SOLUTION AND CRYSTAL STRUCTURES OF ZipA AND ZipA COMPLEX AND USES THEREOF

Abstract: The present invention relates to the three dimensional solution and crystal structures of the C-terminal domain of ZipA ("ZipA185-328"), as well as the three dimensional crystal structure of ZipA185-328 complexed with a C-terminal region of FtsZ. These structures are critical for the design and selection of potent and selective inhibitors of ZipA/FtsZ complex activity, particularly for use as antibiotic agents against Gram negative bacteria. Also provided by the present invention are the inhibitors identified using the three dimensional structures disclosed herein.

Description: SOLUTION AND CRYSTAL STRUCTURES OF ZipA AND ZipA COMPLEX AND USES THEREOF Field of the Invention The present invention relates to the three dimensional solution and crystal structures of the C-terminal domain of ZipA ("ZipA 185 . 328 "), as well as the three dimensional crystal structure of ZipA 185 . 328 complexed with a C-terminal region of FtsZ. These structures are critical for the design and selection of potent and selective inhibitors of ZipA/FtsZ complex activity, particularly for use as antibiotic agents against Gram negative bacteria. Also provided by the present invention are the inhibitors identified using the three dimensional structures disclosed herein. Background of the Invention Bacterial cell division is a complex series of events in which a common feature is the formation of a septum across the middle of the cell (for reviews, see Bramhill, D., Annu. Rev. Cell Dev. Biol 13: 395-424, 1997; Erickson, H.P., Trends Cell Biol. 7: 362-367, 1997; Lutkenhaus and Addinall, Annu. Rev. Biochem 66: 93-116, 1997; Rothfield and Justice, CeU 88: 581-584, 1997). The formation of the septum is driven by the FtsZ ring or "Z ring", a membrane- associated organelle that assembles at the division site well before membrane constriction and remains associated with the ingrowing cell wall until septal closure (Bi and Lutkenhaus, Nature, 354: 161-164, 1991; Lutkenhaus and Addinall, Annu Rev Biochem. 66: 93-116, 1997). This cytoskeleton-like element is believed to be functionally analogous to the contractile ring in eukaryotic cells. During the initial stage of cell division, FtsZ moves from the cytoplasm to accumulate at the division site where it self-assembles into the Z ring. The resulting structure provides a scaffold to recruit other members of the Z ring, which in E. coli involves at least eight additional essential components: FtsA, Ftsl, FtsK, FtsL, FtsN, FtsQ, FtsW and ZipA (for review, see Rothfield and Justice, Cell 88:581-584, 1997). Among them, ZipA (Z interacting protein A) is an integral membrane protein that is recruited to the septum at a very early stage of the division cycle and has been shown to directly bind FtsZ (Hale and de Boer, CeU 88: 175-185, 1997; Hale and De Boer, J, Bacteriol 181: 167-176, 1999; Liu, et al, Mol Microbiol. 31: 1853-1861, 1999). Unlike FtsZ itself, which has a widespread phylogenetic distribution and is conserved among most bacterial cells, ZipA is not that highly conserved and is apparently present in a subset of Gram-negative genomes. No convincing homology is seen in Gram- positive and archaeal genomes. To date, the precise mechanism of the ring assembly and how it affects cell wall invagination remains unknown. A series of experiments have been performed to develop a clearer knowledge of the division cycle in bacteria. On the basis of ZipA depletion studies (Liu et al, Mol Microbiology. 31:1853-1861, 1999), Lutkenhaus and colleagues showed that Z ring formation is independent of ZipA. Their results suggest that ZipA, rather than being a nucleating or a stabilizing factor for the Z ring, functions concurrently with or soon after initial ring formation. As ZipA binds to both the cytoplasmic membrane and FtsZ, it could function as an FtsZ receptor that anchors FtsZ protofilaments to the membrane during invagination of the septum. In addition, two-hybrid experiments and a co-sedimentation assay (Liu et al, Mol. Microbiology, 31:1853-1861, 1999) indicated that the interaction between ZipA and FtsZ is mediated by the C-terminal domains of the proteins. Only the C-terminal domain of ZipA (residues 176-328) is required for interaction with FtsZ, and a region of 63 residues from the C-terminus of FtsZ is required for ZipA binding. Consistent with this, FtsZ mutants missing the last 24 amino acids affect FtsZ localization and cause the formation of punctate aggregates throughout the cell in C. Crescentus (Din et al, Mol. Microbiology. 27:1051-1063, 1998). Similar results on C-terminal deletions were obtained with B. subtilis FtsZ (Wang et al, J, Bacteriol. 179: 5551-5559, 1997). ZipA, a 36.4 kDa protein of 328 amino acids, comprises three domains: a short N-terminal membrane-anchored domain, a central P/Q domain that is rich in proline and glutamine and a C-terminal domain which comprises almost half the protein (residues 185-328). This large domain is implicated to be responsible for interaction with FtsZ. Based on sequence similarity, the majority of FtsZs contain three main regions. A highly conserved N-terminal region of 320 residues has a two domain structure as revealed by X-ray analysis (Lowe and Amos, Nature. 391:203-206, 1998) and is sufficient for polymerization (Wang et al, J. Bacteriol, 179:5551-5559, 1997). It is followed by a variable spacer region and a conserved segment of about ten amino acids at the extreme C-terminus. This C-terminal segment is present in at least 24 organisms in which the FtsZ sequence has been reported. The structure of the C-terminal part of FtsZ has not been determined. To better understand the role of ZipA in cell division and as part of a structure based drug design program, the inventors have determined the high- resolution three dimensional solution and crystal structures of the C-terminal domain of ZipA (hereinafter referred to as "ZipA 185 _ 328 ", having amino acid residues 185-328 of the entire ZipA sequence, where residue 185 corresponds to residue 1 in the crystal and NMR structures), as well as the high resolution three dimensional crystal structure of ZipA 185 . 328 complexed with a C-terminal region of FtsZ. The structures disclosed herein provide the basis with which to design and select new and powerful antimicrobial drugs which are both potent and highly selective for the ZipA/FtsZ complex. Summary of the Invention The present invention relates to the three dimensional structure of the C-terminal domain of ZipA ("ZipA 185 . 328 "), and more specifically, to the crystal and solution structures of the C-terminal domain of ZipA, as determined using crystallography, spectroscopy and various computer modeling techniques. Also provided for is the three dimensional crystal structure of ZipA 185 . 328 complexed with a C-terminal region of FtsZ. Particularly, the invention is further directed to an FtsZ binding active site located on the C-terminal domain of ZipA that provides an attractive target for the rational design of potent and selective ZipA inhibitors which will interfere with bacterial cell division, particularly in Gram negative bacteria. Accordingly, the present invention discloses a solution comprising biologically active ZipA 185 . 328 . Also provided by the present invention is a crystallized ZipA 185 . 328 , alone and complexed with a C-terminal region of FtsZ. The three dimensional structure of ZipA 185 . 328 is provided by the relative atomic structural coordinates of Figure 2, as obtained from spectroscopy data, and Figure 3, as obtained from crystallography data. The three dimensional structure of the crystallized ZipA 185 . 328 :FtsZ complex is provided by the relative atomic structural coordinates of Figure 4. Also provided by the present invention is an FtsZ binding active site of an FtsZ binding protein or peptide, preferably of ZipA 185 . 328 , wherein said active site comprises the relative structural coordinates of amino acid residues VI 0, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved o backbone atoms of said amino acids of not more than 1.5 A. Further provided by the present invention is an FtsZ binding active site of an FtsZ binding protein or peptide, preferably of ZipA 185 . 328 , wherein said active site comprises the relative structural coordinates of amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 according to Figures 2, 3 or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not more than 1.5 A. The solution or crystal structure coordinates of the ZipA 185 . 328 domain or the ZipA 185 . 328 complex (or, in each case, portions thereof, such as an FtsZ or FtsZ-like binding site of the ZipA 185 . 328 domain or complex) as provided by this invention may be stored in a machine-readable form on a machine-readable storage medium, e.g. a computer hard drive, diskette, DAT tape, etc., for display as a three-dimensional shape or for other uses involving computer-assisted manipulation of, or computation based on, the structural coordinates or the three-dimensional structures they define. By way of example, the data defining the three dimensional structure of ZipA 185 . 328 or of a ZipA 185 . 328 complex, or of a portion of ZipA 185 . 328 or of a ZipA 18s _ 328 complex, may be stored in a machine-readable storage medium, and may be displayed as a graphical three-dimensional representation of the relevant structural coordinates, typically using a computer capable of reading the data from said storage medium and programmed with instructions for creating the representation from such data. Accordingly, the present invention provides a machine, such as a computer, programmed in memory with the coordinates of ZipA 185 . 328 or a molecular complex comprising ZipA 185 . 328 , or portions thereof (such as, by way of example, the coordinates of an FtsZ or FtsZ-like binding site of ZipA 185 . 328 ), together with a program capable of converting the coordinates into a three dimensional graphical representation of the structural coordinates on a display connected to the machine. A machine having a memory containing such data aids in the rational design or selection of inhibitors of ZipA/FtsZ activity, including the evaluation of the ability of a particular chemical entity to favorably associate with ZipA or with a ZipA complex as disclosed herein, as well as in the modeling of compounds, proteins, complexes, etc. related by structural or sequence homology to ZipA 185 . 328 , such as various RNA binding proteins comprising a β-α-β split canonical motif (e.g., the U1A spliceosomal protein). The present invention is additionally directed to a method of determining the three dimensional structure of a molecule or molecular complex whose structure is unknown, comprising the steps of first obtaining crystals or a solution of the molecule or molecular complex whose structure is unknown, and then generating X-ray diffraction data from the crystallized molecule or molecular complex and/or generating NMR data from the solution of the molecule or molecular complex. The generated diffraction or spectroscopy data from the molecule or molecular complex can then be compared with the known three dimensional structure of ZipA 185 . 328 as disclosed herein, and the three dimensional structure of the unknown molecule or molecular complex conformed to the known ZipA structure using standard techniques such as molecular replacement analysis, 2D, 3D and 4D isotope filtering, editing and triple resonance NMR techniques, and computer homology modeling. Alternatively, a three dimensional model of the unknown molecule may be generated by generating a sequence alignment between ZipA 185 . 328 and the unknown molecule, based on any or all of amino acid sequence identity, secondary structure elements or tertiary folds, and then generating by computer modeling a three dimensional structure for the molecule using the three dimensional structure of, and sequence alignment with, ZipA 185 . 328 . The present invention further provides a method for identifying a potential inhibitor of ZipA or ZipA/FtsZ activity, comprising the steps of using a three dimensional structure of ZipA 185 . 328 as defined by the relative structural coordinates of amino acids encoding ZipA 185 . 328 to design or select a potential inhibitor, and synthesizing or obtaining said potential inhibitor. The inhibitor may be selected by screening an appropriate database, may designed de novo by analyzing the steric configurations and charge potentials of an empty ZipA 185 . 328 active site in conjunction with the appropriate software programs, or may be designed using characteristics of known inhibitors of ZipA or the ZipA/FtsZ complex in order to create "hybrid" inhibitors. Also provided by the present invention are the inhibitors designed or selected using the methods disclosed herein. Brief Description of the Figures Figure 1A depicts the 144 amino acid sequence encoding the C-terminal domain of E. coli ZipA, which comprises residues 185-328 of E. coli ZipA (referred to herein as "ZipA 185 . 328 "). Figure IB depicts various sequence alignments for the entire ZipA molecule. Figure 2 lists the atomic structure coordinates for the restrained minimized mean structure of ZipA 185 . 328 as derived by NMR spectroscopy. "Atom type" refers to the atom whose coordinates are being measured. "Residue" refers to the type of residue of which each measured atom is a part - i.e., amino acid, cofactor, ligand or solvent. The "x, y and z" coordinates indicate the Cartesian o coordinates of each measured atom's location (A). The last column indicates the temperature factor field, representing the rms deviation of the 30 individual NMR structures about the restrained minimized mean structure. All non-protein atoms are listed as HETATM instead of atoms using PDB conventions. Figure 3 lists the atomic structure coordinates for ZipA 185 . 328 as derived by X-ray diffraction of crystallized ZipA 185 . 328 . Figure headings are as noted above, except "Occ" indicates the occupancy factor, and "B" indicates the "B- value", which is a measure of how mobile the atom is in the atomic structure o (A ). "MOL" indicates the segment identification used to uniquely identify each molecule in the crystal. Each crystallographic asymmetric unit contains two copies of ZipA 185 . 328 . Under "MOL", "A" identifies the first copy of ZipA 185 . 328 , "B" identifies the second copy of ZipA 185 . 328 and "W" identifies water molecules. Figure 4 lists the atomic structure coordinates for a ZipA 185 . 328 :FtsZ- peptide complex as derived by X-ray diffraction of a crystallized ZipA 185 . 328 :FtsZ- peptide complex. Figure headings are as noted for Figure 3, except under "MOL", "A" identifies FtsZ peptide, "B" identifies ZipA 185 . 328 , and "W" identifies water molecules. Detailed Description of the Invention As used herein, the following terms and phrases shall have the meanings set forth below: Unless otherwise noted, "ZipA 185 . 328 " includes both the C-terminal domain of ZipA as encoded by the amino acid sequence of Figure 1A (including conservative substitutions thereof), as well as "ZipA 185 . 328 analogues", defined herein as proteins comprising an FtsZ or FtsZ-like binding active site as defined by the present invention, including, but not limited to, an active site characterized by a three dimensional structure comprising the relative structural coordinates of amino acid residues V10, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 according to Figures 2, 3, or 4, or more preferably, further comprising the relative structural coordinates of amino acid residues A16, D41, V65, K66, and Q87 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone atoms (N, Cct, C, and O) of said o o amino acids of not more than 1.5 A, or more preferably, not more than 1.0 A, or o most preferably, not more than 0.5 A. Alternatively, a ZipA 185 . 328 analogue of the present invention comprises an FtsZ or FtsZ-like binding active site characterized by a three dimensional structure comprising the relative structural coordinates of amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 according to Figures 2, 3 or 4, ± a root mean square deviation o from the conserved backbone atoms of said amino acids of not more than 1.5 A, o or more preferably, not more than 1.0 A, or most preferably, not more than 0.5A. FtsZ includes the C-terminal region of FtsZ, and more particularly, as defined herein, FtsZ includes a 17 amino acid peptide which encompasses the conserved C-terminal region of E. coli FtsZ ( 367 KEPDYLDIPAFLRKQAD 383 ). Unless otherwise indicated, "protein" or "molecule" shall include a protein, protein domain, polypeptide or peptide. "Structural coordinates" are the Cartesian coordinates corresponding to an atom's spatial relationship to other atoms in a molecule or molecular complex. Structural coordinates may be obtained using x-ray crystallography techniques or NMR techniques, or may be derived using molecular replacement analysis or homology modeling. Various software programs allow for the graphical representation of a set of structural coordinates to obtain a three dimensional representation of a molecule or molecular complex. The structural coordinates of the present invention may be modified from the original sets provided in Figures 2, 3 or 4 by mathematical manipulation, such as by inversion or integer additions or subtractions. As such, it is recognized that the structural coordinates of the present invention are relative, and are in no way specifically limited by the actual x, y, z coordinates of Figures 2, 3 or 4. Further, it is recognized that the structural coordinates taken from Figure 3 may be from either molecule of ZipA 185 . 328 in the ZipA 185 . 328 crystallographic asymmetric unit (i.e., from molecule "A" or "B"). An "agent" shall include a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound, antibiotic or drug. "Root mean square deviation" is the square root of the arithmetic mean of the squares of the deviations from the mean, and is a way of expressing deviation or variation from the structural coordinates described herein. The present invention includes all embodiments comprising conservative substitutions of the noted amino acid residues resulting in same structural coordinates within the stated root mean square deviation. It will be obvious to the skilled practitioner that the numbering of the amino acid residues in the various isoforms of ZipA 185 . 328 or in ZipA 185 . 328 analogues covered by the present invention may be different than that set forth herein, or may contain certain conservative amino acid substitutions that yield the same three dimensional structures as those defined by Figures 2, 3 or 4 herein. Corresponding amino acids and conservative substitutions in other isoforms or analogues are easily identified by visual inspection of the relevant amino acid sequences or by using commercially available homology software programs. "Conservative substitutions" are those amino acid substitutions which are functionally equivalent to the substituted amino acid residue, either by way of having similar polarity, steric arrangement, or by belonging to the same class as the substituted residue (e.g., hydrophobic, acidic or basic), and includes substitutions having an inconsequential effect on the three dimensional structure of ZipA 185 . 328 with respect to the use of said structure for the identification and design of ZipA or ZipA/FtsZ complex inhibitors, for molecular replacement analyses and/or for homology modeling. An "active site" refers to a region of a molecule or molecular complex that, as a result of its shape and charge potential, favorably interacts or associates with another agent (including, without limitation, a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound, antibiotic or drug) via various covalent and/or non-covalent binding forces. As such, an active site of the present invention may include both the actual site of FtsZ binding with ZipA 185 . 328 , as well as accessory binding sites adjacent or proximal to the actual site of FtsZ binding that nonetheless may affect ZipA or ZipA/FtsZ activity upon interaction or association with a particular agent, either by direct interference with the actual site of FtsZ binding or by indirectly affecting the steric conformation or charge potential of the ZipA molecule and thereby preventing or reducing FtsZ binding to ZipA 185 . 328 at the actual site of FtsZ binding. As used herein, an "active site" also includes ZipA or ZipA analog residues which exhibit observable NMR perturbations in the presence of a binding ligand, such as FtsZ protein. While such residues exhibiting observable NMR perturbations may not necessarily be in direct contact with or immediately proximate to ligand binding residues, they may be critical ZipA residues for rational drug design protocols. The present invention relates to the three dimensional structure of ZipA 185 . 328 or of a ZipA 185 . 328 analogue, and more specifically, to the crystal and solution structures of ZipA 185 . 328 as determined using crystallography, spectroscopy and various computer modeling techniques. Also provided is the three dimensional structure of ZipA 185 . 328 complexed with an FtsZ C-terminal peptide as determined using crystallography and various computer modeling techniques. The three dimensional solution and crystal structures of uncomplexed ZipA 185 . 328 (disclosed herein at Figures 2 and 3, respectively) and the three dimensional structure of the ZipA 185 . 328 :FtsZ complex (disclosed herein at Figure 4) are useful for a number of applications, including, but not limited to, the visualization, identification and characterization of ZipA 185 . 328 active sites, including the site of FtsZ binding. The active site structures may then be used to predict the orientation and binding affinity of a designed or selected inhibitor of ZipA or the ZipA/FtsZ complex. Accordingly, the invention is particularly directed to the three dimensional structure of a ZipA 185 . 328 active site, including but not limited to the FtsZ binding site. As used herein, ZipA comprises the C-terminal domain of ZipA, and more specifically comprises amino acid residues 185-328 of the ZipA protein ("ZipA 185 . 328 ") or conservative substitutions thereof. The present invention provides a solution comprising a C-terminal domain of ZipA. Preferably, the solution provided for herein comprises ZipA 185 . 328 in a buffer comprising 50 mM potassium or sodium phosphate, 2mM NaN 3 , 50mM KC1 and 50mM deuterated DTT, in either 90% H 2 O/10% D 2 O or 100% D 2 O. Alternatively, the solution may further comprise an FtsZ protein, and may more particularly comprise an FtsZ C-terminal peptide comprising the last 17 amino acids of E. coli FtsZ ( 367 KEPDYLDIPAFLRKQAD 383 ) in a roughly five fold excess to ZipA 185 . 328 concentration. In either case, the concentration of protein or protein complex in the solution should be high enough to yield a good signal-to-noise ratio in the NMR spectrum, but not so high as to result in precipitation or aggregation of the protein or protein complex. By way of example, the solutions of the present invention preferably comprise lmM uncomplexed ZipA 185 . 328 , or 1.5 mM FtsZ peptide to roughly 0.3 mM ZipA 185 . 328 . However, it is understood that one of ordinary skill in the art may devise additional solutions using alternate molar concentrations that are still able to obtain a usable NMR spectrum. A preferred solution pH is around 5.5-6.0. Further, the ZipA 185 . 328 of the solutions of the present invention may be either unlabeled, 15 N enriched or 15 N, 13 C enriched, and is preferably biologically active. As exemplified below, NMR spectra from the solutions of the present invention are preferably obtained at a temperature of 25°C. The secondary structure of the ZipA 185 . 328 used in the solutions of the present invention, based on standard PROCHECK analysis (Laskowski, et al, 1 Appl. Cryst. 26: 283-291, 1993) comprises three alpha helices and a beta sheet having 6 anti-parallel beta strands, wherein the alpha helices and the beta strands are configured in the order βl, αl, β2, β3, β4, β5, 0.2, β6 and <X3. The βl strand comprises amino acid residues 9-16 of ZipA 185 . 328 , 0.1 comprises amino acid residues 25-34 of ZipA 185 . 328 , β2 comprises amino acid residues 37- 39 of ZipA 185 . 328 , β3 comprises amino acid residues 45-48 of ZipA 185 . 328 , β4 comprises amino acid residues 57-63 of ZipA 185 . 328 , β5 comprises amino acid residues 81-88 of ZipA 185 . 328 , α2 comprises amino acid residues 94-112 of ZipA 185 . 328 , β6 comprises amino acid residues 115-117 of ZipA 185 . 328 and 063 comprises amino acid residues 126-144 of ZipA 185 . 328 . Additionally, NMR analysis indicates that residues 122-124 contain beta-strand like characteristics based on observable interstrand NOEs and amide exchange rates, but the conformation of these residues do not conform with the definition of a beta strand region based on standard phi and psi torsion angles. The alpha helices and the beta sheet form surfaces directly opposite each other, and the beta sheet incorporates a shallow hydrophobic cavity o extending roughly 20 A across the beta sheet. In a particular embodiment, the hydrophobic cavity comprises amino acid residues V10, 112, A16, M42, 144, A57, A62, M64, V65, P67 and F85 of ZipA 185 . 328 (or conservative substitutions thereof), and is further characterized by the three dimensional structure characterized by the relative structural coordinates of amino acid residues VI 0, 112, A16, M42, 144, A57, A62, M64, V65, P67 and F85 according to the solution or crystal coordinates of Figures 2, 3 or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not more o o than 1.5 A, or more preferably, not more than 1.0 A, or most preferably, not more than 0.5A. The protein used in the solutions of the present invention includes ZipA 18S . 328 , as well as ZipA 185 . 328 analogues, where said protein comprises an active site characterized by the three dimensional structure comprising the relative structural coordinates of amino acid residues V10, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of o o said amino acids of not more than 1.5 A, or preferably, not more than 1.0 A, or o more preferably not more than 0.5 A. These residues are defined by amino acid residues from ZipA in direct van der Waal and/or hydrogen bond and/or salt bridge contact with the amino acid residues from FtsZ. In a preferred embodiment, the protein used in the solutions of the present invention comprises an active site characterized by a three dimensional structure further comprising the relative structural coordinates of amino acid residues A16, D41, V65, K66, and Q87 according to Figures 2, 3, or 4, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not more o o than 1.5 A, or preferably, not more than 1.0 A, or most preferably, not more o than 0.5 A. These residues are defined by amino acid residues from ZipA within o a 4 A probe of ZipA residues in direct van der Waal and/or hydrogen bond and/or salt-bridge contact with the amino acids residues from FtsZ. In another embodiment, the protein used in the solutions of the present invention comprises an active site characterized by the three dimensional structure comprising the relative structural coordinates of amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not o o more than 1.5 A, or preferably, not more than 1.0 A, or most preferably not o more than 0.5 A. These are amino acid residues on ZipA 185 . 328 which incur a chemical shift perturbation by NMR in the presence of FtsZ. In each case, the three dimensional structure comprising the relative structural coordinates of Figure 4 represents the active site in its bound state with an FtsZ peptide, while the three dimensional structure comprising the relative structural coordinates of Figures 2 and 3 represents the active site in its native or unbound state. In the most preferred embodiment, the protein used in the solution of the present invention is characterized by a three dimensional structure comprising the complete structural coordinates of the amino acids according to Figures 2, 3 or 4, in each case, ± a root mean square deviation from the o conserved backbone atoms of said amino acids of not more than 1.5 A (or more o o preferably, not more than 1.0A, and most preferably, not more than 0.5 A). Also provided by the present invention is a crystallized C-terminal domain of ZipA. In a particular embodiment, the C-terminal domain of ZipA comprises the amino acid residues of Figure 1A, or conservative substitutions thereof ("ZipA 185 . 328 "). The crystal of the present invention effectively diffracts X-rays for the determination of the structural coordinates of ZipA 185 . 328 , and is characterized as being in plate form with space group P21, and having unit cell parameters of a=49.89 A, b=41.74 A, c=71.16 Λ and β=98.26°. Further, a crystallographic asymmetric unit of the crystallized C-terminal domain of ZipA contains two molecules of ZipA 185 . 328 , denoted in Figure 2 as Molecule A and Molecule B. The present invention further provides a crystallized complex comprising a C-terminal domain of ZipA and an FtsZ peptide. In a particular embodiment, the C-terminal domain of ZipA comprises the amino acid residues of Figure 1A, or conservative substitutions thereof ("ZipA 185 . 328 "), and the FtsZ peptide is a C-terminal region of FtsZ from E. coli comprising amino acids 367 KEPDYLDIPAFLRKQAD 383 or conservative substitutions thereof. The crystal complex of the present invention effectively diffracts X-rays for the determination of the structural coordinates of the ZipA 185 . 328 :FtsZ complex, and is characterized as being in elongated plate form with space group P21, and o o o having unit cell parameters of a=36.53 A, b=38.9 A, c=54.54 A and β= 75.89°. Further, the crystallized complex of the present invention consists of one molecule of ZipA 185 . 328 :FtsZ peptide complex in the asymmetric crystal unit. The secondary structure of the ZipA 185 . 328 used in the crystals and crystal complexes of the present invention, based on standard PROCHECK analysis, comprises three alpha helices and a beta sheet having 6 anti-parallel beta strands, wherein the alpha helices and the beta strands are configured in the order βl, 061, β2, β3, β4, β5, 062, β6 and 063. The βl strand comprises amino acid residues 9-16 of ZipA 185 . 328 , 061 comprises amino acid residues 25-34 of ZipA 185 . 328 , β2 comprises amino acid residues 37-39 of ZipA 185 . 328 , β3 comprises amino acid residues 45-48 of ZipA 185 . 328 , β4 comprises amino acid residues 57- 63 of ZipA 185 . 328 , β5 comprises amino acid residues 81-88 of ZipA 185 . 328 , 062 comprises amino acid residues 94-112 of ZipA 185 . 328 , β6 comprises amino acid residues 115-117 of ZipA 185 . 328 and 063 comprises amino acid residues 126-144 of ZipA 185 . 328 . Additionally, NMR analysis indicates that residues 122-124 contain beta strand like characteristics based on observable interstrand NOEs and amide exchange rates, but the conformation of these residues do not conform with the definition of a beta strand region based on standard phi and psi torsion angles. The alpha helices and the beta sheet form surfaces directly opposite each other, and the beta sheet incorporates a shallow hydrophobic cavity o extending roughly 20 A across the beta sheet. In a particular embodiment, the hydrophobic cavity comprises amino acid residues V10, 112, A16, M42, 144, A57, A62, M64, V65, P67 and F85 of ZipA 185 . 328 (or conservative substitutions thereof), and is further characterized by the three dimensional structure characterized by the relative structural coordinates of amino acid residues VI 0, 112, A16, M42, 144, A57, A62, M64, V65, P67 and F85 according to the structural coordinates of Figures 2, 3 or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not more o o than 1.5 A, or more preferably, not more than 1.0 A, or most preferably, not o more than 0.5A. The protein used in the crystals and crystal complexes of the present invention includes ZipA 185 . 328 , as well as ZipA 185 . 328 analogues, wherein said protein comprises an active site characterized by the three dimensional structure comprising the relative structural coordinates of amino acid residues V10, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone o atoms of said amino acids of not more than 1.5 A, or preferably, not more than o o 1.0 A, or more preferably not more than 0.5 A. These residues are defined by amino acid residues from ZipA in direct van der Waal and/or hydrogen bond and/or salt bridge contact with the amino acid residues from FtsZ. In a more preferred embodiment, the protein used in the crystals and crystal complexes of the present invention comprises an active site characterized by a three dimensional structure further comprising the relative structural coordinates of amino acid residues A16, D41, V65, K66, and Q87 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone o atoms of said amino acids of not more than 1.5 A,or preferably, not more than o o 1.0 A, or more preferably not more than 0.5 A. These residues are defined by o amino acid residues from ZipA within a 4 A probe of ZipA residues in direct van der Waal and/or hydrogen bond and/or salt-bridge contact with the amino acids residues from FtsZ. In yet another alternate embodiment, the protein used in the crystals and crystal complexes of the present invention comprises an active site characterized by the three dimensional structure comprising the relative structural coordinates of amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the o conserved backbone atoms of said amino acids of not more than 1.5 A, or o o preferably, not more than 1.0 A, or more preferably not more than 0.5 A. These are amino acid residues on ZipA 185 . 328 which incur a chemical shift perturbation by NMR in the presence of FtsZ. In each case, the three dimensional structure comprising the relative structural coordinates of Figure 4 represents the active site in its bound state with an FtsZ peptide, while the three dimensional structure comprising the relative structural coordinates of Figures 2 and 3 represents the active site in its native or unbound state. Finally, in the most preferred embodiment, the protein used in the crystals and crystal complexes of the present invention comprises the complete structural coordinates according to Figures 2 or 3, in each case, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not o o more than 1.5 A (or more preferably, not more than 1.0 A, and most preferably, o not more than 0.5 A). Molecular modeling methods known in the art may be used to identify an active site or binding pocket of ZipA, a ZipA molecular complex, or a ZipA analogue. Specifically, the structural coordinates provided by the present invention may be used to characterize a three dimensional model of the ZipA molecule, molecular complex or ZipA analogue. From such a model, putative active sites may be computationally visualized, identified and characterized based on the surface structure of the molecule, surface charge, steric arrangement, the presence of reactive amino acids, regions of hydrophobicity or hydrophilicity, etc. Such putative active sites may be further refined using chemical shift perturbations of spectra generated from various and distinct ZipA complexes, competitive and non-competitive inhibition experiments, and/or by the generation and characterization of ZipA or ligand mutants to identify critical residues or characteristics of the active site. The identification of putative active sites of a molecule or molecular complex is of great importance, as most often the biological activity of a molecule or molecular complex results from the interaction between an agent and one or more active sites of the molecule or molecular complex. Accordingly, the active sites of a molecule or molecular complex are the best targets to use in the design or selection of inhibitors that affect the activity of the molecule or molecular complex. The present invention is directed to an active site of ZipA, a ZipA complex or of a ZipA analogue, that, as a result of its shape, reactivity, charge potential, etc., favorably interacts or associates with another agent (including, without limitation, a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound, antibiotic or drug). Accordingly, the present invention is directed to an active site of the ZipA molecule characterized by the three dimensional structure comprising the relative structural coordinates of amino acid residues V10, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of o o said amino acids of not more than 1.5 A, or preferably, not more than 1.0 A, or o more preferably not more than 0.5 A. These residues are defined by amino acid residues from ZipA in direct van der Waal and/or hydrogen bond and/or salt bridge contact with the amino acid residues from FtsZ. In a more preferred embodiment, the active site of the ZipA molecule is characterized by three dimensional structure further comprising the relative structural coordinates of amino acid residues A16, D41, V65, K66 and Q87, in each case, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not o o more than 1.5 A, or preferably, not more than 1.0 A, or more preferably not o more than 0.5 A. These residues are defined by amino acid residues from ZipA o within a 4 A probe of ZipA residues in direct van der Waal and/or hydrogen bond and/or salt-bridge contact with the amino acids residues from FtsZ. In yet another alternate embodiment, an active site of the ZipA molecule is characterized by the three dimensional structure comprising the relative structural coordinates of amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the o conserved backbone atoms of said amino acids of not more than 1.5 A, or o o preferably, not more than 1.0 A, or more preferably not more than 0.5 A. These are amino acid residues on ZipA 185 . 328 which incur a chemical shift perturbation by NMR in the presence of FtsZ. In each case, the three dimensional structure comprising the relative structural coordinates of Figure 4 represents the active site in its bound state with an FtsZ peptide, while the three dimensional structure comprising the relative structural coordinates of Figures 2 and 3 represents the active site in its native or unbound state. In order to use the structural coordinates generated for a crystal or solution structure of the present invention as set forth in Figures 2, 3 and 4, respectively, it is often necessary to display the relevant coordinates as, or convert them to, a three dimensional shape or graphical representation, or to otherwise manipulate them. For example, a three dimensional representation of the structural coordinates is often used in rational drug design, molecular replacement analysis, homology modeling, and mutation analysis. This is typically accomplished using any of a wide variety of commercially available software programs capable of generating three dimensional graphical representations of molecules or portions thereof from a set of structural coordinates. Examples of said commercially available software programs include, without limitation, the following: GRID (Oxford University, Oxford, UK); MCSS (Molecular Simulations, San Diego, CA); AUTODOCK (Scripps Research Institute, La Jolla, CA); DOCK (University of California, San Francisco, CA); Flo99 (Thistlesoft, Morris Township, NJ); Ludi (Molecular Simulations, San Diego, CA); QUANTA (Molecular Simulations, San Diego, CA); Insight (Molecular Simulations, San Diego, CA); SYBYL (TRIPOS, Inc., St. Louis. MO); and LEAPFROG (TRIPOS, Inc., St. Louis, MO). For storage, transfer and use with such programs, a machine, such as a computer, is provided for that produces a three dimensional representation of the ZipA molecule, a portion thereof (such as an active site or a binding site), a ZipA molecular complex, or a ZipA analogue. The machine of the present invention comprises a machine-readable data storage medium comprising a data storage material encoded with machine-readable data. Machine-readable storage media comprising data storage material include conventional computer hard drives, floppy disks, DAT tape, CD-ROM, and other magnetic, magneto-optical, optical, floptical and other media which may be adapted for use with a computer. The machine of the present invention also comprises a working memory for storing instructions for processing the machine-readable data, as well as a central processing unit (CPU) coupled to the working memory and to the machine-readable data storage medium for the purpose of processing the machine-readable data into the desired three dimensional representation. Finally, the machine of the present invention further comprises a display connected to the CPU so that the three dimensional representation may be visualized by the user. Accordingly, when used with a machine programmed with instructions for using said data, e.g., a computer loaded with one or more programs of the sort identified above, the machine provided for herein is capable of displaying a graphical three-dimensional representation of any of the molecules or molecular complexes, or portions of molecules of molecular complexes, described herein. In one embodiment of the invention, the machine-readable data comprises the relative structural coordinates of amino acid residues VI 0, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone o atoms of said amino acids of not more than 1.5 A, or preferably, not more than o o 1.0 A, or more preferably not more than 0.5 A. In an alternate preferred embodiment, the machine-readable data further comprises the relative structural coordinates of amino acid residues A16, D41, V65, K66, and Q87 according to Figures 2, 3 or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not more than 1.5 o o o A, or preferably, not more than 1.0 A, or more preferably not more than 0.5 A. In yet another alternate preferred embodiment, the machine-readable data comprises the relative structural coordinates of amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not more o o than 1.5 A, or preferably, not more than 1.0 A, or more preferably not more o than 0.5 A. In the most preferred embodiment, the machine readable data comprises the complete structural coordinates according to Figures 2, 3 or 4, in σ each case, ± a root mean square deviation of not more than 1.5 A (or more o o preferably, not more than 1.0 A, and most preferably, not more than 0.5 A). The structural coordinates of the present invention permit the use of various molecular design and analysis techniques in order to (i) solve the three dimensional structures of related molecules, molecular complexes or ZipA analogues, and (ii) to design, select, and synthesize chemical agents capable of favorably associating or interacting with an active site of a ZipA molecule, molecular complex or ZipA analogue, wherein said chemical agents potentially act as inhibitors of ZipA or ZipA:FtsZ activity. More specifically, the present invention provides a method for determining the molecular structure of a molecule or molecular complex whose structure is unknown, comprising the steps of obtaining crystals or a solution of the molecule or molecular complex whose structure is unknown, and then generating x-ray diffraction data from the crystallized molecule or molecular complex, and/or generating NMR data from the solution of the molecule or molecular complex. The x-ray diffraction data from the molecule or molecular complex whose structure is unknown is then compared to the x-ray diffraction data obtained from the ZipA or ZipA 185 . 328 :FtsZ crystal of the present invention. Alternatively, the NMR data from the molecule or molecular structure whose structure is unknown is then compared with the NMR data obtained from the ZipA 185 . 328 solution of the present invention. Then, molecular replacement analysis is used to conform the three dimensional structure determined from the ZipA 185 . 328 or ZipA 185 . 328 :FtsZ crystals or crystal complexes of the present invention to the x-ray diffraction data from the unknown molecule or molecular complex, or, alternatively, 2D, 3D and 4D isotope filtering, editing and triple resonance NMR techniques are used to conform the three dimensional structure determined from the ZipA 185 . 328 solution of the present invention to the NMR data from the solution molecule or molecular complex. Molecular replacement analysis uses a molecule having a known structure as a starting point to model the structure of an unknown crystalline sample. This technique is based on the principle that two molecules which have similar structures, orientations and positions will diffract x-rays similarly. A corresponding approach to molecular replacement is applicable to modeling an unknown solution structure using NMR technology. The NMR spectra and resulting analysis of the NMR data for two similar structures will be essentially identical for regions of the proteins that are structurally conserved, where the NMR analysis consists of obtaining the NMR resonance assignments and the structural constraint assignments, which may contain hydrogen bond, distance, dihedral angle, coupling constant, chemical shift and dipolar coupling constant constraints. The observed differences in the NMR spectra of the two structures will highlight the differences between the two structures and identify the corresponding differences in the structural constraints. The structure determination process for the unknown structure is then based on modifying the NMR constraints from the known structure to be consistent with the observed spectral differences between the NMR spectra. Accordingly, in one non-limiting embodiment of the invention, the resonance assignments for the ZipA 185 . 328 solution provide the starting point for resonance assignments of ZipA in a new ZipA: "unsolved agent" complex. Chemical shift perturbances in two dimensional 15 N/1H spectra can be observed and compared between the ZipA solution and the new ZipA: agent complex. In this way, the affected residues may be correlated with the three dimensional structure of ZipA as provided by the relevant residues of Figure 2. This effectively identifies the region of the ZipA: agent complex that has incurred a structural change relative to the native ZipA structure. The 1H, 15 N, 13 C and 13 CO NMR resonance assignments corresponding to both the sequential backbone and side-chain amino acid assignments of ZipA may then be obtained and the three dimensional structure of the new ZipA: agent complex may be generated using standard 2D, 3D and 4D triple resonance NMR techniques and NMR assignment methodology, using the ZipA solution structure, resonance assignments and structural constraints as a reference. Various computer fitting analyses of the new agent with the three dimensional model of ZipA may be performed in order to generate an initial three dimensional model of the new agent complexed with ZipA, and the resulting three dimensional model may be refined using standard experimental constraints and energy minimization techniques in order to position and orient the new agent in association with the three dimensional structure of ZipA. The present invention further provides that the structural coordinates of the present invention may be used with standard homology modeling techniques in order to determine the unknown three-dimensional structure of a molecule or molecular complex. Homology modeling involves constructing a model of an unknown structure using structural coordinates of one or more related protein molecules, molecular complexes or parts thereof (i.e., active sites) . Homology modeling may be conducted by fitting common or homologous portions of the protein whose three dimensional structure is to be solved to the three dimensional structure of homologous structural elements in the known molecule, specifically using the relevant (i.e., homologous) structural coordinates provided by Figures 2, 3 and/or 4 herein. Homology may be determined using amino acid sequence identity, homologous secondary structure elements, and/or homologous tertiary folds. Homology modeling can include rebuilding part or all of a three dimensional structure with replacement of amino acids (or other components) by those of the related structure to be solved. , Accordingly, a three dimensional structure for the unknown molecule or molecular complex may be generated using the three dimensional structure of the ZipA molecule or ZipA molecular complex of the present invention, refined using a number of techniques well known in the art, and then used in the same fashion as the structural coordinates of the present invention, for instance, in applications involving molecular replacement analysis, homology modeling, and rational drug design. Determination of the three dimensional structure of ZipA and its FtsZ binding active site as disclosed herein is critical to the rational identification and/or design of antimicrobial agents that may act as inhibitors of ZipA and/or ZipA: FtsZ complex activity. Alternatively, using conventional drug assay techniques, the only way to identify such an agent is to screen thousands of test compounds until an agent having the desired inhibitory effect on a target compound is identified. Necessarily, such conventional screening methods are expensive, time consuming, and do not elucidate the method of action of the identified agent on the target compound. However, advancing X-ray, spectroscopic and computer modeling technologies allow researchers to visualize the three dimensional structure of a targeted compound. Using such a three dimensional structure, researchers identify putative binding sites and then identify or design agents to interact with these binding sites. These agents are then screened for an inhibitory effect upon the target molecule. In this manner, not only are the number of agents to be screened for the desired activity greatly reduced, but the mechanism of action on the target compound is better understood. Accordingly, the present invention further provides a method for identifying a potential inhibitor of ZipA or of a ZipA: FtsZ complex, comprising the steps of using a three dimensional structure of ZipA or the ZipA:FtsZ complex as defined by the relative structural coordinates of Figures 2, 3 and/or 4 to design or select a potential inhibitor, and synthesizing or obtaining said potential inhibitor. The inhibitor may be selected by screening an appropriate database, may be designed de novo by analyzing the steric configurations and charge potentials of an empty ZipA or ZipA:FtsZ complex active site in conjunction with the appropriate software programs, or may be designed using characteristics of known inhibitors of ZipA or ZipA:FtsZ in order to create "hybrid" inhibitors. An agent that interacts or associates with an active site of ZipA, a ZipA:FtsZ complex or a ZipA analogue may be identified by determining an active site from a three dimensional model of ZipA, a ZipA:FtsZ complex or of a ZipA analogue, and performing computer fitting analyses to identify an agent which interacts or associates with said active site. Computer fitting analyses utilize various computer software programs that evaluate the "fit" between the putative active site and the identified agent, by (a) generating a three dimensional model of the putative active site of a molecule or molecular complex using homology modeling or the atomic structural coordinates of the active site, and (b) determining the degree of association between the putative active site and the identified agent. The degree of association may be determined computationally by any number of commercially available software programs, or may be determined experimentally using standard binding assays. Three dimensional models of the putative active site may be generated using any one of a number of methods known in the art, and include, but are not limited to, homology modeling as well as computer analysis of raw structural coordinate data generated using crystallographic or spectroscopy techniques. Computer programs used to generate such three dimensional models and/or perform the necessary fitting analyses include, but are not limited to: GRID (Oxford University, Oxford, UK), MCSS (Molecular Simulations, San Diego, CA), AUTODOCK (Scripps Research Institute, La Jolla, CA), DOCK (University of California, San Francisco, CA), Flo99 (Thistlesoft, Morris Township, NJ), Ludi (Molecular Simulations, San Diego, CA), QUANTA (Molecular Simulations, San Diego, CA), Insight (Molecular Simulations, San Diego, CA), SYBYL (TRIPOS, Inc., St. Louis. MO) and LEAPFROG (TRIPOS, Inc., St. Louis, MO). In a preferred method of the present invention, the identified active site of ZipA, a ZipA complex or of a ZipA analogue comprises amino acid residues V10, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 (or conservative substitutions thereof) according to Figure 1, and more preferably further comprises amino acid residues A16, D41, V65, K66 and Q87 (or conservative substitutions thereof) according to Figure 1. In an alternate preferred embodiment, the identified active site of ZipA, a ZipA complex or of a ZipA analogue comprises amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 (or conservative substitutions thereof). More preferably, the method of the present invention comprises an identified active site characterized by the three dimensional structure comprising the relative structural coordinates of amino acid residues V10, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone o atoms of said amino acids of not more than 1.5 A, or preferably, not more than o o 1.0 A, or more preferably not more than 0.5 A. In an additional embodiment, the identified active site is characterized by three dimensional structure further comprising the relative structural coordinates of amino acid residues A16, D41, V65, K66 and Q87, in each case, ± a root mean square deviation from the o conserved backbone atoms of said amino acids of not more than 1.5 A, or o o preferably, not more than 1.0 A, or more preferably not more than 0.5 A. In yet another alternate embodiment, the identified active site is characterized by the three dimensional structure comprising the relative structural coordinates of amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 according to Figures 2, 3, or 4, in each case, ± a root mean square deviation from the conserved backbone atoms of said o o amino acids of not more than 1.5 A, or preferably, not more than 1.0 A, or more o preferably not more than 0.5 A. In each case, the three dimensional structure comprising the relative structural coordinates of Figure 4 represents the active site in its bound state with an FtsZ peptide, while the three dimensional structure comprising the relative structural coordinates of Figures 2 and 3 represents the active site in its native or unbound state. The method of the present invention includes additional embodiments comprising conservative substitutions of the noted amino acids which result in the same structural coordinates of the corresponding residues in Figures 2, 3 or 4 within the stated root mean square deviation. The effect of such an agent identified by computer fitting analyses on ZipA, ZipA complex or ZipA analogue activity may be further evaluated computationally, or experimentally by contacting the identified agent with ZipA (or a ZipA complex or analogue) and measuring the effect of the agent on the target's activity. Standard enzymatic assays may be performed and the results analyzed to determine whether the agent is an inhibitor of ZipA activity (i.e., the agent may reduce or prevent binding affinity between ZipA and the relevant substrate, such as FtsZ, and thereby reduce the level or rate of ZipA activity compared to baseline). Further tests may be performed to evaluate the selectivity of the identified agent to ZipA with regard to other ZipA analogues or FtsZ binding targets. Agents designed or selected to interact with ZipA or a ZipA complex must be capable of both physically and structurally associating with ZipA via various covalent and/or non-covalent molecular interactions, and of assuming a three dimensional configuration and orientation that complements the relevant active site of the ZipA molecule. Accordingly, using these criteria, the structural coordinates of the ZipA molecule and molecular complex as disclosed herein, and/or structural coordinates derived therefrom using molecular replacement analysis or homology modeling, agents may be designed to increase either or both of the potency and selectivity of known inhibitors, either by modifying the structure of known inhibitors or by designing new agents de novo via computational inspection of the three dimensional configuration and electrostatic potential of a ZipA or ZipA complex active site. Accordingly, in one embodiment of the invention, the structural coordinates of Figures 2, 3 or 4 of the present invention, or structural coordinates derived therefrom using molecular replacement or homology modeling techniques as discussed above, are used to screen a database for agents that may act as potential inhibitors of ZipA or ZipA complex activity. Specifically, the obtained structural coordinates of the present invention are read into a software package and the three dimensional structure is analyzed graphically. A number of computational software packages may be used for the analysis of structural coordinates, including, but not limited to, Sybyl (Tripos Associates), QUANTA and XPLOR (Brunger, A.T., (1993) XPLOR Version 3.1 Manual Yale University, New Haven, CT) . Additional software programs check for the correctness of the coordinates with regard to features such as bond and atom types. If necessary, the three dimensional structure is modified and then energy minimized using the appropriate software until all of the structural parameters are at their equilibrium/optimal values. The energy minimized structure is superimposed against the original structure to make sure there are no significant deviations between the original and the energy minimized coordinates. The energy minimized coordinates of ZipA or a ZipA complex complexed with a "solved" inhibitor are then analyzed and the interactions between the solved ligand and ZipA or the ZipA complex are identified. The final ZipA or ZipA complex structure is modified by graphically removing the solved inhibitor so that only ZipA or the ZipA complex and a few residues of the solved agent are left for analysis of the binding site cavity. QSAR and SAR analysis and/or conformational analysis may be carried out to determine how other inhibitors compare to the solved inhibitor. The solved agent may be docked into the uncomplexed structure's binding site to be used as a template for data base searching, using software to create excluded volume and distance restrained queries for the searches. Structures qualifying as hits are then screened for activity using standard assays and other methods known in the art. Further, once the specific interaction is determined between the solved inhibitor, docking studies with different inhibitors allow for the generation of initial models of new inhibitors bound to ZipA or the ZipA complex. The integrity of these new models may be evaluated a number of ways, including constrained conformational analysis using molecular dynamics methods (i.e., where both ZipA (or the ZipA complex) and the bound inhibitor are allowed to sample different three dimensional conformational states until the most favorable state is reached or found to exist between the protein (or protein complex) and the bound agent). The final structure as proposed by the molecular dynamics analysis is analyzed visually to make sure that the model is in accord with known experimental SAR based on measured binding affinities. Once models are obtained of the original solved agent bound to ZipA or the ZipA complex and computer models of other molecules bound to ZipA or the ZipA complex, strategies are determined for designing modifications into the inhibitors to improve their activity and/or enhance their selectivity. Once a ZipA or ZipA complex binding agent has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its selectivity and binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Such substituted chemical compounds may then be analyzed for efficiency of fit to the ZipA molecule or the ZipA complex by the same computer methods described in detail above. Various molecular analysis and rational drug design techniques are further disclosed in U.S. Patent Nos. 5,834,228, 5,939,528 and 5,865,116, as well as in PCT Application No. PCT/US98/16879, published as WO 99/09148, the contents of which are hereby incorporated by reference. The present invention may be better understood by reference to the following non-limiting Examples. The following Examples are presented in order to more fully illustrate the preferred embodiments of the invention, and should in no way be construed as limiting the scope of the present invention. Example 1 The 1H, 15 N, 13 C, and 13 CO Assignments of ZipA 185 . 328 were determined and the secondary structure of ZipA ascertained. The uniformly 15 N and 13 C- labeled 144 amino-acid C-terminal domain of ZipA was expressed from the plasmid pEG041 in the E. coli strain BL21(1DE3) plysS. pEG041 is a derivative of pET29 (Novagen, Madison, WI) with a gene insert coding for Metl85 through Ala328 of E. coli ZipA. Cells were grown in M63 minimal media supplemented with 1 mM MgSO 4 , 100 mg/L thiamine, and 2 g/L of 13 C or 12 C glucose. For 15 N-labeling, media contained 2 g/L ( 15 NH 4 ) 2 SO 4 . Cells were grown at 37 °C to an OD 600 of 0.6 to 1.0 and induced with 2 mM IPTG. Two hours after induction, the cells were harvested and resuspended in 50 mM Tris, pH 8.0, 50 mM KCl, 10% glycerol. After addition of 1 mM EDTA and 0.1 mM PMSF, cells were lysed with in a French Press at 16,000 psi and the cell extract was clarified by centrifugation at 100,000 x g for 1 hour. The supernatant was fractionated by a 50% ammonium sulfate cut and the pellet was resuspended in 50 mM Tris, pH 8.0, 10 mM NaCl, 10% glycerol, and dialyzed against the same buffer overnight. The sample was subsequently purified on a Mono Q column using a NaCl gradient in 50 mM Tris, pH 8.0. Fractions containing the C- terminal domain of ZipA were collected, concentrated using a Centriprep-10 filtration device, and passed over a Superosel2 size exclusion column equilibrated in 50 mM Tris, pH 8.0. The yield was 7-10 mg/L of cell culture. The NMR samples contained 1 mM of ZipA 185 . 328 in a buffer containing 50 mM potassium phosphate, 2 mM NaN 3 , 50 mM deuterated DTT, in either 90% H2O/ 10% D2O or 100% D2O at pH 6.0. All NMR spectra were recorded at 25°C on a Bruker DRX 600 spectrometer equipped with a triple-resonance gradient probe. Spectra were processed using the NMRPipe software package (Delaglio et al, Biomol. NMR 6: 277-293, 1995) and analyzed with PIPP (Garrett et al, J, Magn. Reson. 95: 214-20, 1991), NMRPipe and PEAK-SORT, an in-house software package. The assignments of the J H, 15 N, 13 CO, and 13 C resonances were based on the following experiments: CBCA(CO)NH, CBCANH, C(CO)NH, HC(CO)NH, HBHA(CO)NH, HNCO, HNHA, HNCA, HCCH-COSY and HCCH-TOCSY (for reviews, see Bax et al, Methods Enzvmol 239: 79-105, 1994; Clore, G. M., & Gronenborn, Methods Enzvmol. 239: 349-362, 1994). The accuracy of the ZipA 185 . 328 NMR assignments was further confirmed by sequential NOEs in the 15 N-edited NOESY-HMQC spectra and by NOEs between the β-strands observed in the 13 C-edited NOESY-HMQC and 15 N-edited NOESY-HMQC spectra. Since the ZipA 185 . 328 structure was determined to be 06/β topology, the sequential NH r NH i+1 NOEs in the 06-helical regions and the inter-strand NHj-NH j , NH r Cθ6 j and Cα j -CoC j were extremely beneficial in verifying the ZipA 185 . 328 backbone assignments. The secondary structure of ZipA 185 . 328 is based on characteristic NOE data involving the NH, H06 and Hβ protons from 15 N-edited NOESY-HMQC and 13 C -edited NOESY-HMQC spectra, 3J HNC6 coupling constants from HNHA, slowly exchanging NH protons and 13 Cθ6 and 13 Cβ secondary chemical shifts (for reviews, see Wishart & Sykes, Methods Enzymol 239. 1994; Wuthrich, K., NMR of proteins and nucleic acids. John Wiley & Sons, Inc., New York, 1986) . It was determined that the ZipA 185 . 328 NMR structure is composed of three helical regions corresponding to residues 24-34 (06 x ); 94-111 (06 2 ) and 126-144 (06 3 ); and a seven stranded β-sheet region corresponding to residues 11-17 (βi); 38- 40 (β 2 ); 44-47 (β 3 ); 59-64 (β 4 ); 81-86 (β 5 ); 114-119 (β 6 ) and 122-124 (β 7 ). The ZipA 185 . 328 protein was extremely well behaved and provided high- quality NMR data resulting in the complete assignment of the backbone resonances for the C-terminal domain of ZipA. In fact the quality of the NMR data was sufficient to allow for an initial backbone assignment for the protein from just the CBCACONH and CBCANH experiments. There were no observable regions of the protein with significantly sharper or broader line widths or missing resonances. This observation along with the complete assignments for ZipA 185 . 328 implies a well-packed ordered structure and the lack of disordered loops, - or C-terminal regions. Similarly, the side-chain assignments are essentially complete (>95%) where the few missing assignments occurs in residues with long side-chains which are potentially solvent exposed. Example 2 The solution structure of ZipA 185 . 328 was obtained (Figure 2) and the site of FtsZ binding determined. Methods: Uniformly (>95%) 15 N- and 15 N/13 c -labeled recombinant ZipA 185 . 328 was expressed in E. coli and purified as described above. The NMR samples contained 1 mM of ZipA 185 . 328 in a buffer containing 50 mM sodium Phosphate, 2 mM NaN 3 , 50 mM KCl, in either 90% H 2 O/ 10% D 2 O or 100% D 2 O at pH 5.5. All NMR spectra were recorded at 25°C on a Bruker DRX 600 spectrometer equipped with a triple-resonance gradient probe. Spectra were processed using the NMRPipe software package (Delaglio/F. et al, J, Biomol. NMR 6: 277-293, 1995) and analyzed with PIPP (Garrett, et al, J, Magn. Reson. 95: 214-20, 1991). The nearly complete ZipA 185 . 328 assignments of the IH, 15 N, 13 CO, and 13 C resonances were determined as above. For the 2D X H- 15 N HSQC chemical shift perturbation studies, the FtsZ C-terminal peptide, KΕPDYLDIPAFLRKQAD, was in ~ 5-fold excess relative to a ZipA 185 . 328 concentration of 0.3 mM where buffer conditions were as described above (Marion, D. et al, Biochemistry 28: 6150-6, 1989; Zuiderweg, Ε.R.P. & Fesik, S.W. Biochemistry 28: 2387-91, 1989). The present structure is based on the following series of spectra: HNHA (Vuister and Bax, J. AΠ Chem. Soc 115: 7772-7, 1993), HNHB (Archer, et al, J, Magn. Reson. 95: 636-41, 1991), HACAHB-COSY (Grzesiek, et al, J, Am. Chem. Soc 117: 5312-15, 1995), 3D 15 N- (Marion, D. et al. Biochemistry 28: 6150-6, 1989; Zuiderweg, E.R.P. & Fesik, S.W., Biochemistry 28: 2387-91, 1989) and 13 C- edited NOESY (Zuiderweg, et al, J, Magn. Reson 86: 210-16, 1990; Ikura, et al, __ Magn. Reson 86: 204-9, 1990). The 15 N-edited NOESY, and 13 C-edited NOESY experiments were collected with 100 msec and 120 msec mixing times, respectively. The β-methylene stereospecific assignments and χi torsion angle restraints were obtained primarily from a qualitative estimate of the magnitude of 3 J αβ coupling constants from the HACAHB-COSY experiment (Grzesiek, et al, J. AΠ Chem. Soc 117: 5312-15, 1995) and 3 J Nβ coupling constants from the HNHB experiment (Archer, et al, J, Magn. Reson. 95: 636-41, 1991). Val γ-methyl stereospecific assignments were made from the relative intensity of intraresidue NH-CγH and Cθ6H-CγH NOEs (Zuiderweg, et al, Biopolvmers 24: 601-11, 1985). Leu and He %2 torsion angle restraints and Leu δ-methyl stereospecific assignments were obtained primarily from 13 C- 13 C-long range coupling constants (Bax and Pochapsky, 1 Magn. Reson 99: 638-643, 1992) and the relative intensity of intra-molecular NOEs (Powers, R. et al, Biochemistry 32: 6744-62, 1993). The ψ and φ torsion angle restraints were obtained from 3 JN Hβ coupling constants measured from the HNHA experiment (Vuister and Bax, J, Am, Chem. Soc 115: 7772-7, 1993) and from chemical shift analysis using the TALOS program (Cornilescu, et al, J Biomol. NMR 13: 289- 302, 1999). The minimum ranges employed for the ψ, φ, and χ torsion angle restraints were ± 30°, ± 50°, and ± 20°, respectively (Kraulis, P.J. et Z., Biochemistry 28: 7241-57, 1989). The NOEs assigned from the 3D 15 N- and 13 C- edited NOESY experiments were classified into strong, medium, weak and very- weak corresponding to interproton distance restraints (Williamson, et al, J, Mol Biol 182: 295-315, 1985; Clore, G.M. et al, EMBO JL 5: 2729-35, 1986) where non-stereospecifically assignments were corrected appropriately for center averaging (Wuthrich, et al, J. Mol Biol. 169: 949-961, 1983). The structures were calculated using the hybrid distance geometry- dynamical simulated annealing method of Nilges et al (Protein Eng 2: 27-38, 1988) with minor modifications (Clore, et al, Biochemistry 29: 1689-96, 1990) using the program XPLOR (Brunger, A.T. X-PLOR Version 3.1 Manual Yale University, New Haven, CT, 1993), adapted to incorporate pseudopotentials for 3 J NHa coupling constants (Garrett, D.S., et al, J. Magn. Reson., Ser. B 104: 99- 103, 1994), secondary 13 Cθ6/ 13 Cβ chemical shift restraints (Kuszewski, et al, J. Magn. Reson.. Ser. B 106: 92-6, 1995) and a conformational database potential (Kuszewski, et al, Protein Sci. 5: 1067-1080, 1996; Kuszewski, et al, J. Magn. Reson. 125: 171-177, 1997). The target function that is minimized during restrained minimization and simulated annealing comprises only quadratic harmonic terms for covalent geometry, 3 J NHa coupling constants and secondary 13 CC6/ 13 Cβ chemical shift restraints, square-well quadratic potentials for the experimental distance and torsion angle restraints, and a quartic van der Waals term for non-bonded contacts. All peptide bonds were constrained to be planar and trans. There were no hydrogen-bonding, electrostatic, or 6-12 Lennard- Jones empirical potential energy terms in the target function. Competition of the 17 amino-acid peptide with FtsZ for binding to ZipA 18S . 328 was determined in an ELISA format. ZipA 185 . 328 was bound non- specifically to the well of an Immulon 4HBX plate at 1 mg/ml. After removing unbound ZipA 185 . 328 and blocking with BSA, the peptide (1-1000 μM) and FtsZ with an N-terminal FLAG epitope tag (2 μg/ml) were added to the wells for 2 hrs. at room temperature. Unbound FtsZ was washed away and the bound FtsZ was detected via FLAG monoclonal antibody and an anti-mouse IgG horseradish peroxidase conjugate, o-phenylenediamine was used as a substrate for horseradish peroxidase and after the reaction was stopped with diluted sulfuric acid the absorbance at 490 nm was read. Results: The solution structure of ZipA 185 . 328 was obtained (Figure 2). The ZipA 185 . 328 structure is well defined by the NMR data where a total of 2758 constraints were used to refine the structure. This is evident by a best fit superposition of the backbone atoms where the atomic rms distribution of the 30 simulated annealing structures about the mean coordinate positions for residues 5-142 is 0.37 ± 0.04 A for the backbone atoms. The high quality of the ZipA 185 . 328 NMR structure is also evident by the results of the PROCHECK analysis where an overall G-factor of 0.12, a hydrogen bond energy of 0.80 and only 6.9 bad contacts per 100 residues are consistent with a good quality structure comparable to ~1 A X-ray structure. Additionally, most of the backbone torsion angles for non-glycine residues lie within expected regions of the Ramachandran plot where 91.1% of the residues lie within the most favored region of the Ramachandran ψ, φ plot and 8.9% in the additionally allowed region. The ZipA 185 . 328 protein adopts an 06-β fold composed of three 06-helices and a β-sheet consisting of six anti-parallel β-strands. The three helical regions corresponding to residues 25-34 (α 2 ); 94-112 (06 2 ) and 126-144 (06 3 ); and the β-sheet region corresponds to residues 9-16 (β 2 ); 37-39 (β 2 ); 45-48 (β 3 ); 57-63 (β 4 ); 81-88 (β 5 ); and 115-117 (β 6 ). Residues 122-124 were previously assigned (see Example 1) as a seventh β strand based on observable interstrand NOEs and amide exchange rates, but the conformation of these residues do not conform with the definition of a β sheet region based on standard φ and ψ torsion angles. Therefore, the overall topology for ZiρA 185 . 328 is βαββββαβα where the β-sheet and 06 -helices form distinct surfaces directly opposite each other. The short β-strand (β 2 ) and residues 122-124 are located at both edges of the β-sheet and directly follow βj and β πr type turns, respectively. The β- strand β 2 and residues 122-124 effectively enter and exit the β-sheet where a 1 precedes β 2 and 06 3 follows residues 122-124. Thus, the short β-strand (β 2 ) and residues 122-124 occur at the transition point between the β-sheet surface and the α-helical surface. In fact, the β-sheet as a whole does not form a perfectly flat surface, there is an effective twist about the axis perpendicular to the β- strands allowing for the transition from the β-sheet surface to the α-helical surface. This twist is most pronounced for β-strand β 2 and accounts for residues 122-124 not conforming to a standard beta-strand conformation. Another feature of the ZipA 185 . 328 structure are the loops between strands β 4 and β 5 and between strand β x and helix r These loops come in close contact to nearly form a short β-sheet. A short helical region also occurs in the loop between β 4 and β 5 . The combination of the potentially short β-sheet and helical region results in these two loops being relatively well defined. An additional feature of the ZipA 185 . 328 structure is the observation that all of the major loops of the structure effectively protrude from the surface composed of the β-sheet. This has the resulting effect of creating "channels" on the ZipA 185 . 328 surface. This is significantly different from the surface created by the three α-helices, which does not have any distinguishing features. An electrostatic surface potential for ZipA 185 . 328 indicates two distinct clusters within the observed "channels" on the β-sheet surface. These clusters correspond to a negative potential patch composed primarily of D118, D119 and E131 and a large hydrophobic patch comprised of residues V10, 112, A16, F39, M42, 144, A57, A62, M64, V65, P67, P80, and F85. The structure of the β-sheet surface is suggestive of a potential binding site for the interaction of ZipA 185 . 328 with FtsZ. A critical stage in E. coli cell division is the recruitment of ZipA to the FtsZ ring at the division site. It has previously been demonstrated that the recruitmentof ZipA occurs through a direct binding interaction of ZipA with FtsZ (Hale and de Boer, Cell 88:175-185, 1997). Furthermore, it has been determined that the FtsZ binding site within the ZipA structure occurs in the C- terminal domain (Liu, et al, Mol Microbiol 31:1853-1861, 1999). The E. coli FtsZ structure is composed of a large 320 amino acid N-terminal domain that is sufficient for ring formation and a small, variable in length C-terminal domain (Wang, et al, J. Bacteriol. 179: 5551-5559, 1997). Similar to ZipA, the binding site on E. coZi FtsZ for ZipA has been identified as part of the 63 amino acid C- terminal region of the protein (Liu, et al, Mol. Microbiol. 31: 1853-1861, 1999). While an X-ray structure of Methanococcus jannischii FtsZ has been solved, the structure lacks the C-terminal region identified to bind ZipA (Lowe, J., J. Struct. Biol. 124: 235-243, 1998; Lowe and Amos, Nature 391: 203-206, 1998). As a result, there is a lack of structural information pertaining to the interaction of ZipA with FtsZ. The NMR solution structure described herein provides some insight into the nature of the interaction of ZipA with FtsZ since the details of the ZipA 185 . 328 surface suggests a potential FtsZ binding site among the observed "channels" within the β-sheet surface. These results along with the identification that the ZipA binding site in FtsZ is located in the C-terminus led to the exploration of the peptides from FtsZ for the ability to bind ZipA 185 . 328 and disrupt the binding of ZipA 185 . 328 with FtsZ. In order to examine this possibility, a peptide encompassing the last 17 amino acids of E. coli FtsZ ( 367 KEPDYLDIPAFLRKQAD 383 ) was synthesized. Competition experiments demonstrated that this sequence is sufficient to inhibit binding of FtsZ to ZipA 185 . 328 . As a control, a mutation was introduced into the fairly well conserved DIP sequence, which occurs near the end of the C-terminal region of FtsZ. An Asp373Gly mutation within this peptide (using the numbering of the full length protein) led to an approximately 60-fold decrease in inhibition. The same mutation in the full length FtsZ results in a greater than 100-fold increase in the apparent dissociation constant (E. Glasfeld, unpublished results) . The 17 amino acid peptide from the C-terminus of E. coli FtsZ was found to directly bind ZipA 185 . 328 from chemical shift perturbations observed in a 2D X H- 1S N HSQC spectra. It is readily apparent from the 2D WN HSQC spectra that a considerable number of ZipA 185 . 328 residues are perturbed by the presence of the FtsZ C-terminal peptide (A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122). The residues that were significantly perturbed and readily assigned were mapped onto the ZipA 185 . 328 surface and found to occur on the β-sheet surface in the vicinity of the observed "channels". The majority of these residues are located in β-strands β l3 β 2 , β 4 and β 5 and the loops between β-strands β 1 -β 2 and β 4 -β 5 . These results support the identification of the β-sheet as the primary FtsZ binding site on ZipA 185 . 328 . The observed fold for ZipA 185 . 328 has similarities to the split β-06-β fold observed in the ribonucleoprotein motif (RNP) which corresponds to an 06/β sandwich composed of a four-stranded antiparallel β-sheet packed against two 06-helices (Oubridge, et al, Nature 372: 432-8, 1994; Lu and Hall, Biochemistry 36: 10393-10405, 1997; Nagai, et al, Nature 348: 515-20, 1990; Avis, et al, Mol. Biol. 257: 398-411, 1996; Wittekind, et al, Biochemistry 31: 6254-65, 1992; Lee, et al, Biochemistry 33: 13775-86, 1994; and Garrett, et al, Biochemistry 33: 2852-8, 1994). The RNP domain is a very common eukaryotic protein domain that is involved in the recognition of a wide range of RNA structures. The crystal structure of U1A spliceosomal protein complexed with a 21 residue snRNA hairpin turn indicates that the interaction between U1A and the RNA molecule occurs exclusively in the β-sheet (Oubridge, et al, Nature 372: 432-8, 1994). A significant component of the binding is a hydrophobic interaction between the RNA bases and two highly conserved U1A aromatic residues (Allain, et al, Embo J. 16: 5764-5774, 1997). Furthermore, the U1A loop 3 plays a crucial role in defining the surface geometry of the binding interface. These features are very reminiscent of the FtsZ binding site on ZipA 185 . 328 identified from the ZipA 185 . 328 NMR structure and the 2D X H- 15 N HSQC chemical shift perturbations. When the U1A structure is aligned with the ZipA 185 . 328 NMR structure based on the common secondary structure elements (not shown), it is readily apparent from that the U1A RNA binding site correlates very well with the observed chemical shift perturbations observed for ZipA 185 . 328 in the presence of the FtsZ C-terminal peptide. The striking correlation between the U1A RNA binding site and the proposed ZipA 185 . 328 FtsZ C-terminal peptide binding site in addition to the similarity between the protein folds provides further insight into the ZipA-FtsZ interaction. Additionally, the observation that a structural motif is adaptable to function as either an RNA or protein binding domain is an intriguing consequence of the determination of the ZipA 185 . 328 NMR structure. The observed fold for the ZipA 185 . 328 protein in conjunction with the identification of the potential FtsZ binding site is an important step toward understanding the details of the ZipA-FtsZ interaction and establishing a structure-based approach to designing inhibitors of the ZipA- FtsZ complex. Example 3 Presented are X-ray structures ofE. coli ZipA 18S _ 328 (residues 185-328) and the E. coli FtsZ-peptide (residues 367-383) bound to ZipA 185 . 328 . ZipA 185 . 328 represents the domain that binds to FtsZ. The peptide is the consensus segment at the C-terminus of FtsZ that competes with the full length FtsZ for binding to ZipA. The 1.5 A structure of ZipA 185 . 328 reveals a domain of an 06/β topology with a β-sheet surrounded by Oi-helices on one side. On the uncovered side of the sheet, a twist in the β-sheet results in a solvent-accessible cavity across the sheet. The cavity is lined with hydrophobic residues and has space to accommodate a ligand. The 1:1 complex structure, determined at 1.95 A resolution, shows that the peptide occupies the entire cavity of ZipA 185 . 328 . Upon binding, two segments of the peptide adopt extended and 06 -helical conformations, respectively. This conformation directs six side chains of the peptide toward interaction with the hydrophobic surface of the cavity. Two hydrogen bonds between main-chain atoms along the peptide and ZipA 185 . 328 residues from the β-sheet provide an anchor that is independent of peptide sequence. The FtsZ-peptide causes small conformational changes in the ZipA 185 . 328 structure and does not appear to bind to other sites on ZipA 185 . 328 . Methods: A) Expression and Purificaήon of ZipA 185 . 328 and Se-Met ZipA 185 . 828 ZipA 185.328 was cloned into a pΕT derived vector and expressed in BL21DΕ2pLysS Escherichia coli. Cells were grown in a Biostat C-10 (10L) vessel (B. Braun Biotech) using rich media at 37°C and induced for 4 hours with 1 mM IPTG. Se-Met labeled expression of ZipA 185 . 328 was carried out in LeMaster media in BL21DE3pLysS Escherichia coli at 37°C. Cultures were induced for 4 hours with 1 mM IPTG. Cells expressing ZipA 185 . 328 were resuspended in buffer containing 25 mM Hepes, pH 7.5, 2 mM DTT, and 0.1 mM PMSF, and lysed by passage through a Microfluidizer (Microfluidics Corporation, Newton, MA). Cleared lysate was loaded onto a QAE Toyopearl column pre-equilibrated with 20 mM Tris, pH 8.0, and ZipA 185 . 328 was then eluted with a linear 0.-0.5 M NaCl gradient. ZipA 185 . 328 containing fractions were passed through a Hydroxyapatite column (BioRad) and dialyzed against 20 mM Tris, pH 8.0 overnight, at 4°C. The dialyzed protein was subjected to FPLC anion exchange chromatography using Mono Q column (Pharmacia). Greater than 93% pure ZipA 185 . 328 was eluted with a linear 0.-0.5 M NaCl gradient. Fractions containing the major peak were pooled and applied to a TSK-G3000SW size exclusion column. The final product was subjected to SDS-PAGE analysis, exchanged into buffer containing 20 mM Tris, pH 8.0, concentrated to 25mg/ml, and used for crystallization. Se-Met ZipA 185 . 328 was purified following the same procedure as native ZipA 185 . 328 . B) Crystallization of ZipA 185 . 328 and Se-Met ZipA 185 . 328 Crystallization conditions for ZipA 185 . 328 were determined from the sparse matrix screens (Hampton Research). Screening was done using hanging drop vapor diffusion by combining lμl of protein solution (25mg/ml in 20 mM Tris, pH 8.0) with lμl of well solution at both 18°C and 4°C. Initially, ill- formed crystals of ZipA 185 . 328 grew spontaneously at 18°C in a mother liquor consisting of 25% PEG 6000 and 100 mM MES, pH 6.0. To produce diffraction quality crystals of native ZipA 185 . 328 , a streak seeding was used to seed pre- equilibrated (~ 3 hours) 1-1 μl drops containing 25 mg/ml ZipA 185 . 328 , 20% PEG 6000, and 100 mM MES, pH 6.0. Monoclinic plate-like crystals (space group P21; a = 49.89 A, b = 41.74 A, c = 71.16 A, β = 98.26°; two molecules per asymmetric unit; 37% solvent content) developed overnight and reached their maximum size (0.5 x 0.8 x 0.3 mm 3 ) in 3-4 days. Se-Met ZipA 185 . 328 crystallized under the same conditions using a similar seeding technique, with the native crystals as seeds. Differences in cell dimensions were less than 0.6%. C) Crystallization of ZipA 185 . 328 with the FtsZ-peptide A 17 amino acid peptide which encompasses the conserved C-terminal region of E. coZi FtsZ ( 367 KEPDYLDIPAFLRKQAD 383 ) was synthesized for co- crystallization trials. To prepare FtsZ-peptide stock solution (20 mM), FtsZ- peptide powder was dissolved in 20 mM Tris, pH 8.0. A molar excess of the FtsZ-peptide was added to the protein (25mg/ml) such that the final mixture contained 1.3:1 FtsZ-peptide vs ZipA 185 . 328 . Crystallization conditions were again found using PEG 6000 as precipitant (PEG 6K Grid Screen, Hampton Research), except that ZipA 185 . 328 :FtsZ-peptide co-crystals appeared under basic pH (30% PEG 6000 and 100 mM Bicine, pH 9.0). Poor quality crystals grew spontaneously in 4-5 days as clusters of thin elongated plates. Since these crystals were not consistently reproducible, a streak seeding technique was used with crystals of the ZipA 185 . 328 alone as seeds. As in the case of ZipA 185 . 328 , 1-1 μl drops (ZipA 185 . 328 :FtsZ-peptide, 25-30% PEG 6000, and 100 mM Bicine, pH 6.0) were pre-equilibrated (~ 3 hours) prior to cross-seeding. The best monocrystals grew over a period of 2-4 days with a maximum size of 0.2 x 0.2 x 1.0 mm 3 . They belonged to space group P21 (a =36.53 A, b =38.9 A, c =54.54 A, β =75.89°) with 1:1 complex per asymmetric unit and 32% solvent content. D) Data collection and processing Prior to data collection, all crystals were cryoprotected and flash cooled under a gaseous nitrogen stream at 100K. Both native and Se-Met crystals of ZipA 185 . 328 were soaked (~1 min) in a solution containing mother liquor (pH 6.0), 15% ethylene glycol and 35% PEG 4000. Using in-house RAXIS IV mounted on a Rigaku RUH2R rotating anode, two data sets were collected for phase determination: the 1.9 A data for the native ZipA 185 . 328 crystals (180 frames with 1° oscillation) and the 1.85 A data for the Se-Met form of the protein (360 frames with 1° oscillation). For each data set, a single crystal was used. For refinement purposes the high resolution native data set (1.5 A) was collected at beamline 5.0.2 at the Advanced Light Source using a Quantum 4 CCD detector (Area Detector Systems). These data were obtained from the same crystal which was used for in-house data collection. As in the case of ZipA 185 . 328 , a co-crystal of ZipA 185 . 328 :FtsZ-peptide was soaked (~1 min) in a solution containing 15% ethylene glycol, 35% PEG 4000 plus the mother liquor at pH 9.0. The 1.95 A data set was collected from a single crystal (180 frames with 1° oscillation) using in house RAXIS IV imaging plate system. All the data were integrated with DENZO and then scaled and merged with SCALEPACK (Otwinowski, Data Collection and Processing. L.Sawyer, et al, eds. (Daresbury, U.K.: Science and Engineering Council): 56- 62, 1993). Most of the subsequent processing used the CCP4 programs (CCP4, Acta Crvstallogr.. D50: 760-763). E) Structure Determination The data from Se-Met derivative were scaled to the native data to a resolution of 1.9 A (SCALEIT in CCP4) and isomorphous difference Patterson synthesis along with the anomalous Patterson were calculated at 2 A. Sixteen selenium sites were located using these Pattersons and from a double difference Fourier analysis (FFT in CCP4). The N-terminal Se-Met in both ZiρA 185 . 328 molecules was disordered. Refinement of occupancies, coordinates, as well as anomalous scatterer parameters, and phase calculation were performed with MLPHARE (Otwinowski, Data Collection and Processing, L.Sawyer, et al, eds. (Daresbury, U.K.: Science and Engineering Council): 56-62, 1993). Phasing statistics were generated by MLPHARE (not shown). The initial SIRAS map calculated at 2 A was solvent-flattened using DM (Cowtan and Main, Acta Crvstallogr.. D42: 43-48, 1996), assuming 35% solvent- content. Experimental maps were calculated using SHARP (de la Fortelle and Bricogne, Methods Enzymol. 276: 494-523, 1997) and subsequent density modification by SOLOMON (CCP4, Acta Crvstallogr., D50: 760-763). The maps were calculated using all sixteen sites that were identified with MLPHARE phases. The final map was significantly better in terms of connectivity and resolution than that obtained by MLPHARE and DM. Because both algorithms produced correlated and clearly interpretable maps, all density-modified and unmodified SIRAS maps were used to build 100% complete model using X-AUTOFIT within QUANTA (MSI). This model was then used as the initial model for refinement against the 1.5 A resolution native data set. Refinement and map calculations were done in CNS (Brunger et al, Acta Crvstallogr.. D54: 905-921, 1998). At all stages, data from 20.0 to 1.5 A, with | F obs | > 0, where included, with 5% of omitted reflections for R^ calculation. The minimization included a bulk- solvent correction coupled with simulated annealing, positional and individual B factor refinement. Water molecules were located from electron density > 3σ in F 0 -F c maps. The final model (R work = 19.8%, and R^ = 21.7%) contains residues A6-A144, B5-B144 and 422 water molecules (Figure 2). All non- glycine φ and ψ angles lie in the allowed regions of the Ramachandran plot, with 93.7% in the most favored regions and 6.3% in additional allowed regions. Residues A1-A5, B1-B4 were not detected in the electron density maps because of disordering. ZipA 185 . 328 was located using the final model of the ZipA 185 . 328 monomer (residues B6-B144) in rotation and translation searches with AmoRe (Nevaza, Acta Crvstallogr.. A50: 157-163, 1994). All residues of ZipA 185 . 328 were used without truncation, and all the B factors were used without alterations. This model provided unambiguous rotation and translation function solutions. The rigid body refined model gave R factor of 44.2% and correlation coefficient of 55.6% for all data between 12-3 A. The search model was immediately subjected to simulated annealing refinement coupled with a bulk solvent correction as implemented in CNS (Brunger et at, Acta Crvstallogr.. D54: 905- 921, 1998). This resulted in R^ = 32% and R free = 38.7% for 25-1.95 A data, with 10% randomly selected reflections for R^ calculation. This refined model was used to calculate the 1.95 A F 0 -F c map which showed clear electron density for the bound FtsZ-peptide (not shown). All 17 amino acid residues of the FtsZ-peptide were fitted into this map and the refined model of ZipA 185 . 328 was rebuilt using the 1.95 A 3F 0 -2F C map. After three cycles of rebuilding, minimization (positional plus individual B factors refinement) converged to R^ of 20.5% and R^ = 25.1%. The final model contains ZipA 185 . 328 residues 1-144, FtsZ-peptide residues 1-17 and 204 water molecules (Figure 3). All non- glycine φ and ψ angles lie in the allowed regions of the Ramachandran plot, with 94.1% in the most favored regions and 5.9% in additional allowed regions. Side chains for peptide residues 1-2 have weak electron densities, therefore the polyalanines represent this region in the model. The N-terminus of ZipA 185 . 328 (residues 1-5), instead, stabilized and was clearly visible in all electron-density maps, probably because of the tighter crystal packing. Results: Unless stated otherwise, residues 185-328 of the full length ZipA are equivalent to residues 1-144 of ZipA 185 . 328 , and residues 367-383 of the full length FtsZ are equivalent to residues 1-17 of the FtsZ-peptide. A) Structure Determination The C-terminal domain of ZipA (ZipA 185 . 328 , residues 185-328) was expressed in E. coli and purified to homogeneity as described above. Crystals were grown in hanging drops from PEG 6000, and 100 mM MES at pH 6.0. Plate-like crystals (0.5 x 0.8 x 0.3 mm 3 ) diffracted to 1.9 A resolution in-house and to 1.5 A using synchrotron radiation. The crystals belonged to space group P21 (a = 49.89 A, b = 41.74 A, c = 71.16 A, β = 98.26°) with two molecules per asymmetric unit and 37% solvent content. Diffraction data were obtained from a crystal of the native protein and from a crystal using protein in which selenomethionine (Se-Met) had been substituted for methionine. The Se-Met form of the protein crystallized under the same conditions using the native crystals as seeds. Both the native and the selenomethionine data were collected at 1 = 1.5418 A on an in-house Rigaku RAXIS imaging plate system, mounted on a Rigaku rotating anode. The structure was determined to a resolution of 2 A by single isomorphous replacement with anomalous scattering (SIRAS). Initial experimental SIRAS phases were subsequently improved by density modification, and resulted in an electron density map of superior quality. The atomic model has been refined using a high resolution native data set (1.5 A) collected at beamline 5.0.2 at the Advanced Light Source. The final model of ZipNi 85 - 328 (R work = 19.8%, R ftee = 21.7%) contains two copies of the protein: residues A6-A144 and B5-B144 and 422 water molecules (Figure 2). A mixture of ZipA 185 . 328 with a synthetic peptide corresponding to the E. coli FtsZ residues 367-383 (KEPDYLDIPAFLRKQAD) was prepared for co- crystallization trials as described. As with the crystals obtained without the FtsZ-peptide, the material crystallized using PEG 6000 as a precipitant, except that the reservoir consisted of 100 mM Bicine basic buffer, pH 9.0. The 1:1 complex crystallized as elongated plates (0.2 x 0.2 x 1.0 mm 3 ) in a space group P21 (a = 36.53 A, b = 38.9 A, c = 54.54 A, β = 75.89°) with one copy per asymmetric unit and a solvent content of 32%. To produce diffraction quality crystals, crystals of the ZipA 185 . 328 alone were used to seed drops containing the FtsZ-peptide :ZipA 185 . 328 mixture. A high resolution (1.95 A) data set was collected from a single crystal using in-house R-Axis IV and a Rigaku rotating anode. The structure was determined by molecular replacement, with ZipA 185 . 328 as a search model. This model was used to calculate the difference Fourier map which showed unambiguous density for the bound FtsZ-peptide. The structure was refined to 1.95 A (R^ = 20.5%, R free = 25.1%), and the final model contains ZipA 185 . 328 residues 1-144, FtsZ-peptide residues 1-17 and 204 water molecules (Figure 3). B) Overall structure of ZipA 185 . 328 The overall structure of the Zip 185 . 328 monomer is of 06/β topology. The domain (residues 5-144) is a six-stranded antiparallel β-sheet packed against three 06-helices. The core of the domain represents a well known structural motif, the split β-α-β fold (Orengo and Thorton, Structure. 1: 105-120, 1993). The motif consists of a three-stranded antiparallel β-sheet (βl, β5, β6) and one 06-helix (062), with topology βl, β5, 062, β6. This fold, found in a dozen of ribosomal proteins, is the 'common' motif for RNA-binding domains (Yonath and Franceschi, Nature Str. Biology. 4: 3-5, 1997). In these domains, the connection between the first (βl) and the second strand (β5) is variable and sometimes constitutes a separate domain (Nikonov et al, EMBO , 15: 1350- 1359, 1996). In the structure of ZipA 185 . 328 , the insert between βl and β5 (residues 23-80) is composed of one 06-helix (061) and three antiparallel strands (β2, β3, β4) directly adjacent to strand β5, thus extending the β-sheet of the motif. The third 06-helix (063) is found C-terminal to the motif. The connectivity scheme for the whole domain is βl-06l-β2-β3-β4~β5-C62-β6-α3. The connections between the secondary structural elements are mostly reverse 3- turns except for the linkages between the split motif and the insert. These linkages are long irregular loops (residues 16-25 and 64-80) at the bottom of the domain, which pack together through two antiparallel mini-strands along their courses. As in many proteins sharing the canonical split motif, one side of the β- sheet of ZipA 185 . 328 is covered by the α-helices and the opposite side is open to solvent. The interior where the β -strands make extensive contacts with the three helices (061, 062 and 063), as well as the interfaces where the helices contact each other, are exceptionally hydrophobic. Likewise, the exposed sides of the 06 -helices are of polar and hydrophilic residues, with electrostatic potential on their surface dominated by an acidic patch. The uncovered side of the sheet incorporates a large but shallow solvent-exposed cavity which extends to 20 A across the sheet. The ends of the strands, together with their adjacent loops, fold inward the surface of the sheet, forming walls on both sides of the cavity. This surface is lined by side chains from four strands (β3, β4, β5, βl) and from the β2-β3, β4-β5 and β6-063 connections. Much of this cluster is nonpolar residues which, together with the backbone, determine the shape and surface properties of the cavity. Lys 66 and Argl21 are the only charged residues on both walls that interrupt the hydrophobic integrity of the cavity. While Lys 66 is projecting away, the side chain of Arg 121 is oriented across the cavity, thereby closing off part of the left entrance to the hydrophobic volume. In both ZipA 185 . 32S monomers, the volume within the cavity contains moderate number of water molecules, with which the terminal amides of this same Arg 121 form extensive network of hydrogen bonds. In the crystals, ZipA 185 . 328 molecules pack tightly together. The two monomers in asymmetric unit are not symmetry related and, when superimposed, are very close in structure (a root-mean-square (r.m.s.) deviation is 0.79 A for 139 Cθ6 pairs). Each monomer reveals different crystal contacts, with modest interaction between ZipA 185 . 328 copies in the vicinity of surface areas of the cavity. C) Structure of the FtsZ-peptide bound to ZipA l85 . 328 A 17-residue FtsZ-peptide (consensus sequence 3 XD(E)XLD(E)I(V)PXFL 12 ) is bound by the hydrophobic surface of the ZipA 185 . 328 cavity, on the solvent-exposed side of the β-sheet. In complex with the recognition surface of the ZipA 185 . 328 domain the peptide adopts mostly 06-helical (residues 8-17) but partially extended (residues 1-7) conformation (Figure 3a). The peptide conformation includes two patterns of internal hydrogen bonding apart from those that are within the peptide helical region. This conformation directs six side chains of the 30 A long peptide towards interactions with the hydrophobic surface of the ZipA 185 . 328 cavity. The solvent accessible area buried upon peptide binding is 536.4 A 2 for ZipA/M186 and 660 A 2 for the peptide, using a probe radius of 1.4 A in SURFACE (CCP4, Acta Crvstallogr.. D50: 760- 763, 1994). Direct interatomic contacts are made between eleven ZipA 185 . 328 residues and seven peptide amino acid residues. Most of these are hydrophobic contacts but include also two hydrogen bonds. Residues in contact are concentrated in the span from 4 to 15 of the peptide and are distributed over six segments of ZipA 185 . 328 (βl, β3, β4, β5, β2-β3, βl-β5 and β6-α3). Between the peptide side chains buried upon the interaction, there are four (Tyr 5, He 8, Leu 12, Gin 15) that project across and two (Leu 6, Phe 11) oriented down into the cavity, with the peptide backbone rotation of about 90°. Leu 6 and Phe 11 are deeply buried and account for 30% of the total contacts. With respect to ZipA 185 . 328 , one segment (residues 62-69) contributes 48% of the total. The peptide residues close to its -N and C-termini (residues 1-3 and 13-17) extend on either ends of the binding site and make no contacts with the ZipA 185 . 328 domain. The exception is Gin 15, which contacts the cavity through the hydrophobic methylene groups of its side chain. As a result, approximately 55% of the peptide surface (818 A 2 ) remains solvent accessible in the complex. Although the overall structure of ZipA 185 . 328 is unchanged in this complex, small but significant local changes do occur (140 pairs of C06 atoms can be aligned with an r.m.s. deviation of 0.93 A). Such changes are restricted to the binding site. In particular, the intercalation of peptide residue Tyr 5 into the hydrophobic volume of the cavity is accompanied by a slight displacement (~ 0.8 A ) of the β6-063 loop toward the peptide. Upon this rearrangement, the side chain of Arg 121 is swung out of the cavity and into solvent, such that the guanidinium group of Arg 121 is optimally positioned to be stacked on the Tyr 5 ring (3.2 A). A much larger structural change occurs in the segment of the β4- β5 loop. Although the hydrogen-bonding pattern between the strands is maintained, residues 64-66 rotate as a rigid group by ~ 2.5 A towards the floor of the cavity. Here Lys 66 is still exposed, but its side chain flips to avoid a close contact introduced by the peptide. This conformational adjustment in the ZipA 18s _ 328 structure wedges the position of the peptide backbone at this point, by forming two hydrogen bonds to the peptide. These two bonds are made between main chain atoms of peptide residues Asp 4 and Leu 6 and ZipA 185 . 328 residues Lys 66 and Met 64. The aligned sequences of ZipA 185 . 328 domains and those of FtsZ-peptides and the structure of this complex show that most of the side chains in the ZipA 185 . 328 -Fts-peptide interface are conserved within each subset, and the few differences there are appear consistent with the observed packing. Likewise, peptide side chains that project away from the binding site are variable, excluding two consensus residues Asp (or Glu) 7 and Pro 9. A preference for the acidic residue and proline at these positions has an important effect on the conformation of the bound peptide. Pro 9, which is often observed at N-terminal ends of 06 -helices, can account for the hinge point, where the course of the peptide is altered away from the extended conformation. At the same time, the proline ring, which adopts restricted conformations, is likely to decrease a flexibility of the peptide helix at this point. As for Asp 7, although this aspartic acid is located near Lys 66 of ZipA 185 _ 328 , it does not make a hydrogen bond to the electropositive residue. Instead, the side chain of Asp 7 flips towards the helical region of the peptide, where it forms a hydrogen bond with the main chain amino group of Ala 10. As Ala 10 is at the N-terminal end of the α-helix, its NH group is not hydrogen-bonded within the helix. To compensate for the lack of this bond without altering the structure of the peptide backbone, an acidic residue at position 7 should be favored over other side chains. An additional hydrogen bond within the peptide is observed at its N-terminal region, just below the point where the peptide backbone is anchored to ZipA 185 . 328 . This main chain-main chain hydrogen bond is formed between the carbonyl oxygen of Pro 3 and the NH group of Tyr 5. The internal hydrogen bonding among the peptide residues, observed in this structure, is apparently to stabilize the conformation of the ZipA 185 . 328 -bound peptide. Besides the interactions described above, there are some other indirect contacts between the bound peptide and ZipA 185 . 328 . Most of them involve hydrophilic and polar residues interacting through well-ordered water molecules. In the cavity itself only a few water molecules are seen in the complex. D) Structural similarities with other proteins In a large number of proteins sharing the β-06-β split fold, ZipA 185 . 328 represents the first example of this structural class observed among cell division proteins. Although this structural motif is the most abundant element in RNA binding proteins and is associated with their common function as RNA interacting proteins, in ZipA 185 . 328 , this motif is involved in a protein-protein interaction. Comparison of the ZipA 185 . 32 g domain with the RNA-binding domain of the U1A spliceosomal protein (Burd and Dreyfuss, Science. 265: 615-621, 1994) reveals that they are quite close topologically: the insert in the split motif of ZipA 185 . 328 and that of U1A are in similar location. Moreover, the RNA fragment, as seen in the U1A-RNA complex (Outbridge et al, Nature. 372: 432- 438, 1994), is bound by residues on the surface of the β-sheet involving the connecting loops of the split β-06-β motif. When the ZipA 185 . 328 and U1A domains are superimposed, the FtsZ-peptide and the RNA-fragment occupy similar positions on the uncovered sides of their β-sheets. In addition, the RNA- binding loop, which is an α-helical turn connecting two β strands in U1A, has a structural equivalent in ZipA 185 . 328 which anchors the peptide backbone in the complex. As expected, the specific features involved in nucleotide binding are not observed in ZipA 185 . 328 . As the protein-peptide interactions observed in this complex are purely hydrophobic in nature, except for those involving hydrogen bonds, shape complementary between the peptide and cavity rather than the orientation of individual atoms is more important. Example 4 A) Alanine-scanning analysis of the FtsZ-peptide. To further characterize the binding of the FtsZ fragment to ZipA/M185 we used a surface plasmon resonance (SPR) based assay in which ZipA/M185 was covalently immobilized to a biosensor chip (see Biosensor-based assay). As detected by a 100 fold difference in the dissociation constants, the FtsZ-peptide shows less binding to immobilized ZipA/M185 (K D ~ 20 μ; Table 1) than the full length FtsZ for soluble ZipA (K D ~ 0.2 μM). In order to determine which contact side chains of the FtsZ peptide maintain the binding affinity for ZipA/M185, we designed and analyzed 10 single-site alanine substitutions in the FtsZ-peptide using the structure of the complex as a guideline. By measuring binding affinities of these mutants relative to the wild type, we calculated the relative reduction in binding to ZipA/M185 as a consequence of introduced mutations (Table 1). This analysis identified seven side chains that when converted to alanine disrupt binding affinity by factor ranging from 3- to 70-fold (Table 1). Five of these buried side chains (Tyr 5, Leu 6, Ileδ, Phe 11 and Leul2) are in direct contact with ZipA/M185, but only three of them (Ile8, Phe 11 and Leul2) were found to account for virtually all the binding affinity, as each of these mutants individually caused a 50- to 70- fold reduction in binding. Mutants at less conservative positions (Tyr 5 and Leu 6) cause 4- to 5-fold reductions in binding or, as in the case of Gin 15, do not affect the binding at all. Overall this analysis identified four most disruptive alanine mutants: three at hydrophobic residues (Ile8, Phe 11 and Leul2), which form extensive well-packed hydrophobic contacts with ZipA/M185, and one at acidic residue (Asp 7), which is part of the helical capping motif within the structure of the bound FtsZ-peptide. B. Biosensor-based assay. A BIAcore 2000 biosensor system (Pharmacia Biosensor, Upsala) was used to assay interactions between ZipA/M185 and variants of the FtsZ-peptide. Soluble ZipA/M185 molecules were immobilized to the biosensor CM5 chip by standard amine coupling chemistry. The peptide was injected over the chip in 10 mM Hepes (pH 7.5), 150 mM NaCl, 3 mM EDTA and 0.005% polysorbate 20 v/v, at a flow rate of 10 μl/min. Binding between ZipA/M185 and the peptide resulted in changes in the SPR signal that are read out in real time as resonance units (RU). The equilibrium dissociation constants (K D column in Table 1) were derived from sensorgram data using steady affinity model by fitting the plots of R eq (the equilibrium binding response) versus the concentration of the injected peptide. Table 1. Alanine-scanning mutation analysis of FtsZ peptide FtsZ peptides residues 367-383 K D (μM) relative K D 1. Wild type KΞPDYLDIPAFLRKQAD 21 6 1.0 2 D4A ---A 69 4 3.2 3 Y5A A 93 7 4.3 4 L6A A 103 0 4.7 5 D7A A 403 0 18.7 6 I8A A 1510 0 70.0 7 P9A A 19 6 -1.0 8 F11A A 1340 0 62.0 9 L12A A 1040 0 48.0 1C ). K14A A--- 20 1 -1.0 1] L. Q15A A-- 20 0 -1.0 All publications mentioned herein above, whether to issued patents, pending applications, published articles, protein structure deposits, or otherwise, are hereby incorporated by reference in their entirety. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

Claims: What is claimed is: 1. A solution comprising a C-terminal domain of ZipA. 2. The solution of Claim 1, wherein the C-terminal domain of ZipA comprises the amino acid residues 185-328 of Figure 1 (ZipA 185 . 328 ). 3. The solution of Claim 2, comprising ImM ZipA 185 . 328 in a buffer comprising 50 mM sodium or potassium phosphate, 2mM NaN 3 , and 50mM deuterated DTT, in either 90% H 2 O/10% D 2 O or 100% D 2 O. 4. The solution of Claim 3, wherein the ZipA 185 . 328 is either unlabeled, 15 N enriched or 15 N, 13 C enriched. 5. The solution of Claim 4, wherein the ZipA 185 . 328 is biologically active. 6. The solution of Claim 5, further comprising a FtsZ peptide. 7. The solution of Claim 1, wherein the secondary structure of ZipA 185 . 328 comprises three alpha helices and a beta sheet having 6 anti-parallel beta strands. 8. The solution of Claim 7, wherein the alpha helices and the beta strands are configured in the order βl, αl, β2, β3, β4, β5, 062, β6 and 063. 9. The solution of Claim 8, wherein βl comprises amino acid residues 9-16 of ZipA 185 . 328 , 061 comprises amino acid residues 25-34 of ZipA 185 . 328 , β2 comprises amino acid residues 37-39 of ZipA 185 . 328 , β3 comprises amino acid residues 45-48 of ZipA 185 . 328 , β4 comprises amino acid residues 57-63 of ZipA 185 . 328 , β5 comprises amino acid residues 81-88 of ZipA 185 . 328 , 062 comprises amino acid residues 94-112 of ZipA 185 . 328 , βδ comprises amino acid residues 115-117 of ZipA 185 . 328 and 063 comprises amino acid residues 126-144 of ZipA 18S . 3 2 8- 10. The solution of Claim 9, wherein the alpha helices and the beta sheet form surfaces directly opposite each other, and the beta sheet incorporates o a shallow hydrophobic cavity extending roughly 20 A across the beta sheet. 11. The solution of Claim 10, wherein the hydrophobic cavity comprises amino acid residues V10, 112, A16, M42, 144, A57, A62, M64, V65, P67 and F85 of ZipA 185 . 328 . 12. A crystallized C-terminal domain of ZipA. 13. The crystallized C-terminal domain of Claim 12, wherein the C- terminal domain of ZipA comprises the amino acid residues 185-328 of Figure 1 (ZιpA 185 . 328 ) . 14. The crystallized C-terminal domain of Claim 13, characterized as being in plate form with space group P21, and having unit cell parameters of a=49.89 A, b=41.74 A, c=71.16 A and β=98.26°. 15. The crystallized C-terminal domain of Claim 14, wherein a crystallographic asymmetric unit contains two molecules of ZipA 185 . 328 . 16. The crystallized C-terminal domain of Claim 15, wherein the secondary structure of ZipA 185 . 328 comprises three alpha helices and a beta sheet having 6 anti-parallel beta strands. 17. The crystallized C-terminal domain of Claim 16, wherein the alpha helices and the beta strands are configured in the order βl, 061, β2, β3, β4, β5, 062, β6 and 063. 18. The crystallized C-terminal domain of Claim 17, wherein βl comprises amino acid residues 9-16 of ZipA 185 . 328 , 061 comprises amino acid residues 25-34 of ZipA 185 . 328 , β2 comprises amino acid residues 37-39 of ZipA 185 . 328 , β3 comprises amino acid residues 45-48 of ZipA 185 . 328 , β4 comprises amino acid residues 57-63 of ZipA 185 . 328 , β5 comprises amino acid residues 81-88 of ZipA 185 . 328 , 062 comprises amino acid residues 94-112 of ZipA 185 . 328 , β6 comprises amino acid residues 115-117 of ZipA 185 . 328 and 063 comprises amino acid residues 126-144 of ZipA 185 . 328 . 19. The crystallized C-terminal domain of Claim 18, wherein the alpha helices and the beta sheet form surfaces directly opposite each other, and the o beta sheet incorporates a shallow hydrophobic cavity extending roughly 20 A across the beta sheet. 20. The crystallized C-terminal domain of Claim 19, wherein the hydrophobic cavity comprises amino acid residues VI 0, 112, A16, M42, 144, A57, A62, M64, V65, P67 and F85 of ZipA 185 . 328 . 21. A crystallized complex comprising a C-terminal domain of ZipA and an FtsZ peptide. 22. The crystallized complex of Claim 21, wherein the C-terminal domain of ZipA comprises amino acid residues 185-328 of Figure 1 (ZipA 185 . 328 ). 23. The crystallized complex of Claim 22, characterized as being in elongated plate form with space group P21, and having unit cell parameters of a=36.53 A, b=38.9 A, c=54.54 A and β=75.89°. 24. The crystallized complex of Claim 23, further characterized as consisting of one molecule of ZipA 185 . 328 :FtsZ peptide in the asymmetric unit. 25. An active site of an FtsZ binding protein or peptide, wherein said active site comprises the relative structural coordinates of amino acid residues V10, 112, M42, 144, A62, M64, G68, K66, T83, F85, and R121 according to Figures 2, 3, or 4, ± a root mean square deviation from the conserved backbone o atoms of said amino acids of not more than 1.5 A. 26. The active site of Claim 25, wherein said active site further comprises the relative structural coordinates of amino acid residues A16, D41, V65, K66, and Q87 according to Figures 2, 3, or 4, ± a root mean square deviation from the conserved backbone atoms of said amino acids of not more than 1.5 A. 27. An active site of an FtsZ binding protein or peptide, wherein said active site comprises the relative structural coordinates of amino acid residues A9, 112, M13, N14, V15, A17, H19, G25, F37, F39, G40, D41, M42, N43, H48, S60, A62, N63, K66, G68, T69, E73, M74, T78, G81, V82, T83, 184, M86, Q87, S90 and R122 according to Figures 2, 3, or 4, ± a root mean square deviation o from the conserved backbone atoms of said amino acids of not more than 1.5 A. 28. A method for identifying a potential inhibitor of ZipA, comprising the steps of: (a) using a three dimensional structure of ZipA as defined by the relative structural coordinates of amino acids encoding the C-terminal domain of ZipA according to Figures 2, 3, or 4, ± a root mean square deviation from the o conserved backbone atoms of said amino acids of not more than 1.5 A; (b) employing said three-dimensional structure to design or select a potential inhibitor; and (c) synthesizing or obtaining said potential inhibitor. 29. The method according to Claim 28, wherein the potential inhibitor is designed de novo. 30. The method according to Claim 28, wherein the potential inhibitor is designed from a known inhibitor. 31. The method of Claim 29, further comprising the step of contacting the potential inhibitor with the C-terminal domain of ZipA in the presence of the C-terminal region of FtsZ to determine the ability of the potential inhibitor to inhibit ZipA. 32. The method of Claim 30, further comprising the step of contacting the potential inhibitor with the C-terminal domain of ZipA in the presence of the C-terminal region of FtsZ to determine the ability of the potential inhibitor to inhibit ZipA. 33. The method according to Claim 28, wherein the step of employing the three dimensional structure to design or select the potential inhibitor comprises the steps of: (a) identifying chemical entities or fragments capable of associating with the C-terminal domain of ZipA; and (b) assembling the identified chemical entities or fragments into a single molecule to provide the structure of the potential inhibitor. 34. The method according to Claim 33, wherein the potential inhibitor is designed de novo. 35. The method according to Claim 33, wherein the potential inhibitor is designed from a known inhibitor. 36. The method of Claim 34, further comprising the step of contacting the potential inhibitor with the C-terminal domain of ZipA in the presence of the C-terminal region of FtsZ to determine the ability of the potential inhibitor to inhibit ZipA. 37. The method of Claim 35, further comprising the step of contacting the potential inhibitor with the C-terminal domain of ZipA in the presence of the C-terminal region of FtsZ to determine the ability of the potential inhibitor to inhibit ZipA. 38. An inhibitor identified or designed by the method of Claim 28. 39. An inhibitor identified or designed by the method of Claim 33.

Inventors: GLASFELD ELIZABETH, MOSYAK LIDIA, MOY FRANKLIN J, POWERS ROBERT, SOMERS WILLIAM S, GLASFELD, ELIZABETH, MOSYAK, LIDIA, MOY, FRANKLIN, J., POWERS, ROBERT, SOMERS, WILLIAM, S.
Applicants: AMERICAN HOME PROD, AMERICAN HOME PRODUCTS CORPORATION
Application year: 2001
Main classification: G01N33
IPC codes: C07K14