WO2012068053A1

WO2012068053A1 - Crystal structure of pseudomonas aeruginosa murg enzyme

Info

Publication number: WO2012068053A1
Application number: PCT/US2011/060705
Authority: WO
Inventors: Kieron Brown; Neesha Dedi; Sarah Vial; Graham Cheetham
Original assignee: Vertex Pharmaceuticals Incorporated
Priority date: 2010-11-16
Filing date: 2011-11-15
Publication date: 2012-05-24

Abstract

P. a. MurG crystals include a polypeptide comprising amino acids according to SEQ ID NO: 1. Methods of forming such P. a. MurG crystals employ crystallizable compositions. The crystallizable compositions include such a polypeptide, a buffer having a pKa of 6.5 to 8.0, and a polyethylene glycol in a concentration from 5% to 50% by volume. Data storage media comprise the structure coordinates of molecules and molecular complexes that comprise all or part of a binding pocket of P. a. MurG protein. Methods for designing, evaluating and identifying compounds, such as potential inhibitors of P. a. MurG protein or its homologues, which bind to the aforementioned binding pocket employ at least a part of the atom coordinates of the P.a. MurG crystals.

Description

CRYSTAL STRUCTURE OF PSEUDOMONAS AERUGINOSA MurG ENZYME

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S. C. § 119 to United

States Provisional Application No. 61/488,935, filed May 23, 2011, entitled "CRYSTAL STRUCTURE OF PSEUDOMONAS AERUGINOSA MURG ENZYME", and United States Provisional Application No. 61/414,164, filed November 16, 2010, entitled "CRYSTAL STRUCTURE OF PSEUDOMONAS AERUGINOSA MURG ENZYME", the entire contents of which is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention is in the field of structural biology, especially X- ray structural determination.

BACKGROUND OF THE INVENTION

[0003] Pseudomonas aeruginosa (P. a .) is a ubiquitous environmental gram- negative bacterium that is one of the top three causes of opportunistic human infections. P. a. is highly resistant to known antibiotics. One strategy to develop novel and potent antibacterial agents is to target enzymes involved in synthesis of bacterial membranes. The peptidoglycan layer surrounds and protects bacterial cells is essential for survival. Thus, enzymes involved in peptidoglycan biosynthesis are attractive targets for the design of new antibiotics. Many are integral membrane proteins or membrane-associated proteins and are thus difficult to express, purify, crystallize and also develop biochemical assays for. The P. a. enzyme MurG is a glycosyltransferase that is anchored to the cytoplasmic surface of the bacterial cell membrane. Functioning in close collaboration with MraY, it catalyses the rate-limiting step of bacterial peptidoglycan biosynthesis. Specifically, MurG catalyzes the transfer of N-acetyl glucosamine (GlcNAc) from UDP- GlcNAc to Lipid I. The GlcNAc-MurNAc product of the MurG reaction is the minimal subunit of the peptidoglycan polymer.

[0004] The search for new therapeutic agents can be greatly aided by better understanding of the structure of the enzymes.

 SUMMARY OF THE INVENTION

[ 0005] The present invention provides for the first time, crystallizable compositions, crystals, and the crystal structures of P.a. MurG protein and a P.a. MurG protein-inhibitor complex. The crystal structure of P.a. MurG potein has allowed the applicants to determine the key structural features of P.a. MurG protein, particularly the shape of its substrate and P.a. MurG protein-binding pockets.

[ 0006] Thus, in one aspect, the present invention provides molecules or molecular complexes comprising all or parts of the binding pockets, or homologues of the binding pockets that have similar three-dimensional shapes.

[ 0007 ] In another aspect, the present invention further provides crystals of P.a.

MurG protein complexed with a ligand, substrate or inhibitor, such as UDP-N- acetylglucosamine, and methods for producing these crystals.

[ 0008 ] In a further aspect, the present invention provides crystallizable compositions from which crystals of P.a. MurG protein, and complexes of P.a. MurG protein with a chemical entity, such as its inhibitor, ligand, or substrat, may be obtained.

[ 0009] In another aspect, the invention provides a data storage medium that comprises the structure coordinates of molecules and molecular complexes that comprise all or part of a binding pocket of P.a. MurG protein. Such storage medium encoded with these data when read and utilized by a computer programmed with appropriate software displays, on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex comprising such a binding pocket or similarly shaped homologous binding pocket.

[ 0010 ] In yet another aspect, the invention provides computational methods of using structure coordinates of the P.a. MurG protein and P.a. MurG protein complex to screen for and design compounds, including inhibitory compounds and antibodies that interact with P. a. MurG protein or homologues thereof. In certain embodiments, the invention provides methods for designing, evaluating and identifying compounds, which bind to the aforementioned binding pocket. In certain embodiments, such compounds are potential inhibitors of P.a. MurG protein or its homologues.

[ 0011 ] In a further aspect, the invention provides a method for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to P.a. MurG. In certain embodiments, this is achieved by using at least some of the structural coordinates obtained from the P.a. MurG protein and P. a. MurG protein complexed with UDP-N- acetylglucosamine.

BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 lists the atomic structure coordinates of X-ray structure of P.a.

MurG protein as derived by X-ray diffraction from its crystal. The P.a. MurG protein in the crystal comprises amino acid residues of SEQ ID NO : 1. Certain amino acid residues of SEQ ID NO: 1 are not shown in FIG.1 due to disorder in their atom coordinates.

[0013] FIG. 2 lists the atomic structure coordinates of X-ray structure of P.a.

MurG protein and UDP-N-acetylglucosamine complex as derived by X-ray diffraction from its crystal. The P.a. MurG protein in the crystal comprises amino acid residues of SEQ ID NO: 1. Certain amino acid residues of SEQ ID NO: 1 are not shown in FIG.2 due to disorder in their atom coordinates.

[0014 ] The following abbreviations are used in FIGs. 1 and 2:

[0015] "Atom type" refers to the identity of the element whose coordinates are measured. The first letter in the column defines the actual type of atom (i.e., C for carbon, N for nitrogen, O for oxygen, etc.).

[0016] "Resid" refers to the amino acid residue or molecule identity in the molecular model.

[0017] "#" refers to the amino acid residue number.

[0018] "X, Y, Z" crystallographically define the atomic position of the element measured.

[0019] "B" is a thermal factor that measures movement of the atom around its atomic center.

[0020] "Occ" is an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates. A value of "1" indicates that each atom has the same conformation, i.e., the same position, in all molecules of the crystal. [0021] FIG. 3 shows a diagram of a system used to carry out the instructions encoded by the storage medium of Figures 4 and 5.

[0022] FIG. 4 shows a cross section of a magnetic storage medium.

[0023] FIG. 5 shows a cross section of an optically-readable data storage medium.

[0024] FIG. 6 shows UDP-N-acetylglucosamine bound in the binding pocket of

P. a. MurG protein.

[0025] FIG. 7 shows enzymatic characterization of P. a. MurG. Change in fluorescence with time (slope) observed at various concentrations of enzyme.

[0026] FIG. 8 shows residues involved in binding UDP-GlcNAc and forming the Lipid I binding pocket.

[0027 ] FIG. 9 shows graphs for thermal stabilization of the P. a. MurG:UDP-

GlcNAc complex measured by Differential Scanning Fluorimetry Thermal Melt.

DETAILED DESCRIPTION OF THE INVENTION

General Definitions

The following abbreviations are used throughout the application:

A = Ala = Alanine T = Thr = Threonine

V = Val = Valine C = Cys = Cysteine

L = Leu = Leucine Y = Tyr = Tyrosine

1 = Ile = Isoleucine N = Asn = Asparagine

P = Pro = Proline Q = Gln = Glutamine

F = Phe = Phenylalanine D = Asp = Aspartic Acid

W = Trp = Tryptophan E = Glu = Glutamic Acid

M = Met = Methionine K = Lys = Lysine

G = Gly = Glycine Pv = Arg = Arginine

S = Ser = Serine H = His = Histidine

[0028] Throughout the specification, the word "comprise", or variations such as

"comprises" or "comprising" will be understood to imply the inclusion of a stated integer or groups of integers but not exclusion of any other integer or groups of integers. [0029] The term "associating with" refers to a condition of proximity between a chemical entity or compound, or portions thereof, and a binding pocket or binding site on a protein. The association may be non-covalent— wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions - - or it may be covalent.

[0030] The term "binding pocket", as used herein, refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, favourably associates with another chemical entity or compound. The term "pocket" includes, but is not limited to, cleft, channel or site. P. a MurG protein may have binding pockets which include, but are not limited to, substrate-binding and antibody-binding pockets.

[0031] The term "chemical entity", as used herein, refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds or complexes. The chemical entity may be, for example, a ligand, a substrate, a nucleotide triphosphate, a nucleotide diphosphate, phosphate, a nucleotide, an agonist, antagonist, inhibitor, antibody, drug, peptide, protein or compound.

[0032] "Conservative substitutions" refers to residues that are physically or functionally similar to the corresponding reference residues. That is, a conservative substitution and its reference residue have similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like. Preferred conservative substitutions are those fulfilling the criteria defined for an accepted point mutation in Dayhoff et al, Atlas of Protein Sequence and Structure, 5, pp. 345-352 (1978 & Supp.), which is incorporated herein by reference. Examples of conservative substitutions are substitutions including but not limited to the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine.

[0033] The term "corresponding amino acid" or "residue which corresponds to" refers to a particular amino acid or analogue thereof in a P. a MurG protein homologue that corresponds to an amino acid in the P. a MurG protein structure. The corresponding amino acid may be an identical, mutated, chemically modified, conserved, conservatively substituted, functionally equivalent or homologous amino acid when compared to the P. a MurG amino acid to which it corresponds.

[ 0034 ] Methods for identifying a corresponding amino acid are known in the art and are based upon sequence, structural alignment, its functional position or a

combination thereof as compared to the P. a MurG protein structure. For example, corresponding amino acids may be identified by superimposing the backbone atoms of the amino acids in P. a MurG protien and P. a MurG protein homologue using well known software applications, such as QUANTA [Molecular Simulations, Inc., San Diego, CA ©1998,2000]. The corresponding amino acids may also be identified using sequence alignment programs such as the "bestfit" program available from the Genetics Computer Group which uses the local homology algorithm described by Smith and Waterman in Adv. Appl. Math., 2, 482 (1981), which is incorporated herein by reference.

[ 0035 ] The term "domain" refers to a portion of the P. a MurG protein or homologue that can be separated according to its biological function, for example, catalysis. The domain is usually conserved in sequence or structure when compared to other related proteins. The domain can comprise a binding pocket, or a sequence or structural motif.

[ 0036 ] The term "sub-domain" refers to a portion of the domain as defined above in the P. a MurG protein or homologue.

[ 0037 ] The "MurG substrate -binding pocket" of a molecule or molecular complex is defined by the structure coordinates of a certain set of amino acid residues present in the P. a MurG protein structure, as described below. In general, the substrate for the P. a MurG protein is a nucleotide such as UDP, TDP or GDP. This MurG substrate-binding pocket is in the catalytic active site of the MurG protein.

[ 0038 ] The term "part of a MurG substrate-binding pocket" or "part of a MurG- like substrate-binding pocket" refers to less than all of the amino acid residues that define the MurG or MurG-like substrate-binding pocket. The structure coordinates of residues that constitute part of MurG or MurG-like substrate-binding pocket may be specific for defining the chemical environment of the binding pocket, or useful in designing fragments of an inhibitor that may interact with those residues. For example, the portion of residues may be key residues that play a role in substrate binding, or may be residues that are spatially related and define a three-dimensional compartment of the binding pocket. The residues may be contiguous or non-contiguous in primary sequence. In one embodiment, part of the MurG or MurG-like substrate-binding pocket is at least five amino acid residues, preferably, R143, F242, 1243, M246 and E267. In another embodiment, the amino acids are selected from the group consisting of G 14, HI 5, F 17, N124, R143, L186, S189, F242, 1243, M246, A262, L263, T264 and E267.

[ 0039 ] The "catalytic domain of P. a. MurG protein" includes, for example, the catalytic active site which comprises the catalytic residues, the lipid I binding site and the UDP-GlcNAc binding site. The catalytic domain in the MurG protein comprises residues from 1M to 357G (see SEQ ID NO: 1).

[ 0040 ] The term "part of a P. a. MurG catalytic domain" or "part of a MurG-like catalytic domain" refers to a portion of the MurG or MurG-like catalytic domain. The structure coordinates of residues that constitute part of a MurG or MurG-like catalytic domain may be specific for defining the chemical environment of the domain, or useful in designing fragments of an inhibitor that may interact with those residues. For example, the portion of residues may be key residues that play a role in substrate or ligand binding, or may be residues that are spatially related and define a three- dimensional compartment of the domain. The residues may be contiguous or noncontiguous in primary sequence. For example, part of a MurG catalytic domain can be the active site, the glycine-rich loop, the activation loop, or the catalytic loop.

[ 0041 ] The term "homologue of P. a. MurG" refers to a molecule or molecular complex that is homologous to MurG by three-dimensional structure or sequence.

Examples of homologues include a MurG protein from other species than P. a., a protein comprising a P. a. MurG-like substrate-binding pocket, a P. a. MurG catalytic domain, or a binding site of P. a. MurG protein.

[ 0042 ] The term "sufficiently homologous to P. a. MurG" refers to a protein that has a sequence homology of at least 35% compared to P. a. MurG protein. In one embodiment, the sequence homology is at least 40%, at least 60%, at least 80%, at least 90% or at least 95%.

[ 0043 ] The term "part of a P. a. MurG protein" or "part of a P. a. MurG homologue" refers to a portion of the amino acid residues of a P.a. MurG protein or homologue. In one embodiment, part of a P.a. MurG protein or homologue defines the binding pockets, domains, sub-domains, and motifs of the protein or homologue. The structure coordinates of residues that constitute part of a P.a. MurG protein or homologue may be specific for defining the chemical environment of the protein, or useful in designing fragments of an inhibitor that may interact with those residues. The portion of residues may also be residues that are spatially related and define a three-dimensional compartment of a binding pocket, motif or domain. The residues may be contiguous or non-contiguous in primary sequence. For example, the portion of residues may be key residues that play a role in ligand or substrate binding, peptide binding, antibody binding, catalysis, structural stabilization or degradation.

[ 0044 ] The term "P.a. MurG protein complex" or "P.a. MurG homologue complex" refers to a molecular complex formed by associating the P.a. MurG protein or P.a. MurG homologue with a chemical entity, for example, a ligand, a substrate, nucleotide triphosphate, an agonist or antagonist, inhibitor, drug or compound. In one embodiment, the chemical entity is a nucleotide (e.g., UDP, TDP or GDP) and an inhibitor (e.g., a small organic molecule) for the ATP-binding pocket.

[ 0045 ] The term "motif refers to a portion of the P.a. MurG protein or homologue that defines a structural compartment or carries out a function in the protein, for example, catalysis, structural stabilization, or phosphorylation. The motif may be conserved in sequence, structure and function when compared to other related proteins. The motif can be contiguous in primary sequence or three-dimensional space. The motif can comprise a-helices and/or β-sheets. Examples of a motif include but are not limited to a binding pocket, active site, phosphorylation lip or activation loop, the glycine-rich phosphate anchor loop, the catalytic loop [See, Xie et ah, Structure, 6, pp. 983-991 (1998); R. Giet and C. Prigent, J. Cell Sci., 112, pp. 3591-3601 (1999)], and the degradation box.

[ 0046 ] The term "root mean square deviation" or "RMSD" means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the "root mean square deviation" defines the variation in the backbone of a protein from the backbone of P.a. MurG protein, a binding pocket, a motif, a domain, or portion thereof, as defined by the structure coordinates of P. a. MurG protein described herein.

[ 0047 ] The term "soaked" refers to a process in which the crystal is transferred to a solution containing the compound of interest. In certain embodiments, the compound is diffused into the crystal.

[ 0048 ] The term "structure coordinates" refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a protein or protein complex in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the positions of the individual atoms of the molecule or molecular complex. It would be readily apparent to those skilled in the art that all or part of the structure coordinates of FIGs. 1 and 2 may have a RMSD deviation of ± 0.1-0.5 A because of standard error.

[ 0049 ] The term "crystallization solution" refers to a solution that promotes crystallization. The solution comprises at least one agent, and may include a buffer, one or more salts, a precipitating agent, one or more detergents, sugars or organic compounds, lanthanide ions, a poly-ionic compound and/or a stabilizer.

[ 0050 ] The term "homologue of .a.MurG" or "P.a. MurG homologue" refers to a molecule that is homologous to P.a. MurG by three-dimensional structure or sequence and retains the biological activity of P.a. MurG. Examples of homologues include, but are not limited to, P.a. MurG having one or more amino acid residues that are chemically modified, mutated, conservatively substituted, added, deleted or a combination thereof. Examples of homologues include P.a. MurG proteins from other species than P.a. and proteins comprising a P.a. MurG-like binding pocket (e.g., sunstrate-binding pocket) or a P.a. MurG-like catalytic domain.

[ 0051 ] The term "homology model" refers to a structural model derived from known three-dimensional structure(s). Generation of the homology model, termed "homology modeling", can include sequence alignment, residue replacement, residue conformation adjustment through energy minimization, or a combination thereof. [ 0052 ] The term "three-dimensional structural information" refers to information obtained from the structure coordinates. Structural information generated can include the three-dimensional structure or graphical representation of the structure. Structural information can also be generated when subtracting distances between atoms in the structure coordinates, calculating chemical energies for a P.a. MurG protein or homologues thereof, calculating or minimizing energies for an association of a P.a. MurG protein or homologues thereof to a chemical entity. The three-dimensional structure may be displayed as a graphical representation or used to perform computer modeling or fitting operations. In addition, the structure coordinates themselves may be used to perform computer modeling and fitting operations.

Crystallizable Compositions and Crystals ofP.a. MurG Protein and P.a. MurG Complex

[ 0053 ] FIG In one embodiment, the present invention is directed to P.a. MurG crystals. P.a. MurG crystals include crystals of P.a. MurG protein and crystals of P.a. MurG protein complexed with a chemical entity (e.g., ligand, substrate, or inhibitor of MurG protein). The P.a. MurG protein is a single chain polypeptide and comprises a MurG polypeptide comprising amino acids according to SEQ ID NO: l :

MKGNVLIMAGGTGGHVFPALACAREFQARGYAVHWLGTP

RGIENDLVPKAGLPLHLIQVSGLRGKGLKSLVKAPLELLKSL

FQALRVIRQLRPVCVLGLGGYVTGPGGLAARLNGVPLVIHE

QNAVAGTANRSLAPIARRVCEAFPDTFPASDKRLTTGNPVR

GELFLDAHARAPLTGRRVNLLVLGGSLGAEPLNKLLPEALA

QVPLEIRPAIRHQAGRQHAEITAERYRTVAVEADVAPFISD

MAAAYAWADLVICRAGALTVSELTAAGLPAFLVPLPHAID

DHQTRNAEFLVRSGAGRLLPQKSTGAAELAAQLSEVLMHP

ETLRSMADQARSLAKPEATRTVVDACLEVARG.

[ 0054 ] FIG As used herein, the terms 'crystalline P.a. MurG' and 'P.a.

crystal' both refer to crystallised P.a. MurG protein and are intended to be interchangeable. The term "crystal" means that the crystal has an X-ray diffraction quality to provide X-ray diffraction data for determination of atomic coordinates to a resolution of 4 angstroms or less. A typical crystal of the present invention provides X- ray diffraction data for determination of atomic coordinates to a resolution of 3 angstroms or less, specifically 2.4 angstroms or less, and more specifically 1.8 angstroms or less. More typically, the crystal of the present invention provide X-ray diffraction data for determination of atomic coordinates to a resolution of between 4 angstroms and 1 angstrom, specifically between 3 angstroms and 1 angstrom, more specifically between 3 angstroms and 1.5 angstroms.

[ 0055 ] Typically the substrate of P. a. MurG protein include a nucleotide, such as a UDP, TDP, or GDP. Specifically, the substrate of MurG protein is UDP-N- acetylglucosamine (UDP-GlcNAc).

[ 0056 ] In one specific embodiment, the P. a. MurG crystals of the invention are in a tetragonal space group. In another specific embodiment, the amino acids of P. a. MurG protein of the P. a. MurG crystals have atomic coordinates according to FIG. 1 or FIG. 2. In yet another specific embodiment, the MurG crystals of the invention is in space group P4(l)2(l)2 and have unit cell dimensions a=49.9 angstroms ± 1-2 angstroms, b=49.9 angstroms ± 1-2 angstroms, c=278.6 angstroms ± 1-2 angstroms, alpha=90 degree, beta=90 degree, gamma=90 degree. In another specific embodiment, the amino acids of P. a. MurG protein of the MurG crystals have atomic coordinates according to FIG. 1. In yet another specific embodiment, the amino acids of P. a. MurG protein of the MurG crystals have atomic coordinates according to FIG. 2.

[ 0057 ] In another embodiment, the present invention is directed to crystallizable compositions of P. a. MurG protein. The crystallizable compositions comprise a MurG protein concentrate and a reservoir solution. Typically, the MurG protein concentrate includes a polypeptide comprising amino acid residues according to SEQ. ID. NO. l . Optionally, the MurG protein concentrate further includes a chemical entity (e.g., ligand, substrate, or inhibitor of MurG protein) that forms a complex with the polypeptide according to SEQ. ID. NO. l . Typically, the polypeptide is in a concentration of 5 mg/ml to 20mg/ml. More typically, the polypeptide is in a concentration of 5 mg/ml to 15 mg/ml or 8 mg/ml to 14 mg/ml. More typically, the polypeptide is in a concentration of 10 mg/ml.

[ 0058 ] The reservoir includes a buffer having a pKa of 6.0 and 8.0. Any suitable buffer known in the art can be employed in the present invention. Typical examples include a HEPES (4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic acid, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) buffer and a Tris

(Tris(hydroxymethyl)aminomethane) buffer. In a specific embodiment, the buffer has a pKa ranged from 6.5 to 8.0. In another specific embodiment, the buffer has a pKa ranged from 6.5 to 7.5. In yet another specific embodiment, the buffer is a HEPES buffer, specifically HEPES buffer having a pH ranged from 6.8 to 7.5, more specifically HEPES buffer having a pH of 7.0.

[ 0059 ] The reservoir further includes a polyethylene glycol (PEG) as a precipitant. A molecular weight of the polyethylene glycol can range from 200 to 20,000 Daltons. Typically, the molecular weight of the polyethylene glycol has a range of 1,000 to 20,000 Daltons. In a specific embodiment, the polyethylene glycol has a molecular weight of 2,500 to 4,000 Daltons. In another specific embodiment, the polyethylene glycol has a molecular weight of 3,000 to 4,000 Daltons. In another specific

embodiment, the polyethylene glycol has a molecular weight of 3,350 Daltons. A concentration of polyethylene glycol can range from 5% to 50% by volume. In one specific embodiment, the concentration of polyethylene glycol can vary from 8%-35% by volume. In another specific embodiment, the concentration of polyethylene glycol can vary from 10%-30% by volume. In yet another specific embodiment, the concentration of polyethylene glycol can vary from 8%-22% by volume. In yet another specific embodiment, the polyethylene glycol has a concentration of 20% by volume.

[ 0060 ] The reservoir can further include one or more other additives . In one specific embodiment, the reservoir further includes an additional precipitant. A typical example of such additional precipitant is ammonium sulphate. In another specific embodiment, the reservoir further includes one or more reducing agents. Typical examples of suitable reducing agents include DTT (tAreo-l,4-Dimercapto-2,3-butanediol) and TCEP (Tris(2-carboxyethyl)phosphine hydrochloride). Typically the reducing agent is in a concentration of 1 mM to 20 mM. More typically, the reducing agent is in a concentration of 5 mM to 15 mM. Even more typically, the reducing agent is in a concentration of 10 mM.

[0061] In some specific embodiments, the MurG protein concentrate of the crystallizable compositions further comprise a chemical entity, such as a ligand (e.g., a nucleotide) or substrate of P. a. MurG protein, an analogue thereof, or an inhibitor of P. a. MurG protein (e.g., a small organic compound), which forms a complex with the MurG protein. In a more specific embodiment, the crystallizable compositions further comprise UDP-GlcNAc. Typically, UDP-GlcNAc is in a molar ratio of 1 :600 to 1 :2,000 P. a. MurG protein to UDP-GlcNAc. More typically, UDP-GlcNAc is in a molar ratio of 1 :600 to 1 : 1 ,400 P. a. MerG protein to UDP-GlcNAc. More typically, UDP-GlcNAc is in a molar ratio of 1 : 1 ,000 P. a. MerG protein to UDP-GlcNAc. Such crystallizable compositions can produce crystals of P. a. MurG complex with UDP-GlcNAc.

[0062] In a particular embodiment, the crystallizable compositions of the invention comprise a polypeptide comprising amino acid residues according to SEQ. ID. NO. l of P. a. MurG protein in a concentration of 5 mg/ml to 15 mg/ml, a HEPES buffer having a pKa of 6.8 to 7.2 (e.g., 7.0), and polyethylene glycol having a molecular weight of 3,350 Daltons in a concentration of 15% to 25% by volume. In another particular embodiment, the crystallizable compositions of the invention comprise a polypeptide comprising amino acid residues according to SEQ. ID. NO. l of P. a. MurG protein in a concentration of 5 mg/ml to 15 mg/ml; a HEPES buffer having a pKa of 6.8 to 7.2 (e.g., 7.0); polyethylene glycol having a molecular weight of 3,350 Daltons in a concentration of 15% to 25% by volume; and UDP-GlcNAc in a molar ratio of 1 :500 to 1 :700 MurG protein to UDP-GlcNAc. More specifically, the molar ratio of MurG protein to UDP- GlcNAc is 1 :600.

[0063] In another particular embodiment, the crystallizable compositions of the invention comprise a polypeptide comprising amino acid residues according to SEQ. ID. NO.1 of P. a. MurG protein in a concentration of 10 mg/ml in a buffer of 25mM HEPES having a pH of 7.0, NaCl (e.g., in 500 mM), DTT (e.g., 2.5mM), Decyl Maltopyranoside (e.g., 0.11%) by volume), and glycerol (e.g., 10%> by volume). [ 0064 ] In another particular embodiment, the reservoir of the crystallizable composition include 0.1M HEPES having a pH of 7.0, 20 %(v/v) PEG having a molecular weight of 3,350 Daltons, and 10 mM DTT.

[ 0065 ] In some embodiments, the reservoir of the crystallizable composition further includes ammonium sulfate. A typical concentration of ammonium sulfate is in a range of 80 mM to 120 mM, such as 100 mM.

[ 0066 ] In some embodiments, the polypeptides of the P. a. MurG crystals and the polypeptides included in the MurG protein concentrate for the crystallizable compositions can further comprise non- .α. MurG amino acids, such as amino acids from a tag. Typically, a P.a. MurG protein is overexpressed with a C-terminal tag, such as his- tag (SEQ ID NO:2: LEHHHHHH), to facilitate its purification using an affinity column. In one specific embodiment, the polypeptides of the MurG crystals and crystallizable compositions of the invention further comprise amino acids of his-tag at the C-terminal. Specifically, the amino acids of his-tag consist of LEHHHHHH (SEQ ID NO:2). The sequence listing of the amino acids of such MurG crystals and crystallizable

compositions of the invention that further comprises amino acids of the his-tag is as follows (SEQ ID NO:3):

MKGNVLIMAGGTGGHVFPALACAREFQARGYAVHWLGTP

PvGIENDLVPKAGLPLHLIQVSGLPvGKGLKSLVKAPLELLKSL

FQALPvVIPvQLRPVCVLGLGGYVTGPGGLAARLNGVPLVIHE

QNAVAGTANRSLAPIARRVCEAFPDTFPASDKPvLTTGNPVPv

GELFLDAHARAPLTGRRVNLLVLGGSLGAEPLNKLLPEALA

QVPLEIRPAIRHQAGRQHAEITAERYRTVAVEADVAPFISD

MAAAYAWADLVICRAGALTVSELTAAGLPAFLVPLPHAID

DHQTRNAEFLVRSGAGRLLPQKSTGAAELAAQLSEVLMHP

ETLRSMADQARSLAKPEATRTVVDACLEVARGLEHHHHH

H.

[ 0067 ] FIG The present invention further provides methods of forming P.a.

MurG crystals (e.g., crystals of P.a. MurG protein and crystals of P.a. MurG complexes with a chemical entity). In one aspect, the methods comprise the steps of: a) combining a MurG protein concentrate with a reservoir solution; and b) inducing crystal formation to produce crystals by subjecting the resulting mixture to devices or conditions, which promote crystallization. In another aspect, the methods comprise the steps of : a) preparing a crystallizable composition as described above; and inducing crystal formation to produce crystals by subjecting the resulting mixture to devices or conditions which promote crystallization. The features, including the specific features, of the MurG protein concentrate and reservoir solution (including its components, such as buffer and polyethylene glycol) are each and independently as described above.

[ 0068 ] In one specific embodiment, the method comprises the steps of: a) combining a polypeptide comprising amino acid residues according to SEQ ID NO: l of P.a. MurG protein with UDP-GlcNAc in a molar ratio of 1 :600 to 1 :2,000 MurG protein to UDP-GlacNac to result in a mixture of MurG protein and UDP-GlcNAc, b) combining the mixture of MurG protein and UDP-GlcNAc with a reservoir solution that includes a buffer having a pKa of 6.5 to 8.0 and a polyethylene glycol in a concentration from 5% to 50% by volume, and c) inducing crystal formation to produce a crystal of MurG protein. The features, including the specific features, of the polypeptide, UDP-GlcNAc, and reservoir solution (including its components, such as buffer and polyethylene glycol) are each and independently as described above. In a more specific embodiment, the UDP- GlcNAc is in a molar ratio of 1 :600 to 1 : 1 ,400 MurG protein to UDP-GlcNAc. In another more specific embodiment, the UDP-GlcNAc is in a molar ratio of 1 : 1000 MurG protein to UDP-GlcNAc. In another more specific embodiment, the polypeptide further comprise additional amino acids of a tag consisting of LEHHHHHH (SEQ ID NO:2) at the C-terminal.

[ 0069 ] In another specific embodiment, the methods comprise: a) concentrating a MurG protein solution that includes a MurG polypeptide as described above in a buffer solution; b) mixing the MurG protein solution with UDP-GlcNAc in a 1 :600 - 1 :2,000 molar ratio, such as 1 :600 to 1 : 1 ,400 molar ratio or 1 : 1000 molar ratio, to generate a mixture of the MurG polypeptide and UDP-GlcNAc; c) mixing equal volumes of the mixture of the MurG polypeptide and UDP-GlcNAc with a reservoir solution as described above; and d) inducing crystal formation using hanging drop vapour diffusion. In a particular embodiment, the reservoir solution comprises 0.1M HEPES having pH7.0, 20% (v/v) PEG (polyethylene glycol) having a molecular weight of 3,350 Daltons, and lOmM DTT. In another particular embodiment, the MurG polypeptide is concentrated to lOmg/ml in a buffer that includes 25mM HEPES pH7.0, 500mM NaCl, 2.5mM DTT, 0.11% Decyl Maltopyranoside and 10% glycerol.

[ 0070 ] In yet another specific embodiment, the MurG crystals are produced using the crystal formation method described in Example 1.

[ 0071 ] The concentrated solutions of MurG protein can be induced to crystallise by several methods including, but not limited to, vapour diffusion, liquid diffusion, batch crystallisation, dialysis or a combination thereof. In a specific embodiment, vapour diffusion is employed.

[ 0072 ] In a vapour diffusion method, typically, the concentrated solutions of

MurG protein become supersaturated and form MurG crystals at a constant temperature by diffusion of solvent(s), in which the MurG protein is not generally soluble, into the MurG protein solutions. In one specific embodiment, vapour diffusion is performed under a controlled temperature in a range of 12°C±1 °C to 37°C±1 °C, specifically from 16°C±1 °C to 24°C±1 °C, more specifically at a constant temperature of 20°C±1 °C.

[ 0073 ] Devices for promoting crystallization can include but are not limited to the hanging-drop, sitting-drop, dialysis or microtube batch devices. [U.S. patent

4,886,646, 5,096,676, 5,130,105, 5,221,410 and 5,400,741; Pav et al, Proteins:

Structure, Function, and Genetics, 20, pp. 98-102 (1994), incorporated herein by reference]. The hanging-drop or sitting-drop methods produce crystals by vapor diffusion. The hanging-drop, sitting-drop, and some adaptations of the microbatch methods [D'Arcy et al, J. Cryst. Growth, 168, pp. 175-180 (1996) and Chayen, J. Appl. Cryst., 30, pp. 198-202 (1997)] produce crystals by vapor diffusion. The hanging drop and sitting drop containing the crystallizable composition is equilibrated in a reservoir containing a higher or lower concentration of the precipitant. As the drop approaches equilibrium with the reservoir, the saturation of protein in the solution leads to the formation of crystals.

[ 0074 ] Microseeding or seeding may be used to obtain larger, or better quality

{i.e., crystals with higher resolution diffraction or single crystals) crystals from initial micro-crystals. Microseeding involves the use of crystalline particles to provide nucleation under controlled crystallization conditions. Microseeding is used to increase the size and quality of crystals. In this instance, micro -crystals are crushed to yield a stock seed solution. The stock seed solution is diluted in series. Using a needle, glass rod or strand of hair, a small sample from each diluted solution is added to a set of equilibrated drops containing a protein concentration equal to or less than a concentration needed to create crystals without the presence of seeds. The aim is to end up with a single seed crystal that will act to nucleate crystal growth in the drop.

[ 0075 ] In some embodiments, the methods further include the steps of producing and purifying P. a. MurG protein. In a particular embodiment, the

characterization step includes a) size-exclusion chromatography and dynamic light scattering, b) mass spectrometry analysis, c) differential scanning fluorescence study of UDP-GlcNAc binding to P. a. MurG protein, and/or d) enzymatic characterization of P. a. MurG protein.

[ 0076 ] In general, the P. a. MurG protein can be produced by any well-known method, including synthetic methods, such as solid phase, liquid phase and combination solid phase/liquid phase syntheses; recombinant DNA methods, including cDNA cloning, optionally combined with site directed mutagenesis; and/or purification of the natural products. In a certain embodiment, the protein is overexpressed from E. coli system.

[ 0077 ] Applicants have demonstrated herein that it is possible to express P. a.

MurG in E. coli cells (see the experimental section below). This fining facilitates obtaining sufficient quantities of isolated and/or purified P. a. MurG protein. The expression of P.a. MurG protein in E. coli host cells can be performed, for example by expressing the P.a. MurG gene cloned into pET21b expression vector and transformed into an E. coli host cell. The P.a. MurG protein can be overexpressed with a C-terminal his-tag (LEHHHHHH) which allows the protein to be purified using a His-tag affinity column. The protein is then crystallised as described above and demonstrated in the experimental section. The atomic coordinates can then be determined using X-ray diffraction and methods to those skilled in the art.

[ 0078 ] The present invention also provides crystals of mutants and homo logs of

P.a. MurG protein, and fragments thereof, and crystals of molecular complexes of mutants and homo logs of P.a. MurG protein, and fragments thereof with a chemical entity such as a nucleotide or substrate, or analogue thereof. The present invention also provides crystallizable composition for, and methods of, making such crystals.

[ 0079 ] It would be readily apparent to one of skill in the art following the teachings of the specification to vary the crystallization conditions disclosed herein to identify other crystallization conditions that would produce crystals of P.a. MurG homologue or P. a. MurG homologue complex. Such variations include, but are not limited to, adjusting pH, protein concentration and/or crystallization temperature, changing the identity or concentration of salt and/or precipitant used, using a different method of crystallization, or introducing additives such as detergents {e.g., TWEEN 20 (monolaurate), LDAO, Brij 30 (4 lauryl ether)), sugars {e.g., glucose, maltose), organic compounds {e.g., dioxane, dimethylformamide), lanthanide ions or polyionic compounds that aid in crystallization. High throughput crystallization assays may also be used to assist in finding or optimizing the crystallization conditions.

Active Sites and Binding Pockets of P.a. MurG Protein

[ 0080 ] The x-ray structure of the P.a. MurG consist of two α/β domains separated by a cleft 20 angstroms deep and 16 angstroms across at its widest point. Individual N and C domains are similar in the presence and absence of substrate, although the C-terminal domain of the Apo structure is significantly disordered without substrate bound (see Fig6). There is a slight change in the relative orientation of the two domains so that in the presence of UDP-GlcNAc, MurG adopts a more closed conformation. The conformational change results mostly from a rigid body domain movement. Binding sites for Lipid I and UDP-GlcNAc are contained within the cleft between the N- and C-terminal domains of MurG.

[ 0081 ] The structure of the UDP-GlcNAc:MurG complex (FIG. 6) shows that the UDP-GlcNAc moiety mostly contacts the C-terminal domain. UDP-GlcNAc makes several contacts to these helices as well as to the loops connecting them to the adjacent β strands of the N- and C-terminal domains. The uracil base is bound in a pocket flanked on one edge by a helix and the strand connecting the - and C-terminal domains. [ 0082 ] Those of skill in the art understand that a set of structure coordinates for a molecule or a molecular-complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates will have little effect on overall shape. In terms of binding pockets, these variations would not be expected to significantly alter the nature of ligands that could associate with those pockets.

[ 0083 ] The variations in coordinates discussed above may be generated because of mathematical manipulations of the P. a. MurG protein structure coordinates. For example, the structure coordinates set forth in FIGs. 1 and 2 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

[ 0084 ] Alternatively, modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates. If such variations are within a certain root mean square deviation as compared to the original coordinates, the resulting three-dimensional shape is considered encompassed by this invention. Thus, for example, a ligand or substrate that bound to the binding pocket of P. a. MurG protein would also be expected to bind to another binding pocket whose structure coordinates defined a shape that fell within the acceptable root mean square deviation.

[ 0085 ] Various computational analyses maybe necessary to determine whether a binding pocket, motif, domain or portion thereof of a molecule or molecular complex is sufficiently similar to the binding pocket, motif, domain or portion thereof of P. a. MurG protein Such analyses may be carried out in well known software applications, such as ProFit [A. C.R. Martin, SciTech Software, ProFit version 1.8, University College

London, http://www.bioinf.org.uk/software], Swiss-Pdb Viewer [Guex et al.,

Electrophoresis, 18, pp. 2714-2723 (1997)], the Molecular Similarity application of QUANTA® [Molecular Simulations, Inc., San Diego, CA; now part of Accelrys, San Diego, CA] and as described in the accompanying User's Guide, which are incorporated herein by reference.

[ 0086 ] The above programs permit comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in QUANTA® [Molecular Simulations, Inc., San Diego, CA; now part of Accelrys, San Diego, CA] and Swiss-Pdb Viewer to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation on the structures; and 4) analyze the results. The procedure used in ProFit to compare structures includes the following steps: 1) load the structures to be compared; 2) specify selected residues of interest; 3) define the atom equivalences in the selected residues; 4) perform a fitting operation on the selected residues; and 5) analyze the results.

[ 0087 ] Each structure in the comparison is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). Since atom equivalency within the above programs is defined by user input, for the purpose of this invention we will define equivalent atoms as protein backbone atoms (N, Ca, C and O) for P.a. MurG protein amino acids and corresponding amino acids in the structures being compared.

[ 0088 ] The corresponding amino acids may be identified by sequence alignment programs such as the "bestfit" program available from the Genetics Computer Group which uses the local homology algorithm described by Smith and Waterman in Advances in Applied Mathematics 2, 482 (1981), which is incorporated herein by reference. A suitable amino acid sequence alignment will require that the proteins being aligned share minimum percentage of identical amino acids. Generally, a first protein being aligned with a second protein should share in excess of about 35% identical amino acids with the second protein [Hanks et ah, Science, 241, 42 (1988); Hanks and Quinn, Meth. Enzymol, 200, 38 (1991)]. The identification of equivalent residues can also be assisted by secondary structure alignment, for example, aligning the a-helices, β-sheets in the structure. The program Swiss-Pdb Viewer has its own best fit algorithm that is based on secondary sequence alignment. [ 0089 ] When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by the above programs. The Swiss-Pdb Viewer program sets an RMSD cutoff for eliminating pairs of equivalent atoms that have high RMSD values. For programs that calculate an average of the individual RMSD values of the backbone atoms, an RMSD cutoff value can be used to exclude pairs of equivalent atoms with extreme individual RMSD values. In the program ProFit, the RMSD cutoff value can be specified by the user.

[ 0090 ] For the purpose of this invention, any molecule, molecular complex, binding pocket, motif, domain thereof or portion thereof that is within a root mean square deviation for backbone atoms (N, Ca, C, O) when superimposed on the relevant backbone atoms described by structure coordinates listed in FIGs. 1 and 2 are

encompassed by this invention.

Computer Systems

[ 0091 ] According to another embodiment of this invention is provided a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data comprises all or part of P.a. MurG binding pocket defined by structure coordinates of P.a. MurG amino acids G14, H15, F17, N124, R143, LI 86, SI 89, F242, 1243, M246, A262, L263, T264 and E267, according to FIG. 1 or FIG. 2; or a molecule or molecular complex comprising all or part of a P.a. MurG-like binding pocket defined by structure coordinates of corresponding amino acids that are identical to said MurG amino acids, wherein the root mean square deviation of the backbone atoms between said corresponding amino acids and said MurG amino acids is not more than 3.0 A±0.1 A, 2.5 A±0.1 A, 2.0 A±0.1 A, 1.5 A±0.1 A, or 1.0 A±0.1 A; or a molecule or molecular complex comprising all or part of a P.a. MurG-like binding pocket defined by structure coordinates of a set of corresponding amino acids, wherein the root mean square deviation of the backbone atoms between said set of corresponding amino acids and said MurG amino acids is not more than 1.1, 0.9, 0.7 or 0.5 A, and wherein at least one of said corresponding amino acids is not identical to the MurG amino acid to which it corresponds.

[ 0092 ] In other embodiments of this invention is provided a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data comprises all or part of any molecule or molecular complex discussed in the above paragraphs.

[ 0093 ] In one embodiment of this invention is provided a computer comprising:

a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data comprises all or part of a P.a. MurG substrate-binding pocket defined by structure coordinates of P. a. MurG amino acids R143, F242, 1243, M246 and E267, according to FIG. 1 or FIG. 2; or a molecule or molecular complex comprising all or part of a P. a. MurG-like substrate-binding pocket defined by structure coordinates of corresponding amino acids that are identical to said P.a. MurG amino acids, wherein the root mean square deviation of the backbone atoms between said corresponding amino acids and said P.a. MurG amino acids is not more than 3.0 A±0.1 A, 2.5 A±0.1 A, 2.0 A±0.1 A, 1.5 A±0.1 A, or 1.0 A±0.1 A; or a molecule or molecular complex comprising all or part of a P.a. MurG-like substrate-binding pocket defined by structure coordinates of a set of corresponding amino acids, wherein the root mean square deviation of the backbone atoms between said set of corresponding amino acids and said P.a. MurG amino acids is not more than 0.5 A±0.1 A, and wherein at least one of said corresponding amino acids is not identical to the P.a. MurG amino acid to which it corresponds.

[ 0094 ] In one embodiment, a computer according to this invention comprises a working memory for storing instructions for processing the machine-readable data, a central-processing unit coupled to the working memory and to said machine -readable data storage medium for processing said machine-readable data into the three- dimensional structure. In one embodiment, the computer further comprises a display for displaying the three-dimensional structure as a graphical representation. In another embodiment, the computer further comprises commercially available software program to display the graphical representation. Examples of software programs include but are not limited to QUANTA® [Molecular Simulations, Inc., San Diego, CA; now part of Accelrys, San Diego, CA], O [Jones et al., Acta Cryst. A, 47, pp. 110-119 (1991)] and RIBBONS [M. Carson, J. Appl. Cryst., 24, pp. 958-961 (1991)], which are incorporated herein by reference.

[ 0095 ] In another embodiment, a computer according to this invention comprises executable code for:

(a) using structural coordinates (all or part of) according to FIG. 1 or FIG. 2 as a 3-dimensional model of a catalytic domain of P. a. MurG protein;

(b) analyzing a binding site of the 3-dimensional model; and

(c) screening in silico a library for small molecules that fit into said binding site. In another embodiment, the computer further comprises executable code for: (d) controlling a unit for assaying the small molecules determined in step (c) in a protein binding assay. Using structural coordinates may include displaying the coordinates graphically or manipulating the structure coordinates with computational programs.

[ 0096 ] This invention also provides a computer comprising:

a) a machine -readable data storage medium comprising a data storage material encoded with machine-readable data, wherein the data defines any one of the above binding pockets or protein of the molecule or molecular complex;

b) a working memory for storing instructions for processing said machine-readable data;

c) a central processing unit (CPU) coupled to the working memory and to the machine-readable data storage medium for processing said machine readable data as well as an instruction or set of instructions for generating three-dimensional structural information of said binding pocket or protein; and

d) output hardware coupled to the CPU for outputting three- dimensional structural information of the binding pocket or protein, or information produced by using the three-dimensional structural information of said binding pocket or protein. The output hardware may include monitors, touchscreens, printers, facsimile machines, modems, disk drives, CD-ROMs, etc. [ 0097 ] Three-dimensional data generation may be provided by an instruction or set of instructions such as a computer program or commands for generating a three- dimensional structure or graphical representation from structure coordinates, or by subtracting distances between atoms, calculating chemical energies for a P.a. MurG protein or homologues thereof, or for complexes of such P. a. MurG protein or homologues thereof with a chemical entity; or calculating or minimizing energies for an association of a P. a. MurG protein or homologues thereof to a chemical entity. The graphical representation can be generated or displayed by commercially available software programs. Examples of software programs include but are not limited to QUANTA® [Molecular Simulations, Inc., San Diego, CA; now part of Accelrys, San Diego, CA], O [Jones et al., Acta Crystallogr. A47, pp. 110-119 (1991)] and RIBBONS [Carson, J. Appl. Crystallogr., 24, pp. 9589-961 (1991)], Coot [Emsley, P. and Cowtan, K. Acta Crystallogr. D60, pp. 2126-2132 Part 12 Sp. Iss. 1 (Dec 2004)], and PyMol™ [DeLano, W.L. The PyMOL Molecular Graphics System (2002) DeLano Scientific™, Palo Alto, CA, USA.], which are incorporated herein by reference. Certain software programs may imbue this representation with physico-chemical attributes which are known from the chemical composition of the molecule, such as residue charge, hydrophobicity, torsional and rotational degrees of freedom for the residue or segment, etc. Examples of software programs for calculating chemical energies are described in the Rational Drug Design section.

[ 0098 ] Information about said binding pocket or information produced by using said binding pocket can be outputted through display terminals, touchscreens, printers, modems, facsimile machines, CD-ROMs or disk drives. The information can be in graphical or alphanumeric form.

[ 0099 ] FIG. 3 demonstrates one version of these embodiments. System 10 includes a computer 11 comprising a central processing unit ("CPU") 20, a working memory 22 which may be, e.g., RAM (random-access memory) or "core" memory, mass storage memory 24 (such as one or more disk drives or CD-ROM drives), one or more cathode -ray tube ("CRT") display terminals 26, one or more keyboards 28, one or more input lines 30, and one or more output lines 40, all of which are interconnected by a conventional bi-directional system bus 50. [00100] Input hardware 36, coupled to computer 11 by input lines 30, may be implemented in a variety of ways. Machine -readable data of this invention may be inputted via the use of a modem or modems 32 connected by a telephone line or dedicated data line 34. Alternatively or additionally, the input hardware 36 may comprise CD-ROM drives or disk drives 24. In conjunction with display terminal 26, keyboard 28 may also be used as an input device.

[00101] Output hardware 46, coupled to computer 11 by output lines 40, may similarly be implemented by conventional devices. By way of example, output hardware 46 may include CRT display terminal 26 for displaying a graphical representation of a binding pocket of this invention using a program such as QUANTA® [Molecular Simulations, Inc., San Diego, CA; now part of Accelrys, San Diego, CA] as described herein. Output hardware might also include a printer 42, so that hard copy output may be produced, or a disk drive 24, to store system output for later use. Output hardware may also include a display terminal, a CD or DVD recorder, ZIP™ or JAZ™ drive, or other machine-readable data storage device.

[00102] In operation, CPU 20 coordinates the use of the various input and output devices 36, 46, coordinates data accesses from mass storage 24 and accesses to and from working memory 22, and determines the sequence of data processing steps. A number of programs may be used to process the machine -readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system 10 are included as appropriate throughout the following description of the data storage medium.

[00103] FIG. 4 shows a cross section of a magnetic data storage medium 100 which can be encoded with a machine -readable data that can be carried out by a system such as system 10 of FIG. 3. Medium 100 can be a conventional floppy diskette or hard disk, having a suitable substrate 101, which may be conventional, and a suitable coating 102, which may be conventional, on one or both sides, containing magnetic domains (not visible) whose polarity or orientation can be altered magnetically. Medium 100 may also have an opening (not shown) for receiving the spindle of a disk drive or other data storage device 24. [ 00104 ] The magnetic domains of coating 102 of medium 100 are polarized or oriented so as to encode in a manner that may be conventional, machine readable data such as that described herein, for execution by a system such as system 10 of FIG. 3.

[ 00105] FIG. 5 shows a cross section of an optically-readable data storage medium 110 which also can be encoded with such a machine-readable data, or set of instructions, which can be carried out by a system such as system 10 of FIG. 3. Medium 110 can be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk that is optically readable and magneto-optically writable. Medium 100 preferably has a suitable substrate 111, which may be

conventional, and a suitable coating 112, which may be conventional, usually of one side of substrate 111.

[ 00106] In the case of CD-ROM, as is well known, coating 112 is reflective and is impressed with a plurality of pits 113 to encode the machine -readable data. The arrangement of pits is read by reflecting laser light off the surface of coating 112. A protective coating 114, which preferably is substantially transparent, is provided on top of coating 112.

[00107 ] In the case of a magneto-optical disk, as is well known, coating 112 has no pits 113, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser (not shown). The orientation of the domains can be read by measuring the polarization of laser light reflected from coating 112. The arrangement of the domains encodes the data as described above.

[ 00108 ] In one embodiment, the data defines the above-mentioned binding pockets by comprising the structure coordinates of said amino acid residues according to FIG. 1 or FIG. 2.

[ 00109] To use the structure coordinates generated for P. a. MurG protein or a homologue thereof, one of its binding pockets, motifs, domains, or portion thereof, it is at times necessary to convert them into a three-dimensional shape or to generate three- dimensional structural information from them. This is achieved through the use of commercially or publicly available software that is capable of generating a three- dimensional structure of molecules or portions thereof from a set of structure coordinates. In one embodiment, the three-dimensional structure may be displayed as a graphical representation.

[00110] Therefore, according to another embodiment, this invention provides a machine-readable data storage medium comprising a data storage material encoded with machine readable data. In one embodiment, a machine programmed with instructions for using said data, is capable of generating a three-dimensional structure of any of the molecule or molecular complexes, or binding pockets thereof, that are described herein.

[00111] In certain embodiment, this invention also provides a computer for producing a three-dimensional structure of:

a) a molecule or molecular complex

comprising all or part of a P. a. MurG binding pocket defined by structure coordinates of P. a. MurG amino acids G14, H15, F17, N124, R143, L186, S189, F242, 1243, M246, A262, L263, T264 and E267, according to FIG. 1 or FIG. 2;

b) a molecule or molecular complex

comprising all or part of a P. a. MurG-like binding pocket defined by structure coordinates of corresponding amino acids that are identical to said P. a. MurG amino acids, wherein the root mean square deviation of the backbone atoms between said corresponding amino acids and said P. a. MurG amino acids is not more than 3.0 A±0.1 A, 2.5 A±0.1 A, 2.0 A±0.1 A, 1.5 A±0.1 A, or 1.0 A±0.1 A; and/or

c) a molecule or molecular complex

comprising all or part of a P. a. MurG-like binding pocket defined by structure coordinates of a set of corresponding amino acids, wherein the root mean square deviation of the backbone atoms between said set of corresponding amino acids and said P. a. MurG amino acids is not more than 0.6 A, 0.5 A or 0.4 A, and wherein at least one of said corresponding amino acids is not identical to the P. a. MurG amino acid to which it corresponds,

comprising:

i) a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data comprises all or part of a P. a. MurG binding pocket defined by structure coordinates of P. a. MurG amino acids R143, F242, 1243, M246 and E267, according to FIG. 1 or FIG. 2; all or part of a P.a. MurG -like binding pocket defined by structure coordinates of corresponding amino acids that are identical to said P. a. MurG amino acids, wherein the root mean square deviation of the backbone atoms between said corresponding amino acids and said P. a. MurG amino acids is not more than 3.0 A±0.1 A, 2.5 A±0.1 A, 2.0 A±0.1 A, 1.5 A±0.1 A, or 1.0 A±0.1 A; or all or part of a P. a. MurG -like binding pocket defined by structure coordinates of a set of corresponding amino acids, wherein the root mean square deviation of the backbone atoms between said set of corresponding amino acids and said P.a. MurG amino acids is not more than 0.6 A, 0.5 A or 0.4 A, and wherein at least one of said corresponding amino acids is not identical to the P.a. MurG amino acid to which it corresponds; and

ii) instructions for processing said machine-readable data into said three-dimensional structure.

[00112] FIG According to other embodiments, the computer is also for producing the three-dimensional structure of the aforementioned molecules and molecular complexes and comprises the corresponding machine-readable data storage mediums. In one embodiment, the three-dimensional structure is displayed as a graphical representation.

[00113] In one embodiment, the structure coordinates of said molecules or molecular complexes are produced by homology modeling of at least a portion of the structure coordinates of FIG. 1 or FIG. 2. Homology modeling can be used to generate structural models of P.a. MurG homologues or other homologous proteins based on the known structure of P.a. MurG. This can be achieved by performing one or more of the following steps: performing sequence alignment between the amino acid sequence of an unknown molecule against the amino acid sequence of P.a. MurG; identifying conserved and variable regions by sequence or structure; generating structure co-ordinates for structurally conserved residues of the unknown structure from those of P.a. MurG;

generating conformations for the structurally variable residues in the unknown structure; replacing the non-conserved residues of P.a. MurG with residues in the unknown structure; building side chain conformations; and refining and/or evaluating the unknown structure.

[00114] For example, since the protein sequence of the catalytic domains of P.a.

MurG and homologues thereof can be aligned relative to each other, it is possible to construct models of the structures of P. a. MurG homologues, particularly in the regions of the active site, using the P. a. MurG structure. Software programs that are useful in homology modeling include XALIGN [Wishart, D. S. et al, Comput. Appl. BioscL, 10, pp. 687-88 (1994)] and CLUSTAL W Alignment Tool [Higgins D. G. et al., Methods Enzymol, 266, pp. 383-402 (1996)]. See also, U.S. Patent No. 5,884,230. These references are incorporated herein by reference.

[00115] To perform the sequence alignment, programs such as the "bestfit" program available from the Genetics Computer Group [Waterman in Advances in Applied Mathematics 2, 482 (1981), which is incorporated herein by reference] and CLUSTAL W Alignment Tool [Higgins D. G. et al, Methods Enzymol, 266, pp. 383-402 (1996), which is incorporated by reference] can be used. To model the amino acid side chains of homologous P. a. MurG proteins, the amino acid residues in P.a. MurG proteins can be replaced, using a computer graphics program such as "O" [Jones et al, (1991) Acta Cryst. Sect. A, 47: 110-119], by those of the homologous protein, where they differ. The same orientation or a different orientation of the amino acid can be used. Insertions and deletions of amino acid residues may be necessary where gaps occur in the sequence alignment.

[00116] Homology modeling can be performed using, for example, the computer programs SWISS-MODEL available through Glaxo Wellcome Experimental Research in Geneva, Switzerland; WHATIF available on EMBL servers; Schnare et al, J. Mol. Biol, 256: 701-719 (1996); Blundell et al, Nature 326: 347-352 (1987); Fetrow and Bryant, Bio/Technology 11 :479-484 (1993); Greer, Methods in Enzymology 202: 239-252 (1991); and Johnson et al, Crit. Rev. Biochem. Mol Biol. 29: 1-68 (1994). An example of homology modeling can be found, for example, in Szklarz G.D., Life Sci. 61 : 2507-2520 (1997). These references are incorporated herein by reference.

[00117] Thus, in accordance with the present invention, data capable of generating the three dimensional structure of, for example, the above molecules or molecular complexes, or binding pockets thereof, can be stored in a machine-readable storage medium, which is capable of displaying three-dimensional structural information or a graphical three-dimensional representation of the structure. Drug Design

[00118] The P. a. MurG structure coordinates or the three-dimensional graphical representation generated from these coordinates may be used in conjunction with a computer for a variety of purposes, including drug discovery. In certain embodiments, the computer is programmed with software to translate those coordinates into the three- dimensional structure of P. a. MurG.

[00119] For example, the structure encoded by the data may be computationally evaluated for its ability to associate with chemical entities. Chemical entities that associate with P. a. MurG may inhibit or activate P. a. MurG or its homologues, and are potential drug candidates. Alternatively, the structure encoded by the data may be displayed in a graphical three-dimensional representation on a computer screen. This allows visual inspection of the structure, as well as visual inspection of the structure's association with chemical entities.

[00120] Thus, according to another embodiment, the invention provides a method for designing, selecting and/or optimizing a chemical entity that binds to the molecule or molecular complex comprising the steps of:

(a) providing the structure coordinates of said molecule or molecular complex on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; and

(b) designing, selecting and/or optimizing said chemical entity by employing means for performing a fitting operation between said chemical entity and said three-dimensional structural information of said molecule or molecular complex.

[00121] Three-dimensional structural information in step (a) may be generated by instructions such as a computer program or commands that can generate a three- dimensional structure or graphical representation; subtract distances between atoms; calculate chemical energies for a P. a. MurG protein or homologues thereof, or for a complex of P. a. MurG protein or homologues thereof with a chemical entity; or calculate or minimize energies of an association of P. a. MurG protein or homologues thereof to a chemical entity. These types of computer programs are known in the art. The graphical representation can be generated or displayed by commercially available software programs. Examples of software programs include but are not limited to QUANTA® [Molecular Simulations, Inc., San Diego, CA; now part of Accelrys, San Diego, CA], O [Jones et al., Acta Crystallogr. A47, pp. 110-119 (1991)] and RIBBONS [Carson, J. Appl. Crystallogr., 24, pp. 9589-961 (1991)], which are incorporated herein by reference. Certain software programs may imbue this representation with physico-chemical attributes which are known from the chemical composition of the molecule, such as residue charge, hydrophobicity, torsional and rotational degrees of freedom for the residue or segment, etc. Examples of software programs for calculating chemical energies are described below.

[00122] Another embodiment of the invention provides a method for evaluating the potential of a chemical entity to associate with the molecule or molecular complex as described previously.

[00123] This method comprises the steps of: a) employing computational means to perform a fitting operation between the chemical entity and the molecule or molecular complex described before; b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the molecule or molecular complex; and, optionally, c) outputting said quantified association to a suitable output hardware, such as a CRT display terminal, a printer, a CD or DVD recorder, ZIP™ or JAZ™ drive, a disk drive, or other machine-readable data storage device, as described previously. The method may further comprise generating a three-dimensional structure, graphical representation thereof, or both, of the molecule or molecular complex prior to step a). In one embodiment, the method is for evaluating the ability of a chemical entity to associate with the binding pocket of a molecule or molecular complex.

[00124] In another embodiment, the method comprises the steps of:

a) constructing a computer model of a binding pocket of the molecule or molecular complex;

b) selecting a chemical entity to be evaluated by a method selected from the group consisting of assembling said chemical entity; selecting a chemical entity from a small molecule database; de novo ligand design of said chemical entity; and modifying a known agonist or inhibitor, or a portion thereof, of a P. a. MurG protein or homologues thereof; c) employing computational means to perform a fitting program operation between computer models of said chemical entity to be evaluated and said binding pocket in order to provide an energy-minimized configuration of said chemical entity in the binding pocket; and

d) evaluating the results of said fitting operation to quantify the association between said chemical entity and the binding pocket model, thereby evaluating the ability of said chemical entity to associate with said binding pocket.

[00125] In another embodiment, the invention provides a method of using a computer for evaluating the ability of a chemical entity to associate with the molecule or molecular complex, wherein said computer comprises a machine-readable data storage medium comprising a data storage material encoded with said structure coordinates defining said binding pocket and means for generating a three-dimensional graphical representation of the binding pocket, and wherein said method comprises the steps of:

(a) positioning a first chemical entity within all or part of said binding pocket using a graphical three-dimensional representation of the structure of the chemical entity and the binding pocket;

(b) performing a fitting operation between said chemical entity and said binding pocket by employing computational means;

(c) analyzing the results of said fitting operation to quantitate the association between said chemical entity and all or part of the binding pocket; and

(d) outputting said quantitated association to a suitable output hardware.

[00126] The above method may further comprise the steps of:

(e) repeating steps (a) through (d) with a second chemical entity; and

(f) selecting at least one of said first or second chemical entity that associates with said all or part of said binding pocket based on said quantitated association of said first or second chemical entity.

[00127 ] Alternatively, the structure coordinates of the P. a. MurG binding pockets may be utilized in a method for identifying an agonist or antagonist of a molecule comprising a binding pocket of P. a. MurG. In certain embodiments, the method comprises steps of: a) using a three-dimensional structure of the molecule or molecular complex to design, select or optimize a chemical entity;

b) contacting the chemical entity with the molecule or molecular complex;

c) monitoring the catalytic activity of the molecule or molecular complex; and

d) classifying the chemical entity as an agonist or antagonist based on the effect of the chemical entity on the catalytic activity of the molecule or molecular complex.

[00128 ] In one embodiment, step a) is performed using a graphical representation of the binding pocket or portion thereof of the molecule or molecular complex.

[00129] In one embodiment, the three-dimensional structure is displayed as a graphical representation.

[00130 ] In another embodiment, the method comprises the steps of:

b) selecting a chemical entity to be evaluated by a method selected from the group consisting of assembling said chemical entity; selecting a chemical entity from a small molecule database; de novo ligand design of said chemical entity; and modifying a known agonist or inhibitor, or a portion thereof, of a P. a. MurG protein or homologue thereof;

c) employing computational means to perform a fitting program operation between computer models of said chemical entity to be evaluated and said binding pocket in order to provide an energy-minimized configuration of said chemical entity in the binding pocket; and

d) evaluating the results of said fitting operation to quantify the

association between said chemical entity and the binding pocket model, thereby evaluating the ability of said chemical entity to associate with said binding pocket;

e) obtaining said chemical entity; and

f) contacting said chemical entity with said molecule or molecular complex to determine the ability of said compound to activate or inhibit said molecule. Obtaining said chemical entity includes synthesizing the chemical entity, obtaining a commercially available product, or isolating the chemical entity.

[00131] For the first time, the present invention permits the use of molecular design techniques to identify, select and design chemical entities, including inhibitory compounds, capable of binding to P.a. MurG or P.a. MurG-like binding pockets, motifs and domains.

[00132] Applicants ' elucidation of binding pockets on P.a. MurG can provide the necessary information for designing new chemical entities and compounds that may interact with P.a. MurG or P.a. MurG-like substrate in whole or in part.

[00133] Assays to determine if a compound binds to P.a. MurG protein are well known in the art and are exemplified below.

[00134 ] The design of chemical entities that bind to or inhibit P.a. MurG binding pockets according to this invention generally involves consideration of two factors. First, the entity must be capable of physically and structurally associating with parts or all of the P.a. MurG binding pockets. Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions and electrostatic interactions.

[00135] Second, the entity must be able to assume a conformation that allows it to associate with the P.a. MurG binding pockets directly. Although certain portions of the entity will not directly participate in these associations, those portions of the entity 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 entity in relation to all or a portion of the binding pocket, or the spacing between functional groups of an entity comprising several chemical entities that directly interact with the P.a. MurG or P.a. MurG -like binding pockets.

[00136] The potential inhibitory or binding effect of a chemical entity on P.a. MurG binding pockets may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques. If the theoretical structure of the given entity suggests insufficient interaction and association between it and the P.a. MurG binding pockets, testing of the entity is obviated. However, if computer modeling indicates a strong interaction, the compound may then be synthesized and tested for its ability to bind to a P. a. MurG binding pocket. This may be achieved by testing the ability of the molecule to inhibit P. a. MurG protein using the assays described in Example 4. In this manner, synthesis of inoperative compounds may be avoided.

[00137] A potential inhibitor of a P. a. MurG binding pocket may be

computationally evaluated by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the P. a. MurG binding pockets.

[00138 ] One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a P. a. MurG binding pocket. This process may begin by visual inspection of, for example, a P. a. MurG binding pocket on the computer screen based on the P. a. MurG structure coordinates in FIG. 1 or FIG.2, or other coordinates which define a similar shape generated from the machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within that binding pocket as defined supra. Docking may be accomplished using software such as QUANTA® [Molecular Simulations, Inc., San Diego, CA; now part of Accelrys, San Diego, CA] and SYBYL® [Tripos Associates, St. Louis, MO], followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMm® [Accelrys, San Diego, CA] and AMBER.

[00139] Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:

1. GRID [P. J. Goodford, "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem., 28, pp. 849-857 (1985)]. GRID is available from Oxford University, Oxford, UK.

2. MCSS [A. Miranker et al, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method." Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)]. MCSS is available from Molecular Simulations, San Diego, CA. 3. AUTODOCK [D. S. Goodsell et al, "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)]. AUTODOCK is available from Scripps Research Institute, La Jolla, CA.

4. DOCK [I. D. Kuntz et al, "A Geometric Approach to Macromolecule-Ligand Interactions", J. Mol. Biol, 161, pp. 269-288 (1982)]. DOCK is available from University of California, San Francisco, CA.

5. GLIDE [Schrodinger, Portland, Oregon 97204, USA; Thomas A. Halgren, Robert B. Murphy, Richard A. Friesner, Hege S. Beard, Leah L. Frye, W. Thomas Pollard, and Jay L. Banks "Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening", J. Med. Chem., 47 (7), pp. 1750 -1759 (2004)]

[00140] Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or complex of compounds. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of P. a. MurG protein. This would be followed by manual model building using software such as QUANTA® and DISCOVERY STUDIO® [Molecular

Simulations, Inc., San Diego, CA; now part of Accelrys, San Diego, CA], SYBYL®

[00141] Useful programs to aid one of skill in the art in building an inhibitor of a P. a. MurG binding pocket in a step-wise fashion, including one fragment or chemical entity at a time, include:

1. CAVEAT [P. A. Bartlett et al, "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, pp. 182-196 (1989); G. Lauri and P. A. Bartlett, "CAVEAT: a Program to Facilitate the Design of Organic Molecules", J. Comput. Aided Mol. Des. , 8, pp. 51-66 (1994)]. CAVEAT is available from the University of California, Berkeley, CA. 2. 3D Database systems such as ISIS (MDL Information Systems, San Leandro, CA). This area is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992).

3. HOOK [M. B. Eisen et al. , "HOOK: A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site", Proteins: Struct., Fund, Genet., 19, pp. 199-221 (1994)]. HOOK is available from Molecular Simulations, San Diego, CA.

[00142] Instead of proceeding to build an inhibitor of a P. a. MurG binding pocket in a step-wise fashion one fragment or chemical entity at a time as described above, inhibitory or other P. a. MurG binding compounds may be designed as a whole or "de novo" using either an empty binding pocket or optionally including some portion(s) of a known inhibitor(s). There are many de novo ligand design methods including:

1. LUDI [H.-J. Bohm, "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)]. LUDI is available from Molecular Simulations Incorporated, San Diego, CA; now Accelrys, San Diego,CA.

2. LEGEND [Y. Nishibata et al, Tetrahedron, 47, p. 8985 (1991)]. LEGEND is available from Molecular Simulations Incorporated, San Diego, CA; now Acclerys, San Diego, CA.

3. LEAPFROG® [available from Tripos Associates, St. Louis, MO].

4. SPROUT [V. Gillet et al, "SPROUT: A Program for Structure Generation)", J. Comput. Aided Mol Design, 7, pp. 127-153 (1993)]. SPROUT is available from the University of Leeds, UK.

5. NEWLEAD (V. Tschinke and N.C. Cohen, "The NEWLEAD Program: A New Method for the Design of Candidate Structures from Pharmacophoric Hypotheses", J. Med. Chem., 36, 3863-3870 (1993)).

[00143] Other molecular modeling techniques may also be employed in accordance with this invention [see, e.g., N. C. Cohen et al, "Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990); see also, M. A. Navia and M. A. Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202-210 (1992); L. M. Balbes et al., "A Perspective of Modern Methods in Computer- Aided Drug Design", Reviews in Computational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds., VCH, New York, pp. 337-380 (1994); see also, W. C. Guida, "Software For Structure-Based Drug Design", Curr. Opin. Struct. Biology, 4, pp. 777-781 (1994)].

[00144] Once a chemical entity has been designed or selected by the above methods, the efficiency with which that chemical entity may bind to a P. a. MurG binding pocket may be tested and optimized by computational evaluation. For example, an effective P. a. MurG binding pocket inhibitor must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient P.a. MurG binding pocket inhibitors should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, more preferably, not greater than 7 kcal/mole. P.a. MurG binding pocket inhibitors may interact with the binding pocket 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 entity and the average energy of the conformations observed when the inhibitor binds to the protein.

[00145] An entity designed or selected as binding to a P.a. MurG binding pocket may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme and with the surrounding water molecules. Such non-complementary electrostatic interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.

[00146] Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include: Gaussian 94, revision C [M. J. Frisch, Gaussian, Inc., Pittsburgh, PA ©1995]; AMBER, version 4.1 [P. A. Kollman, University of California at San Francisco, ©1995]; QUANTA®/ CHARMm® [Accelrys, San Diego, CA]; Insight II®/Discovery Studio® [Accelrys, San Diego, CA ©2001, 2002]; DelPhi [Accelrys, San Diego, CA ©2001, 2002]; and AMSOL [Quantum Chemistry Program Exchange, Indiana

University]. These programs may be implemented, for instance, using a Silicon

Graphics® workstation such as an INDIG02 with "IMPACT™" graphics. Other hardware systems and software packages will be known to those skilled in the art. [00147] Another approach enabled by this invention, is the computational screening of small molecule databases for chemical entities or compounds that can bind in whole, or in part, to a .a.MurG binding pocket. In this screening, the quality of fit of such entities to the binding pocket may be judged either by shape complementarity or by estimated interaction energy [E. C. Meng et al., J. Comp. Chem., 13, pp. 505-524 (1992)].

[00148] Another particularly useful drug design technique enabled by this invention is iterative drug design. Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three- dimensional structures of successive sets of protein/compound complexes.

[00149] According to another embodiment, the invention provides compounds which associate with a P. a. MurG binding pocket produced or identified by the method set forth above.

[00150] Another particularly useful drug design technique enabled by this invention is iterative drug design. Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three- dimensional structures of successive sets of protein/compound complexes.

[00151 ] In iterative drug design, crystals of a series of protein or protein complexes are obtained and then the three-dimensional structures of each crystal is solved. Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/compound complex, solving the three-dimensional structure of the complex, and comparing the associations between the new protein/compound complex and previously solved protein/compound complexes. By observing how changes in the compound affected the protein/compound associations, these associations may be optimized.

[00152 ] In some cases, iterative drug design is carried out by forming successive protein-compound complexes and then crystallizing each new complex. Alternatively, a pre-formed protein crystal is soaked in the presence of an inhibitor, thereby forming a protein/compound complex and obviating the need to crystallize each individual protein/compound complex. [ 00153 ] In one embodiment, this invention provides for a method of designing a compound which binds to a catalytic domain of a P. a. MurG protein comprising a P. a. MurG binding pocket, wherein said catalytic domain is characterized by:

(i) the atomic coordinates of amino acids 1-357 of SEQ ID NO: l shown in FIG. 1 positioned within a rmsd of 1.2 A or the atomic coordinates of amino acids 1-357 of SEQ ID NO: 1 shown in FIG. 2 positioned within a rmsd of 1.2 A;

(ii) the atomic coordinates of said binding pocket defined by amino acids G14, H15, F17, N124, R143, L186, S189, F242, 1243, M246, A262, L263, T264 and E267 of FIG. 1 or FIG.2 and which are within a rmsd of 1.2 A;

(iii) the atomic coordinates of said binding pocket defined by amino acids G14, HI 5, F17, N124, R143, LI 86, SI 89, F242, 1243, M246, A262, L263, T264 and E267 of FIG. 1 or FIG.2 and which are within a rmsd of 1.2 A;

(iv) the atomic coordinates of said binding pocket defined by amino acids R143, F242, 1243, M246 and E267 of FIG. 1 or FIG.2 and and which are within a rmsd of 1.0 A; or

(v) the atomic coordinates of said binding pocket defined by amino acids R143, F242, 1243, M246 and E267 of FIG. 1 or FIG.2 and which are within a rmsd of 1.0 A;

said method comprising the steps of:

(a) using the atomic coordinates of FIG. 1 or FIG. 2 to build a 3-D computer model of a compound interaction region of said protein comprising at least one of (i) - (v);

(b) assessing the stereochemical complementarity between a compound and said interaction region;

(c) optimizing said stereochemical complementarity in an iterative approach by observing changes in the protein or compound that affect the

protein/compound associations; and

(d) designing a compound which optimizes said protein/compound stereochemical complementarity. [00154] In another embodiment, the invention provides for a method of identifying a potential inhibitor of a P. a. MurG protein comprising a P. a. MurG binding pocket, wherein said method comprising the steps of:

(a) using the structure coordinates of P. a. MurG protein according to FIG. 1 or FIG. 2 to generate a three-dimensional model;

(b) identifying said binding pocket residues, and using said residues to generate a specific three-dimensional (3-D) target;

(c) employing said 3-D target of (b) to design or select said potential inhibitor;

(d) obtaining said potential inhibitor; and

(e) contacting said potential inhibitor with said P. a. MurG protein in vitro to determine the ability of said potential inhibitor to interact with said P. a. MurG protein; whereby the ability to interact is an indication that said potential inhibitor of said P. a. MurG protein is identified. Obtaining said potential inhibitor includes synthesizing the potential inhibitor, obtaining a commercially available product or isolating the potential inhibitor.

[00155] In another embodiment, this invention provides for a method of identifying a candidate inhibitor of a molecule or molecular complex comprising a binding pocket or domain selected from the group consisting of:

(i) a set of amino acid residues that are identical to P. a. MurG amino acid residues of a P. a. MurG binding pocket according to FIG.l or FIG. 2 and as described above, wherein the root mean square deviation of the backbone atoms between said set of amino acid residues and said P. a. MurG amino acid residues is not greater than about 1.0 A; and

(ii) a set of amino acid residues that are identical to P. a. MurG binding pocket amino acid residues according to FIG. 1 or FIG. 2, wherein the root mean square deviation between said set of amino acid residues and said P. a. MurG binding pocket amino acid residues is not more than about 1.0 A;

comprising the steps of:

(a) using a three-dimensional structure of the binding pocket or domain to design, select or optimize a plurality of chemical entities; (b) contacting each chemical entity with the molecule or the molecular complex;

(c) monitoring the inhibition to the catalytic activity of the molecule or molecular complex by each chemical entity; and

(d) selecting a chemical entity based on the inhibitory effect of the chemical entity on the catalytic activity of the molecule or molecular

[00156] In one embodiment, this invention provides for a method for identifying a candidate inhibitor that interacts with a binding site of a P. a. MurG protein or a homologue thereof, comprising the steps of:

(a) obtaining a crystal comprising said P. a. MurG protein, wherein the crystal is in space group P4(l)2(l)2 and has unit cell dimensions a=b= 49.9 angstroms ± 1-2 angstroms, c= 277.4 angstroms ± 1-2 angstroms, alpha= 90 degree, beta= 90 degree, and gamma= 90 degree;

(b) obtaining the structure coordinates of amino acids of the crystal of step

(a) ;

(c) generating a three-dimensional model of said P. a. MurG protein or said homologue thereof using the structure coordinates of the amino acids generated in step

(b) , a root mean square deviation from backbone atoms of said amino acids of not more than 1.0 A;

(d) determining a binding site of said P. a. MurG protein or said homologue thereof from said three-dimensional model; and

(e) performing computer fitting analysis to design or identify the candidate inhibitor which interacts with said binding site.

(f) contacting the designed or identified candidate inhibitor with said P. a. MurG protein or said homologue thereof in vitro in order to determine the effect of the inhibitor on P. a. MurG protein activity.

[00157] In another embodiment, this invention provides the methods of identifying above, wherein the binding site of said P. a. MurG protein or said homologue thereof determined in step (d) comprises the structure coordinates of P. a. MurG protein according to FIG. 1 or FIG.2 of a set of amino acid residues that are identical to P.a. MurG amino acid residues G14, H15, F17, N124, R143, L186, S189, F242, 1243, M246, A262, L263, T264 and E267 wherein the root mean square deviation from the backbone atoms of said amino acids is not more than 1.0 A.

[00158] One embodiment of this invention provides for a method of identifying compounds that bind P. a. MurG protein comprising:

(a) obtaining a 3-D molecular model of P. a. MurG protein;

(b) reducing said model to a 3-D molecular model of a P. a. MurG binding pocket of the P. a. MurG protein;

(c) using the model of (b) in a method of rational drug design to identify candidate compounds that can bind P. a. MurG protein; and

(d) assaying for P P. a. MurG protein activity in the presence of the binding candidate compounds identified in step (c);

to thereby identify compounds that bind P. a. MurG protein. In this invention, the 3-D molecular model of P. a. MurG protein is represented by the structure coordinates of P. a. MurG protein according to FIG. 1 or FIG. 2. In one embodiment, the 3-D molecular model of P. a. MurG protein comprises the structure coordinates of P. a. MurG protein according to FIG. l or FIG.2. In another embodiment, the 3-D molecular model comprises amino acid residues 1-357 of SEQ ID NO: l .

[00159] Another embodiment of this invention provides for a method of identifying compounds that bind a P. a. MurG protein comprising:

(a) obtaining a 3-D molecular model of a P. a. MurG protein;

(c) comparing the model of (b) with a library of 3-D molecular models representing structures of candidate compounds to electronically screen said library;

(d) identifying candidate compounds whose structures electronically fit in model of (b) as compounds that can bind P. a. MurG protein; and

(e) assaying for P. a. MurG protein activity in the presence of the binding candidate compounds identified in step (d);

to thereby identify compounds that bind P. a. MurG protein. In this invention, the 3-D molecular model of P. a. MurG protein is represented by the structure coordinates of P. a. MurG protein according to FIG. 1 or FIG.2. In one embodiment, the 3-D molecular model of P.a. MurG protein comprises the structure coordinates of P.a. MurG protein according to FIG. l or FIG.2. In another embodiment, the 3-D molecular model comprises amino acid residues 1-357 of SEQ ID NO: l .

Structure Determination of Other Molecules

[00160] The structure coordinates of P.a. MurG protein according to FIG. 1 or FIG.2 can also be used to aid in obtaining structural information about another crystallized molecule or molecular complex. This may be achieved by any of a number of well-known techniques, including molecular replacement.

[00161] According to an alternate embodiment, the machine -readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of at least a portion of the structure coordinates of P.a. MurG protein according to FIG. 1 or FIG. 2, or homology model thereof, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.

[00162] In another embodiment, the invention provides a computer for determining at least a portion of the structure coordinates corresponding to X-ray diffraction data obtained from a molecule or molecular complex, wherein said computer comprises:

a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structural coordinates of P.a. MurG according to FIG. 1 or FIG.2, or homology model thereof;

b) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises X-ray diffraction data obtained from said molecule or molecular complex; and c) instructions for performing a Fourier transform of the machine readable data of (a) and for processing said machine readable data of (b) into structure coordinates.

[00163] For example, the Fourier transform of at least a portion of the structure coordinates of P.a. MurG protein according to FIG. 1 or FIG. 2, or homology model thereof may be used to determine at least a portion of the structure coordinates of P.a. MurG homologues.

[00164] Therefore, in another embodiment this invention provides a method of utilizing molecular replacement to obtain structural information about a molecule or molecular complex whose structure is unknown comprising the steps of:

a) crystallizing said molecule or molecular complex of unknown structure;

b) generating an X-ray diffraction pattern from said crystallized molecule or molecular complex;

c) applying at least a portion of the structure coordinates of P.a. MurG protein according to FIG. 1 or FIG.2, or homology model thereof to the X-ray diffraction pattern to generate a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown; and

d) generating a structural model of the molecule or molecular complex from the three-dimensional electron density map.

[00165] In one embodiment, the method is performed using a computer. In another embodiment, the molecule is selected from the group consisting of P.a. MurG and P.a. MurG homologues. In another embodiment, the molecule is a P.a. MurG molecular complex or homologue thereof.

[00166] By using molecular replacement, all or part of the structure coordinates of P.a. MurG protein provided by this invention (and set forth in FIG. 1 or FIG. 2) can be used to determine the structure of a crystallized molecule or molecular complex whose structure is unknown more quickly and efficiently than attempting to determine such information ab initio.

[00167] Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that can not be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures. However, when the crystal structure of a protein containing at least a homologous portion has been solved, the phases from the known structure provide a satisfactory estimate of the phases for the unknown structure.

[00168] Thus, this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of the P.a. MurG protein according to FIG. 1 or FIG. 2, or homology model thereof within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed X-ray diffraction pattern of the crystal of the molecule or molecular complex whose structure is unknown. Phases can then be calculated from this model and combined with the observed X-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown. This, in turn, can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallized molecule or molecular complex [E. Lattman, "Use of the Rotation and Translation Functions", in Meth. Enzymol, 115, pp. 55-77 (1985); M. G. Rossmann, ed., "The Molecular Replacement Method", Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York (1972)].

[00169] The structure of any portion of any crystallized molecule or molecular complex that is sufficiently homologous to any portion of the P.a. MurG can be resolved by this method.

[00170] In a particular embodiment, the method of molecular replacement is utilized to obtain structural information about a P.a. MurG homologue. The structure coordinates of P.a. MurG as provided by this invention are particularly useful in solving the structure of P.a. MurG complexes that are bound by ligands, substrates and inhibitors.

[00171] Furthermore, the structure coordinates of P.a. MurG protein as provided by this invention are useful in solving the structure of MurG proteins that have amino acid substitutions, additions and/or deletions (referred to collectively as "P.a. MurG mutants", as compared to naturally occurring P.a. MurG). These P.a. MurG mutants may optionally be crystallized in co-complex with a chemical entity, such as UDP-N- acetylglucosamine. The crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of wild-type P. a. MurG protein. Potential sites for modification within the various binding pockets of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between P. a. MurG protein and a chemical entity.

[00172] The structure coordinates are also particularly useful in solving the structure of crystals of P. a. MurG protein or P. a. MurG homologues, which is co- complexed with a variety of chemical entities. This approach enables the determination of the optimal sites for interaction between chemical entities, including candidate P. a. MurG inhibitors. For example, high resolution X-ray diffraction data collected from crystals exposed to different types of solvent allows the determination of where each type of solvent molecule resides. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their P. a. MurG inhibition activity.

[00173] All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 1.5-3.4A resolution X-ray data to an R value of about 0.30 or less using computer software, such as X-PLOR (Yale

University, ©1992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth. EnzymoL, vol. 114 & 115, H. W. Wyckoff et ah, eds., Academic Press (1985)), CNS (Brunger et al., Acta Crystallogr. D. Biol. Crystallogr., 54, pp. 905- 921, (1998)) or CNX (Accelrys, ©2000,2001). This information may thus be used to design new P. a. MurG inhibitors.

[00174] In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.

EXEMPLIFICATION

Example 1 : Expression and Purification of P. a. MurG [ 00175 ] Pseudomonas Aeruginosa MurG (M 1 -G357) was cloned into the pET21b vector (Novagen), The c-terminal his-tagged protein was overexpressed using BL21(DE3)pLysS cells (Novagen) in E.coli 2xYT media supplemented with lOOug/ml carbenicillin and 0.34ug/mL chloramphenicol. When an OD₆oo_nm of 0.8 was reached, IPTG was added to a final concentration of ImM. The cell culture was then harvested 3.5 hours post induction and cell pellet stored at -80°C.

[ 00176 ] The MurG cell pellets were thawed at 4°C and resuspended in Buffer 1 : 50mM Tris, pH8.0, 500mM NaCl, lOmM Imidazole, 5mM B-Me, 3% Triton X-100, 10% Glycerol and Protease Inhibitor tablets. (Good protein solubilisation was achieved only in the presence of high salt and triton concentration). Cells were initially disrupted by dounce homogenization and sonicated (3x 30seconds). Resuspended cells were further mechanically disrupted using a Micro fluidizer (Micro fluidics, Newton, MA). The cell debris was removed by centrifugation (24,000rpm, 15min at 4°C) and the supernatant decanted. Protein was purified by Ni/NTA agarose metal affinity chromatography (Novagen).

[ 00177 ] Protein supernatant was incubated for 2 hours at 4°C with pre- equilibrated Nickel-NTA metal affinity resin in Buffer 1. The NiNTA resin was collected by centrifugation (4000g, 4 min) and the non-specifically bound protein was removed by extensively washing with Buffer 2: 50mM Tris, pH 8.0, 500mM NaCl, lOmM Imidazole, 5mM B-Me and 10% Glycerol. MurG protein was eluted using a step imidazole gradient of 5%, 10%, 20% and 30% of 1M Imidazole . Clean MurG eluted at 20% and 30%. MurG was further purified by size-exclusion on a Superdex 200(26/60) column

(Amersham Biotech, Sweden) pre-equilibrated in Buffer 3 : 25mM Hepes (pH 7.0), 500 mM NaCl, 10% (v/v) glycerol, 2 mM DTT. The protein eluted as a symmetric peak at an estimated molecular weight of 76kDa (dimer). Purity of the enzyme was greater than >95%> from Coomassie blue-stained SDS-Polyacrylamide gel and Mass Spec analysis. Purified MurG was buffer exchanged using PD10 columns (GE Healthcare) into 25mM Hepes (pH 7.0), 500 mM NaCl, 10% (v/v) glycerol, 2 mM DTT and 2.24mM decyl-b-d maltopyranoside (Anatrace) at 4°C and concentrated to 10 mg/ml for crystallisation. [ 00178 ] High quality crystals, high quality diffraction and subsequent crystal structure determination of P. a MurG was achieved only from the protein that was eluted at 30% imidazole elution fraction pool.

Example 2: Analysis and characterization of P.a. MurG

[ 00179] Protein was analysed on DLS and Coomassie gel Electrospray Mass Spectrometry data, acquired on a Q-TOF premier (Waters) confirmed the protein identity and the homogeneity of the crystallography sample. The mass was in good agreement with expected values. Peptidic digest confirmed identity of MurG.

Example 3: DSF analysis of UDP-GlcNAc binding to P.a. MurG

[ 00180 ] With an aim for producing substrate-bound crystal structure of P. a MurG and assessing functionality and stability changes on binding UDP-GlcNAc, Differential Scanning Fluorimetry (DSF) measurements were carried out using MurG (25.7uM) and a range of UDP-GlcNAc concentrations (0.41mM to 41mM). The dissociation curve showed a stabilising effect of MurG on substrate binding which is apparent using 0.4 luM UDP-GlcNAc (15-fold protein/substrate). Generally, an excess of 1,000-fold substrate was used to observe a full stabilisation of MurG.

Example 4: Enzymatic characterization of P.a. MurG

[ 00181 ] Materials and reagents: HEPES, UDP-GlcNAc, NaCl, KCl, MgCl₂, glycerol, NADH, Triton X-100 and methanol were supplied by Sigma- Aldrich. DTT was from Melford Laboratories. L-Lactate dehydrogenase (LDH), pyruvate kinase (PK) and phosphoenolpyruvate (PEP) were purchased from Roche. Pseudamonas aeruginosa MurG was as a 2.6 mg/mL solution in 25 mM HEPES (pH 7.0), 500 mM NaCl, 2 mM DTT and 10 % glycerol. Lipid I was supplied as a 0.6 mg/mL solution in a

water:chloroform:methanol mix (kind gift of Prof. Timothy Bugg, Warwick University, UK). The Lipid I solvent was evaporated using a stream of nitrogen gas. Lipid I was reconstituted in 50 mM HEPES (pH 7.9), 15 mM KCl, 5 mM MgCl₂, 0.1% Triton X-100 and 10 % methanol by vortexing for 3 minutes, to give a 1.7 mg/mL (1 mM) solution. Assay: MurG activity was tested using a continuous enzyme-coupled UV absorbance assay as described previously (Biochem. (2002) 41 6829-6833; Chem. Bio. Chem. (2003) 4 603-609). All following solutions were prepared in 50 mM HEPES (pH 7.9), 15 mM KC1, 5 mM MgCl₂, 0.01 % Triton X-100 and 15 % methanol. An assay mix consisting of LDH, PK, UDP-GlcNAc and PEP was prepared. To 30 μΕ of this was added 15 μΕ of a solution that contained Lipid I and NADH. This was equilibrated to 37 °C for 10 minutes. Finally, the reaction was initiated by the addition of 15 MurG solution (various concentrations) and the change in fluorescence due to NADH turnover (l_ex 340 nm, l_em 465 nm) was monitored for 15 minutes at 37 °C using a Spectramax Gemini fluorescence reader (Molecular Devices). Background change in fluorescence (enzyme null experiment) was subtracted from all other data to determine rate of turnover due to MurG. Final concentrations in the assay were as follows: 0.056 to 1.5 μΜ MurG, 40 μΜ Lipid I, 170 μΜ UDP-GlcNAc, 200 μΜ NADH, 500 μΜ PEP, 30 μg/mL PK and 15 μg/mL LDH. Specific activity of MurG was calculated by undertaking a standard curve of NADH and converting change in fluorescence into moles of substrate turned over per second per active site.

[ 00182 ] Enzymatic characterization of P. a. MurG. change in fluorescence with time (slope) observed at various concentrations of enzyme is shown in FIG. 7. Each experiment was carried out in triplicate. Calculated turnover number of MurG (168 nM data point) was 1.0 s^"1 molecules of Lipid II produced per molecule of enzyme per unit time (s ¹). Non-linearity observed in this assay most likely results from overestimation of the Lipid I concentration and substrate depletion.

Example 5: Crystallization of P.a. MurG and Structure determination of P.a. MurG

[ 00183] Crystallization and structure determination. apo-MmG crystals were grown at room temperature using the hanging-drop vapour diffusion method using decyl maltoside as a solubilising detergent, combined with several cycles of streak seeding. Typical crystallisation conditions were 18% PEG3350, 0.1M HEPES pH7.0 and lOmM DTT. For the UDP-GlcNAc complexed crystals, MurG protein at a concentration of 12.5mg/ml was mixed with UDP-GlcNAc (at a final concentration of 50mM) and incubated on ice for 30 minutes. Crystallisation conditions were similar to the apo-fovm. Prior to data collection crystals were soaked in a step-wise procedure in mother liquor containing 50mM UDP-GlcNAc. Crystals of both apo- and substrate -bound MurG were cryo-protected in 30% glycerol and belong to the tetragonal space group P4( 1)2(1)2, with unit cell dimensions a=b=49.9A, c=277.4A. Data were collected on beamline 103 at the Diamond Light Source at a wavelength of 0.9795 A. Following integration and reduction by MOSFLM (31) and SCALA (32), respectively, the apo-MurG crystal structure was solved by PHASER (33) using the as search model a truncated form of the E.coli enzyme (21; residues 7 to 162). Iterative cycles of refinement using Buster (34; 35) and manual rebuilding in COOT (36) led to the final model. Due to structural disorder of the apo- complex, the electron density was missing for several regions of the protein and peptide chain for these regions were not built. The final R-factor and R-free (37) were 0.31 for all data 20-2.2A resolution (98% completeness). The MurG:UDP-GlcNAc complex was solved using the refined apo- crystal structure as an initial model using a similar refinement procedure. The final R-factor and R-free for this complex were 0.23 and 0.29, respectively, for all data 20-2.2A resolution (84% completeness). Both structures have good geometry with Rms deviations in bond lengths and angles equal to 0.01 A and 1.3°, respectively.

[ 00184 ] The crystals structures of apo MurG and the MurG- UDP-N- acetylglucosamine (UDP-GlcNAc) complex, corresponding to FIG's 1 and 2, are shown in Fig 6. The data and refinement statistics for the apo MurG and the MurG- UDP-N- acetylglucosamine (UDP-GlcNAc) complex are shown in Table 1.

Table 1 : Summary of data collection and structure refinement

[ 00185] Results. In general, P. a. MurG enzyme could not be purified using known reagents and methods as for the E.coli enzyme (Ha S, Walker D, Shi Y, Walker S (2000), Protein Sci. 9: 1045-52). We characterized protein preparations prior to crystallization using dynamic light scattering and mass spectrometry (data not shown) to verify the protein sequence and monodispersity and measured UDP-GlcNAc substrate binding and enzymatic activity using dynamic scanning fluorimetry and a UV

fluorescence intensity enzyme assay (FIG. 7).

[ 00186] The 2.2 A resolution X-ray structure of UDP-GlcNAc bound to P. a MurG has many features in common with the E.coli MurG:UDP-GlcNAc crystal structure previously published (Ha S, Walker D, Shi Y, Walker S (2000), Protein Sci. 9: 1045-52; PDB code: INLM). The overall structure of MurG consists of and N-terminal and a C-terminal domain linked by a hinge region (residues 162-166 and 339-341) and separated by a deep cleft. Although N- and C-domains have minimal sequence homology they share high structural homology, and both have an α-β Rossmann-like fold, which is characteristic of domains that bind nucleotides. Incorporated within these domains, consistent with binding negatively charged phosphates from co-factors, are three

Glycine-rich motifs (G-loops), which have the consensus sequence GXGXXG. These G- loops are located at a turn between the carboxyl end of one β-strand and the amino terminus of the adjacent a-helix. These features are shared by the P. a. and E.coli MurG structures.

[ 00187 ] In a structural superposition of the E. coli and P. a. MurG UDP-GlcNAc complexes, although overall sequence similarity between these two related enzymes is only 45%, generally they closely overlap. Almost all of the invariant residues are located at or near the cleft between the two domains. Key residues involved in substrate binding are shown in Figure 8. By superimposing the N-terminal domains of the P.a. and E.coli UDP-GlcNAc complexes, it is clear that the relative orientation of the N- and C-terminal domains is not conserved. In comparison to the P.a. enzyme, the C-terminal domain of E.coli MurG swings away from the N-terminal domain as a rigid-body by an angle of approximately 15° about the inter-domain hinge. This means the cleft is significantly wider in E.coli and consequently the UDP-GlcNAc substrate, which remains in contact with the C-terminal domain, is displaced by approximately 6A.

[ 00188 ] Comparing the P. a. MurG :UDP-GlcN Ac complex structure with the apo-MurG structure indicates that a large degree of flexibility is present in MurG when its donor substrate is not bound. This flexibility is characterized by a large number of loops and complete helices that could not be located in weighted 2Fo-Fc electron density maps, composite simulated annealing omit maps for the apo- crystal structure, even at high resolution. Most flexibility is contained within the C-terminal domain, though analysis of atomic temperature factors (not shown) is consistent with inherent conformational flexibility throughout the apo- structure. According to a previously established numbering scheme (Ha S, Walker D, Shi Y, Walker S (2000), Protein Sci. 9: 1045-52), helices a2 (N-terminal domain) and a5 (C-terminal domain), in addition to several flexible or unstructured regions could not be modelled in the apo-structure. G- loop2 and G-loop3 are highly mobile and also unstructured in the apo- crystal structure. This degree of disorder and flexibility was not observed for a similar pair of E.coli MurG crystal structures (Ha S, Walker D, Shi Y, Walker S (2000), Protein Sci. 9: 1045-52; PDB's INLM and IFOK). Upon binding of the donor sugar UDP-GlcNAc to P.a. MurG the three G-loops become visible in experimental electron density. Therefore it seems that substrate binding induces a more stable enzyme conformation and productive organization of the catalytic machinery. This conformational stabilization is consistent with an observed thermal stabilization upon substrate binding, as observed using differential scanning fluorimetry thermal melt (see FIG. 9), which suggests that a large excess of UDP-GlcNAc is required. [ 00189] Discussion. The crystal structures of P.a. MurG that we have determined contrast to those previously reported for E. coli MurG in several respects. Enzyme kinetic studies suggests that MurG follows a sequential ordered binding of its two substrates to achieve activation, in which the donor substrate binds before the Lipid I acceptor. We have demonstrated that in P.a. binding of the donor UDP-GlcNAc brings about large-scale structural changes to MurG that lead to a catalytic conformation and the formation of the acceptor Lipid I binding pocket (although the putative proximal membrane association site remains poorly resolved). It has been shown in E. coli that MurG associates at the cytoplasmic membrane with MreB, a homologue of actin, which polymerizes to form filaments and is also involved in both cell elongation and division through association with RodA, PBP2, FtsA, FtsQ, FtsW and PBP3 proteins

(Mohammadi T, Karczmarek A, Crouvoisier M, Bouhss A, Mengin-Lecreulx D, den Blaauwen T (2007), Mol Microbiol. 65: 1106-21). Conformational entropy is important in facilitating protein-protein interactions and the inherent flexibility and disorder observed in crystals of apo- MurG may reflect the number and variety of complexes in which it participates. Upon binding UDP-GlcNAc, its three dimensional structure becomes primed for binding lipid I and catalysis at the cytoplasmic membrane.

[ 00190 ] MurG associates with the cytoplasmic surface of bacterial membranes and it has been proposed that the membrane association site is comprised of a

hydrophobic patch of residues in the N-domain, which is surrounded by basic residues, located next to the Lipid I binding site. These residues (63-72 in P.a. MurG) are disordered in both apo- and the UDPGlcNAc complex, suggesting that an interaction with the acceptor Lipid I substrate is required to stabilize the protein in this region.

Residues T 16, HI 9 and Y106 identified in the E.coli enzyme by enzymology as being important for binding Lipid I are present in our crystal structures (residues T12, HI 5 and Y102 in P.a. MurG) and point towards the proposed Lipid I binding site. These residues are invariant in MurG enzymes from all known bacteria. Positioning of residue HI 9 so close to the bound UDP-GlcNAc donor substrate and proposed Lipid I binding site suggests it has a key role in anchoring the lipid acceptor tail.

Claims

CLAIMS What is claimed is:

1. A MurG crystal comprising a polypeptide comprising amino acid residues according to SEQ. ID. NO: l of P. a. MurG protein.

2. The MurG crystal of claim 1 further comprising a ligand or substrate of P. a. MurG protein complexed with the polypeptide.

3. The MurG crystal of claim 2 wherein the ligand or substrate is UDP-N- acetylglucosamine.

4. The MurG crystal of claim 3 wherein the amino acid residues according to SEQ. ID. NO: l have atomic coordinates according to FIG. 1 or FIG. 2.

5. The MurG crystal of any one of claims 1-4 wherein the crystal is in space group P4(l)2(l)2 and has unit cell dimensions a=b= 49.9 angstroms ± 1-2 angstroms, c= 277.4 angstroms ± 1-2 angstroms, alpha= 90 degree, beta= 90 degree, and gamma= 90 degree.

6. The MurG crystal of any one of claims 1-5 wherein the polypeptide further comprises additional amino acids of a tag consisting of LEHHHHHH (SEQ ID: 2) at the C-terminal of the polypeptide.

7. The MurG crystal of any one of claims 1-6 wherein the crystal has an X-ray diffraction quality to provide X-ray diffraction data for determination of atomic coordinates to a resolution of less than or equal to 3 angstroms.

8. A crystallizable composition comprising:

a) a polypeptide comprising amino acid residues according to SEQ. ID. NO: 1 of P. a. MurG protein in a concentration of 5 mg/ml to 20mg/ml;

b) a buffer having a pKa of 6.0 to 8.0; and c) a polyethylene glycol in a concentration of 5% to 50% by volume.

9. The crystallizable composition of claim 8 wherein the buffer is a HEPES (4-(2- Hydroxyethyl)piperazine- 1 -ethanesulfonic acid, N-(2 -Hydroxy ethyl)piperazine-N'-(2- ethanesulfonic acid) buffer or a Tris (Tris(hydroxymethyl)aminomethane) buffer.

10. The crystallizable composition of claim 9 wherein the buffer is a HEPES buffer.

11. The crystallizable composition of any one of claims 1-10 wherein the buffer has a pKa of 6.5 to 7.5.

12. The crystallizable composition of any one of claims 1-10 wherein the buffer has a pKa of 7.0.

13. The crystallizable composition of any one of claims 1-12 wherein the polyethylene glycol has a molecular weight of 1,000 to 20,000 Daltons.

14. The crystallizable composition of claim 13 wherein the polyethylene glycol has a molecular weight of 1,000 to 5,000 Daltons.

15. The crystallizable composition of claim 14 wherein the polyethylene glycol has a molecular weight of 2,500 to 4,000 Daltons.

16. The crystallizable composition of claim 15 wherein the polyethylene glycol has a molecular weight of 3350 Daltons.

17. The crystallizable composition of any one of claims 1-16 wherein the polyethylene glycol is in a concentration from 8% to 22% by volume.

18. The crystallizable composition of claim 17 wherein the polyethylene glycol is in a concentration of 20% by volume.

19. The crystallizable composition of any one of claims 1-18 further comprising a reducing agent in a concentration from 1 mM to 20 mM.

20. The crystallizable composition of claim 19, wherein the reducing agent is in a concentration of 10 mM.

21. The crystallizable composition of claim 19 or 20 wherein the reducing agent is DTT (threo- l,4-Dimercapto-2,3-butanediol) or TCEP (Tris(2-carboxyethyl)phosphine hydrochloride).

22. The crystallizable composition of claim 21 wherein the reducing agent is DTT.

23. The crystallizable composition of any one of claims 8-22 further comprising UDP- N-acetylglucosamine (UDP-GlcNAc) in a molar ratio of 1 :600 to 1 :2,000 P.a. MerG protein to UDP-GlcNAc.

24. The crystallizable composition of any one of claims 8-22 the UDP-GlcNAc is in a molar ratio of 1 : 1 ,000 P.a. MerG protein to UDP-GlcNAc.

25. The crystallizable composition of any one of claims 8-22 wherein the polypeptide further comprises additional amino acids of a tag consisting of LEHHHHHH (SEQ ID: 2) at the C-terminal of the polypeptide.

26. A crystallizable composition comprising:

a) a polypeptide comprising amino acid residues according to SEQ. ID. NO: 1 of P.a. MurG protein in a concentration from 5 mg/ml to 15 mg/ml;

b) a (4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic acid, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) buffer having a pKa of 7.0; and c) a polyethylene glycol having a molecular weight of 3350 Daltons in a concentration from 15% to 22% by volume.

27. The crystallizable composition of claim 26 further comprising UDP-N- acetylglucosamine in a molar ratio of 1 :600 to 1 :2,000 P.a. MerG protein to UDP- GlcNAc.

28. The crystallizable composition of claim 26 further comprising UDP-N- acetylglucosamine in a molar ratio of 1 : 1 ,000 .a.MerG protein to UDP-GlcNAc.

29. The crystallizable composition of any one of claims 26-28 wherein the polypeptide further comprises additional amino acids of a tag consisting of LEHHHHHH (SEQ ID: 2) at the C -terminal.

30. The crystallizable composition of any one of claims 8-29, further comprising ammonium sulfate.

31. The crystallizable composition of claim 30, wherein said ammonium sulfate is in a concentration of 80 mM to 120 mM.

32. A method of forming a crystal of .a.MurG protein comprising the steps of:

a) combining a polypeptide comprising amino acid residues according to SEQ. ID. NO: l of P.a. MurG protein with a reservoir solution, wherein the polypeptide is in a concentration from 5 mg/ml to 20 mg/ml, and wherein the reservoir solution includes a buffer having a pKa of 6.5 to 8.0 and a polyethylene glycol in a concentration from 5% to 50% by volume; and

b) inducing crystal formation to produce a crystal of P.a. MurG protein.

33. A method of forming a crystal of P. a. MurG protein comprising the steps of:

a) combining a polypeptide comprising amino acid residues according to SEQ. ID. NO: l of P.a. MurG protein with UDP-N-acetylglucosamine (UDP-GlcNAc) in a molar ratio of 1 :600 to 1 :2,000 .a.MerG protein to UDP-GlcNAc to result in a mixture of P. a. MurG protein and UDP-GlcNAc; b) combining the mixture of P. a. MurG protein and UDP-GlcNAc with a reservoir that includes a buffer having a pKa of 6.5 to 8.0 and a polyethylene glycol in a concentration from 5% to 50% by volume; and

c) inducing crystal formation to produce a crystal of P. a. MurG protein.

34. The method of claim 33 wherein the UDP-GlcNAc is in a molar ratio of 1 : 1 ,000 P. a. MerG protein to UDP-GlcNAc.

35. The method of claim 33 or 34 wherein the polypeptide further comprises additional amino acids of a tag consisting of LEHHHHHH (SEQ ID: 2) at the C-terminal.

36. The method of any one of claims 32-35, wherein the reservoir further includes ammonium sulfate.

37. The method of claim 36, wherein said ammonium sulfate is in a concentration of 80 mM to 120 mM.

38. A computer comprising executable code for:

a) using structural coordinates of P. a MurG protein according to FIG. 1 or FIG. 2 as a 3-dimensional model of a catalytic domain of P. a MurG protein;

b) analyzing a binding pocket of the 3-dimensional model; and

c) screening in silico library for small molecules that fit into said binding site.

39. The computer of claim 38, further comprising executable code for:

d) controlling a unit for assaying the small molecules determined in step c) in a protein binding assay.

40. A method of preparing a 3-D computer model of a binding pocket of P. a. MurG protein, comprising the step of building a 3-D computer model of a binding pocket of P. a. MurG protein using the atomic coordinates of P. a MurG protein according to FIG. 1 or FIG. 2.

41. A method of identifying a potential inhibitor of P. a MurG protein, comprising the steps of:

a) using the structure coordinates of P. a MurG protein according to FIG. 1 or FIG. 2 to generate a three-dimensional model of a catalytic domain of P. a MurG protein; b) identifying residues of a binding pocket of the P. a MurG protein;

c) generating a specific 3-D target using the binding site residues;

d) employing the specific 3-D target to design or select a potential inhibitor of P. a MurG protein;

e) obtaining the potential inhibitor; and

f) contacting the potential inhibitor with P. a. MurG protein in vitro to determine the ability of said potential inhibitor to interact with said P. a MurG protein, whereby the ability to interact is an indication that said potential inhibitor of P. a MurG protein is determined.

42. A method of designing a compound which binds to a catalytic domain of P. a MurG protein comprising the steps of:

a) using the atomic coordinates of P. a MurG protein according to FIG. 1 or FIG. 2 to build a 3-D computer model of a binding pocket of P. a MurG protein;

b) assessing the stereochemical complementarity between a compound and the binding pocket of MurG protein;

c) optimizing stereochemical complementarity in an iterative approach by observing changes in the protein or compound that affect the protein/compound associations; and

d) designing a compound which optimize said protein/compound

stereochemical complementarity.

43. A method of identifying a potential inhibitor of P. a MurG protein, comprising the steps of: a) using the structure coordinates of P. a MurG protein according to FIG. 1 or FIG. 2 to generate a three-dimensional model;

b) identifying residues of a binding pocket of P. a. MurG protein;

c) generating a specific 3-D target using the binding site residues;

d) employing the 3-D target to design or select a potential inhibitor of P. a MurG protein;

e) obtaining the potential inhibitor; and

f) contacting the potential inhibitor with P. a MurG protein in vitro to determine the ability of said potential inhibitor to interact with said P. a MurG protein, whereby the ability to interact is an indication that said potential inhibitor of P. a MurG protein is determined.

44. A method for identifying a candidate inhibitor that interacts with a binding pocket of P. a MurG protein or homologues thereof, comprising the steps of:

a) obtaining a crystal comprising amino acid residues according to SEQ. ID. NO: l of P. a MurG protein, wherein the crystal is in space group P4( 1)2(1)2 and has unit cell dimensions a=b= 49.9 angstroms ± 1-2 angstroms, c= 277.4 angstroms! 1-2 angstroms, alpha= 90 degree, beta= 90 degree, and gamma= 90 degree;

b) obtaining the structure coordinates of amino acids of the crystal of step a); c) generating a 3-D model of P. a. MurG protein or homologues thereof using the structure coordinates of the amino acids generated in step b), a root mean square deviation from backbone atoms of said amino acids of not more than plus/minus 1.0 angstrom;

d) determining a binding pocket of P. a. MurG protein or homologues thereof from the 3-D model;

e) performing computer fitting analysis to design or identify the candidate inhibitor which interacts with the binding pocket; and

f) contacting the designed or identified candidate inhibitor with P. a. MurG protein or homologues in vitro to determine the effect of the inhibitor on P. a. MurG protein activity.

45. A method of identifying compounds that bind P. a. MurG protein, comprising the steps of:

a) obtaining a 3-D molecular model of P. a. MurG protein according to the method of claim 26;

b) reducing the model to a 3-D molecular model of the binding pocket of P. a. MurG protein ;

c) using the model of b) in a method of rational drug design to identify candidate compounds that can bind P. a. MurG protein; and

d) assaying for P. a. MurG protein activity in the presence of the binding candidate compounds identified in step c) to thereby identify compounds that bind P. a. MurG protein.

46. A method of identifying compounds that bind P. a. MurG protein, comprising the steps of:

a) obtaining a 3-D molecular model of a binding pocket of P. a. MurG protein according to the method of claim 31 ;

b) comparing the model of a) with a library 3-D moelcular models representing structures of candidate compounds to electronically screen the library;

c) identifying candidate compounds whose structures electronically fit in the model of a) as compounds that can bind P. a. MurG protein; and

d) assaying for P. a. MurG protein activity in the presence of the binding candidate compounds identified in step c),

to thereby identify compounds that bind P. a. MurG protein.

47. A method of utilizing molecular replacement to obtain structural information about a molecule or a molecular-complex of unknown structure, comprising the steps of:

a) applying at least a portion of the structure coordinates of P. a MurG protein according to FIG. 1 or FIG. 2, or a homology model thereof to an X-ray diffraction pattern of crystals of the molecule or molecular complex of unknown structure to generate a three-dimensional electron density map of at least a portion of said molecule or molecular complex of unknown structure; and b) generating a structural model of the molecule or molecular complex from the three-dimensional electron density map,

wherein the molecule is sufficiently homologous to P.a. MurG protein comprising amino acid residues according to SEQ. ID. NO: l

48. The method of claim 47, further comprising the steps of:

a) crystallizing said molecule or molecular complexes; and

b) generating an X-ray diffraction pattern from said crystallized molecule or molecular complex.

49. The method of claim 47 or 48, wherein a binding pocket of P.a MurG protein is applied to said to an X-ray diffraction pattern of crystals of the molecule or molecular complex of unknown structure.

50. The method of any one of claims 38-46 and 49, wherein the binding pocket P.a MurG protein comprises R143, F242, 1243, M246 and E267.

51. The method of claim 50 wherein the binding pocket P.a MurG protein comprises G14, H15, F17, N124, R143, L186, S189, F242, 1243, M246, A262, L263, T264 and E267.