM
441
CD
GLU
E
53
27.233
52.551
44.161
1.00
0.97
C


ATOM
442
OE1
GLU
E
53
26.138
51.999
44.110
1.00
1.90
O


ATOM
443
OE2
GLU
E
53
27.485
53.518
44.863
1.00
0.66
O


ATOM
444
C
GLU
E
53
28.710
48.135
44.556
1.00
0.02
C


ATOM
445
O
GLU
E
53
29.753
47.485
44.413
1.00
0.02
O


ATOM
446
N
GLN
E
54
27.757
47.785
45.404
1.00
0.02
N


ATOM
447
CA
GLN
E
54
27.966
46.673
46.318
1.00
0.02
C


ATOM
448
CB
GLN
E
54
27.271
46.961
47.638
1.00
0.43
C


ATOM
449
CG
GLN
E
54
27.861
48.197
48.301
1.00
1.21
C


ATOM
450
CD
GLN
E
54
27.266
48.346
49.696
1.00
2.05
C


ATOM
451
OE1
GLN
E
54
26.470
49.255
49.963
1.00
2.88
O


ATOM
452
NE2
GLN
E
54
27.660
47.434
50.568
1.00
2.60
N


ATOM
453
C
GLN
E
54
27.445
45.381
45.714
1.00
0.02
C


ATOM
454
O
GLN
E
54
28.010
44.324
46.003
1.00
0.02
O


ATOM
455
N
ILE
E
55
26.590
45.488
44.710
1.00
0.03
N


ATOM
456
CA
ILE
E
55
26.129
44.292
43.998
1.00
0.02
C


ATOM
457
CB
ILE
E
55
24.909
44.640
43.153
1.00
0.37
C


ATOM
458
CG2
ILE
E
55
24.485
43.455
42.291
1.00
0.53
C


ATOM
459
CG1
ILE
E
55
23.747
45.093
44.024
1.00
0.44
C


ATOM
460
CD1
ILE
E
55
22.538
45.435
43.163
1.00
1.13
C


ATOM
461
C
ILE
E
55
27.225
43.737
43.093
1.00
0.02
C


ATOM
462
O
ILE
E
55
27.435
42.517
43.089
1.00
0.02
O


ATOM
463
N
TYR
E
56
28.066
44.611
42.560
1.00
0.03
N


ATOM
464
CA
TYR
E
56
29.199
44.144
41.757
1.00
0.03
C


ATOM
465
CB
TYR
E
56
29.859
45.340
41.076
1.00
0.28
C


ATOM
466
CG
TYR
E
56
31.114
44.982
40.278
1.00
0.83
C


ATOM
467
CD1
TYR
E
56
32.268
45.744
40.417
1.00
1.24
C


ATOM
468
CE1
TYR
E
56
33.412
45.408
39.703
1.00
1.92
C


ATOM
469
CZ
TYR
E
56
33.398
44.310
38.853
1.00
2.21
C


ATOM
470
OH
TYR
E
56
34.572
43.876
38.280
1.00
2.88
O


ATOM
471
CE2
TYR
E
56
32.244
43.556
38.700
1.00
1.96
C


ATOM
472
CD2
TYR
E
56
31.103
43.894
39.414
1.00
1.29
C


ATOM
473
C
TYR
E
56
30.228
43.425
42.623
1.00
0.02
C


ATOM
474
O
TYR
E
56
30.562
42.269
42.330
1.00
0.01
O


ATOM
475
N
GLN
E
57
30.482
43.964
43.805
1.00
0.03
N


ATOM
476
CA
GLN
E
57
31.484
43.359
44.687
1.00
0.01
C


ATOM
477
CB
GLN
E
57
31.898
44.387
45.727
1.00
0.12
C


ATOM
478
CG
GLN
E
57
32.582
45.573
45.059
1.00
0.66
C


ATOM
479
CD
GLN
E
57
32.925
46.630
46.100
1.00
1.12
C


ATOM
480
OE1
GLN
E
57
33.903
46.506
46.846
1.00
1.47
O


ATOM
481
NE2
GLN
E
57
32.098
47.658
46.147
1.00
1.44
N


ATOM
482
C
GLN
E
57
30.963
42.106
45.375
1.00
0.03
C


ATOM
483
O
GLN
E
57
31.686
41.102
45.413
1.00
0.02
O


ATOM
484
N
LYS
E
58
29.663
42.061
45.613
1.00
0.02
N


ATOM
485
CA
LYS
E
58
29.040
40.898
46.243
1.00
0.02
C


ATOM
486
CB
LYS
E
58
27.633
41.298
46.672
1.00
0.02
C


ATOM
487
CG
LYS
E
58
26.971
40.270
47.581
1.00
0.02
C


ATOM
488
CD
LYS
E
58
27.746
40.113
48.884
1.00
0.03
C


ATOM
489
CE
LYS
E
58
26.967
39.293
49.905
1.00
0.01
C


ATOM
490
NZ
LYS
E
58
26.648
37.957
49.385
1.00
0.02
N


ATOM
491
C
LYS
E
58
28.966
39.743
45.257
1.00
0.03
C


ATOM
492
O
LYS
E
58
29.351
38.625
45.616
1.00
0.03
O


ATOM
493
N
ALA
E
59
28.804
40.071
43.986
1.00
0.03
N


ATOM
494
CA
ALA
E
59
28.785
39.048
42.942
1.00
0.02
C


ATOM
495
CB
ALA
E
59
28.272
39.689
41.659
1.00
0.02
C


ATOM
496
C
ALA
E
59
30.175
38.471
42.711
1.00
0.03
C


ATOM
497
O
ALA
E
59
30.326
37.241
42.675
1.00
0.03
O


ATOM
498
N
GLU
E
60
31.184
39.316
42.855
1.00
0.02
N


ATOM
499
CA
GLU
E
60
32.569
38.860
42.736
1.00
0.02
C


ATOM
500
CB
GLU
E
60
33.492
40.073
42.733
1.00
0.15
C


ATOM
501
CG
GLU
E
60
33.225
40.966
41.529
1.00
0.77
C


ATOM
502
CD
GLU
E
60
34.035
42.255
41.627
1.00
1.48
C


ATOM
503
OE1
GLU
E
60
33.563
43.173
42.287
1.00
2.34
O


ATOM
504
OE2
GLU
E
60
35.036
42.351
40.931
1.00
1.92
O


ATOM
505
C
GLU
E
60
32.941
37.950
43.900
1.00
0.02
C


ATOM
506
O
GLU
E
60
33.356
36.811
43.651
1.00
0.03
O


ATOM
507
N
ARG
E
61
32.517
38.312
45.100
1.00
0.02
N


ATOM
508
CA
ARG
E
61
32.840
37.510
46.283
1.00
0.02
C


ATOM
509
CB
ARG
E
61
32.539
38.346
47.520
1.00
0.15
C


ATOM
510
CG
ARG
E
61
33.556
39.473
47.662
1.00
0.49
C


ATOM
511
CD
ARG
E
61
33.247
40.385
48.845
1.00
0.42
C


ATOM
512
NE
ARG
E
61
32.076
41.235
48.578
1.00
0.40
N


ATOM
513
CZ
ARG
E
61
31.287
41.726
49.535
1.00
0.52
C


ATOM
514
NH1
ARG
E
61
30.388
42.666
49.237
1.00
0.91
N


ATOM
515
NH2
ARG
E
61
31.515
41.406
50.811
1.00
1.11
N


ATOM
516
C
ARG
E
61
32.077
36.189
46.342
1.00
0.03
C


ATOM
517
O
ARG
E
61
32.692
35.162
46.662
1.00
0.02
O


ATOM
518
N
ILE
E
62
30.878
36.150
45.783
1.00
0.03
N


ATOM
519
CA
ILE
E
62
30.134
34.887
45.748
1.00
0.02
C


ATOM
520
CB
ILE
E
62
28.667
35.157
45.429
1.00
0.02
C


ATOM
521
CG2
ILE
E
62
27.889
33.850
45.342
1.00
0.02
C


ATOM
522
CG1
ILE
E
62
28.023
36.053
46.474
1.00
0.03
C


ATOM
523
CD1
ILE
E
62
26.587
36.385
46.085
1.00
0.03
C


ATOM
524
C
ILE
E
62
30.721
33.941
44.702
1.00
0.01
C


ATOM
525
O
ILE
E
62
30.989
32.777
45.034
1.00
0.02
O


ATOM
526
N
ARG
E
63
31.188
34.487
43.588
1.00
0.02
N


ATOM
527
CA
ARG
E
63
31.811
33.640
42.566
1.00
0.02
C


ATOM
528
CB
ARG
E
63
31.922
34.401
41.252
1.00
0.13
C


ATOM
529
CG
ARG
E
63
30.546
34.607
40.639
1.00
1.18
C


ATOM
530
CD
ARG
E
63
30.626
35.286
39.278
1.00
1.07
C


ATOM
531
NE
ARG
E
63
31.281
36.598
39.379
1.00
1.43
N


ATOM
532
CZ
ARG
E
63
30.660
37.749
39.114
1.00
2.56
C


ATOM
533
NH1
ARG
E
63
31.355
38.888
39.080
1.00
2.76
N


ATOM
534
NH2
ARG
E
63
29.370
37.744
38.774
1.00
3.57
N


ATOM
535
C
ARG
E
63
33.186
33.150
42.993
1.00
0.03
C


ATOM
536
O
ARG
E
63
33.435
31.944
42.901
1.00
0.02
O


ATOM
537
N
GLU
E
64
33.883
33.956
43.773
1.00
0.02
N


ATOM
538
CA
GLU
E
64
35.204
33.561
44.268
1.00
0.03
C


ATOM
539
CB
GLU
E
64
35.948
34.823
44.688
1.00
0.27
C


ATOM
540
CG
GLU
E
64
36.224
35.732
43.495
1.00
0.87
C


ATOM
541
CD
GLU
E
64
36.803
37.062
43.969
1.00
1.38
C


ATOM
542
OE1
GLU
E
64
38.020
37.177
43.990
1.00
2.45
O


ATOM
543
OE2
GLU
E
64
36.019
37.962
44.244
1.00
1.02
O


ATOM
544
C
GLU
E
64
35.137
32.597
45.454
1.00
0.03
C


ATOM
545
O
GLU
E
64
36.136
31.933
45.752
1.00
0.02
O


ATOM
546
N
GLU
E
65
33.969
32.436
46.051
1.00
0.02
N


ATOM
547
CA
GLU
E
65
33.852
31.506
47.167
1.00
0.03
C


ATOM
548
CB
GLU
E
65
33.084
32.211
48.277
1.00
0.03
C


ATOM
549
CG
GLU
E
65
32.988
31.364
49.540
1.00
0.03
C


ATOM
550
CD
GLU
E
65
32.241
32.150
50.611
1.00
0.03
C


ATOM
551
OE1
GLU
E
65
31.520
33.065
50.238
1.00
0.02
O


ATOM
552
OE2
GLU
E
65
32.426
31.846
51.781
1.00
0.01
O


ATOM
553
C
GLU
E
65
33.146
30.211
46.776
1.00
0.03
C


ATOM
554
O
GLU
E
65
33.623
29.131
47.148
1.00
0.02
O


ATOM
555
N
PHE
E
66
32.103
30.303
45.966
1.00
0.02
N


ATOM
556
CA
PHE
E
66
31.296
29.115
45.660
1.00
0.02
C


ATOM
557
CB
PHE
E
66
29.841
29.381
46.050
1.00
0.03
C


ATOM
558
CG
PHE
E
66
29.546
29.844
47.474
1.00
0.02
C


ATOM
559
CD1
PHE
E
66
29.575
28.937
48.524
1.00
0.01
C


ATOM
560
CE1
PHE
E
66
29.293
29.360
49.815
1.00
0.02
C


ATOM
561
CZ
PHE
E
66
28.969
30.689
50.055
1.00
0.02
C


ATOM
562
CE2
PHE
E
66
28.923
31.593
49.002
1.00
0.03
C


ATOM
563
CD2
PHE
E
66
29.208
31.170
47.712
1.00
0.02
C


ATOM
564
C
PHE
E
66
31.247
28.720
44.184
1.00
0.01
C


ATOM
565
O
PHE
E
66
30.674
27.665
43.881
1.00
0.01
O


ATOM
566
N
LEU
E
67
31.726
29.550
43.273
1.00
0.01
N


ATOM
567
CA
LEU
E
67
31.390
29.299
41.859
1.00
0.02
C


ATOM
568
CB
LEU
E
67
30.551
30.449
41.307
1.00
0.84
C


ATOM
569
CG
LEU
E
67
29.097
30.485
41.788
1.00
0.56
C


ATOM
570
CD1
LEU
E
67
28.490
29.089
41.897
1.00
0.99
C


ATOM
571
CD2
LEU
E
67
28.900
31.268
43.076
1.00
0.46
C


ATOM
572
C
LEU
E
67
32.579
29.103
40.922
1.00
0.03
C


ATOM
573
O
LEU
E
67
32.430
28.457
39.874
1.00
0.03
O


ATOM
574
N
ALA
E
68
33.710
29.700
41.253
1.00
0.02
N


ATOM
575
CA
ALA
E
68
34.923
29.561
40.443
1.00
0.02
C


ATOM
576
CB
ALA
E
68
35.999
30.481
41.007
1.00
0.82
C


ATOM
577
C
ALA
E
68
35.401
28.121
40.484
1.00
0.02
C


ATOM
578
O
ALA
E
68
35.068
27.393
41.423
1.00
0.02
O


ATOM
579
N
GLN
E
69
36.128
27.705
39.463
1.00
0.02
N


ATOM
580
CA
GLN
E
69
36.611
26.320
39.414
1.00
0.02
C


ATOM
581
CB
GLN
E
69
37.028
26.009
37.980
1.00
0.34
C


ATOM
582
CG
GLN
E
69
36.847
24.533
37.632
1.00
1.25
C


ATOM
583
CD
GLN
E
69
35.403
24.262
37.209
1.00
1.97
C


ATOM
584
OE1
GLN
E
69
34.445
24.615
37.912
1.00
2.61
O


ATOM
585
NE2
GLN
E
69
35.269
23.690
36.025
1.00
2.11
N


ATOM
586
C
GLN
E
69
37.808
26.157
40.350
1.00
0.02
C


ATOM
587
O
GLN
E
69
38.944
26.482
39.983
1.00
0.02
O


ATOM
588
N
GLY
E
70
37.539
25.645
41.541
1.00
0.02
N


ATOM
589
CA
GLY
E
70
38.558
25.554
42.586
1.00
0.03
C


ATOM
590
C
GLY
E
70
38.224
26.507
43.734
1.00
0.02
C


ATOM
591
O
GLY
E
70
39.109
26.918
44.496
1.00
0.02
O


ATOM
592
N
ALA
E
71
36.958
26.881
43.823
1.00
0.03
N


ATOM
593
CA
ALA
E
71
36.509
27.788
44.886
1.00
0.02
C


ATOM
594
CB
ALA
E
71
35.150
28.347
44.487
1.00
0.02
C


ATOM
595
C
ALA
E
71
36.414
27.042
46.217
1.00
0.03
C


ATOM
596
O
ALA
E
71
36.007
25.873
46.241
1.00
0.03
O


ATOM
597
N
PRO
E
72
36.708
27.731
47.313
1.00
0.02
N


ATOM
598
CA
PRO
E
72
36.912
27.058
48.610
1.00
0.03
C


ATOM
599
CB
PRO
E
72
37.475
28.114
49.512
1.00
0.12
C


ATOM
600
CG
PRO
E
72
37.518
29.444
48.776
1.00
0.08
C


ATOM
601
CD
PRO
E
72
37.050
29.156
47.360
1.00
0.07
C


ATOM
602
C
PRO
E
72
35.640
26.465
49.228
1.00
0.03
C


ATOM
603
O
PRO
E
72
35.722
25.573
50.079
1.00
0.02
O


ATOM
604
N
ALA
E
73
34.487
26.917
48.769
1.00
0.02
N


ATOM
605
CA
ALA
E
73
33.213
26.336
49.170
1.00
0.02
C


ATOM
606
CB
ALA
E
73
32.525
27.286
50.141
1.00
0.30
C


ATOM
607
C
ALA
E
73
32.352
26.147
47.928
1.00
0.02
C


ATOM
608
O
ALA
E
73
31.152
26.439
47.957
1.00
0.01
O


ATOM
609
N
GLN
E
74
32.967
25.678
46.853
1.00
0.03
N


ATOM
610
CA
GLN
E
74
32.266
25.531
45.573
1.00
0.02
C


ATOM
611
CB
GLN
E
74
33.261
25.028
44.527
1.00
0.55
C


ATOM
612
CG
GLN
E
74
32.664
25.036
43.122
1.00
0.46
C


ATOM
613
CD
GLN
E
74
33.724
24.744
42.065
1.00
0.54
C


ATOM
614
OE1
GLN
E
74
34.929
24.705
42.359
1.00
1.12
O


ATOM
615
NE2
GLN
E
74
33.259
24.627
40.833
1.00
1.25
N


ATOM
616
C
GLN
E
74
31.064
24.591
45.682
1.00
0.02
C


ATOM
617
O
GLN
E
74
31.129
23.517
46.293
1.00
0.02
O


ATOM
618
N
VAL
E
75
29.940
25.082
45.189
1.00
0.01
N


ATOM
619
CA
VAL
E
75
28.698
24.307
45.172
1.00
0.01
C


ATOM
620
CB
VAL
E
75
27.524
25.277
45.121
1.00
0.02
C


ATOM
621
CG1
VAL
E
75
27.512
26.140
46.373
1.00
0.02
C


ATOM
622
CG2
VAL
E
75
27.572
26.154
43.878
1.00
0.03
C


ATOM
623
C
VAL
E
75
28.673
23.354
43.982
1.00
0.01
C


ATOM
624
O
VAL
E
75
29.497
23.465
43.064
1.00
0.02
O


ATOM
625
N
ASN
E
76
27.790
22.372
44.042
1.00
0.01
N


ATOM
626
CA
ASN
E
76
27.689
21.413
42.931
1.00
0.02
C


ATOM
627
CB
ASN
E
76
27.169
20.079
43.457
1.00
0.02
C


ATOM
628
CG
ASN
E
76
27.241
19.008
42.369
1.00
0.02
C


ATOM
629
OD1
ASN
E
76
28.042
19.103
41.434
1.00
0.02
O


ATOM
630
ND2
ASN
E
76
26.422
17.982
42.524
1.00
0.01
N


ATOM
631
C
ASN
E
76
26.781
21.960
41.832
1.00
0.02
C


ATOM
632
O
ASN
E
76
25.552
21.980
41.960
1.00
0.01
O


ATOM
633
N
VAL
E
77
27.415
22.427
40.770
1.00
0.02
N


ATOM
634
CA
VAL
E
77
26.702
23.048
39.646
1.00
0.02
C


ATOM
635
CB
VAL
E
77
26.855
24.561
39.760
1.00
2.69
C


ATOM
636
CG1
VAL
E
77
25.709
25.202
40.535
1.00
3.69
C


ATOM
637
CG2
VAL
E
77
28.199
24.920
40.381
1.00
3.06
C


ATOM
638
C
VAL
E
77
27.210
22.559
38.288
1.00
0.02
C


ATOM
639
O
VAL
E
77
28.392
22.249
38.112
1.00
0.02
O


ATOM
640
N
ASP
E
78
26.297
22.505
37.331
1.00
0.02
N


ATOM
641
CA
ASP
E
78
26.632
22.057
35.967
1.00
0.02
C


ATOM
642
CB
ASP
E
78
25.343
21.728
35.206
1.00
0.01
C


ATOM
643
CG
ASP
E
78
24.532
22.986
34.894
1.00
0.01
C


ATOM
644
OD1
ASP
E
78
23.762
23.408
35.743
1.00
0.02
O


ATOM
645
OD2
ASP
E
78
24.819
23.587
33.866
1.00
0.01
O


ATOM
646
C
ASP
E
78
27.451
23.117
35.222
1.00
0.02
C


ATOM
647
O
ASP
E
78
27.212
24.323
35.374
1.00
0.02
O


ATOM
648
N
ASN
E
79
28.293
22.668
34.303
1.00
0.02
N


ATOM
649
CA
ASN
E
79
29.229
23.582
33.622
1.00
0.01
C


ATOM
650
CB
ASN
E
79
30.362
22.776
33.000
1.00
0.19
C


ATOM
651
CG
ASN
E
79
31.170
22.100
34.100
1.00
0.21
C


ATOM
652
OD1
ASN
E
79
31.104
20.877
34.269
1.00
0.31
O


ATOM
653
ND2
ASN
E
79
31.900
22.909
34.848
1.00
0.27
N


ATOM
654
C
ASN
E
79
28.618
24.480
32.546
1.00
0.02
C


ATOM
655
O
ASN
E
79
29.200
25.533
32.267
1.00
0.02
O


ATOM
656
N
ARG
E
80
27.388
24.226
32.130
1.00
0.02
N


ATOM
657
CA
ARG
E
80
26.739
25.140
31.184
1.00
0.02
C


ATOM
658
CB
ARG
E
80
25.464
24.487
30.672
1.00
0.22
C


ATOM
659
CG
ARG
E
80
24.692
25.417
29.741
1.00
1.30
C


ATOM
660
CD
ARG
E
80
23.300
24.865
29.465
1.00
1.26
C


ATOM
661
NE
ARG
E
80
22.551
24.723
30.726
1.00
1.42
N


ATOM
662
CZ
ARG
E
80
21.985
23.580
31.119
1.00
2.19
C


ATOM
663
NH1
ARG
E
80
22.068
22.493
30.348
1.00
2.85
N


ATOM
664
NH2
ARG
E
80
21.332
23.524
32.282
1.00
2.79
N


ATOM
665
C
ARG
E
80
26.382
26.437
31.901
1.00
0.02
C


ATOM
666
O
ARG
E
80
26.874
27.510
31.522
1.00
0.01
O


ATOM
667
N
THR
E
81
25.868
26.266
33.109
1.00
0.02
N


ATOM
668
CA
THR
E
81
25.501
27.402
33.949
1.00
0.02
C


ATOM
669
CB
THR
E
81
24.600
26.891
35.069
1.00
0.02
C


ATOM
670
OG1
THR
E
81
23.516
26.178
34.489
1.00
0.02
O


ATOM
671
CG2
THR
E
81
24.029
28.029
35.905
1.00
0.02
C


ATOM
672
C
THR
E
81
26.754
28.024
34.551
1.00
0.02
C


ATOM
673
O
THR
E
81
26.899
29.251
34.537
1.00
0.03
O


ATOM
674
N
LEU
E
82
27.754
27.196
34.796
1.00
0.02
N


ATOM
675
CA
LEU
E
82
29.022
27.697
35.326
1.00
0.01
C


ATOM
676
CB
LEU
E
82
29.886
26.507
35.707
1.00
0.19
C


ATOM
677
CG
LEU
E
82
29.546
25.977
37.089
1.00
1.08
C


ATOM
678
CD1
LEU
E
82
30.396
24.760
37.426
1.00
1.07
C


ATOM
679
CD2
LEU
E
82
29.759
27.064
38.132
1.00
1.73
C


ATOM
680
C
LEU
E
82
29.794
28.564
34.338
1.00
0.02
C


ATOM
681
O
LEU
E
82
30.224
29.656
34.718
1.00
0.02
O


ATOM
682
N
ASP
E
83
29.797
28.205
33.068
1.00
0.02
N


ATOM
683
CA
ASP
E
83
30.582
28.963
32.092
1.00
0.02
C


ATOM
684
CB
ASP
E
83
30.977
28.018
30.957
1.00
1.00
C


ATOM
685
CG
ASP
E
83
31.816
26.848
31.479
1.00
1.95
C


ATOM
686
OD1
ASP
E
83
32.507
27.038
32.473
1.00
2.53
O


ATOM
687
OD2
ASP
E
83
31.785
25.795
30.854
1.00
2.36
O


ATOM
688
C
ASP
E
83
29.816
30.157
31.522
1.00
0.02
C


ATOM
689
O
ASP
E
83
30.437
31.082
30.984
1.00
0.02
O


ATOM
690
N
GLN
E
84
28.509
30.184
31.723
1.00
0.02
N


ATOM
691
CA
GLN
E
84
27.694
31.297
31.235
1.00
0.03
C


ATOM
692
CB
GLN
E
84
26.378
30.695
30.760
1.00
0.79
C


ATOM
693
CG
GLN
E
84
25.353
31.759
30.392
1.00
1.50
C


ATOM
694
CD
GLN
E
84
23.996
31.093
30.212
1.00
1.49
C


ATOM
695
OE1
GLN
E
84
23.142
31.142
31.107
1.00
1.62
O


ATOM
696
NE2
GLN
E
84
23.854
30.392
29.101
1.00
1.90
N


ATOM
697
C
GLN
E
84
27.383
32.325
32.319
1.00
0.02
C


ATOM
698
O
GLN
E
84
27.343
33.534
32.050
1.00
0.03
O


ATOM
699
N
THR
E
85
27.247
31.856
33.542
1.00
0.02
N


ATOM
700
CA
THR
E
85
26.799
32.725
34.623
1.00
0.02
C


ATOM
701
CB
THR
E
85
25.507
32.108
35.162
1.00
0.01
C


ATOM
702
OG1
THR
E
85
24.553
32.025
34.108
1.00
0.02
O


ATOM
703
CG2
THR
E
85
24.886
32.925
36.283
1.00
0.02
C


ATOM
704
C
THR
E
85
27.823
32.878
35.753
1.00
0.02
C


ATOM
705
O
THR
E
85
28.027
34.001
36.223
1.00
0.02
O


ATOM
706
N
LEU
E
86
28.602
31.839
36.016
1.00
1.29
N


ATOM
707
CA
LEU
E
86
29.427
31.812
37.243
1.00
1.82
C


ATOM
708
CB
LEU
E
86
29.148
30.530
38.018
1.00
1.96
C


ATOM
709
CG
LEU
E
86
27.748
30.409
38.623
1.00
2.93
C


ATOM
710
CD1
LEU
E
86
27.181
31.760
39.063
1.00
3.57
C


ATOM
711
CD2
LEU
E
86
26.782
29.645
37.728
1.00
3.69
C


ATOM
712
C
LEU
E
86
30.942
31.877
37.024
1.00
1.44
C


ATOM
713
O
LEU
E
86
31.498
32.940
36.727
1.00
1.68
O


ATOM
714
N
GLU
E
87
31.550
30.699
37.131
1.00
1.98
N


ATOM
715
CA
GLU
E
87
33.013
30.436
37.165
1.00
2.32
C


ATOM
716
CB
GLU
E
87
33.377
29.353
36.152
1.00
3.37
C


ATOM
717
CG
GLU
E
87
32.959
27.956
36.599
1.00
4.08
C


ATOM
718
CD
GLU
E
87
33.583
26.915
35.670
1.00
4.73
C


ATOM
719
OE1
GLU
E
87
34.702
27.159
35.240
1.00
5.27
O


ATOM
720
OE2
GLU
E
87
32.937
25.906
35.398
1.00
4.98
O


ATOM
721
C
GLU
E
87
33.951
31.617
36.931
1.00
2.43
C


ATOM
722
O
GLU
E
87
34.047
32.530
37.757
1.00
2.58
O


ATOM
723
N
CYS
E
88
34.813
31.436
35.942
1.00
2.78
N


ATOM
724
CA
CYS
E
88
35.847
32.432
35.632
1.00
3.22
C


ATOM
725
CB
CYS
E
88
37.198
31.728
35.622
1.00
3.41
C


ATOM
726
SG
CYS
E
88
37.640
30.871
37.153
1.00
4.58
S


ATOM
727
C
CYS
E
88
35.628
33.107
34.278
1.00
2.78
C


ATOM
728
O
CYS
E
88
36.412
33.979
33.884
1.00
2.56
O


ATOM
729
N
ILE
E
89
34.617
32.665
33.549
1.00
2.87
N


ATOM
730
CA
ILE
E
89
34.357
33.238
32.223
1.00
2.85
C


ATOM
731
CB
ILE
E
89
34.102
32.092
31.239
1.00
3.27
C


ATOM
732
CG2
ILE
E
89
34.465
32.517
29.820
1.00
3.74
C


ATOM
733
CG1
ILE
E
89
34.922
30.865
31.622
1.00
3.86
C


ATOM
734
CD1
ILE
E
89
34.648
29.702
30.677
1.00
4.56
C


ATOM
735
C
ILE
E
89
33.115
34.111
32.336
1.00
1.87
C


ATOM
736
O
ILE
E
89
33.185
35.262
32.774
1.00
2.29
O


ATOM
737
N
SER
E
90
32.002
33.537
31.914
1.00
0.02
N


ATOM
738
CA
SER
E
90
30.661
34.038
32.195
1.00
0.03
C


ATOM
739
CB
SER
E
90
30.466
33.901
33.689
1.00
0.02
C


ATOM
740
OG
SER
E
90
30.719
32.530
33.972
1.00
0.02
O


ATOM
741
C
SER
E
90
30.333
35.454
31.739
1.00
0.02
C


ATOM
742
O
SER
E
90
30.848
36.456
32.255
1.00
0.02
O


ATOM
743
N
LYS
E
91
29.180
35.486
31.101
1.00
0.02
N


ATOM
744
CA
LYS
E
91
28.578
36.719
30.611
1.00
0.02
C


ATOM
745
CB
LYS
E
91
27.496
36.302
29.625
1.00
0.28
C


ATOM
746
CG
LYS
E
91
28.031
35.240
28.672
1.00
0.77
C


ATOM
747
CD
LYS
E
91
26.917
34.620
27.845
1.00
0.79
C


ATOM
748
CE
LYS
E
91
27.449
33.495
26.970
1.00
1.97
C


ATOM
749
NZ
LYS
E
91
26.363
32.848
26.221
1.00
1.97
N


ATOM
750
C
LYS
E
91
27.948
37.469
31.777
1.00
0.02
C


ATOM
751
O
LYS
E
91
28.080
38.696
31.857
1.00
0.02
O


ATOM
752
N
ALA
E
92
27.586
36.718
32.809
1.00
0.02
N


ATOM
753
CA
ALA
E
92
27.086
37.324
34.047
1.00
0.02
C


ATOM
754
CB
ALA
E
92
26.232
36.300
34.779
1.00
0.35
C


ATOM
755
C
ALA
E
92
28.205
37.829
34.963
1.00
0.02
C


ATOM
756
O
ALA
E
92
27.962
38.729
35.775
1.00
0.02
O


ATOM
757
N
LYS
E
93
29.441
37.440
34.687
1.00
0.03
N


ATOM
758
CA
LYS
E
93
30.593
38.021
35.383
1.00
0.02
C


ATOM
759
CB
LYS
E
93
31.747
37.023
35.357
1.00
0.35
C


ATOM
760
CG
LYS
E
93
33.064
37.670
35.780
1.00
1.21
C


ATOM
761
CD
LYS
E
93
34.241
36.717
35.614
1.00
1.35
C


ATOM
762
CE
LYS
E
93
34.174
35.566
36.608
1.00
2.56
C


ATOM
763
NZ
LYS
E
93
34.409
36.048
37.977
1.00
3.03
N


ATOM
764
C
LYS
E
93
31.023
39.302
34.670
1.00
0.03
C


ATOM
765
O
LYS
E
93
31.378
40.291
35.322
1.00
0.03
O


ATOM
766
N
ASP
E
94
30.704
39.355
33.385
1.00
0.02
N


ATOM
767
CA
ASP
E
94
30.990
40.534
32.561
1.00
0.02
C


ATOM
768
CB
ASP
E
94
30.969
40.101
31.097
1.00
0.27
C


ATOM
769
CG
ASP
E
94
31.992
39.006
30.813
1.00
0.50
C


ATOM
770
OD1
ASP
E
94
31.692
38.164
29.975
1.00
0.76
O


ATOM
771
OD2
ASP
E
94
33.103
39.126
31.311
1.00
0.52
O


ATOM
772
C
ASP
E
94
29.951
41.640
32.759
1.00
0.02
C


ATOM
773
O
ASP
E
94
30.205
42.794
32.397
1.00
0.02
O


ATOM
774
N
ALA
E
95
28.830
41.300
33.376
1.00
0.02
N


ATOM
775
CA
ALA
E
95
27.790
42.285
33.679
1.00
0.02
C


ATOM
776
CB
ALA
E
95
26.846
42.394
32.487
1.00
0.64
C


ATOM
777
C
ALA
E
95
27.007
41.864
34.917
1.00
0.02
C


ATOM
778
O
ALA
E
95
25.788
41.635
34.839
1.00
0.03
O


ATOM
779
N
SER
E
96
27.708
41.823
36.043
1.00
0.03
N


ATOM
780
CA
SER
E
96
27.131
41.359
37.316
1.00
0.03
C


ATOM
781
CB
SER
E
96
28.193
41.422
38.403
1.00
1.65
C


ATOM
782
OG
SER
E
96
28.434
42.790
38.690
1.00
2.08
O


ATOM
783
C
SER
E
96
25.926
42.192
37.737
1.00
0.02
C


ATOM
784
O
SER
E
96
25.887
43.419
37.574
1.00
0.02
O


ATOM
785
N
GLN
E
97
24.927
41.482
38.225
1.00
0.02
N


ATOM
786
CA
GLN
E
97
23.644
42.085
38.562
1.00
0.02
C


ATOM
787
CB
GLN
E
97
22.879
42.279
37.263
1.00
1.35
C


ATOM
788
CG
GLN
E
97
22.797
40.959
36.506
1.00
2.41
C


ATOM
789
CD
GLN
E
97
22.005
41.149
35.231
1.00
3.21
C


ATOM
790
OE1
GLN
E
97
20.783
40.936
35.216
1.00
3.45
O


ATOM
791
NE2
GLN
E
97
22.713
41.553
34.190
1.00
4.08
N


ATOM
792
C
GLN
E
97
22.830
41.172
39.472
1.00
0.03
C


ATOM
793
O
GLN
E
97
23.333
40.176
40.003
1.00
0.02
O


ATOM
794
N
MET
E
98
21.564
41.521
39.604
1.00
0.02
N


ATOM
795
CA
MET
E
98
20.601
40.708
40.346
1.00
0.03
C


ATOM
796
CB
MET
E
98
19.331
41.549
40.555
1.00
0.44
C


ATOM
797
CG
MET
E
98
19.223
42.782
39.635
1.00
1.26
C


ATOM
798
SD
MET
E
98
18.756
42.530
37.899
1.00
1.41
S


ATOM
799
CE
MET
E
98
18.753
44.250
37.342
1.00
2.02
C


ATOM
800
C
MET
E
98
20.281
39.373
39.658
1.00
0.03
C


ATOM
801
O
MET
E
98
21.118
38.454
39.623
1.00
0.03
O


ATOM
802
N
ARG
E
99
19.121
39.335
39.018
1.00
0.02
N


ATOM
803
CA
ARG
E
99
18.528
38.119
38.450
1.00
0.02
C


ATOM
804
CB
ARG
E
99
17.374
38.525
37.543
1.00
1.64
C


ATOM
805
CG
ARG
E
99
16.409
39.473
38.232
1.00
2.02
C


ATOM
806
CD
ARG
E
99
15.306
39.893
37.270
1.00
2.78
C


ATOM
807
NE
ARG
E
99
14.484
40.969
37.842
1.00
4.09
N


ATOM
808
CZ
ARG
E
99
14.399
42.179
37.284
1.00
4.97
C


ATOM
809
NH1
ARG
E
99
13.592
43.103
37.813
1.00
5.86
N


ATOM
810
NH2
ARG
E
99
15.082
42.450
36.169
1.00
5.20
N


ATOM
811
C
ARG
E
99
19.486
37.287
37.607
1.00
0.01
C


ATOM
812
O
ARG
E
99
19.971
36.253
38.079
1.00
0.02
O


ATOM
813
N
PHE
E
100
19.950
37.847
36.504
1.00
0.14
N


ATOM
814
CA
PHE
E
100
20.704
37.047
35.528
1.00
0.21
C


ATOM
815
CB
PHE
E
100
20.622
37.740
34.177
1.00
0.58
C


ATOM
816
CG
PHE
E
100
19.195
37.822
33.647
1.00
0.93
C


ATOM
817
CD1
PHE
E
100
18.411
36.674
33.594
1.00
1.12
C


ATOM
818
CE1
PHE
E
100
17.104
36.744
33.117
1.00
1.69
C


ATOM
819
CZ
PHE
E
100
16.587
37.963
32.693
1.00
2.30
C


ATOM
820
CE2
PHE
E
100
17.372
39.104
32.742
1.00
2.50
C


ATOM
821
CD2
PHE
E
100
18.673
39.035
33.216
1.00
1.84
C


ATOM
822
C
PHE
E
100
22.163
36.756
35.899
1.00
0.35
C


ATOM
823
O
PHE
E
100
22.845
36.051
35.147
1.00
0.74
O


ATOM
824
N
ALA
E
101
22.622
37.205
37.057
1.00
0.03
N


ATOM
825
CA
ALA
E
101
23.969
36.832
37.469
1.00
0.02
C


ATOM
826
CB
ALA
E
101
24.673
38.016
38.108
1.00
0.45
C


ATOM
827
C
ALA
E
101
23.960
35.661
38.447
1.00
0.03
C


ATOM
828
O
ALA
E
101
24.975
34.963
38.552
1.00
0.03
O


ATOM
829
N
PHE
E
102
22.842
35.401
39.111
1.00
0.03
N


ATOM
830
CA
PHE
E
102
22.849
34.294
40.077
1.00
0.01
C


ATOM
831
CB
PHE
E
102
23.204
34.843
41.452
1.00
0.02
C


ATOM
832
CG
PHE
E
102
24.692
35.096
41.628
1.00
0.03
C


ATOM
833
CD1
PHE
E
102
25.585
34.031
41.587
1.00
0.02
C


ATOM
834
CE1
PHE
E
102
26.945
34.256
41.741
1.00
0.03
C


ATOM
835
CZ
PHE
E
102
27.413
35.545
41.931
1.00
0.01
C


ATOM
836
CE2
PHE
E
102
26.530
36.612
41.964
1.00
0.02
C


ATOM
837
CD2
PHE
E
102
25.167
36.389
41.809
1.00
0.02
C


ATOM
838
C
PHE
E
102
21.571
33.465
40.182
1.00
0.02
C


ATOM
839
O
PHE
E
102
21.627
32.369
40.757
1.00
0.02
O


ATOM
840
N
TYR
E
103
20.505
33.852
39.502
1.00
0.02
N


ATOM
841
CA
TYR
E
103
19.239
33.110
39.624
1.00
0.02
C


ATOM
842
CB
TYR
E
103
18.130
33.927
38.970
1.00
0.55
C


ATOM
843
CG
TYR
E
103
16.831
33.153
38.751
1.00
0.93
C


ATOM
844
CD1
TYR
E
103
16.432
32.829
37.460
1.00
1.87
C


ATOM
845
CE1
TYR
E
103
15.262
32.115
37.255
1.00
2.82
C


ATOM
846
CZ
TYR
E
103
14.491
31.730
38.342
1.00
2.98
C


ATOM
847
OH
TYR
E
103
13.389
30.922
38.148
1.00
4.01
O


ATOM
848
CE2
TYR
E
103
14.877
32.065
39.633
1.00
2.47
C


ATOM
849
CD2
TYR
E
103
16.049
32.781
39.837
1.00
1.55
C


ATOM
850
C
TYR
E
103
19.282
31.721
38.985
1.00
0.02
C


ATOM
851
O
TYR
E
103
18.803
30.766
39.611
1.00
0.02
O


ATOM
852
N
HIS
E
104
20.135
31.559
37.986
1.00
0.02
N


ATOM
853
CA
HIS
E
104
20.273
30.263
37.308
1.00
0.02
C


ATOM
854
CB
HIS
E
104
21.136
30.471
36.061
1.00
0.29
C


ATOM
855
CG
HIS
E
104
20.763
31.653
35.183
1.00
1.03
C


ATOM
856
ND1
HIS
E
104
21.622
32.506
34.587
1.00
1.52
N


ATOM
857
CE1
HIS
E
104
20.919
33.433
33.902
1.00
2.51
C


ATOM
858
NE2
HIS
E
104
19.606
33.152
34.053
1.00
2.89
N


ATOM
859
CD2
HIS
E
104
19.493
32.053
34.831
1.00
2.10
C


ATOM
860
C
HIS
E
104
20.998
29.261
38.207
1.00
0.02
C


ATOM
861
O
HIS
E
104
20.574
28.105
38.337
1.00
0.02
O


ATOM
862
N
SER
E
105
21.828
29.818
39.070
1.00
0.02
N


ATOM
863
CA
SER
E
105
22.693
29.018
39.923
1.00
0.02
C


ATOM
864
CB
SER
E
105
23.903
29.879
40.239
1.00
0.14
C


ATOM
865
OG
SER
E
105
24.232
30.583
39.052
1.00
0.90
O


ATOM
866
C
SER
E
105
21.969
28.686
41.216
1.00
0.02
C


ATOM
867
O
SER
E
105
21.979
27.532
41.661
1.00
0.01
O


ATOM
868
N
GLU
E
106
21.134
29.619
41.644
1.00
0.02
N


ATOM
869
CA
GLU
E
106
20.357
29.430
42.867
1.00
0.02
C


ATOM
870
CB
GLU
E
106
19.819
30.783
43.316
1.00
0.14
C


ATOM
871
CG
GLU
E
106
19.028
30.664
44.614
1.00
0.19
C


ATOM
872
CD
GLU
E
106
18.371
31.998
44.948
1.00
1.03
C


ATOM
873
OE1
GLU
E
106
18.248
32.299
46.125
1.00
1.38
O


ATOM
874
OE2
GLU
E
106
18.029
32.707
44.008
1.00
1.80
O


ATOM
875
C
GLU
E
106
19.190
28.481
42.634
1.00
0.02
C


ATOM
876
O
GLU
E
106
18.889
27.667
43.514
1.00
0.02
O


ATOM
877
N
GLU
E
107
18.703
28.417
41.407
1.00
0.01
N


ATOM
878
CA
GLU
E
107
17.628
27.477
41.099
1.00
0.02
C


ATOM
879
CB
GLU
E
107
16.971
27.919
39.797
1.00
0.14
C


ATOM
880
CG
GLU
E
107
15.771
27.050
39.442
1.00
1.12
C


ATOM
881
CD
GLU
E
107
15.125
27.577
38.165
1.00
1.48
C


ATOM
882
OE1
GLU
E
107
15.328
28.748
37.873
1.00
1.96
O


ATOM
883
OE2
GLU
E
107
14.484
26.794
37.479
1.00
1.73
O


ATOM
884
C
GLU
E
107
18.170
26.057
40.964
1.00
0.02
C


ATOM
885
O
GLU
E
107
17.573
25.125
41.522
1.00
0.01
O


ATOM
886
N
HIS
E
108
19.401
25.939
40.493
1.00
0.02
N


ATOM
887
CA
HIS
E
108
20.012
24.617
40.367
1.00
0.02
C


ATOM
888
CB
HIS
E
108
21.232
24.723
39.457
1.00
0.27
C


ATOM
889
CG
HIS
E
108
21.936
23.400
39.227
1.00
0.70
C


ATOM
890
ND1
HIS
E
108
21.624
22.479
38.297
1.00
0.85
N


ATOM
891
CE1
HIS
E
108
22.470
21.434
38.399
1.00
1.41
C


ATOM
892
NE2
HIS
E
108
23.327
21.699
39.411
1.00
1.84
N


ATOM
893
CD2
HIS
E
108
23.010
22.907
39.931
1.00
1.50
C


ATOM
894
C
HIS
E
108
20.435
24.078
41.729
1.00
0.02
C


ATOM
895
O
HIS
E
108
20.112
22.930
42.055
1.00
0.02
O


ATOM
896
N
VAL
E
109
20.903
24.958
42.598
1.00
0.02
N


ATOM
897
CA
VAL
E
109
21.329
24.522
43.930
1.00
0.02
C


ATOM
898
CB
VAL
E
109
22.318
25.551
44.465
1.00
0.05
C


ATOM
899
CG1
VAL
E
109
22.603
25.357
45.947
1.00
0.15
C


ATOM
900
CG2
VAL
E
109
23.611
25.510
43.659
1.00
0.13
C


ATOM
901
C
VAL
E
109
20.161
24.328
44.898
1.00
0.02
C


ATOM
902
O
VAL
E
109
20.204
23.389
45.706
1.00
0.01
O


ATOM
903
N
PHE
E
110
19.041
24.983
44.639
1.00
0.02
N


ATOM
904
CA
PHE
E
110
17.867
24.768
45.486
1.00
0.01
C


ATOM
905
CB
PHE
E
110
16.885
25.920
45.306
1.00
0.03
C


ATOM
906
CG
PHE
E
110
15.657
25.823
46.208
1.00
0.02
C


ATOM
907
CD1
PHE
E
110
15.778
26.076
47.568
1.00
0.03
C


ATOM
908
CE1
PHE
E
110
14.665
25.984
48.393
1.00
0.01
C


ATOM
909
CZ
PHE
E
110
13.431
25.636
47.859
1.00
0.02
C


ATOM
910
CE2
PHE
E
110
13.310
25.381
46.499
1.00
0.02
C


ATOM
911
CD2
PHE
E
110
14.424
25.473
45.673
1.00
0.02
C


ATOM
912
C
PHE
E
110
17.190
23.449
45.137
1.00
0.02
C


ATOM
913
O
PHE
E
110
16.865
22.675
46.047
1.00
0.02
O


ATOM
914
N
THR
E
111
17.236
23.081
43.866
1.00
0.03
N


ATOM
915
CA
THR
E
111
16.690
21.778
43.470
1.00
0.01
C


ATOM
916
CB
THR
E
111
16.354
21.781
41.981
1.00
0.36
C


ATOM
917
OG1
THR
E
111
17.522
22.111
41.239
1.00
0.82
O


ATOM
918
CG2
THR
E
111
15.277
22.809
41.654
1.00
1.28
C


ATOM
919
C
THR
E
111
17.664
20.647
43.797
1.00
0.01
C


ATOM
920
O
THR
E
111
17.217
19.562
44.185
1.00
0.01
O


ATOM
921
N
LEU
E
112
18.940
20.979
43.912
1.00
0.02
N


ATOM
922
CA
LEU
E
112
19.944
20.010
44.356
1.00
0.02
C


ATOM
923
CB
LEU
E
112
21.314
20.652
44.167
1.00
0.02
C


ATOM
924
CG
LEU
E
112
22.450
19.748
44.627
1.00
0.01
C


ATOM
925
CD1
LEU
E
112
22.541
18.500
43.756
1.00
0.02
C


ATOM
926
CD2
LEU
E
112
23.769
20.509
44.610
1.00
0.02
C


ATOM
927
C
LEU
E
112
19.750
19.659
45.829
1.00
0.01
C


ATOM
928
O
LEU
E
112
19.645
18.472
46.168
1.00
0.02
O


ATOM
929
N
MET
E
113
19.456
20.662
46.642
1.00
0.02
N


ATOM
930
CA
MET
E
113
19.211
20.403
48.060
1.00
0.02
C


ATOM
931
CB
MET
E
113
19.175
21.722
48.820
1.00
0.02
C


ATOM
932
CG
MET
E
113
20.518
22.436
48.786
1.00
0.01
C


ATOM
933
SD
MET
E
113
20.587
23.967
49.742
1.00
0.03
S


ATOM
934
CE
MET
E
113
19.290
24.902
48.903
1.00
0.02
C


ATOM
935
C
MET
E
113
17.887
19.685
48.274
1.00
0.02
C


ATOM
936
O
MET
E
113
17.893
18.607
48.887
1.00
0.02
O


ATOM
937
N
ALA
E
114
16.869
20.089
47.530
1.00
0.02
N


ATOM
938
CA
ALA
E
114
15.525
19.532
47.716
1.00
0.02
C


ATOM
939
CB
ALA
E
114
14.531
20.435
46.997
1.00
0.15
C


ATOM
940
C
ALA
E
114
15.357
18.100
47.210
1.00
0.01
C


ATOM
941
O
ALA
E
114
14.607
17.330
47.819
1.00
0.01
O


ATOM
942
N
LYS
E
115
16.136
17.705
46.217
1.00
0.02
N


ATOM
943
CA
LYS
E
115
16.040
16.335
45.708
1.00
0.02
C


ATOM
944
CB
LYS
E
115
16.190
16.379
44.193
1.00
0.02
C


ATOM
945
CG
LYS
E
115
15.117
17.260
43.565
1.00
0.01
C


ATOM
946
CD
LYS
E
115
15.305
17.384
42.058
1.00
0.02
C


ATOM
947
CE
LYS
E
115
14.276
18.332
41.454
1.00
0.01
C


ATOM
948
NZ
LYS
E
115
12.908
17.869
41.736
1.00
0.02
N


ATOM
949
C
LYS
E
115
17.097
15.404
46.299
1.00
0.02
C


ATOM
950
O
LYS
E
115
17.033
14.189
46.083
1.00
0.02
O


ATOM
951
N
ASP
E
116
18.043
15.949
47.046
1.00
0.02
N


ATOM
952
CA
ASP
E
116
19.098
15.103
47.610
1.00
0.02
C


ATOM
953
CB
ASP
E
116
20.443
15.662
47.157
1.00
0.02
C


ATOM
954
CG
ASP
E
116
21.605
14.847
47.715
1.00
0.02
C


ATOM
955
OD1
ASP
E
116
21.754
13.706
47.302
1.00
0.01
O


ATOM
956
OD2
ASP
E
116
22.372
15.415
48.482
1.00
0.02
O


ATOM
957
C
ASP
E
116
19.045
15.028
49.133
1.00
0.01
C


ATOM
958
O
ASP
E
116
18.713
13.979
49.698
1.00
0.02
O


ATOM
959
N
SER
E
117
19.284
16.152
49.786
1.00
0.02
N


ATOM
960
CA
SER
E
117
19.451
16.123
51.238
1.00
0.03
C


ATOM
961
CB
SER
E
117
20.534
17.116
51.635
1.00
0.01
C


ATOM
962
OG
SER
E
117
21.769
16.664
51.095
1.00
0.02
O


ATOM
963
C
SER
E
117
18.165
16.448
51.978
1.00
0.01
C


ATOM
964
O
SER
E
117
17.952
15.924
53.075
1.00
0.03
O


ATOM
965
N
TYR
E
118
17.244
17.122
51.314
1.00
0.02
N


ATOM
966
CA
TYR
E
118
15.955
17.448
51.943
1.00
0.01
C


ATOM
967
CB
TYR
E
118
15.130
18.308
50.998
1.00
0.02
C


ATOM
968
CG
TYR
E
118
13.720
18.586
51.501
1.00
0.02
C


ATOM
969
CD1
TYR
E
118
13.526
19.156
52.753
1.00
0.02
C


ATOM
970
CE1
TYR
E
118
12.238
19.402
53.209
1.00
0.02
C


ATOM
971
CZ
TYR
E
118
11.152
19.076
52.409
1.00
0.02
C


ATOM
972
OH
TYR
E
118
9.888
19.437
52.802
1.00
0.03
O


ATOM
973
CE2
TYR
E
118
11.342
18.498
51.162
1.00
0.02
C


ATOM
974
CD2
TYR
E
118
12.629
18.251
50.708
1.00
0.02
C


ATOM
975
C
TYR
E
118
15.136
16.232
52.419
1.00
0.03
C


ATOM
976
O
TYR
E
118
14.843
16.212
53.622
1.00
0.02
O


ATOM
977
N
PRO
E
119
14.866
15.198
51.620
1.00
0.02
N


ATOM
978
CA
PRO
E
119
14.082
14.085
52.174
1.00
0.01
C


ATOM
979
CB
PRO
E
119
13.755
13.207
51.006
1.00
0.20
C


ATOM
980
CG
PRO
E
119
14.467
13.719
49.766
1.00
0.34
C


ATOM
981
CD
PRO
E
119
15.184
14.985
50.194
1.00
0.16
C


ATOM
982
C
PRO
E
119
14.824
13.295
53.259
1.00
0.03
C


ATOM
983
O
PRO
E
119
14.201
12.948
54.268
1.00
0.03
O


ATOM
984
N
ARG
E
120
16.148
13.301
53.216
1.00
0.03
N


ATOM
985
CA
ARG
E
120
16.938
12.592
54.221
1.00
0.02
C


ATOM
986
CB
ARG
E
120
18.292
12.291
53.594
1.00
0.01
C


ATOM
987
CG
ARG
E
120
19.230
11.555
54.540
1.00
0.03
C


ATOM
988
CD
ARG
E
120
20.473
11.082
53.797
1.00
0.02
C


ATOM
989
NE
ARG
E
120
21.102
12.188
53.057
1.00
0.03
N


ATOM
990
CZ
ARG
E
120
21.340
12.143
51.744
1.00
0.02
C


ATOM
991
NH1
ARG
E
120
20.969
11.075
51.034
1.00
0.01
N


ATOM
992
NH2
ARG
E
120
21.920
13.178
51.134
1.00
0.02
N


ATOM
993
C
ARG
E
120
17.088
13.414
55.504
1.00
0.02
C


ATOM
994
O
ARG
E
120
17.152
12.837
56.594
1.00
0.02
O


ATOM
995
N
PHE
E
121
16.852
14.712
55.397
1.00
0.01
N


ATOM
996
CA
PHE
E
121
16.797
15.593
56.565
1.00
0.03
C


ATOM
997
CB
PHE
E
121
16.887
17.033
56.069
1.00
0.02
C


ATOM
998
CG
PHE
E
121
16.510
18.091
57.100
1.00
0.02
C


ATOM
999
CD1
PHE
E
121
17.353
18.358
58.171
1.00
0.02
C


ATOM
1000
CE1
PHE
E
121
17.001
19.321
59.106
1.00
0.02
C


ATOM
1001
CZ
PHE
E
121
15.807
20.017
58.973
1.00
0.02
C


ATOM
1002
CE2
PHE
E
121
14.964
19.749
57.904
1.00
0.01
C


ATOM
1003
CD2
PHE
E
121
15.315
18.787
56.967
1.00
0.02
C


ATOM
1004
C
PHE
E
121
15.492
15.405
57.320
1.00
0.02
C


ATOM
1005
O
PHE
E
121
15.517
15.300
58.551
1.00
0.01
O


ATOM
1006
N
VAL
E
122
14.451
15.059
56.580
1.00
0.03
N


ATOM
1007
CA
VAL
E
122
13.138
14.801
57.173
1.00
0.01
C


ATOM
1008
CB
VAL
E
122
12.100
14.970
56.063
1.00
0.27
C


ATOM
1009
CG1
VAL
E
122
10.684
14.647
56.528
1.00
0.14
C


ATOM
1010
CG2
VAL
E
122
12.154
16.381
55.491
1.00
0.44
C


ATOM
1011
C
VAL
E
122
13.063
13.398
57.784
1.00
0.02
C


ATOM
1012
O
VAL
E
122
12.244
13.152
58.676
1.00
0.03
O


ATOM
1013
N
ARG
E
123
13.995
12.536
57.411
1.00
0.02
N


ATOM
1014
CA
ARG
E
123
14.084
11.206
58.020
1.00
0.03
C


ATOM
1015
CB
ARG
E
123
14.541
10.224
56.949
1.00
0.29
C


ATOM
1016
CG
ARG
E
123
13.632
10.232
55.730
1.00
0.95
C


ATOM
1017
CD
ARG
E
123
14.219
9.356
54.630
1.00
1.08
C


ATOM
1018
NE
ARG
E
123
13.417
9.429
53.401
1.00
0.69
N


ATOM
1019
CZ
ARG
E
123
13.957
9.610
52.194
1.00
0.68
C


ATOM
1020
NH1
ARG
E
123
13.181
9.596
51.108
1.00
1.02
N


ATOM
1021
NH2
ARG
E
123
15.280
9.751
52.068
1.00
0.80
N


ATOM
1022
C
ARG
E
123
15.109
11.166
59.152
1.00
0.02
C


ATOM
1023
O
ARG
E
123
15.123
10.221
59.950
1.00
0.02
O


ATOM
1024
N
SER
E
124
15.955
12.180
59.219
1.00
0.02
N


ATOM
1025
CA
SER
E
124
17.040
12.186
60.202
1.00
0.02
C


ATOM
1026
CB
SER
E
124
18.236
12.951
59.651
1.00
0.02
C


ATOM
1027
OG
SER
E
124
17.909
14.333
59.663
1.00
0.02
O


ATOM
1028
C
SER
E
124
16.625
12.836
61.509
1.00
0.02
C


ATOM
1029
O
SER
E
124
15.723
13.683
61.558
1.00
0.01
O


ATOM
1030
N
GLN
E
125
17.511
12.666
62.475
1.00
0.01
N


ATOM
1031
CA
GLN
E
125
17.316
13.184
63.833
1.00
0.03
C


ATOM
1032
CB
GLN
E
125
18.330
12.466
64.719
1.00
0.38
C


ATOM
1033
CG
GLN
E
125
18.156
12.773
66.201
1.00
1.43
C


ATOM
1034
CD
GLN
E
125
19.232
12.038
66.992
1.00
2.28
C


ATOM
1035
OE1
GLN
E
125
20.431
12.248
66.776
1.00
3.19
O


ATOM
1036
NE2
GLN
E
125
18.790
11.131
67.845
1.00
2.30
N


ATOM
1037
C
GLN
E
125
17.505
14.703
63.942
1.00
0.01
C


ATOM
1038
O
GLN
E
125
17.043
15.297
64.920
1.00
0.03
O


ATOM
1039
N
ILE
E
126
17.899
15.352
62.856
1.00
0.01
N


ATOM
1040
CA
ILE
E
126
18.065
16.802
62.888
1.00
0.01
C


ATOM
1041
CB
ILE
E
126
19.004
17.185
61.746
1.00
0.31
C


ATOM
1042
CG2
ILE
E
126
19.341
18.668
61.795
1.00
0.72
C


ATOM
1043
CG1
ILE
E
126
20.298
16.379
61.813
1.00
0.82
C


ATOM
1044
CD1
ILE
E
126
21.140
16.765
63.026
1.00
1.53
C


ATOM
1045
C
ILE
E
126
16.697
17.475
62.721
1.00
0.03
C


ATOM
1046
O
ILE
E
126
16.443
18.526
63.320
1.00
0.02
O


ATOM
1047
N
TYR
E
127
15.773
16.766
62.089
1.00
0.01
N


ATOM
1048
CA
TYR
E
127
14.395
17.249
61.982
1.00
0.02
C


ATOM
1049
CB
TYR
E
127
13.920
16.977
60.561
1.00
0.02
C


ATOM
1050
CG
TYR
E
127
12.449
17.270
60.290
1.00
0.02
C


ATOM
1051
CD1
TYR
E
127
12.001
18.583
60.209
1.00
0.02
C


ATOM
1052
CE1
TYR
E
127
10.660
18.841
59.963
1.00
0.01
C


ATOM
1053
CZ
TYR
E
127
9.772
17.786
59.797
1.00
0.02
C


ATOM
1054
OH
TYR
E
127
8.438
18.040
59.561
1.00
0.02
O


ATOM
1055
CE2
TYR
E
127
10.218
16.475
59.875
1.00
0.02
C


ATOM
1056
CD2
TYR
E
127
11.559
16.218
60.123
1.00
0.01
C


ATOM
1057
C
TYR
E
127
13.494
16.534
62.986
1.00
0.01
C


ATOM
1058
O
TYR
E
127
12.598
17.145
63.584
1.00
0.01
O


ATOM
1059
N
LYS
E
128
13.877
15.312
63.317
1.00
0.02
N


ATOM
1060
CA
LYS
E
128
13.112
14.492
64.266
1.00
0.03
C


ATOM
1061
CB
LYS
E
128
13.450
13.028
64.006
1.00
0.23
C


ATOM
1062
CG
LYS
E
128
12.916
12.555
62.660
1.00
1.16
C


ATOM
1063
CD
LYS
E
128
11.393
12.600
62.632
1.00
1.39
C


ATOM
1064
CE
LYS
E
128
10.849
12.077
61.309
1.00
2.20
C


ATOM
1065
NZ
LYS
E
128
11.327
10.709
61.051
1.00
2.66
N


ATOM
1066
C
LYS
E
128
13.380
14.792
65.743
1.00
0.02
C


ATOM
1067
O
LYS
E
128
12.624
14.299
66.594
1.00
0.04
O


ATOM
1068
N
ALA
E
129
14.306
15.691
66.041
1.00
0.04
N


ATOM
1069
CA
ALA
E
129
14.668
15.978
67.435
1.00
0.02
C


ATOM
1070
CB
ALA
E
129
15.855
16.935
67.433
1.00
0.43
C


ATOM
1071
C
ALA
E
129
13.524
16.607
68.222
1.00
0.02
C


ATOM
1072
O
ALA
E
129
13.183
16.124
69.307
1.00
0.02
O


ATOM
1073
N
VAL
E
130
12.826
17.545
67.600
1.00
0.02
N


ATOM
1074
CA
VAL
E
130
11.665
18.170
68.246
1.00
0.02
C


ATOM
1075
CB
VAL
E
130
11.790
19.687
68.115
1.00
1.13
C


ATOM
1076
CG1
VAL
E
130
10.878
20.411
69.104
1.00
1.60
C


ATOM
1077
CG2
VAL
E
130
13.231
20.121
68.367
1.00
1.34
C


ATOM
1078
C
VAL
E
130
10.358
17.641
67.638
1.00
0.02
C


ATOM
1079
O
VAL
E
130
9.318
18.310
67.656
1.00
0.04
O


ATOM
1080
N
LEU
E
131
10.421
16.436
67.098
1.00
0.01
N


ATOM
1081
CA
LEU
E
131
9.262
15.860
66.422
1.00
0.02
C


ATOM
1082
CB
LEU
E
131
9.290
16.320
64.964
1.00
1.51
C


ATOM
1083
CG
LEU
E
131
7.970
16.030
64.260
1.00
2.24
C


ATOM
1084
CD1
LEU
E
131
6.818
16.742
64.957
1.00
3.13
C


ATOM
1085
CD2
LEU
E
131
8.033
16.435
62.793
1.00
2.99
C


ATOM
1086
C
LEU
E
131
9.325
14.339
66.501
1.00
0.00
C


ATOM
1087
O
LEU
E
131
8.307
13.643
66.354
1.00
0.02
O


END


















TABLE 4







REMARK
4
Human RGS7 protein modeled on Rat RGS-4 (E. chain from PDB


REMARK
4
entry 1AGR. See: Tesmer J.J., D.M. Berman, A.G. Gilman, S.R. Sprang


REMARK
4
Structure of G(I RGS4 Bound to ALF4 (−)-Activated Alpha1):


REMARK
4
Stabilization of the Transition State for GTP Hydrolysis,


REMARK
4
Cell(Cambridge, Mass.) V. 89 251 1997)


REMARK
4







HEADER seqmod.18424, Minimized 100 + 2 * 200















ATOM
1
N
GLY E 1
−0.821
27.251
73.130
1.00
1.02


ATOM
5
CA
GLY E 1
−1.017
28.084
71.932
1.00
0.85


ATOM
6
C
GLY E 1
0.229
28.103
71.055
1.00
0.53


ATOM
7
O
GLY E 1
0.240
27.532
69.957
1.00
0.56


ATOM
8
N
LEU E 2
1.319
28.585
71.628
1.00
0.41


ATOM
10
CA
LEU E 2
2.568
28.728
70.871
1.00
0.33


ATOM
11
CB
LEU E 2
3.481
29.677
71.640
1.00
0.41


ATOM
12
CG
LEU E 2
4.741
30.020
70.853
1.00
1.13


ATOM
13
CD1
LEU E 2
4.392
30.672
69.518
1.00
1.51


ATOM
14
CD2
LEU E 2
5.658
30.926
71.666
1.00
1.25


ATOM
15
C
LEU E 2
3.267
27.385
70.658
1.00
0.30


ATOM
16
O
LEU E 2
3.900
27.185
69.615
1.00
0.28


ATOM
17
N
VAL E 3
2.970
26.415
71.506
1.00
0.29


ATOM
19
CA
VAL E 3
3.486
25.055
71.297
1.00
0.25


ATOM
20
CB
VAL E 3
3.274
24.223
72.560
1.00
0.37


ATOM
21
CG1
VAL E 3
3.839
22.815
72.395
1.00
0.52


ATOM
22
CG2
VAL E 3
3.903
24.907
73.768
1.00
0.41


ATOM
23
C
VAL E 3
2.871
24.385
70.049
1.00
0.25


ATOM
24
O
VAL E 3
3.659
24.046
69.155
1.00
0.20


ATOM
25
N
PRO E 4
1.547
24.262
69.903
1.00
0.31


ATOM
26
CA
PRO E 4
1.018
23.763
68.626
1.00
0.30


ATOM
27
CB
PRO E 4
−0.459
23.617
68.822
1.00
0.41


ATOM
28
CG
PRO E 4
−0.841
24.122
70.201
1.00
0.67


ATOM
29
CD
PRO E 4
0.459
24.535
70.862
1.00
0.40


ATOM
30
C
PRO E 4
1.310
24.663
67.418
1.00
0.26


ATOM
31
O
PRO E 4
1.500
24.114
66.325
1.00
0.23


ATOM
32
N
ARG E 5
1.503
25.963
67.613
1.00
0.27


ATOM
34
CA
ARG E 5
1.906
26.834
66.501
1.00
0.23


ATOM
35
CB
ARG E 5
1.928
28.285
66.962
1.00
0.50


ATOM
36
CG
ARG E 5
0.526
28.844
67.153
1.00
1.05


ATOM
37
CD
ARG E 5
0.603
30.281
67.651
1.00
1.48


ATOM
38
NE
ARG E 5
1.499
31.069
66.788
1.00
2.28


ATOM
39
CZ
ARG E 5
1.073
31.965
65.894
1.00
3.07


ATOM
40
NH1
ARG E 5
−0.233
32.216
65.768
1.00
3.88


ATOM
41
NH2
ARG E 5
1.953
32.622
65.136
1.00
3.52


ATOM
42
C
ARG E 5
3.286
26.456
65.982
1.00
0.15


ATOM
43
O
ARG E 5
3.409
26.024
64.827
1.00
0.14


ATOM
44
N
GLY E 6
4.237
26.382
66.897
1.00
0.03


ATOM
46
CA
GLY E 6
5.615
25.999
66.575
1.00
0.02


ATOM
47
C
GLY E 6
5.683
24.627
65.916
1.00
0.03


ATOM
48
O
GLY E 6
6.220
24.509
64.809
1.00
0.03


ATOM
49
N
SER E 7
4.962
23.666
66.475
1.00
0.03


ATOM
51
CA
SER E 7
4.994
22.307
65.930
1.00
0.04


ATOM
52
CB
SER E 7
4.323
21.354
66.916
1.00
0.42


ATOM
53
OG
SER E 7
2.975
21.762
67.101
1.00
0.35


ATOM
54
C
SER E 7
4.346
22.166
64.546
1.00
0.02


ATOM
55
O
SER E 7
4.929
21.453
63.721
1.00
0.02


ATOM
56
N
HIS E 8
3.363
22.982
64.186
1.00
0.04


ATOM
58
CA
HIS E 8
2.817
22.836
62.830
1.00
0.03


ATOM
59
CB
HIS E 8
1.327
23.184
62.745
1.00
0.45


ATOM
60
CG
HIS E 8
0.909
24.638
62.874
1.00
0.78


ATOM
61
ND1
HIS E 8
0.188
25.165
63.879
1.00
1.80


ATOM
63
CE1
HIS E 8
−0.009
26.478
63.652
1.00
2.59


ATOM
64
NE2
HIS E 8
0.589
26.784
62.478
1.00
2.35


ATOM
65
CD2
HIS E 8
1.145
25.658
61.980
1.00
1.43


ATOM
66
C
HIS E 8
3.631
23.635
61.819
1.00
0.02


ATOM
67
O
HIS E 8
3.708
23.227
60.654
1.00
0.01


ATOM
68
N
ARG E 9
4.432
24.571
62.301
1.00
0.02


ATOM
70
CA
ARG E 9
5.359
25.268
61.414
1.00
0.04


ATOM
71
CB
ARG E 9
5.750
26.596
62.046
1.00
0.23


ATOM
72
CD
ARG E 9
4.511
27.454
62.272
1.00
0.96


ATOM
73
CD
ARG E 9
4.851
28.794
62.910
1.00
1.69


ATOM
74
NE
ARG E 9
5.662
29.615
61.996
1.00
2.00


ATOM
75
CZ
ARG E 9
6.745
30.291
62.386
1.00
2.66


ATOM
76
NH1
ARG E 9
7.163
30.206
63.650
1.00
3.47


ATOM
77
NH2
ARG E 9
7.421
31.034
61.506
1.00
3.00


ATOM
78
C
ARG E 9
6.587
24.400
61.165
1.00
0.01


ATOM
79
O
ARG E 9
7.055
24.343
60.024
1.00
0.02


ATOM
80
N
VAL E 10
6.873
23.512
62.107
1.00
0.03


ATOM
82
CA
VAL E 10
7.910
22.498
61.900
1.00
0.00


ATOM
83
CB
VAL E 10
8.203
21.791
63.220
1.00
0.02


ATOM
84
CG1
VAL E 10
9.122
20.592
63.012
1.00
0.00


ATOM
85
CG2
VAL E 10
8.791
22.749
64.248
1.00
0.01


ATOM
86
C
VAL E 10
7.450
21.468
60.874
1.00
0.02


ATOM
87
O
VAL E 10
8.192
21.203
59.923
1.00
0.02


ATOM
88
N
LYS E 11
6.160
21.163
60.878
1.00
0.01


ATOM
90
CA
LYS E 11
5.606
20.239
59.881
1.00
0.02


ATOM
91
CB
LYS E 11
4.180
19.895
60.288
1.00
0.02


ATOM
92
CG
LYS E 11
4.144
19.259
61.670
1.00
0.01


ATOM
93
CD
LYS E 11
2.718
19.146
62.192
1.00
0.01


ATOM
94
CE
LYS E 11
2.696
18.664
63.638
1.00
0.00


ATOM
95
NZ
LYS E 11
1.324
18.668
64.171
1.00
0.03


ATOM
96
C
LYS E 11
5.597
20.861
58.487
1.00
0.03


ATOM
97
O
LYS E 11
5.997
20.188
57.526
1.00
0.02


ATOM
98
N
ARG E 12
5.482
22.179
58.433
1.00
0.02


ATOM
100
CA
ARG E 12
5.538
22.903
57.159
1.00
0.03


ATOM
101
CB
ARG E 12
5.029
24.320
57.376
1.00
0.15


ATOM
102
CG
ARG E 12
3.538
24.347
57.672
1.00
1.14


ATOM
103
CD
ARG E 12
3.071
25.776
57.915
1.00
1.50


ATOM
104
NE
ARG E 12
1.606
25.847
58.008
1.00
2.77


ATOM
105
CZ
ARG E 12
0.845
26.323
57.019
1.00
3.81


ATOM
106
NH1
ARG E 12
−0.483
26.328
57.140
1.00
4.89


ATOM
107
NH2
ARG E 12
1.412
26.769
55.894
1.00
4.15


ATOM
108
C
ARG E 12
6.942
22.994
56.565
1.00
0.02


ATOM
109
O
ARG E 12
7.047
23.084
55.336
1.00
0.02


ATOM
110
N
TRP E 13
7.973
22.717
57.350
1.00
0.03


ATOM
112
CA
TRP E 13
9.340
22.720
56.813
1.00
0.03


ATOM
113
CB
TRP E 13
10.345
22.869
57.953
1.00
0.01


ATOM
114
CG
TRP E 13
10.191
24.083
58.847
1.00
0.02


ATOM
115
CD1
TRP E 13
9.558
25.273
58.554
1.00
0.01


ATOM
116
NE1
TRP E 13
9.636
26.079
59.642
1.00
0.03


ATOM
118
CE2
TRP E 13
10.317
25.487
60.641
1.00
0.02


ATOM
119
CZ2
TRP E 13
10.655
25.896
61.920
1.00
0.02


ATOM
120
CH2
TRP E 13
11.391
25.049
62.743
1.00
0.01


ATOM
121
CZ3
TRP E 13
11.784
23.796
62.289
1.00
0.03


ATOM
122
CE3
TRP E 13
11.442
23.374
61.008
1.00
0.03


ATOM
123
CD2
TRP E 13
10.709
24.217
60.185
1.00
0.01


ATOM
124
C
TRP E 13
9.635
21.400
56.106
1.00
0.02


ATOM
125
O
TRP E 13
10.437
21.353
55.166
1.00
0.02


ATOM
126
N
GLY E 14
8.835
20.396
56.426
1.00
0.00


ATOM
128
CA
GLY E 14
8.977
19.069
55.824
1.00
0.00


ATOM
129
C
GLY E 14
7.986
18.890
54.683
1.00
0.02


ATOM
130
O
GLY E 14
8.148
18.000
53.840
1.00
0.02


ATOM
131
N
PHE E 15
6.965
19.731
54.668
1.00
0.01


ATOM
133
CA
PHE E 15
6.025
19.743
53.542
1.00
0.02


ATOM
134
CB
PHE E 15
4.646
20.162
54.044
1.00
0.62


ATOM
135
CG
PHE E 15
3.981
19.179
55.004
1.00
1.08


ATOM
136
CD1
PHE E 15
3.200
19.657
56.049
1.00
1.84


ATOM
137
CE1
PHE E 15
2.594
18.769
56.927
1.00
2.25


ATOM
138
CZ
PHE E 15
2.763
17.401
56.760
1.00
1.96


ATOM
139
CE2
PHE E 15
3.536
16.921
55.711
1.00
1.56


ATOM
140
CD2
PHE E 15
4.143
17.809
54.832
1.00
1.19


ATOM
141
C
PHE E 15
6.483
20.706
52.444
1.00
0.01


ATOM
142
O
PHE E 15
6.033
20.599
51.297
1.00
0.01


ATOM
143
N
GLY E 16
7.390
21.604
52.787
1.00
0.02


ATOM
145
CA
GLY E 16
7.974
22.512
51.799
1.00
0.01


ATOM
146
C
GLY E 16
9.338
23.012
52.263
1.00
0.00


ATOM
147
O
GLY E 16
9.454
23.688
53.296
1.00
0.03


ATOM
148
N
MET E 17
10.320
22.871
51.386
1.00
0.00


ATOM
150
CA
MET E 17
11.694
23.241
51.749
1.00
0.01


ATOM
151
CB
MET E 17
12.680
22.583
50.790
1.00
0.97


ATOM
152
CG
MET E 17
14.106
22.721
51.313
1.00
1.67


ATOM
153
SD
MET E 17
15.410
21.988
50.302
1.00
2.86


ATOM
154
CE
MET E 17
15.289
23.064
48.858
1.00
3.38


ATOM
155
C
MET E 17
11.902
24.752
51.729
1.00
0.02


ATOM
156
O
MET E 17
12.708
25.264
52.512
1.00
0.02


ATOM
157
N
ASP E 18
10.999
25.474
51.085
1.00
0.02


ATOM
159
CA
ASP E 18
11.069
26.935
51.130
1.00
0.02


ATOM
160
CB
ASP E 18
10.342
27.543
49.928
1.00
0.18


ATOM
161
CG
ASP E 18
8.901
27.050
49.805
1.00
0.80


ATOM
162
OD1
ASP E 18
8.020
27.718
50.330
1.00
0.95


ATOM
163
OD2
ASP E 18
8.710
26.023
49.166
1.00
1.43


ATOM
164
C
ASP E 18
10.495
27.475
52.438
1.00
0.03


ATOM
165
O
ASP E 18
10.982
28.499
52.923
1.00
0.02


ATOM
166
N
GLU E 19
9.720
26.666
53.143
1.00
0.01


ATOM
168
CA
GLU E 19
9.205
27.083
54.445
1.00
0.00


ATOM
169
CB
GLU E 19
7.916
26.328
54.781
1.00
0.36


ATOM
170
CG
GLU E 19
6.677
26.894
54.079
1.00
1.11


ATOM
171
CD
GLU E 19
6.337
26.194
52.758
1.00
2.06


ATOM
172
OE1
GLU E 19
5.191
26.300
52.349
1.00
3.03


ATOM
173
OE2
GLU E 19
7.219
25.561
52.186
1.00
2.42


ATOM
174
C
GLU E 19
10.265
26.816
55.504
1.00
0.02


ATOM
175
O
GLU E 19
10.451
27.638
56.412
1.00
0.02


ATOM
176
N
ALA E 20
11.104
25.833
55.223
1.00
0.03


ATOM
178
CA
ALA E 20
12.261
25.572
56.071
1.00
0.01


ATOM
179
CB
ALA E 20
12.892
24.248
55.654
1.00
0.13


ATOM
180
C
ALA E 20
13.281
26.690
55.917
1.00
0.02


ATOM
181
O
ALA E 20
13.484
27.437
56.881
1.00
0.01


ATOM
182
N
LEU E 21
13.603
27.012
54.673
1.00
0.01


ATOM
184
CA
LEU E 21
14.678
27.972
54.377
1.00
0.02


ATOM
185
CB
LEU E 21
15.125
27.761
52.936
1.00
0.19


ATOM
186
CG
LEU E 21
15.669
26.358
52.701
1.00
0.28


ATOM
187
CD1
LEU E 21
16.085
26.180
51.245
1.00
0.37


ATOM
188
CD2
LEU E 21
16.837
26.055
53.631
1.00
0.47


ATOM
189
C
LEU E 21
14.317
29.450
54.554
1.00
0.02


ATOM
190
O
LEU E 21
15.229
30.279
54.656
1.00
0.02


ATOM
191
N
LYS E 22
13.039
29.788
54.619
1.00
0.02


ATOM
193
CA
LYS E 22
12.665
31.178
54.907
1.00
0.03


ATOM
194
CB
LYS E 22
11.398
31.525
54.135
1.00
0.45


ATOM
195
CG
LYS E 22
11.648
31.485
52.632
1.00
0.74


ATOM
196
CD
LYS E 22
10.349
31.643
51.853
1.00
1.17


ATOM
197
CE
LYS E 22
10.588
31.506
50.353
1.00
1.93


ATOM
198
NZ
LYS E 22
9.323
31.601
49.609
1.00
3.00


ATOM
199
C
LYS E 22
12.439
31.382
56.401
1.00
0.02


ATOM
200
O
LYS E 22
12.401
32.516
56.892
1.00
0.02


ATOM
201
N
ASP E 23
12.305
30.279
57.114
1.00
0.01


ATOM
203
CA
ASP E 23
12.219
30.326
58.658
1.00
0.00


ATOM
204
CB
ASP E 23
11.425
29.097
58.996
1.00
0.79


ATOM
205
CG
ASP E 23
11.125
29.086
60.488
1.00
1.19


ATOM
206
OD1
ASP E 23
9.950
29.095
60.823
1.00
1.81


ATOM
207
OD2
ASP E 23
12.059
28.874
61.251
1.00
1.63


ATOM
208
C
ASP E 23
13.635
30.277
59.133
1.00
0.01


ATOM
209
O
ASP E 23
14.391
29.357
58.807
1.00
0.03


ATOM
210
N
PRO E 24
13.940
31.161
60.071
1.00
0.02


ATOM
211
CA
PRO E 24
15.297
31.235
60.627
1.00
0.02


ATOM
212
CB
PRO E 24
15.239
32.324
61.654
1.00
0.23


ATOM
213
CG
PRO E 24
13.846
32.938
61.663
1.00
0.18


ATOM
214
CD
PRO E 24
13.042
32.182
60.619
1.00
0.14


ATOM
215
C
PRO E 24
15.761
29.909
61.235
1.00
0.01


ATOM
216
O
PRO E 24
16.699
29.305
60.698
1.00
0.02


ATOM
217
N
VAL E 25
14.937
29.318
62.086
1.00
0.02


ATOM
219
CA
VAL E 25
15.309
28.064
62.750
1.00
0.02


ATOM
220
CB
VAL E 25
14.340
27.835
63.903
1.00
0.22


ATOM
221
CG1
VAL E 25
14.667
26.547
64.651
1.00
0.23


ATOM
222
CG2
VAL E 25
14.351
29.024
64.858
1.00
0.32


ATOM
223
C
VAL E 25
15.269
26.868
61.800
1.00
0.02


ATOM
224
O
VAL E 25
16.223
26.080
61.804
1.00
0.02


ATOM
225
N
GLY E 26
14.380
26.916
60.820
1.00
0.03


ATOM
227
CA
GLY E 26
14.275
25.856
59.809
1.00
0.01


ATOM
228
C
GLY E 26
15.524
25.788
58.934
1.00
0.02


ATOM
229
O
GLY E 26
16.172
26.949
58.485
1.00
0.02


ATOM
230
N
ARG E 27
15.973
26.949
58.485
1.00
0.02


ATOM
232
CA
ARG E 27
17.163
27.042
57.639
1.00
0.01


ATOM
233
CB
ARG E 27
17.158
28.445
57.029
1.00
0.18


ATOM
234
CG
ARG E 27
18.198
28.676
55.931
1.00
0.52


ATOM
235
CD
ARG E 27
19.543
29.138
56.477
1.00
1.05


ATOM
236
NE
ARG E 27
19.363
30.333
57.313
1.00
2.02


ATOM
237
CZ
ARG E 27
20.116
30.590
58.383
1.00
2.94


ATOM
238
NH1
ARG E 27
19.829
31.633
59.165
1.00
3.86


ATOM
239
NH2
ARG E 27
21.102
29.754
58.717
1.00
3.32


ATOM
240
C
ARG E 27
18.434
26.784
58.447
1.00
0.03


ATOM
241
O
ARG E 27
19.341
26.117
57.937
1.00
0.03


ATOM
242
N
GLU E 28
18.390
27.049
59.744
1.00
0.02


ATOM
244
CA
GLU E 28
19.529
26.739
60.618
1.00
0.02


ATOM
245
CB
GLU E 28
19.305
27.411
61.965
1.00
0.14


ATOM
246
CG
GLU E 28
19.442
28.923
61.891
1.00
0.41


ATOM
247
CD
GLU E 28
18.765
29.539
63.108
1.00
0.96


ATOM
248
OE1
GLU E 28
18.277
30.655
62.987
1.00
1.43


ATOM
249
OE2
GLU E 28
18.576
28.808
64.072
1.00
1.42


ATOM
250
C
GLU E 28
19.682
25.241
60.863
1.00
0.03


ATOM
251
O
GLU E 28
20.807
24.734
60.792
1.00
0.03


ATOM
252
N
GLN E 29
18.575
24.516
60.921
1.00
0.02


ATOM
254
CA
GLN E 29
18.662
23.076
61.174
1.00
0.01


ATOM
255
CB
GLN E 29
17.326
22.568
61.711
1.00
0.11


ATOM
256
CG
GLN E 29
16.820
23.351
62.922
1.00
0.22


ATOM
257
CD
GLN E 29
17.743
23.242
64.133
1.00
0.64


ATOM
258
OE1
GLN E 29
17.908
22.166
64.716
1.00
1.46


ATOM
259
NE2
GLN E 29
18.229
24.391
64.575
1.00
1.11


ATOM
262
C
GLN E 29
19.006
22.333
59.888
1.00
0.02


ATOM
263
O
GLN E 29
19.815
21.397
59.919
1.00
0.02


ATOM
264
N
PHE E 30
18.629
22.913
58.758
1.00
0.02


ATOM
266
CA
PHE E 30
18.998
22.307
57.477
1.00
0.02


ATOM
267
CB
PHE E 30
18.062
22.807
56.383
1.00
0.02


ATOM
268
CG
PHE E 30
18.237
22.077
55.053
1.00
0.01


ATOM
269
CD1
PHE E 30
18.452
20.705
55.037
1.00
0.01


ATOM
270
CE1
PHE E 30
18.610
20.038
53.830
1.00
0.02


ATOM
271
CZ
PHE E 30
18.551
20.743
52.635
1.00
0.02


ATOM
272
CE2
PHE E 30
18.333
22.115
52.649
1.00
0.03


ATOM
273
CD2
PHE E 30
18.175
22.781
53.857
1.00
0.01


ATOM
274
C
PHE E 30
20.442
22.643
57.117
1.00
0.02


ATOM
275
O
PHE E 30
21.148
21.772
56.596
1.00
0.03


ATOM
276
N
LEU E 31
20.994
23.753
57.632
1.00
0.02


ATOM
278
CA
LEU E 31
22.351
24.101
57.430
1.00
0.02


ATOM
279
CB
LEU E 31
22.534
25.589
57.710
1.00
0.21


ATOM
280
CG
LEU E 31
23.953
26.055
57.400
1.00
1.03


ATOM
281
CD1
LEU E 31
24.316
25.781
55.944
1.00
1.18


ATOM
282
CD2
LEU E 31
24.120
27.536
57.722
1.00
1.18


ATOM
283
C
LEU E 31
23.245
23.288
58.358
1.00
0.04


ATOM
284
O
LEU E 31
24.325
22.867
57.934
1.00
0.02


ATOM
285
N
LYS E 32
22.701
22.849
59.481
1.00
0.04


ATOM
287
CA
LYS E 32
23.441
21.956
60.377
1.00
0.02


ATOM
288
CB
LYS E 32
22.737
21.958
61.728
1.00
0.16


ATOM
289
CG
LYS E 32
23.431
21.043
62.728
1.00
1.06


ATOM
290
CD
LYS E 32
22.649
20.966
64.032
1.00
1.39


ATOM
291
CE
LYS E 32
21.231
20.460
63.787
1.00
2.08


ATOM
292
NZ
LYS E 32
20.506
20.302
65.056
1.00
2.62


ATOM
293
C
LYS E 32
23.482
20.533
59.819
1.00
0.01


ATOM
294
O
LYS E 32
24.543
19.892
59.843
1.00
0.03


ATOM
295
N
PHE E 33
22.448
20.173
59.075
1.00
0.02


ATOM
297
CA
PHE E 33
22.428
18.873
58.405
1.00
0.01


ATOM
298
CB
PHE E 33
20.993
18.573
57.998
1.00
0.02


ATOM
299
CG
PHE E 33
20.828
17.222
57.318
1.00
0.03


ATOM
300
CD1
PHE E 33
21.116
16.059
58.019
1.00
0.02


ATOM
301
CE1
PHE E 33
20.976
14.824
57.400
1.00
0.04


ATOM
302
CZ
PHE E 33
20.548
14.756
56.082
1.00
0.02


ATOM
303
CE2
PHE E 33
20.257
15.920
55.383
1.00
0.02


ATOM
304
CD2
PHE E 33
20.397
17.154
56.000
1.00
0.02


ATOM
305
C
PHE E 33
23.333
18.871
57.172
1.00
0.03


ATOM
306
O
PHE E 33
24.066
17.899
56.951
1.00
0.01


ATOM
307
N
LEU E 34
23.472
20.025
56.543
1.00
0.02


ATOM
309
CA
LEU E 34
24.417
20.166
55.436
1.00
0.02


ATOM
310
CB
LEU E 34
24.013
21.390
54.628
1.00
0.03


ATOM
311
CG
LEU E 34
22.711
21.174
53.867
1.00
0.02


ATOM
312
CD1
LEU E 34
22.148
22.492
53.361
1.00
0.02


ATOM
313
CD2
LEU E 34
22.894
20.198
52.714
1.00
0.02


ATOM
314
C
LEU E 34
25.858
20.311
55.927
1.00
0.03


ATOM
315
O
LEU E 34
26.783
19.892
55.220
1.00
0.03


ATOM
316
N
GLU E 35
26.047
20.699
57.178
1.00
0.02


ATOM
318
CA
GLU E 35
27.394
20.714
57.760
1.00
0.03


ATOM
319
CB
GLU E 35
27.405
21.510
59.064
1.00
0.23


ATOM
320
CG
GLU E 35
27.198
23.011
58.880
1.00
0.80


ATOM
321
CD
GLU E 35
28.375
23.663
58.160
1.00
1.78


ATOM
322
OE1
GLU E 35
28.183
24.758
57.651
1.00
2.50


ATOM
323
OE2
GLU E 35
29.465
23.112
58.221
1.00
2.17


ATOM
324
C
GLU E 35
27.844
19.294
58.072
1.00
0.03


ATOM
325
O
GLU E 35
28.949
18.910
57.672
1.00
0.03


ATOM
326
N
SER E 36
26.905
18.466
58.504
1.00
0.02


ATOM
328
CA
SER E 36
27.227
17.066
58.815
1.00
0.01


ATOM
329
CB
SER E 36
26.213
16.525
59.819
1.00
0.02


ATOM
330
OG
SER E 36
24.933
16.508
59.204
1.00
0.01


ATOM
331
C
SER E 36
27.259
16.171
57.572
1.00
0.02


ATOM
332
O
SER E 36
27.652
15.003
57.664
1.00
0.02


ATOM
333
N
GLU E 37
26.841
16.701
56.432
1.00
0.02


ATOM
335
CA
GLU E 37
27.033
16.008
55.154
1.00
0.02


ATOM
336
CB
GLU E 37
25.808
16.238
54.283
1.00
0.02


ATOM
337
CG
GLU E 37
24.541
15.642
54.871
1.00
0.01


ATOM
338
CD
GLU E 37
23.363
16.140
54.046
1.00
0.01


ATOM
339
OE1
GLU E 37
23.162
17.348
54.018
1.00
0.00


ATOM
340
OE2
GLU E 37
22.707
15.319
53.425
1.00
0.02


ATOM
341
C
GLU E 37
28.242
16.545
54.389
1.00
0.02


ATOM
342
O
GLU E 37
28.584
16.000
53.332
1.00
0.03


ATOM
343
N
PHE E 38
28.860
17.591
54.924
1.00
0.03


ATOM
345
CA
PHE E 38
29.927
18.344
54.248
1.00
0.03


ATOM
346
CB
PHE E 38
31.179
17.479
54.143
1.00
0.97


ATOM
347
CG
PHE E 38
32.455
18.264
53.857
1.00
1.85


ATOM
348
CD1
PHE E 38
33.454
17.710
53.066
1.00
2.66


ATOM
349
CE1
PHE E 38
34.615
18.429
52.812
1.00
3.84


ATOM
350
CZ
PHE E 38
34.778
19.701
53.348
1.00
4.14


ATOM
351
CE2
PHE E 38
33.778
20.253
54.139
1.00
3.53


ATOM
352
CD2
PHE E 38
32.619
19.535
54.393
1.00
2.52


ATOM
353
C
PHE E 38
29.449
18.812
52.870
1.00
0.02


ATOM
354
O
PHE E 38
30.064
18.541
51.833
1.00
0.01


ATOM
355
N
SER E 39
28.339
19.530
52.900
1.00
0.02


ATOM
357
CA
SER E 39
27.662
20.011
51.696
1.00
0.01


ATOM
358
CB
SER E 39
26.671
18.943
51.257
1.00
0.02


ATOM
359
OG
SER E 39
25.756
18.767
52.331
1.00
0.02


ATOM
360
C
SER E 39
26.897
21.291
52.008
1.00
0.03


ATOM
361
O
SER E 39
25.926
21.645
51.323
1.00
0.00


ATOM
362
N
SER E 40
27.408
22.036
52.975
1.00
0.02


ATOM
364
CA
SER E 40
26.713
23.239
53.458
1.00
0.03


ATOM
365
CB
SER E 40
27.167
23.546
54.877
1.00
1.37


ATOM
366
OG
SER E 40
28.560
23.828
54.861
1.00
2.01


ATOM
367
C
SER E 40
26.939
24.464
52.583
1.00
0.02


ATOM
368
O
SER E 40
26.134
25.404
52.631
1.00
0.02


ATOM
369
N
GLU E 41
27.825
24.324
51.611
1.00
0.02


ATOM
371
CA
GLU E 41
28.142
25.413
50.685
1.00
0.02


ATOM
372
CB
GLU E 41
29.429
25.087
49.910
1.00
0.02


ATOM
373
CG
GLU E 41
29.311
24.045
48.787
1.00
0.02


ATOM
374
CD
GLU E 41
29.334
22.599
49.279
1.00
0.02


ATOM
375
OE1
GLU E 41
29.694
22.433
50.441
1.00
0.02


ATOM
376
OE2
GLU E 41
28.625
21.804
48.680
1.00
0.03


ATOM
377
C
GLU E 41
27.005
25.706
49.707
1.00
0.01


ATOM
378
O
GLU E 41
26.829
26.873
49.346
1.00
0.02


ATOM
379
N
ASN E 42
26.081
24.770
49.546
1.00
0.01


ATOM
381
CA
ASN E 42
24.946
25.003
48.660
1.00
0.02


ATOM
382
CB
ASN E 42
24.331
23.651
48.306
1.00
0.02


ATOM
383
CG
ASN E 42
25.370
22.723
47.676
1.00
0.02


ATOM
384
OD1
ASN E 42
26.036
23.070
46.690
1.00
0.02


ATOM
385
ND2
ASN E 42
25.529
21.562
48.288
1.00
0.02


ATOM
388
C
ASN E 42
23.904
25.896
49.335
1.00
0.03


ATOM
389
O
ASN E 42
23.426
26.850
48.708
1.00
0.00


ATOM
390
N
LEU E 43
23.800
25.801
50.653
1.00
0.02


ATOM
392
CA
LEU E 43
22.818
26.629
51.358
1.00
0.02


ATOM
393
CB
LEU E 43
22.340
25.906
52.609
1.00
0.29


ATOM
394
CG
LEU E 43
21.182
26.649
53.267
1.00
0.88


ATOM
395
CD1
LEU E 43
20.067
26.916
52.263
1.00
1.15


ATOM
396
CD2
LEU E 43
20.646
25.883
54.469
1.00
1.57


ATOM
397
C
LEU E 43
23.422
27.976
51.732
1.00
0.02


ATOM
398
O
LEU E 43
22.722
28.997
51.695
1.00
0.02


ATOM
399
N
ARG E 44
24.741
28.017
51.821
1.00
0.01


ATOM
401
CA
ARG E 44
25.407
29.296
52.049
1.00
0.03


ATOM
402
CB
ARG E 44
26.802
29.041
52.602
1.00
0.17


ATOM
403
CG
ARG E 44
26.713
28.416
53.990
1.00
0.48


ATOM
404
CD
ARG E 44
28.083
28.249
54.636
1.00
0.81


ATOM
405
NE
ARG E 44
28.908
27.253
53.934
1.00
0.67


ATOM
406
CZ
ARG E 44
30.132
27.512
53.468
1.00
0.63


ATOM
407
NH1
ARG E 44
30.606
28.759
53.492
1.00
1.43


ATOM
408
NH2
ARG E 44
30.844
26.540
52.894
1.00
0.62


ATOM
409
C
ARG E 44
25.467
30.113
50.762
1.00
0.02


ATOM
410
O
ARG E 44
25.283
31.335
50.815
1.00
0.03


ATOM
411
N
PHE E 45
25.431
29.433
49.628
1.00
0.02


ATOM
413
CA
PHE E 45
25.319
30.124
48.345
1.00
0.02


ATOM
414
CB
PHE E 45
25.718
29.155
47.241
1.00
0.03


ATOM
415
CG
PHE E 45
25.400
29.666
45.843
1.00
0.02


ATOM
416
CD1
PHE E 45
26.153
30.691
45.287
1.00
0.02


ATOM
417
CE1
PHE E 45
25.852
31.161
44.016
1.00
0.02


ATOM
418
CZ
PHE E 45
24.799
30.608
43.302
1.00
0.01


ATOM
419
CE2
PHE E 45
24.045
29.585
43.861
1.00
0.02


ATOM
420
CD2
PHE E 45
24.342
29.116
45.131
1.00
0.02


ATOM
421
C
PHE E 45
23.894
30.606
48.099
1.00
0.02


ATOM
422
O
PHE E 45
23.705
31.755
47.678
1.00
0.01


ATOM
423
N
TRP E 46
22.922
29.852
48.587
1.00
0.01


ATOM
425
CA
TRP E 46
21.522
30.256
48.446
1.00
0.03


ATOM
426
CB
TRP E 46
20.634
29.103
48.906
1.00
0.02


ATOM
427
CG
TRP E 46
19.149
29.390
48.807
1.00
0.01


ATOM
428
CD1
TRP E 46
18.354
29.208
47.696
1.00
0.02


ATOM
429
NE1
TRP E 46
17.094
29.604
48.001
1.00
0.03


ATOM
431
CE2
TRP E 46
17.009
30.033
49.274
1.00
0.02


ATOM
432
CZ2
TRP E 46
15.964
30.546
50.027
1.00
0.02


ATOM
433
CH2
TRP E 46
16.183
30.915
51.350
1.00
0.02


ATOM
434
CZ3
TRP E 46
17.443
30.772
51.920
1.00
0.02


ATOM
435
CE3
TRP E 46
18.498
30.263
51.170
1.00
0.01


ATOM
436
CD2
TRP E 46
18.286
29.899
49.848
1.00
0.03


ATOM
437
C
TRP E 46
21.223
31.496
49.282
1.00
0.02


ATOM
438
O
TRP E 46
20.467
32.459
48.757
1.00
0.02


ATOM
439
N
LEU E 47
21.821
31.570
50.461
1.00
0.02


ATOM
441
CA
LEU E 47
21.613
32.723
51.337
1.00
0.03


ATOM
442
CB
LEU E 47
22.000
32.308
52.752
1.00
0.12


ATOM
443
CG
LEU E 47
21.653
33.380
53.777
1.00
0.93


ATOM
444
CD1
LEU E 47
20.161
33.699
53.748
1.00
1.23


ATOM
445
CD2
LEU E 47
22.078
32.945
55.175
1.00
1.20


ATOM
446
C
LEU E 47
22.446
33.925
50.892
1.00
0.03


ATOM
447
O
LEU E 47
21.938
35.055
50.919
1.00
0.02


ATOM
448
N
ALA E 48
23.546
33.658
50.208
1.00
0.01


ATOM
450
CA
ALA E 48
24.376
34.738
49.673
1.00
0.02


ATOM
451
CB
ALA E 48
25.724
34.159
49.260
1.00
0.05


ATOM
452
C
ALA E 48
23.719
35.418
48.477
1.00
0.02


ATOM
453
O
ALA E 48
23.702
36.655
48.432
1.00
0.01


ATOM
454
N
VAL E 49
22.949
34.670
47.700
1.00
0.02


ATOM
456
CA
VAL E 49
22.219
35.291
46.590
1.00
0.03


ATOM
457
CB
VAL E 49
21.892
34.233
45.547
1.00
0.09


ATOM
458
CG1
VAL E 49
21.246
34.866
44.318
1.00
0.10


ATOM
459
CG2
VAL E 49
23.151
33.483
45.141
1.00
0.20


ATOM
460
C
VAL E 49
20.940
35.974
47.080
1.00
0.02


ATOM
461
O
VAL E 49
20.598
37.044
46.563
1.00
0.02


ATOM
462
N
GLU E 50
20.445
35.549
48.235
1.00
0.03


ATOM
464
CA
GLU E 50
19.328
36.260
48.877
1.00
0.02


ATOM
465
CB
GLU E 50
18.840
35.486
50.099
1.00
0.02


ATOM
466
CG
GLU E 50
18.260
34.117
49.769
1.00
0.01


ATOM
467
CD
GLU E 50
16.978
34.240
48.955
1.00
0.02


ATOM
468
OE1
GLU E 50
17.013
33.813
47.809
1.00
0.01


ATOM
469
OE2
GLU E 50
15.951
34.501
49.564
1.00
0.03


ATOM
470
C
GLU E 50
19.793
37.630
49.360
1.00
0.02


ATOM
471
O
GLU E 50
19.144
38.640
49.063
1.00
0.02


ATOM
472
N
ASP E 51
21.028
37.680
49.837
1.00
0.03


ATOM
474
CA
ASP E 51
21.621
38.939
50.297
1.00
0.02


ATOM
475
CB
ASP E 51
22.898
38.617
51.070
1.00
0.17


ATOM
476
CG
ASP E 51
22.627
37.683
52.248
1.00
0.59


ATOM
477
OD1
ASP E 51
21.603
37.861
52.895
1.00
0.77


ATOM
478
OD2
ASP E 51
23.518
36.905
52.567
1.00
0.92


ATOM
479
C
ASP E 51
21.987
39.843
49.122
1.00
0.03


ATOM
480
O
ASP E 51
21.710
41.048
49.173
1.00
0.02


ATOM
481
N
LEU E 52
22.318
39.233
47.994
1.00
0.03


ATOM
483
CA
LEU E 52
22.688
39.978
46.786
1.00
0.03


ATOM
484
CB
LEU E 52
23.367
39.010
45.827
1.00
0.49


ATOM
485
CG
LEU E 52
23.870
39.725
44.580
1.00
0.86


ATOM
486
CD1
LEU E 52
25.021
40.664
44.926
1.00
1.11


ATOM
487
CD2
LEU E 52
24.297
38.723
43.519
1.00
1.06


ATOM
488
C
LEU E 52
21.483
40.607
46.084
1.00
0.02


ATOM
489
O
LEU E 52
21.630
41.665
45.462
1.00
0.02


ATOM
490
N
LYS E 53
20.286
40.131
46.391
1.00
0.03


ATOM
492
CA
LYS E 53
19.075
40.764
45.863
1.00
0.01


ATOM
493
CB
LYS E 53
17.960
39.735
45.738
1.00
0.02


ATOM
494
CG
LYS E 53
18.252
38.771
44.594
1.00
0.03


ATOM
495
CD
LYS E 53
17.030
37.924
44.258
1.00
0.01


ATOM
496
CE
LYS E 53
16.689
36.945
45.374
1.00
0.02


ATOM
497
NZ
LYS E 53
17.734
35.918
45.485
1.00
0.02


ATOM
498
C
LYS E 53
18.609
41.970
46.687
1.00
0.02


ATOM
499
O
LYS E 53
17.542
42.529
46.408
1.00
0.02


ATOM
500
N
LYS E 54
19.392
42.365
47.679
1.00
0.02


ATOM
502
CA
LYS E 54
19.103
43.579
48.450
1.00
0.02


ATOM
503
CB
LYS E 54
19.252
43.255
49.936
1.00
0.02


ATOM
504
CG
LYS E 54
18.442
42.025
50.341
1.00
0.03


ATOM
505
CD
LYS E 54
16.941
42.234
50.167
1.00
0.02


ATOM
506
CE
LYS E 54
16.168
40.960
50.487
1.00
0.03


ATOM
507
NZ
LYS E 54
14.726
41.159
50.273
1.00
0.02


ATOM
508
C
LYS E 54
20.073
44.712
48.091
1.00
0.03


ATOM
509
O
LYS E 54
20.043
45.768
48.734
1.00
0.03


ATOM
510
N
ARG E 55
20.902
44.499
47.081
1.00
0.02


ATOM
512
CA
ARG E 55
22.030
45.401
46.790
1.00
0.02


ATOM
513
CB
ARG E 55
22.857
44.774
45.667
1.00
2.33


ATOM
514
CG
ARG E 55
22.081
44.477
44.385
1.00
3.12


ATOM
515
CD
ARG E 55
21.984
45.663
43.430
1.00
4.25


ATOM
516
NE
ARG E 55
21.497
45.229
42.114
1.00
4.96


ATOM
517
CZ
ARG E 55
20.777
46.008
41.306
1.00
5.88


ATOM
518
NH1
ARG E 55
20.343
45.530
40.137
1.00
6.52


ATOM
519
NH2
ARG E 55
20.464
47.249
41.680
1.00
6.43


ATOM
520
C
ARG E 55
21.689
46.848
46.438
1.00
0.03


ATOM
521
O
ARG E 55
20.662
47.164
45.828
1.00
0.03


ATOM
522
N
PRO E 56
22.523
47.731
46.962
1.00
0.02


ATOM
523
CA
PRO E 56
22.876
48.956
46.243
1.00
0.03


ATOM
524
CB
PRO E 56
23.742
49.727
47.188
1.00
1.92


ATOM
525
CG
PRO E 56
24.147
48.807
48.327
1.00
2.37


ATOM
526
CD
PRO E 56
23.457
47.480
48.056
1.00
1.91


ATOM
527
C
PRO E 56
23.627
48.622
44.955
1.00
0.03


ATOM
528
O
PRO E 56
24.429
47.678
44.915
1.00
0.03


ATOM
529
N
ILE E 57
23.530
49.526
43.993
1.00
0.01


ATOM
531
CA
ILE E 57
24.080
49.295
42.646
1.00
0.02


ATOM
532
CB
ILE E 57
23.575
50.426
41.754
1.00
0.40


ATOM
533
CG2
ILE E 57
24.005
50.216
40.306
1.00
0.91


ATOM
534
CG1
ILE E 57
22.057
50.539
41.838
1.00
1.03


ATOM
535
CD1
ILE E 57
21.531
51.645
40.931
1.00
1.87


ATOM
536
C
ILE E 57
25.613
49.264
42.595
1.00
0.02


ATOM
537
O
ILE E 57
26.179
48.504
41.806
1.00
0.02


ATOM
538
N
LYS E 58
26.272
49.903
43.548
1.00
0.03


ATOM
540
CA
LYS E 58
27.737
49.861
43.576
1.00
0.03


ATOM
541
CB
LYS E 58
28.249
51.182
44.131
1.00
0.17


ATOM
542
CG
LYS E 58
27.861
52.339
43.219
1.00
1.06


ATOM
543
CD
LYS E 58
28.407
53.665
43.733
1.00
1.21


ATOM
544
CE
LYS E 58
28.050
54.807
42.788
1.00
1.95


ATOM
545
NZ
LYS E 58
28.607
54.571
41.445
1.00
1.86


ATOM
546
C
LYS E 58
28.285
48.704
44.411
1.00
0.02


ATOM
547
O
LYS E 58
29.479
48.393
44.319
1.00
0.02


ATOM
548
N
GLU E 59
27.422
48.012
45.137
1.00
0.02


ATOM
550
CA
GLU E 59
27.910
46.915
45.969
1.00
0.02


ATOM
551
CB
GLU E 59
27.212
46.962
47.321
1.00
0.29


ATOM
552
CG
GLU E 59
27.863
46.010
48.317
1.00
1.18


ATOM
553
CD
GLU E 59
27.117
46.057
49.644
1.00
1.75


ATOM
554
OE1
GLU E 59
25.908
46.242
49.604
1.00
2.15


ATOM
555
OE2
GLU E 59
27.763
45.922
50.672
1.00
2.09


ATOM
556
C
GLU E 59
27.658
45.578
45.285
1.00
0.02


ATOM
557
O
GLU E 59
28.402
44.613
45.513
1.00
0.02


ATOM
558
N
VAL E 60
26.744
45.572
44.328
1.00
0.03


ATOM
560
CA
VAL E 60
26.507
44.334
43.580
1.00
0.02


ATOM
561
CB
VAL E 60
25.225
44.428
42.751
1.00
0.39


ATOM
562
CG1
VAL E 60
25.210
45.557
41.727
1.00
1.04


ATOM
563
CG2
VAL E 60
24.895
43.099
42.087
1.00
0.78


ATOM
564
C
VAL E 60
27.703
43.831
42.740
1.00
0.02


ATOM
565
O
VAL E 60
27.964
42.635
42.920
1.00
0.02


ATOM
566
N
PRO E 61
28.544
44.624
42.062
1.00
0.03


ATOM
567
CA
PRO E 61
29.681
43.994
41.377
1.00
0.03


ATOM
568
CB
PRO E 61
30.381
45.089
40.632
1.00
0.17


ATOM
569
CG
PRO E 61
29.695
46.412
40.913
1.00
0.22


ATOM
570
CD
PRO E 61
28.542
46.084
41.841
1.00
0.09


ATOM
571
C
PRO E 61
30.647
43.293
42.331
1.00
0.02


ATOM
572
O
PRO E 61
30.868
42.090
42.144
1.00
0.01


ATOM
573
N
SER E 62
30.896
43.891
43.487
1.00
0.03


ATOM
575
CA
SER E 62
31.872
43.315
44.415
1.00
0.01


ATOM
576
CB
SER E 62
32.308
44.380
45.420
1.00
0.13


ATOM
577
OG
SER E 62
31.179
44.795
46.180
1.00
0.38


ATOM
578
C
SER E 62
31.328
42.093
45.149
1.00
0.03


ATOM
579
O
SER E 62
32.072
41.121
45.324
1.00
0.02


ATOM
580
N
ARG E 63
30.023
42.021
45.356
1.00
0.02


ATOM
582
CA
ARG E 63
29.488
40.829
46.010
1.00
0.02


ATOM
583
CB
ARG E 63
28.252
41.177
46.821
1.00
0.27


ATOM
584
CG
ARG E 63
28.633
42.085
47.981
1.00
0.98


ATOM
585
CD
ARG E 63
27.522
42.149
49.017
1.00
1.31


ATOM
586
NE
ARG E 63
26.269
42.632
48.423
1.00
2.08


ATOM
587
CZ
ARG E 63
25.104
42.549
49.065
1.00
2.89


ATOM
588
NH1
ARG E 63
25.037
41.945
50.253
1.00
3.46


ATOM
589
NH2
ARG E 63
23.997
43.022
48.498
1.00
3.50


ATOM
590
C
ARG E 63
29.194
39.715
45.016
1.00
0.03


ATOM
591
O
ARG E 63
29.378
38.544
45.368
1.00
0.03


ATOM
592
N
VAL E 64
29.039
40.063
43.749
1.00
0.03


ATOM
594
CA
VAL E 64
28.936
39.034
42.714
1.00
0.02


ATOM
595
CB
VAL E 64
28.452
39.665
41.412
1.00
0.06


ATOM
596
CG1
VAL E 64
28.719
38.764
40.213
1.00
0.14


ATOM
597
CG2
VAL E 64
26.977
40.037
41.484
1.00
0.07


ATOM
598
C
VAL E 64
30.297
38.389
42.499
1.00
0.03


ATOM
599
O
VAL E 64
30.398
37.156
42.519
1.00
0.03


ATOM
600
N
GLN E 65
31.338
39.194
42.617
1.00
0.02


ATOM
602
CA
GLN E 65
32.692
38.672
42.490
1.00
0.02


ATOM
603
CB
GLN E 65
33.618
39.866
42.326
1.00
0.16


ATOM
604
CG
GLN E 65
33.284
40.551
41.008
1.00
0.35


ATOM
605
CD
GLN E 65
33.871
41.954
40.937
1.00
1.24


ATOM
606
OE1
GLN E 65
34.014
42.648
41.952
1.00
1.87


ATOM
607
NE2
GLN E 65
34.069
42.406
39.711
1.00
1.48


ATOM
610
C
GLN E 65
33.070
37.820
43.693
1.00
0.02


ATOM
611
O
GLN E 65
33.380
36.341
43.490
1.00
0.03


ATOM
612
N
GLU E 66
32.677
38.247
44.883
1.00
0.02


ATOM
614
CA
GLU E 66
32.994
37.463
46.085
1.00
0.02


ATOM
615
CB
GLU E 66
32.658
38.287
47.323
1.00
0.18


ATOM
616
CG
GLU E 66
33.752
39.500
47.461
1.00
0.66


ATOM
617
CD
GLU E 66
35.027
39.065
47.607
1.00
0.69


ATOM
618
OE1
GLU E 66
35.402
38.713
48.717
1.00
1.39


ATOM
619
OE2
GLU E 66
35.748
39.120
46.620
1.00
1.13


ATOM
620
C
GLU E 66
32.250
36.128
46.153
1.00
0.03


ATOM
621
O
GLU E 66
32.900
35.101
46.392
1.00
0.02


ATOM
622
N
ILE E 67
31.005
36.092
45.703
1.00
0.03


ATOM
624
CA
ILE E 67
30.250
34.835
45.738
1.00
0.02


ATOM
625
CB
ILE E 67
28.765
35.161
45.611
1.00
0.02


ATOM
626
CG2
ILE E 67
27.932
33.887
45.544
1.00
0.02


ATOM
627
CG1
ILE E 67
28.292
36.031
46.769
1.00
0.03


ATOM
628
CD1
ILE E 67
26.855
36.495
46.557
1.00
0.03


ATOM
629
C
ILE E 67
30.675
33.895
44.611
1.00
0.01


ATOM
630
O
ILE E 67
30.878
32.698
44.865
1.00
0.02


ATOM
631
N
TRP E 68
31.095
34.464
43.492
1.00
0.02


ATOM
633
CA
TRP E 68
31.540
33.646
42.365
1.00
0.02


ATOM
634
CB
TRP E 68
31.590
34.524
41.120
1.00
0.15


ATOM
635
CG
TRP E 68
31.962
33.799
39.841
1.00
0.21


ATOM
636
CD1
TRP E 68
31.104
33.158
38.974
1.00
0.38


ATOM
637
NE1
TRP E 68
31.831
32.648
37.948
1.00
0.44


ATOM
639
CE2
TRP E 68
33.141
32.917
38.100
1.00
0.42


ATOM
640
CZ2
TRP E 68
34.257
32.609
37.337
1.00
0.60


ATOM
641
CH2
TRP E 68
35.513
33.033
37.751
1.00
0.73


ATOM
642
CZ3
TRP E 68
35.656
33.766
38.926
1.00
0.72


ATOM
643
CE3
TRP E 68
34.542
34.078
39.695
1.00
0.50


ATOM
644
CD2
TRP E 68
33.288
33.654
39.287
1.00
0.33


ATOM
645
C
TRP E 68
32.915
33.051
42.642
1.00
0.03


ATOM
646
O
TRP E 68
33.085
31.838
42.482
1.00
0.02


ATOM
647
N
GLN E 69
33.754
33.797
43.338
1.00
0.02


ATOM
649
CA
GLN E 69
35.089
33.303
43.686
1.00
0.03


ATOM
650
CB
GLN E 69
35.948
34.509
44.045
1.00
0.34


ATOM
651
CG
GLN E 69
36.126
35.457
42.865
1.00
1.17


ATOM
652
CD
GLN E 69
36.423
36.855
43.398
1.00
2.07


ATOM
653
OE1
GLN E 69
36.271
37.860
42.691
1.00
2.43


ATOM
654
NE2
GLN E 69
36.740
36.904
44.680
1.00
3.04


ATOM
657
C
GLN E 69
35.073
32.347
44.878
1.00
0.03


ATOM
658
O
GLN E 69
36.022
31.573
45.050
1.00
0.02


ATOM
659
N
GLU E 70
33.990
32.330
45.635
1.00
0.02


ATOM
661
CA
GLU E 70
33.925
31.429
46.779
1.00
0.03


ATOM
662
CB
GLU E 70
33.224
32.158
47.917
1.00
0.03


ATOM
663
CG
GLU E 70
33.151
31.305
49.177
1.00
0.03


ATOM
664
CD
GLU E 70
32.441
32.082
50.280
1.00
0.03


ATOM
665
OE1
GLU E 70
31.731
33.020
49.942
1.00
0.02


ATOM
666
OE2
GLU E 70
32.647
31.748
51.438
1.00
0.01


ATOM
667
C
GLU E 70
33.185
30.136
46.458
1.00
0.03


ATOM
668
O
GLU E 70
33.656
29.063
46.855
1.00
0.02


ATOM
669
N
PHE E 71
32.125
30.217
45.670
1.00
0.02


ATOM
671
CA
PHE E 71
31.300
29.030
45.419
1.00
0.02


ATOM
672
CB
PHE E 71
29.853
29.356
45.795
1.00
0.03


ATOM
673
CG
PHE E 71
29.584
29.788
47.236
1.00
0.02


ATOM
674
CD1
PHE E 71
29.568
28.846
48.256
1.00
0.01


ATOM
675
CE1
PHE E 71
29.315
29.240
49.564
1.00
0.02


ATOM
676
CZ
PHE E 71
29.067
30.575
49.851
1.00
0.02


ATOM
677
CE2
PHE E 71
29.068
31.516
48.829
1.00
0.03


ATOM
678
CD2
PHE E 71
29.323
31.121
47.522
1.00
0.02


ATOM
679
C
PHE E 71
31.280
28.540
43.968
1.00
0.01


ATOM
680
O
PHE E 71
30.867
27.399
43.736
1.00
0.01


ATOM
681
N
LEU E 72
31.670
29.355
43.003
1.00
0.01


ATOM
683
CA
LEU E 72
31.439
28.959
41.604
1.00
0.02


ATOM
684
CB
LEU E 72
30.612
30.026
40.886
1.00
0.77


ATOM
685
CG
LEU E 72
29.141
30.081
41.296
1.00
0.45


ATOM
686
CD1
LEU E 72
28.897
31.051
42.477
1.00
0.32


ATOM
687
CD2
LEU E 72
28.306
30.523
40.104
1.00
1.01


ATOM
688
C
LEU E 72
32.714
27.897
39.875
1.00
0.03


ATOM
689
O
LEU E 72
32.713
27.897
39.875
1.00
0.03


ATOM
690
N
ALA E 73
33.774
29.428
41.145
1.00
0.02


ATOM
692
CA
ALA E 73
35.068
29.291
40.463
1.00
0.02


ATOM
693
CB
ALA E 73
36.066
30.197
41.182
1.00
0.73


ATOM
694
C
ALA E 73
35.560
27.844
40.483
1.00
0.02


ATOM
695
O
ALA E 73
35.273
27.107
41.431
1.00
0.02


ATOM
696
N
PRO E 74
36.368
27.468
39.500
1.00
0.02


ATOM
697
CA
PRO E 74
36.809
26.066
39.356
1.00
0.02


ATOM
698
CB
PRO E 74
37.439
25.998
37.998
1.00
0.48


ATOM
699
CG
PRO E 74
37.516
27.396
37.402
1.00
0.55


ATOM
700
CD
PRO E 74
36.839
28.318
38.401
1.00
0.24


ATOM
701
C
PRO E 74
37.801
25.580
40.430
1.00
0.02


ATOM
702
O
PRO E 74
38.112
24.386
40.483
1.00
0.02


ATOM
703
N
GLY E 75
38.261
26.481
41.283
1.00
0.02


ATOM
705
CA
GLY E 75
39.087
26.112
42.433
1.00
0.03


ATOM
706
C
GLY E 75
38.586
26.858
43.669
1.00
0.02


ATOM
707
O
GLY E 75
39.367
27.221
44.555
1.00
0.02


ATOM
708
N
ALA E 76
37.284
27.098
43.699
1.00
0.03


ATOM
710
CA
ALA E 76
36.663
27.856
44.788
1.00
0.02


ATOM
711
CB
ALA E 76
35.236
28.181
44.370
1.00
0.02


ATOM
712
C
ALA E 76
36.649
27.063
46.090
1.00
0.03


ATOM
713
O
ALA E 76
36.309
25.874
46.102
1.00
0.03


ATOM
714
N
PRO E 77
36.915
27.756
47.188
1.00
0.02


ATOM
715
CA
PRO E 77
37.094
27.100
48.494
1.00
0.03


ATOM
716
CB
PRO E 77
37.692
28.156
49.373
1.00
0.15


ATOM
717
CG
PRO E 77
37.700
29.487
48.636
1.00
0.11


ATOM
718
CD
PRO E 77
37.188
29.196
47.237
1.00
0.08


ATOM
719
C
PRO E 77
35.796
26.574
49.118
1.00
0.03


ATOM
720
O
PRO E 77
35.853
25.798
50.078
1.00
0.02


ATOM
721
N
SER E 78
34.665
26.989
48.577
1.00
0.02


ATOM
723
CA
SER E 78
33.367
26.486
49.009
1.00
0.02


ATOM
724
CB
SER E 78
32.678
27.572
49.823
1.00
0.15


ATOM
725
OG
SER E 78
33.556
27.947
50.875
1.00
0.16


ATOM
726
C
SER E 78
32.530
26.153
47.781
1.00
0.02


ATOM
727
O
SER E 78
31.328
26.440
47.756
1.00
0.01


ATOM
728
N
ALA E 79
33.173
25.586
46.781
1.00
0.03


ATOM
730
CA
ALA E 79
32.511
25.259
45.507
1.00
0.02


ATOM
731
CB
ALA E 79
33.505
24.543
44.598
1.00
1.29


ATOM
732
C
ALA E 79
31.251
24.409
45.664
1.00
0.02


ATOM
733
O
ALA E 79
31.215
23.417
46.404
1.00
0.02


ATOM
734
N
ILE E 80
30.202
24.881
45.015
1.00
0.01


ATOM
736
CA
ILE E 80
28.907
24.200
45.002
1.00
0.01


ATOM
737
CB
ILE E 80
27.816
25.259
44.892
1.00
1.88


ATOM
738
CG2
ILE E 80
27.805
26.153
46.125
1.00
2.48


ATOM
739
CG1
ILE E 80
27.998
26.091
43.629
1.00
2.64


ATOM
740
CD1
ILE E 80
27.007
27.245
43.575
1.00
3.61


ATOM
741
C
ILE E 80
28.826
23.240
43.821
1.00
0.01


ATOM
742
O
ILE E 80
29.706
23.225
42.949
1.00
0.02


ATOM
743
N
ASN E 81
27.818
22.388
43.828
1.00
0.01


ATOM
745
CA
ASN E 81
27.662
21.463
42.701
1.00
0.02


ATOM
746
CB
ASN E 81
26.991
20.181
43.174
1.00
0.02


ATOM
747
CG
ASN E 81
26.761
19.252
41.986
1.00
0.02


ATOM
748
OD1
ASN E 81
25.617
19.007
41.586
1.00
0.02


ATOM
749
ND2
ASN E 81
27.851
18.756
41.428
1.00
0.01


ATOM
752
C
ASN E 81
26.857
22.101
41.571
1.00
0.02


ATOM
753
O
ASN E 81
25.630
22.231
41.645
1.00
0.01


ATOM
754
N
LEU E 82
27.577
22.566
40.564
1.00
0.02


ATOM
756
CA
LEU E 82
26.935
23.140
39.375
1.00
0.02


ATOM
757
CB
LEU E 82
27.435
24.558
39.135
1.00
0.02


ATOM
758
CG
LEU E 82
26.874
25.557
40.133
1.00
0.02


ATOM
759
CD1
LEU E 82
27.439
26.942
39.849
1.00
0.02


ATOM
760
CD2
LEU E 82
25.351
25.582
40.080
1.00
0.01


ATOM
761
C
LEU E 82
27.229
22.341
38.117
1.00
0.02


ATOM
762
O
LEU E 82
28.259
21.669
37.994
1.00
0.02


ATOM
763
N
ASP E 83
26.282
22.394
37.199
1.00
0.02


ATOM
765
CA
ASP E 83
26.538
21.915
35.840
1.00
0.02


ATOM
766
CB
ASP E 83
25.229
21.487
35.175
1.00
0.01


ATOM
767
CG
ASP E 83
24.286
22.671
34.983
1.00
0.01


ATOM
768
OD1
ASP E 83
23.156
22.953
35.886
1.00
0.02


ATOM
769
OD2
ASP E 83
24.464
23.361
33.987
1.00
0.01


ATOM
770
C
ASP E 83
27.226
23.044
35.071
1.00
0.02


ATOM
771
O
ASP E 83
26.974
24.226
35.344
1.00
0.02


ATOM
772
N
SER E 84
27.975
22.698
34.037
1.00
0.02


ATOM
774
CA
SER E 84
28.790
23.709
33.348
1.00
0.01


ATOM
775
CB
SER E 84
29.778
23.007
32.426
1.00
0.02


ATOM
776
OG
SER E 84
30.510
24.020
31.745
1.00
0.01


ATOM
777
C
SER E 84
27.998
24.724
32.525
1.00
0.02


ATOM
778
O
SER E 84
28.410
25.887
32.849
1.00
0.02


ATOM
779
N
LYS E 85
26.773
24.407
32.140
1.00
0.02


ATOM
781
CA
LYS E 85
25.987
25.367
31.359
1.00
0.02


ATOM
782
CB
LYS E 85
24.804
24.633
30.745
1.00
0.11


ATOM
783
CG
LYS E 85
25.278
23.497
29.849
1.00
1.06


ATOM
784
CD
LYS E 85
24.100
22.757
29.230
1.00
0.76


ATOM
785
CE
LYS E 85
24.571
21.631
28.318
1.00
1.78


ATOM
786
NZ
LYS E 85
23.426
20.928
27.716
1.00
2.43


ATOM
787
C
LYS E 85
25.486
26.513
32.232
1.00
0.02


ATOM
788
O
LYS E 85
25.726
27.679
31.895
1.00
0.01


ATOM
789
N
SER E 86
25.111
26.186
33.459
1.00
0.02


ATOM
791
CA
SER E 86
24.650
27.210
34.401
1.00
0.02


ATOM
792
CB
SER E 86
23.821
26.555
35.505
1.00
0.23


ATOM
793
OG
SER E 86
24.640
25.631
36.213
1.00
0.75


ATOM
794
C
SER E 86
25.824
27.970
35.012
1.00
0.02


ATOM
795
O
SER E 86
25.709
29.182
35.237
1.00
0.03


ATOM
796
N
TYR E 87
26.991
27.342
35.024
1.00
0.02


ATOM
798
CA
TYR E 87
28.194
28.031
35.482
1.00
0.01


ATOM
799
CB
TYR E 87
29.286
27.004
35.763
1.00
0.14


ATOM
800
CG
TYR E 87
30.653
27.636
36.010
1.00
0.20


ATOM
801
CD1
TYR E 87
30.800
28.618
36.982
1.00
0.30


ATOM
802
CE1
TYR E 87
32.039
29.205
37.192
1.00
0.43


ATOM
803
CZ
TYR E 87
33.130
28.806
36.433
1.00
0.48


ATOM
804
OH
TYR E 87
34.352
29.405
36.631
1.00
0.66


ATOM
805
CE2
TYR E 87
32.989
27.820
35.465
1.00
0.48


ATOM
806
CD2
TYR E 87
31.748
27.235
35.254
1.00
0.36


ATOM
807
C
TYR E 87
28.689
29.014
34.428
1.00
0.02


ATOM
808
O
TYR E 87
29.903
30.184
34.758
1.00
0.02


ATOM
809
N
ASP E 88
29.595
28.637
33.163
1.00
0.02


ATOM
811
CA
ASP E 88
29.072
29.505
32.081
1.00
0.02


ATOM
812
CB
ASP E 88
29.252
28.671
30.813
1.00
0.06


ATOM
813
CG
ASP E 88
30.281
27.553
31.002
1.00
0.57


ATOM
814
OD1
ASP E 88
31.201
27.741
31.787
1.00
0.74


ATOM
815
OD2
ASP E 88
30.184
26.569
30.278
1.00
0.97


ATOM
816
C
ASP E 88
28.087
30.637
31.801
1.00
0.02


ATOM
817
O
ASP E 88
28.502
31.754
31.461
1.00
0.02


ATOM
818
N
LYS E 89
26.824
30.413
32.125
1.00
0.02


ATOM
820
CA
LYS E 89
25.829
31.477
32.003
1.00
0.03


ATOM
821
CB
LYS E 89
24.438
30.866
32.105
1.00
0.16


ATOM
822
CG
LYS E 89
23.360
31.939
32.022
1.00
0.84


ATOM
823
CD
LYS E 89
23.442
32.716
30.713
1.00
1.52


ATOM
824
CE
LYS E 89
22.416
33.842
30.677
1.00
2.77


ATOM
825
NZ
LYS E 89
21.054
33.314
30.852
1.00
3.78


ATOM
826
C
LYS E 89
26.022
32.506
33.109
1.00
0.02


ATOM
827
O
LYS E 89
26.221
33.688
32.802
1.00
0.03


ATOM
828
N
THR E 90
26.329
32.018
34.299
1.00
0.02


ATOM
830
CA
THR E 90
26.550
32.907
35.437
1.00
0.02


ATOM
831
CB
THR E 90
26.421
32.072
36.702
1.00
0.01


ATOM
832
OG1
THR E 90
25.060
31.668
36.801
1.00
0.02


ATOM
833
CG2
THR E 90
26.762
32.881
37.943
1.00
0.02


ATOM
834
C
THR E 90
27.909
33.607
35.382
1.00
0.02


ATOM
835
O
THR E 90
28.000
34.763
35.803
1.00
0.02


ATOM
836
N
THR E 91
28.831
33.058
34.615
1.00
0.02


ATOM
838
CA
THR E 91
30.133
33.712
34.406
1.00
0.03


ATOM
839
CB
THR E 91
31.114
32.674
33.870
1.00
0.15


ATOM
840
OG1
THR E 91
31.260
31.662
34.858
1.00
0.15


ATOM
841
CG2
THR E 91
32.490
33.274
33.308
1.00
0.29


ATOM
842
C
THR E 91
30.016
34.881
33.426
1.00
0.02


ATOM
843
O
THR E 91
30.598
35.942
33.684
1.00
0.02


ATOM
844
N
GLN E 92
29.057
34.799
32.515
1.00
0.02


ATOM
846
CA
GLN E 92
28.788
35.927
31.615
1.00
0.02


ATOM
847
CB
GLN E 92
28.058
35.410
30.384
1.00
0.15


ATOM
848
CG
GLN E 92
28.953
34.497
29.557
1.00
0.34


ATOM
849
CD
GLN E 92
28.135
33.852
28.444
1.00
0.70


ATOM
850
OE1
GLN E 92
28.041
34.367
27.325
1.00
1.06


ATOM
851
NE2
GLN E 92
27.561
32.710
28.774
1.00
0.87


ATOM
854
C
GLN E 92
27.931
36.975
32.317
1.00
0.02


ATOM
855
O
GLN E 92
28.160
38.179
32.140
1.00
0.02


ATOM
856
N
ASN E 93
27.205
36.528
33.328
1.00
0.02


ATOM
858
CA
ASN E 93
26.441
37.441
34.180
1.00
0.02


ATOM
859
CB
ASN E 93
25.369
36.638
34.902
1.00
0.02


ATOM
860
CG
ASN E 93
24.353
35.981
33.970
1.00
0.03


ATOM
861
OD1
ASN E 93
24.181
36.374
32.809
1.00
0.03


ATOM
862
ND2
ASN E 93
23.628
35.031
34.536
1.00
0.02


ATOM
865
C
ASN E 93
27.327
38.137
35.224
1.00
0.02


ATOM
866
O
ASN E 93
26.949
39.194
35.741
1.00
0.02


ATOM
867
N
VAL E 94
28.551
37.658
35.402
1.00
0.03


ATOM
869
CA
VAL E 94
29.533
38.339
36.260
1.00
0.02


ATOM
870
CB
VAL E 94
30.554
37.311
36.758
1.00
0.29


ATOM
871
CG1
VAL E 94
31.722
37.950
37.503
1.00
0.50


ATOM
872
CG2
VAL E 94
29.896
36.264
37.644
1.00
0.38


ATOM
873
C
VAL E 94
30.240
39.457
35.490
1.00
0.03


ATOM
874
O
VAL E 94
30.717
40.425
36.096
1.00
0.03


ATOM
875
N
LYS E 95
30.099
39.434
34.172
1.00
0.02


ATOM
877
CA
LYS E 95
30.636
40.511
33.333
1.00
0.02


ATOM
878
CB
LYS E 95
30.840
39.966
31.923
1.00
0.23


ATOM
879
CG
LYS E 95
31.510
40.984
31.010
1.00
0.39


ATOM
880
CD
LYS E 95
31.673
40.439
29.597
1.00
0.34


ATOM
881
CE
LYS E 95
32.344
41.470
28.699
1.00
1.25


ATOM
882
NZ
LYS E 95
31.552
42.710
28.465
1.00
1.85


ATOM
883
C
LYS E 95
29.666
41.696
33.316
1.00
0.02


ATOM
884
O
LYS E 95
30.061
42.839
33.058
1.00
0.02


ATOM
885
N
GLU E 96
28.419
41.428
33.670
1.00
0.02


ATOM
887
CA
GLU E 96
27.474
42.514
33.955
1.00
0.02


ATOM
888
CB
GLU E 96
26.512
42.673
32.781
1.00
0.03


ATOM
889
CG
GLU E 96
25.654
43.930
32.910
1.00
0.03


ATOM
890
CD
GLU E 96
26.532
45.180
32.953
1.00
0.02


ATOM
891
OE1
GLU E 96
26.884
45.586
34.053
1.00
0.03


ATOM
892
OE2
GLU E 96
26.801
45.723
31.892
1.00
0.02


ATOM
893
C
GLU E 96
26.720
42.201
35.252
1.00
0.02


ATOM
894
O
GLU E 96
25.548
41.802
35.215
1.00
0.03


ATOM
895
N
PRO E 97
27.361
42.500
36.375
1.00
0.03


ATOM
896
CA
PRO E 97
26.998
41.884
37.659
1.00
0.03


ATOM
897
CB
PRO E 97
28.209
42.055
38.521
1.00
0.02


ATOM
898
CG
PRO E 97
29.203
42.967
37.825
1.00
0.03


ATOM
899
CD
PRO E 97
28.607
43.270
36.465
1.00
0.02


ATOM
900
C
PRO E 97
25.778
42.514
38.329
1.00
0.02


ATOM
901
O
PRO E 97
25.874
43.555
38.991
1.00
0.02


ATOM
902
N
GLY E 98
24.638
41.870
38.143
1.00
0.02


ATOM
904
CA
GLY E 98
23.403
42.266
38.832
1.00
0.02


ATOM
905
C
GLY E 98
22.873
41.125
39.699
1.00
0.03


ATOM
906
O
GLY E 98
23.540
40.097
39.870
1.00
0.02


ATOM
907
N
ARG E 99
21.613
41.240
40.094
1.00
0.02


ATOM
909
CA
ARG E 99
20.981
40.222
40.954
1.00
0.03


ATOM
910
CB
ARG E 99
19.658
40.783
41.456
1.00
1.36


ATOM
911
CG
ARG E 99
19.835
42.034
42.300
1.00
1.75


ATOM
912
CD
ARG E 99
18.474
42.610
42.673
1.00
2.08


ATOM
913
NE
ARG E 99
18.609
43.744
43.597
1.00
2.09


ATOM
914
CZ
ARG E 99
17.991
44.912
43.418
1.00
2.46


ATOM
915
NH1
ARG E 99
18.128
45.884
44.323
1.00
3.38


ATOM
916
NH2
ARG E 99
17.219
45.101
42.347
1.00
2.32


ATOM
917
C
ARG E 99
20.669
38.928
40.205
1.00
0.03


ATOM
918
O
ARG E 99
20.750
37.837
40.784
1.00
0.03


ATOM
919
N
TYR E 100
20.580
39.039
38.889
1.00
0.02


ATOM
921
CA
TYR E 100
20.263
37.895
38.029
1.00
0.02


ATOM
922
CB
TYR E 100
19.664
38.436
36.733
1.00
0.10


ATOM
923
CG
TYR E 100
18.488
39.391
36.930
1.00
0.73


ATOM
924
CD1
TYR E 100
17.362
38.984
37.636
1.00
1.16


ATOM
925
CE1
TYR E 100
16.299
39.858
37.815
1.00
1.80


ATOM
926
CZ
TYR E 100
16.362
41.139
37.282
1.00
2.07


ATOM
927
OH
TYR E 100
15.350
42.005
37.455
1.00
2.76


ATOM
928
CE2
TYR E 100
17.482
41.548
36.572
1.00
1.87


ATOM
929
CD2
TYR E 100
18.545
40.672
36.394
1.00
1.24


ATOM
930
C
TYR E 100
21.491
37.043
37.699
1.00
0.01


ATOM
931
O
TYR E 100
21.332
35.931
37.176
1.00
0.02


ATOM
932
N
THR E 101
22.655
37.449
38.189
1.00
0.03


ATOM
934
CA
THR E 101
23.903
36.747
37.883
1.00
0.02


ATOM
935
CB
THR E 101
25.056
37.469
38.572
1.00
0.36


ATOM
936
OG1
THR E 101
25.217
38.728
37.938
1.00
0.41


ATOM
937
CG2
THR E 101
26.366
36.708
38.421
1.00
0.26


ATOM
938
C
THR E 101
23.873
35.307
38.359
1.00
0.03


ATOM
939
O
THR E 101
23.917
34.376
37.540
1.00
0.03


ATOM
940
N
PHE E 102
23.497
35.151
39.615
1.00
0.03


ATOM
942
CA
PHE E 102
23.476
33.833
40.234
1.00
0.01


ATOM
943
CB
PHE E 102
23.924
33.964
41.679
1.00
0.02


ATOM
944
CG
PHE E 102
25.347
34.484
41.815
1.00
0.03


ATOM
945
CD1
PHE E 102
26.396
33.780
41.242
1.00
0.02


ATOM
946
CE1
PHE E 102
27.694
34.259
41.349
1.00
0.03


ATOM
947
CZ
PHE E 102
27.940
35.438
42.036
1.00
0.01


ATOM
948
CE2
PHE E 102
26.892
36.136
42.620
1.00
0.02


ATOM
949
CD2
PHE E 102
25.595
35.658
42.511
1.00
0.02


ATOM
950
C
PHE E 102
22.109
33.175
40.185
1.00
0.02


ATOM
951
O
PHE E 102
21.989
32.052
40.680
1.00
0.02


ATOM
952
N
GLU E 103
21.172
33.727
39.431
1.00
0.02


ATOM
954
CA
GLU E 103
19.800
33.208
39.450
1.00
0.02


ATOM
955
CB
GLU E 103
18.934
34.193
38.675
1.00
0.44


ATOM
956
CG
GLU E 103
17.455
33.837
38.726
1.00
0.96


ATOM
957
CD
GLU E 103
16.668
34.859
37.915
1.00
1.34


ATOM
958
OE1
GLU E 103
17.257
35.881
37.590
1.00
1.84


ATOM
959
OE2
GLU E 103
15.507
34.607
37.625
1.00
1.75


ATOM
960
C
GLU E 103
19.705
31.819
38.817
1.00
0.02


ATOM
961
O
GLU E 103
19.091
30.921
39.409
1.00
0.02


ATOM
962
N
ASP E 104
20.561
31.570
37.837
1.00
0.02


ATOM
964
CA
ASP E 104
20.588
30.259
37.181
1.00
0.02


ATOM
965
CB
ASP E 104
21.454
30.362
35.927
1.00
0.28


ATOM
966
CG
ASP E 104
20.989
31.517
35.045
1.00
1.07


ATOM
967
OD1
ASP E 104
19.856
31.462
34.591
1.00
1.74


ATOM
968
OD2
ASP E 104
21.722
32.493
34.961
1.00
1.66


ATOM
969
C
ASP E 104
21.192
29.202
38.103
1.00
0.02


ATOM
970
O
ASP E 104
20.547
28.183
38.387
1.00
0.02


ATOM
971
N
ALA E 105
22.245
29.597
38.800
1.00
0.02


ATOM
973
CA
ALA E 105
22.960
28.661
39.664
1.00
0.02


ATOM
974
CB
ALA E 105
24.355
29.222
39.909
1.00
0.02


ATOM
975
C
ALA E 105
22.251
28.438
40.998
1.00
0.02


ATOM
976
O
ALA E 105
22.234
27.303
41.486
1.00
0.01


ATOM
977
N
GLN E 106
21.460
29.408
41.425
1.00
0.02


ATOM
979
CA
GLN E 106
20.726
29.289
42.684
1.00
0.02


ATOM
980
CB
GLN E 106
20.308
30.684
43.132
1.00
0.02


ATOM
981
CG
GLN E 106
19.621
30.648
44.491
1.00
0.03


ATOM
982
CD
GLN E 106
18.959
31.991
44.765
1.00
0.02


ATOM
983
OE1
GLN E 106
18.592
32.712
43.829
1.00
0.03


ATOM
984
NE2
GLN E 106
18.853
32.330
46.037
1.00
0.02


ATOM
987
C
GLN E 106
19.482
28.431
42.515
1.00
0.02


ATOM
988
O
GLN E 106
19.155
27.649
43.415
1.00
0.02


ATOM
989
N
GLU E 107
18.959
28.381
41.301
1.00
0.01


ATOM
991
CA
GLU E 107
17.819
27.505
41.035
1.00
0.02


ATOM
992
CB
GLU E 107
17.128
27.991
39.770
1.00
0.15


ATOM
993
CG
GLU E 107
16.478
29.347
40.011
1.00
0.61


ATOM
994
CD
GLU E 107
16.077
29.977
38.683
1.00
1.24


ATOM
995
OE1
GLU E 107
16.148
29.281
37.681
1.00
1.81


ATOM
996
OE2
GLU E 107
15.868
31.183
38.673
1.00
1.66


ATOM
997
C
GLU E 107
18.273
26.060
40.870
1.00
0.02


ATOM
998
O
GLU E 107
17.636
25.156
41.428
1.00
0.01


ATOM
999
N
HIS E 108
19.495
25.886
40.392
1.00
0.02


ATOM
1001
CA
HIS E 108
20.055
24.540
40.285
1.00
0.02


ATOM
1002
CB
HIS E 108
21.246
24.581
39.335
1.00
0.16


ATOM
1003
CG
HIS E 108
21.925
23.238
39.160
1.00
1.11


ATOM
1004
ND1
HIS E 108
21.467
22.199
38.438
1.00
1.67


ATOM
1006
CE1
HIS E 108
22.342
21.176
38.524
1.00
2.49


ATOM
1007
NE2
HIS E 108
23.362
21.575
39.317
1.00
2.85


ATOM
1008
CD2
HIS E 108
23.116
22.843
39.719
1.00
2.23


ATOM
1009
C
HIS E 108
20.499
24.020
41.651
1.00
0.02


ATOM
1010
O
HIS E 108
20.213
22.863
41.979
1.00
0.02


ATOM
1011
N
ILE E 109
20.925
24.923
42.519
1.00
0.02


ATOM
1013
CA
ILE E 109
21.329
24.538
43.875
1.00
0.02


ATOM
1014
CB
ILE E 109
22.237
25.635
44.417
1.00
0.02


ATOM
1015
CG2
ILE E 109
22.376
25.554
45.930
1.00
0.03


ATOM
1016
CG1
ILE E 109
23.603
25.568
43.746
1.00
0.02


ATOM
1017
CD1
ILE E 109
24.260
24.211
43.966
1.00
0.02


ATOM
1018
C
ILE E 109
20.137
24.312
44.805
1.00
0.02


ATOM
1019
O
ILE E 109
20.169
23.365
45.603
1.00
0.01


ATOM
1020
N
TYR E 110
19.018
24.953
44.509
1.00
0.02


ATOM
1022
CA
TYR E 110
17.798
24.712
45.284
1.00
0.01


ATOM
1023
CB
TYR E 110
16.802
25.829
44.983
1.00
0.33


ATOM
1024
CG
TYR E 110
15.497
25.761
45.776
1.00
0.50


ATOM
1025
CD1
TYR E 110
15.410
26.379
47.017
1.00
1.45


ATOM
1026
CE1
TYR E 110
14.225
26.320
47.742
1.00
2.06


ATOM
1027
CZ
TYR E 110
13.131
25.643
47.220
1.00
1.75


ATOM
1028
OH
TYR E 110
11.970
25.541
47.956
1.00
2.40


ATOM
1029
CE2
TYR E 110
13.211
25.034
45.975
1.00
1.09


ATOM
1030
CD2
TYR E 110
14.394
25.096
45.251
1.00
0.69


ATOM
1031
C
TYR E 110
17.201
23.361
44.906
1.00
0.02


ATOM
1032
O
TYR E 110
16.864
22.570
45.797
1.00
0.02


ATOM
1033
N
LYS E 111
17.362
22.999
43.644
1.00
0.03


ATOM
1035
CA
LYS E 111
16.900
21.696
43.163
1.00
0.01


ATOM
1036
CB
LYS E 111
16.909
21.754
41.642
1.00
0.14


ATOM
1037
CG
LYS E 111
16.381
20.475
41.008
1.00
1.13


ATOM
1038
CD
LYS E 111
16.475
20.567
39.490
1.00
1.61


ATOM
1039
CE
LYS E 111
15.756
21.809
38.974
1.00
2.77


ATOM
1040
NZ
LYS E 111
15.884
21.926
37.513
1.00
3.61


ATOM
1041
C
LYS E 111
17.820
20.572
43.637
1.00
0.01


ATOM
1042
O
LYS E 111
17.332
19.522
44.075
1.00
0.01


ATOM
1043
N
LEU E 112
19.092
20.899
43.802
1.00
0.02


ATOM
1045
CA
LEU E 112
20.076
19.944
44.314
1.00
0.02


ATOM
1046
CB
LEU E 112
21.454
20.574
44.158
1.00
0.02


ATOM
1047
CG
LEU E 112
22.555
19.675
44.704
1.00
0.01


ATOM
1048
CD1
LEU E 112
22.702
18.417
43.854
1.00
0.02


ATOM
1049
CD2
LEU E 112
23.873
20.432
44.769
1.00
0.02


ATOM
1050
C
LEU E 112
19.842
19.631
45.789
1.00
0.01


ATOM
1051
O
LEU E 112
19.763
18.450
46.155
1.00
0.02


ATOM
1052
N
MET E 113
19.497
20.643
46.568
1.00
0.02


ATOM
1054
CA
MET E 113
19.233
20.407
47.986
1.00
0.02


ATOM
1055
CB
MET E 113
19.182
21.740
48.721
1.00
0.02


ATOM
1056
CG
MET E 113
20.521
22.461
48.673
1.00
0.01


ATOM
1057
SD
MET E 113
20.576
24.026
49.574
1.00
0.03


ATOM
1058
CE
MET E 113
19.254
24.908
48.714
1.00
0.02


ATOM
1059
C
MET E 113
17.912
19.677
48.185
1.00
0.02


ATOM
1060
O
MET E 113
17.906
18.650
48.878
1.00
0.02


ATOM
1061
N
LYS E 114
16.944
19.981
47.335
1.00
0.02


ATOM
1063
CA
LYS E 114
15.612
19.383
47.447
1.00
0.02


ATOM
1064
CB
LYS E 114
14.679
20.196
46.557
1.00
0.27


ATOM
1065
CG
LYS E 114
13.236
19.716
46.638
1.00
1.16


ATOM
1066
CD
LYS E 114
12.338
20.551
45.735
1.00
1.35


ATOM
1067
CE
LYS E 114
10.889
20.081
45.799
1.00
2.33


ATOM
1068
NZ
LYS E 114
10.035
20.894
44.919
1.00
2.92


ATOM
1069
C
LYS E 114
15.576
17.918
47.014
1.00
0.01


ATOM
1070
O
LYS E 114
14.912
17.106
47.668
1.00
0.01


ATOM
1071
N
SER E 115
16.413
17.553
46.058
1.00
0.02


ATOM
1073
CA
SER E 115
16.440
16.167
45.589
1.00
0.02


ATOM
1074
CB
SER E 115
16.758
16.199
44.097
1.00
0.32


ATOM
1075
OG
SER E 115
16.772
14.864
43.611
1.00
1.25


ATOM
1076
C
SER E 115
17.473
15.303
46.315
1.00
0.02


ATOM
1077
O
SER E 115
17.414
14.071
46.234
1.00
0.02


ATOM
1078
N
ASP E 116
18.366
15.924
47.065
1.00
0.02


ATOM
1080
CA
ASP E 116
19.412
15.150
47.736
1.00
0.02


ATOM
1081
CB
ASP E 116
20.756
15.772
47.372
1.00
0.02


ATOM
1082
CG
ASP E 116
21.912
15.035
48.040
1.00
0.02


ATOM
1083
OD1
ASP E 116
22.110
13.873
47.716
1.00
0.01


ATOM
1084
OD2
ASP E 116
22.647
15.688
48.768
1.00
0.02


ATOM
1085
C
ASP E 116
19.251
15.121
49.252
1.00
0.01


ATOM
1086
O
ASP E 116
18.941
14.074
49.833
1.00
0.02


ATOM
1087
N
SER E 117
19.365
16.283
49.871
1.00
0.02


ATOM
1089
CA
SER E 117
19.456
16.322
51.329
1.00
0.03


ATOM
1090
CB
SER E 117
20.447
17.408
51.724
1.00
0.01


ATOM
1091
OG
SER E 117
21.722
17.041
51.214
1.00
0.02


ATOM
1092
C
SER E 117
18.114
16.585
51.991
1.00
0.01


ATOM
1093
O
SER E 117
17.893
16.124
53.114
1.00
0.03


ATOM
1094
N
TYR E 118
17.172
17.131
51.243
1.00
0.02


ATOM
1096
CA
TYR E 118
15.842
17.411
51.800
1.00
0.01


ATOM
1097
CB
TYR E 118
15.001
18.135
50.760
1.00
0.02


ATOM
1098
CG
TYR E 118
13.647
18.589
51.283
1.00
0.02


ATOM
1099
CD1
TYR E 118
13.550
19.145
52.552
1.00
0.02


ATOM
1100
CE1
TYR E 118
12.317
19.560
53.034
1.00
0.02


ATOM
1101
CZ
TYR E 118
11.188
19.419
52.241
1.00
0.02


ATOM
1102
OH
TYR E 118
9.982
19.894
52.690
1.00
0.03


ATOM
1103
CE2
TYR E 118
11.280
18.865
50.972
1.00
0.02


ATOM
1104
CD2
TYR E 118
12.514
18.450
50.491
1.00
0.02


ATOM
1105
C
TYR E 118
15.096
16.175
52.343
1.00
0.03


ATOM
1106
O
TYR E 118
14.795
16.208
53.543
1.00
0.02


ATOM
1107
N
PRO E 119
14.913
15.074
51.611
1.00
0.02


ATOM
1108
CA
PRO E 119
14.197
13.943
52.224
1.00
0.01


ATOM
1109
CB
PRO E 119
13.942
12.982
51.104
1.00
0.20


ATOM
1110
CG
PRO E 119
14.649
13.461
49.848
1.00
0.33


ATOM
1111
CD
PRO E 119
15.280
14.792
50.210
1.00
0.16


ATOM
1112
C
PRO E 119
14.969
13.252
53.358
1.00
0.03


ATOM
1113
O
PRO E 119
14.342
12.828
54.338
1.00
0.03


ATOM
1114
N
ARG E 120
16.285
13.397
53.373
1.00
0.03


ATOM
1116
CA
ARG E 120
17.094
12.786
54.423
1.00
0.02


ATOM
1117
CB
ARG E 120
18.466
12.511
53.826
1.00
0.01


ATOM
1118
CG
ARG E 120
19.371
11.726
54.764
1.00
0.03


ATOM
1119
CD
ARG E 120
20.580
11.185
54.008
1.00
0.02


ATOM
1120
NE
ARG E 120
21.231
12.241
53.217
1.00
0.03


ATOM
1121
CZ
ARG E 120
21.394
12.159
51.893
1.00
0.02


ATOM
1122
NH1
ARG E 120
20.921
11.104
51.227
1.00
0.01


ATOM
1123
NH2
ARG E 120
21.997
13.148
51.231
1.00
0.02


ATOM
1124
C
ARG E 120
17.168
13.684
55.661
1.00
0.02


ATOM
1125
O
ARG E 120
17.311
13.188
56.784
1.00
0.02


ATOM
1126
N
PHE E 121
16.801
14.941
55.478
1.00
0.01


ATOM
1128
CA
PHE E 121
16.645
15.871
56.592
1.00
0.03


ATOM
1129
CB
PHE E 121
16.696
17.285
56.024
1.00
0.02


ATOM
1130
CG
PHE E 121
16.430
18.400
57.028
1.00
0.02


ATOM
1131
CD1
PHE E 121
17.205
18.508
58.175
1.00
0.02


ATOM
1132
CE1
PHE E 121
16.959
19.529
59.083
1.00
0.02


ATOM
1133
CZ
PHE E 121
15.941
20.444
58.843
1.00
0.02


ATOM
1134
CE2
PHE E 121
15.170
20.339
57.693
1.00
0.01


ATOM
1135
CD2
PHE E 121
15.416
19.317
56.785
1.00
0.02


ATOM
1136
C
PHE E 121
15.304
15.647
57.275
1.00
0.02


ATOM
1137
O
PHE E 121
15.244
15.652
58.509
1.00
0.01


ATOM
1138
N
ILE E 122
14.344
15.157
56.507
1.00
0.03


ATOM
1140
CA
ILE E 122
13.019
14.848
57.051
1.00
0.01


ATOM
1141
CB
ILE E 122
12.045
14.755
55.879
1.00
0.15


ATOM
1142
CG2
ILE E 122
10.646
14.367
56.345
1.00
0.37


ATOM
1143
CG1
ILE E 122
11.994
16.072
55.116
1.00
0.15


ATOM
1144
CD1
ILE E 122
11.059
15.976
53.917
1.00
0.13


ATOM
1145
C
ILE E 122
13.024
13.535
57.836
1.00
0.02


ATOM
1146
O
ILE E 122
12.281
13.398
58.813
1.00
0.03


ATOM
1147
N
ARG E 123
13.956
12.649
57.522
1.00
0.02


ATOM
1149
CA
ARG E 123
14.077
11.417
58.307
1.00
0.03


ATOM
1150
CB
ARG E 123
14.377
10.248
57.374
1.00
0.24


ATOM
1151
CG
ARG E 123
15.721
10.392
56.676
1.00
1.09


ATOM
1152
CD
ARG E 123
15.921
9.312
55.623
1.00
1.06


ATOM
1153
NE
ARG E 123
14.879
9.405
54.589
1.00
0.78


ATOM
1154
CZ
ARG E 123
15.143
9.472
53.282
1.00
0.81


ATOM
1155
NH1
ARG E 123
14.143
9.554
52.403
1.00
1.03


ATOM
1156
NH2
ARG E 123
16.407
9.455
52.852
1.00
0.80


ATOM
1157
C
ARG E 123
15.144
11.526
59.401
1.00
0.02


ATOM
1158
O
ARG E 123
15.306
10.594
60.197
1.00
0.02


ATOM
1159
N
SER E 124
15.856
12.641
59.449
1.00
0.02


ATOM
1161
CA
SER E 124
16.876
12.825
60.485
1.00
0.02


ATOM
1162
CB
SER E 124
18.069
13.593
59.928
1.00
0.02


ATOM
1163
OG
SER E 124
17.662
14.931
59.685
1.00
0.02


ATOM
1164
C
SER E 124
16.306
13.578
61.676
1.00
0.02


ATOM
1165
O
SER E 124
15.393
14.405
61.540
1.00
0.01


ATOM
1166
N
SER E 125
17.014
13.470
62.788
1.00
0.01


ATOM
1168
CA
SER E 125
16.570
14.100
64.040
1.00
0.03


ATOM
1169
CB
SER E 125
17.365
13.514
65.202
1.00
0.48


ATOM
1170
OG
SER E 125
18.722
13.908
65.048
1.00
0.36


ATOM
1171
C
SER E 125
16.734
15.623
64.053
1.00
0.01


ATOM
1172
O
SER E 125
15.979
16.281
64.775
1.00
0.03


ATOM
1173
N
ALA E 126
17.438
16.171
63.070
1.00
0.01


ATOM
1175
CA
ALA E 126
17.621
17.623
62.970
1.00
0.01


ATOM
1176
CB
ALA E 126
18.790
17.904
62.034
1.00
0.20


ATOM
1177
C
ALA E 126
16.371
18.321
62.435
1.00
0.03


ATOM
1178
O
ALA E 126
16.245
19.546
62.544
1.00
0.02


ATOM
1179
N
TYR E 127
15.458
17.554
61.865
1.00
0.01


ATOM
1181
CA
TYR E 127
14.142
18.095
61.544
1.00
0.02


ATOM
1182
CB
TYR E 127
13.818
17.822
60.082
1.00
0.02


ATOM
1183
CG
TYR E 127
12.341
18.000
59.756
1.00
0.02


ATOM
1184
CD1
TYR E 127
11.782
19.270
59.742
1.00
0.02


ATOM
1185
CE1
TYR E 127
10.432
19.423
59.471
1.00
0.01


ATOM
1186
CZ
TYR E 127
9.642
18.309
59.218
1.00
0.02


ATOM
1187
OH
TYR E 127
8.292
18.467
58.989
1.00
0.02


ATOM
1188
CE2
TYR E 127
10.198
17.039
59.225
1.00
0.02


ATOM
1189
CD2
TYR E 127
11.550
16.885
59.497
1.00
0.01


ATOM
1190
C
TYR E 127
13.071
17.441
62.403
1.00
0.01


ATOM
1191
O
TYR E 127
12.254
18.133
63.029
1.00
0.01


ATOM
1192
N
GLN E 128
13.195
16.135
62.572
1.00
0.02


ATOM
1194
CA
GLN E 128
12.090
15.361
63.139
1.00
0.03


ATOM
1195
CB
GLN E 128
12.201
13.926
62.628
1.00
1.25


ATOM
1196
CG
GLN E 128
10.929
13.131
62.917
1.00
2.29


ATOM
1197
CD
GLN E 128
9.711
13.837
62.318
1.00
2.89


ATOM
1198
OE1
GLN E 128
9.656
14.096
61.109
1.00
2.82


ATOM
1199
NE2
GLN E 128
8.789
14.213
63.190
1.00
3.73


ATOM
1202
C
GLN E 128
12.004
15.392
64.666
1.00
0.02


ATOM
1203
O
GLN E 128
10.918
15.162
65.214
1.00
0.04


ATOM
1204
N
GLU E 129
13.054
15.841
65.335
1.00
0.04


ATOM
1206
CA
GLU E 129
12.976
16.024
66.787
1.00
0.02


ATOM
1207
CB
GLU E 129
14.374
15.857
67.377
1.00
1.65


ATOM
1208
CG
GLU E 129
14.352
15.596
68.879
1.00
2.63


ATOM
1209
CD
GLU E 129
13.674
14.257
69.154
1.00
3.67


ATOM
1210
OE1
GLU E 129
14.226
13.254
68.721
1.00
4.45


ATOM
1211
OE2
GLU E 129
12.617
14.257
69.767
1.00
3.84


ATOM
1212
C
GLU E 129
12.416
17.408
67.142
1.00
0.02


ATOM
1213
O
GLU E 129
12.213
17.705
68.324
1.00
0.02


ATOM
1214
N
LEU E 130
12.135
18.230
66.143
1.00
0.02


ATOM
1216
CA
LEU E 130
11.589
19.561
66.419
1.00
0.02


ATOM
1217
CB
LEU E 130
12.046
20.519
65.325
1.00
0.02


ATOM
1218
CG
LEU E 130
13.565
20.572
65.203
1.00
0.03


ATOM
1219
CD1
LEU E 130
13.967
21.353
63.961
1.00
0.04


ATOM
1220
CD2
LEU E 130
14.214
21.166
66.450
1.00
0.02


ATOM
1221
C
LEU E 130
10.064
19.513
66.457
1.00
0.02


ATOM
1222
O
LEU E 130
9.429
20.229
67.239
1.00
0.04


TER

## Claims

1. A method of screening to identify a candidate agent that agonizes interaction of a regulator of G protein signaling (RGS) polypeptide and a G protein subunit alpha q (Gαq) polypeptide, said method comprising:
(a) contacting a RGS polypeptide and a Gαq polypeptide with a candidate agent, wherein said RGS polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of human RGS2 (SEQ ID NO.: 26), human RGS6 (SEQ ID NO.:27), human RGS7 (SEQ ID NO.:7), human RGS9 (SEQ ID NO.:11), or human RGS11 (SEQ ID NO.:9), and, wherein said Gαq polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of human Gαq (SEQ ID NO.:13);
(b) detecting an amount of binding of said RGS polypeptide to said Gαq polypeptide in the presence of the candidate agent;
(c) detecting a reference amount of binding of said RGS polypeptide to said Gαq polypeptide in the absence of the candidate agent; and
(d) comparing the amount of binding of said RGS polypeptide to said Gαq polypeptide in the presence of the candidate agent to the reference amount of binding,

wherein an increase in the amount of binding detected in the presence of the candidate agent relative to the reference amount of binding indicates that the candidate agent binds to said RGS polypeptide or Gαq polypeptide, and agonizes interaction of RGS and Gαq, and wherein said candidate agent is not a guanine nucleotide.

2. The method of claim 1 wherein said contacting in (a) comprises administering said candidate agent to cultured host cells that have been genetically engineered to express said RGS polypeptide and said Gαq polypeptide.

3. The method of claim 1 wherein said contacting in (a) comprises administering said candidate agent to a cell-free system comprising purified RGS and Gαq polypeptides.

4. The method of claim 1 wherein the method is used in a high throughput format to screen test agents in a compound library.

5. The method of claim 1 further comprising: testing the candidate agent in an animal model defective for RGS or Gαq, and detecting phenotypic changes relative to control animals.

6. A method for identifying an agent that modulates urinary incontinence, said method comprising:
(a) contacting a RGS polypeptide and a Gαq polypeptide with a candidate agent, wherein said RGS polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of human RGS2 (SEQ ID NO.: 26), human RGS6 (SEQ ID NO.:27), human RGS7 (SEQ ID NO.:7), human RGS9 (SEQ ID NO.:11), or human RGS11 (SEQ ID NO.:9), and wherein said Gαq polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of human Gαq (SEQ ID NO.:13);
(b) detecting an amount of binding of said RGS polypeptide to said Gαq polypeptide in the presence of the candidate agent;
(c) detecting a reference amount of binding of said RGS polypeptide to said Gαq polypeptide in the absence of the candidate agent;
(d) comparing the amount of binding of said RGS polypeptide to said Gαq polypeptide in the presence of the candidate agent to the reference amount of binding,
wherein an increase in the amount of binding detected in the presence of the candidate agent relative to the reference amount of binding indicates that the candidate agent binds to said RGS polypeptide or αq polypeptide, and agonizes interaction of RGS and Gαq; and
(e) testing if the candidate agent that agonizes interaction of RGS and Gαq modulates urinary incontinence, and wherein said candidate agent is not a guanine nucleotide.