TARGET FOR ANTIVIRAL THERAPY

Abstract: A crystallised molecular complex of an E2 N-terminal module (E2NT) dimer protein or homologue thereof, that comprises residues vital for viral transcription and/or replication. The invention also provides for the use of the dimer protein and interactions at its dimerisation surface in rationalised antiviral drug design.

Description: Target for Antiviral Therapy The present invention provides a crystallised module of a nuclear phosphoprotein and an assay and method for determining interactions with human papillomavirus E2 for u:<?e in drug design, for use particularly but not exclusively in designing antiviral agents with potential use in treating warts, proliferative skin lesions and carcinoma of the cervix. Background to the Invention Human papillomaviruses (HPVs) cause warts and proliferative lesions in skin and other epithelia. In a minority of HPV types ("high risk", which include HPVs 16, 18, 31, 33, 45 and 56), further transformation of the wart lesions can produce tumours, most notably carcinoma of the cervix 1 . HPVs have evolved a sophisticated system of control, mediated by protein:DNA and protei protein interactions, that involves both cellular and viral proteins. The 45 kDalton nuclear phosphoprotein, E2, has two central roles in this control. It acts as the principal virally encoded transcription factor and, in association with the viral El protein, it creates the molecular complex at the origin of the viral DNA replication . E2 has three distinct modules. The N-terminal module (E2NT) of about 200 amino acids is responsible for interactions with viral and host cell transcription factors. It is followed by a flexible, proline-rich, linker module and a C-terminal module (E2CT), each of about 100 amino acids 3 (Fig. la). The E2CT binds as a homodimer to DNA sites with a consensus sequence of ACCGN 4 CGGT 4 . In most HPVs a long upstream regulatory region (UR ) precedes the viral genes and contains four spatially conserved E2 binding sites: three sites proximal to the transcription start site (p97 in HPV 16) and one approximately 500bp upstream. The dimer of E2CT serves to anchor E2 protein to its recognition sites on the DNA, the function of the E2NT is to bind and localise at least three cellular transcription factors, Spl, TFIIB and AMF-1, to the transcription initiation complex. In addition, E2 interacts with another viral protein, El, which has ATPase and helicase activities. El itself binds to the viral origin of replication which consists of about 100 bp and is surrounded by the three E2-binding sites, proximal to the transcription start. The E2:E1 interaction greatly increases the rate of HPV genome replication 2,5,6 , Fig. la. An intact E2 is essential for the normal productive (wart) life cycle of HPV, however during malignant progression HPV DNA is integrated into the host cell genome, which usually results in disruption of the E2/E1 ORFs and loss of E2 protein, in turn leading to dysregulated expression of the viral oncogenes E6 and E7 7 . Consistent with its role as a transcription regulator, E2 has been shown to direct the formation of loops in DNA containing E2 binding sites . The loops were only formed with intact E2, and not with the E2CT alone. The E2 binding sites did not function independently and their co-operative effect was mediated by full length E2, leading the authors to suggest that there were specific interactions mediated by E2 that bridged across the set of DNA binding sites through its N-terminal. A similar DNA loop structure could also be achieved with Spl, a cellular transcription factor, which forms a complex with distally bound E2 9 ; Spl/E2 interactions are critical for transcription activation in BPV 10 . Eighty six known E2 proteins from different species and different human subtypes ! are highly conserved, with sequence identities typically of 35% in the N and C- terminal modules (Fig. lb). The crystal structure of the E2CT has been determined both alone and in complex with cognate DNA 12"14 . The module is a dimer with a barrel fold, and induces substantial bending (42-44°) of the DNA from its B-form double helix 14 . The structure of the proteolytic fragment of HPV 18 E2NT, missing 65 N-terminal residues, was recently reported at 2.1 A spacing 15 . This allowed some analysis of mutational effects on function, although the missing 65 amino acids contain residues which are essential for the transcriptional and replication activities of the protein. We report herein the structure of the complete E2NT determined by X-ray analysis at 1.9 A. We have found that it is an L-shaped molecule with the residues vital for transcriptional and replication activities of the protein lying on opposite sides of the N-terminal domain. Surprisingly, our results show that the surface, vital for transcription activation, is in fact involved in association of two E2NT's into a dimer. We suggest that dimerisation of E2NT plays an important and key role in induction of DNA loop formation, the mechanism by which distally bound transcription factors would be brought close to the site of transcription initiation. More importantly, our results raise the possibility that dimer formation serves as a molecular switch between early gene expression and viral genome replication during HPV infection. The process of rationalised drug design requires no explanation or teaching for the skilled person but a brief description is given here of computational design for the lay reader: various computational analyses are necessary to determine whether a molecule is sufficiently similar to the target moiety or structure. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, Mass.) version 3.3, and as described in the accompanying User's Guide, Volume 3 pages. 134-135. The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in Molecular Similarity to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses a least squares fitting algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA. One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a target. Again, these methods require no elucidation for the skilled person but are described here for the benefit of the unskilled reader. The screening process may begin by visual inspection of the target on the computer screen, generated from a machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within that binding pocket as defined supra. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER. Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include: 1. GRID (P. J. Goodford, "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem., 28, pp. 849-857 (1985)). GRID is available from Oxford University, Oxford, UK. 2. MCSS (A. Miranker et al., "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method." Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, Burlington, Mass. 3. AUTODOCK (D. S. Goodsell et al., "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is available from Scripps Research Institute, La Jolla, Calif. 4. DOCK (I. D. Kuntz et al., "A Geometric Approach to Macromolecule-Ligand Interactions", J. Mol. Biol., 161, pp. 269-288 (1982)). DOCK is available from University of California, San Francisco, Calif. Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or complex. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of calcineurin. This would be followed by manual model building using software such as Quanta or Sybyl. Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include: 1. CAVEAT (P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the Structure- Derived Design of Biologically Active Molecules". In Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182- 196 (1989)). CAVEAT is available from the University of California, Berkeley, Calif. 2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif). This area is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992). 3. HOOK (available from Molecular Simulations, Burlington, Mass.). As the skilled reader will already know, instead of proceeding to build ligand for the target in a step-wise fashion, one fragment or chemical entity at a time as described above, inhibitory or other target-binding compounds may be designed as a whole or de novo. These methods include: 1. LUDI (H.-J. Bohm, "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from Biosym Technologies, San Diego, Calif. 2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations, Burlington, Mass. 3. LeapFrog (available from Tripos Associates, St. Louis, Mo.). Other molecular modelling techniques may also be employed. See, e.g., N. C. Cohen et al., "Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990). See also, M. A. Navia et al., "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202-210 (1992). Once a compound has been designed or selected by the above methods, the efficiency with which that entity may bind to a target may be tested and optimized by computational evaluation. For example, an effective ligand will preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient ligands should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, preferably, not greater than 7 kcal/mole. Ligands may interact with the target in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the inhibitor binds to the protein. An entity designed or selected as binding to a target may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the inhibitor or other ligand and the target, when the inhibitor is bound to the target, preferably make a neutral or favourable contribution to the enthalpy of binding. Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include: Gaussian 92, revision C [M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. .COPYRGT.1992]; AMBER, version 4.0 [P. A. Kollman, University of California at San Francisco, .COPYRGT.1994]; QUANTA/CHARMM [Molecular Simulations, Inc., Burlington, Mass. .COPYRGT.1994]; and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif. .COPYRGT.1994). These programs may be implemented, for instance, using a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known to those skilled in the art. Once the ligand 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 binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to a calcineurin-like binding pocket by the same computer methods described in detail, above. Again, all these facts are familiar to the skilled person. Another approach is the computational screening of small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to a target. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy. E. C. Meng et al., J. Comp. Chem., 13, pp. 505-524 (1992). The computational analysis and design of molecules, as well as software and computer systems therefor are described in US Patent No 5,978,740 which is included herein by reference, including specifically but not by way of limitation the computer system diagram described with reference to and illustrated in Fig 3 thereof as well as the data storage media diagram described with reference to and illustrated in Fig 4s and 5 thereof. Statement of the Invention According to a first aspect of the invention there is provided a crystallised molecular complex of an E2 N-terminal module (E2NT) dimer protein or homologue thereof, for use in rationalised drug design. We have found that the dimer comprises residues vital for transcriptional and replicational activities of said protein lying on opposite sides of an N-terminal domain, for use in rationalised drug design. Preferably the E2NT dimer protein is substantially as depicted in any of Figures 2c and/or 3a-d. According to a second aspect of the invention there is provided an in vitro method for identifying and/or selecting a candidate therapeutic agent, the method comprising determining interaction of a E2 N-terminal module (E2NT) dimer in a sample by contacting said sample with said candidate therapeutic agent and measuring DNA loop formation. Preferably, the method is for use in identifying and/or selecting an antiviral candidate therapeutic agent. Preferably, the candidate therapeutic agent interferes or blocks interactions of E2NT so as to interfere or block viral and/or cellular transcription factors. According to a third aspect of the invention there is provided use of an E2NT dimerisation inhibitor in the preparation of a medicament for use in treating warts, proliferative skin lesions and/or cervical cancer. According to a fourth aspect of the invention there is provided a method of monitoring the efficacy of an antiviral therapy in a patient receiving a medicament for the treatment of warts, proliferative skin lesions and/or cervical cancer comprising taking a sample from said patient and measuring E2NT interactions and/or DNA loop formation. Thus it will be appreciated that a patient can be monitored at the start of therapy to test its effectiveness. Alternatively, a patient can be monitored once a therapy has been established so as to monitor its efficacy with a view to altering a therapy if found to be unsatisfactory. The human papillomavirus E2 protein controls the primary transcription and replication of the viral genome. Both activities are governed by a ~200 amino acid N-terminal module (E2NT) which is connected to a DNA binding C-terminal module by a flexible linker. The crystal structure of the E2NT module from high-risk type 16 human papillomavirus reveals an L-shaped molecule with two closely packed domains, each with a novel fold. It forms a dimer in the crystal and in solution. The dimer structure is important in the interactions of E2NT with viral and cellular transcription factors and is the key to induction of DNA loops by E2. These loops may serve to target distal DNA-binding transcription factors to the region proximal to the start of transcription. The structure has implications for antiviral drug design and cervical cancer therapy. The invention includes method for identifying and/or selecting a candidate therapeutic agent, comprising applying rationalised drug design to a crystal structure obtainable by crystallising E2NT, cryogenically freezing the crystals and generating the crystal structure using X-ray diffraction. The method by which the E2NT crystal structure is obtainable may comprise crystallisation using hanging-drop vapour diffusion. The method by which E2NT crystal structure is obtainable may comprise X-ray diffraction using uranium acetate and gold cyanide E2NT derivatives and refining with data extending to 1.9 A spacing. The crystal structure may comprise the portions of amino acids Ile82, Glu90, Trp92, Lysl l2, Tyrl38, Vall45, Prol06, Lysl l l, Phel68, Trpl34, Trp33 and Leu94. The rationalised drug design may comprise designing drugs which interact with the dimerisation surface of E2NT. Further provided is a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises or a three-dimensional representation of a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A , wherein said computer comprises: (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl l2, Tyrl38, Vall45, ProlOό, Lysl 11, Phel68, Trpl34, Trp33 and Leu94 according to Table 3; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central-processing unit coupled to said working memory and to said machine- readable data storage medium for processing said machine readable data into said three-dimensional representation; and (d) a display coupled to said central-processing unit for displaying said three- dimensional representation. In class of embodiments, the three-dimensional representation is of a molecule or molecular complex is defined by the set of structure coordinates according to Table 3, or wherein said three-dimensional representation is of a homologue of said molecule or molecular complex, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. An additional aspect of the invention resides in a computer for determining at least a portion of the structure coordinates corresponding to an X-ray diffraction pattern of a molecule or molecular complex, wherein said computer comprises: (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structural coordinates according to Table 3; (b) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises an X-ray diffraction pattern of said molecule or molecular complex; (c) a working memory for storing instructions for processing said machine-readable data of (a) and (b); (d) a central-processing unit coupled to said working memory and to said machine- readable data storage medium of (a) and (b) for performing a Fourier transform of the machine readable data of (a) and for processing said machine readable data of (b) into structure coordinates; and (e) a display coupled to said central-processing unit for displaying said structure coordinates of said molecule or molecular complex. A yet further aspect of the invention relates to a crystallised molecule or molecular complex comprising a dimerisation surface defined by structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl l2, Tyrl38, Vall45, ProlOό, Lysl l l, Phel68, Trpl34, Trp33 and Leu94 according to Table 3or a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. The molecule or molecular complex may be defined by the set of structure coordinates according to Table 3, or a homologue thereof, wherein said homologue has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. 27. A machine-readable data storage medium (e.g. a magnetic or optical storage medium, for example a hard disc, a floppy disc or a CD-ROM), comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex comprising a dimerisation surface defined by structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vall45, Prol06, Lysl 11, Phel68, Trpl34, Trp33 and Leu94 according to Table 3, or a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. In the machine-readable data storage medium the molecule or molecular complex may be defined by the set of structure coordinates according to Table 3, or a homologue of said molecule or molecular complex, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5A. The invention further provides a machine-readable data storage medium comprising a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, wherein: said first set of data comprises a Fourier transform of at least a portion of the structural coordinates according to Table 3; and said second set of data comprises an x-ray diffraction pattern of a molecule or molecular complex. In another aspect, the invention resides in a method for evaluating the ability of a chemical entity to associate with a molecule or molecular complex according to the invention, comprising the steps of: a. employing computational means to perform a fitting operation between the chemical entity and a dimerisation surface of the molecule or molecular complex; and b. analysing the results of said fitting operation to quantify the association between the chemical entity and the dimerisation surface. Detailed Description of the Invention The invention will now be described by way of example only with reference to the following Figures and Tables wherein: Table 1 illustrates X-ray data and phasing statistics; Table 2 illustrates refinement and model correlation; Table 3 shows the structure coordinates of the E2NT module; Figure la represents functional assignments of HPV 16 E2 protein; Figure lb illustrates sequence alignment of E2NT modules from a subset of HPV types; Figure 2a illustrates a stereo view of electron density with a final model at the dimer interface of the E2NT module, viewed down the crystallographic two-fold axis; Figure 2b represents a stereo ribbon diagram of the E2NT module; Figure 2c represents the E2NT dimer; Figure 3 a illustrates a schematic view of URR; Figure 3b illustrates a schematic view of loop formation induced by binding of E2 proteins to two cognate sites; Figure 3c illustrates a model of E2 dimer formation; Figure 3d illustrates loops within URR as shown in Figure 3b; Figure 4a illustrates the distribution of conserved residues on the E2NT monomer; Figure 4b illustrates a first cluster of conserved residues on the E2NT monomer; Figure 4c illustrates a second cluster of conserved residues on the E2NT monomer; and Figure 4d illustrates conserved residues Gin 12 and Glu39. Those of skill in the art understand that a set of structure coordinates for an enzyme or an enzyme-complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations caused by acceptable errors in the individual coordinates will have little, if any effect on overall shape. In terms of binding pockets, these acceptable variations would not be expected to alter the nature of ligands that could associate with those pockets. The term "associating with" refers to a condition of proximity between a chemical entity or compound, or portions thereof, and a calcineurin molecule or portions thereof. The association may be non-covalent— wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions— or it may be covalent. The invention is also described with reference to US Patent No 5,978,740 which is included herein by reference, including specifically but not by way of limitation the computer system diagram described with reference to and illustrated in Fig 3 thereof as well as the data storage media diagram described with reference to and illustrated • in Fig s 4 and 5 thereof. With reference to Figure la and functional assignments of E2. There is shown in a schematic view of NT, linker and CT modules of E2 indicating known functions of each module. Amino acid numbers which delimit the modules correspond to E2 from HPV 16. In Figure lb, there is shown the sequence alignment of the E2NT modules from a subset of HPV types (HPV16, HPV18, HPV11 and HPV2a) and one BPV type. Shaded blocks above the alignment indicate the experimentally determined secondary structure. Shaded blocks below the sequences indicate the minimal peptide sequences involved in proteimprotein interactions, suggested by mutation studies. Residues with more than 90% identity among 86 PV types are coloured: red for internal structural residues, green for residues within the fulcrum region, blue for surface residues. With reference to the structural features of E2, in Figure 2a there is shown a stereo view of the electron density with the final model, at the dimer interface of the E2NT module, viewed down the crystallographic two-fold axis. The likelihood weighted map is contoured at the 1.5 σ level. Ribbons of two independent monomers are coloured blue and yellow. Side chains of ARG37 and Ile73 which are known to be critical for transactivation 4 ' 31 , are shown in dark green; side chain of other residues at the dimer interface are shown in light green. Oxygen atoms are in red, nitrogen in blue, water molecules are shown as orange spheres and hydrogen bonds as dashed sticks. In Figure 2b, there is shown a stereo ribbon diagram of the E2NT module. The Nl domain is shown in aquamarine and the N2 domain in pink, with the fulcrum in green. In Figure 2c, there is shown the dimer of E2NT, showing the extent of the interface between the two subunits. The view is as in Figure 2a but rotated clockwise by 90°. Side chains of Glnl2 and Glu39 which are critical for interactions with El 31" ' are shown in magenta. Side chains of residues at the dimer interface are coloured as per Figure 2a. With reference to Figures 3a-d there is shown loop formation in the URR of HPV 16. In Figure 3 a, there is shown a schematic view of the URR. The four E2-binding sites are represented by boxes. Numbers in italics indicate distances between individual sites upstream of the p97 promoter. Two possible E2 configurations, with separate or dimeric E2NT modules are shown. In Figure 3b, there is shown a schematic view of loop formation induced by binding of E2 proteins to two cognate sites, based on the experiments reported by Knight et al . In Figure 3d, there is shown the possible DNA loops within the URR as depicted in Figure 3b. In Figure 3c, there is shown a model of the formation of E2 dimers, showing interactions between both the C-terminal and E2NT modules. The C-terminal dimer, with its bound DNA, is based on the crystal structure of this module 12 . The E2NT dimer is proposed from the present work. The relative orientation and position of the E2NT and C-terminal modules is purely schematic. With reference to Figures 4a-d there are shown functionally important residues. In Figure 4a, there is shown the distribution of conserved residues on the E2NT monomer. In Figures 4b and 4c there is shown the two clusters of conserved residues in the fulcrum of E2NT. In Figure 4d, there are shown conserved residues Gin 12 and Glu39. Bonds in ball-and stick models are coloured aquamarine (Nl domain), pink (N2 domain) and green (fulcrum). Hydrogen bonds are shown as dashed lines, water molecules as orange spheres, oxygen atoms are in red, nitrogen atoms in blue and sulphur atoms in yellow. There is convincing evidence that the E2 protein has an extended structure, is flexible and that its functions depend on this property. This is probably the reason why the intact protein has not yet been crystallised in spite of intensive efforts. A major problem is the extended flexible linker module, with around 100 residues. E2NT proved difficult to crystallise, and a number of different constructs were made and overexpressed before crystallisation with residues 1 to 201 was achieved, but even this construct possessed limited stability. The protein had to be crystallised within 2- 3 days of purification; crystals grew within about 48 hours but only retained useful diffraction quality for a further 2-3 days. This necessitated that crystals be rapidly vitrified in cryoprotectant buffer and stored for use as soon as detector time became available 16 . Crystals of E2NT belong to the space group P3]21 with unit-cell dimensions a=b=54.3 A, c=155.5 A. The structure was determined using two heavy atom derivatives and refined with data extending to 1.9 A spacing (Fig. 2a). The main chain is well defined throughout with the exception of residues 125 and 126 which are in an exposed loop and are mobile. There was density for the last residue of the His-tag at the N-terminus, but none for the remainder of this entity. All amino acids lie in the allowed regions of the Ramachandran (φ,ψ) plot 17 with 92.4% in most favoured regions 18 . The transactivation module is composed of two domains, Nl and N2, arranged so as to give it an overall L-shaped appearance. Analysis of the PDB 19 using DALI 20 shows that both have unique organisation of their secondary structures. Domain Nl, which forms the N-terminus of the intact E2, is composed of residues 1 to 92, which fold into three long α-helices, Figure 2 (b,c). There is a tight loop between αl and α2 and a more extended one between α2 and α3. The three helices pack antiparallel to one another in the form of a twisted plane, with angles of about 20° and 25° between the pairs of consecutive helices. DALI indicated a maximum Z-score of 5.7, that could suggest a significant correlation, for colicin la, a membrane protein which contains three 80 A long α-helices arranged more or less coplanar 21 . This is the only other known protein that contains a true domain made up of such a packing of three helices. In addition there were 42 other structures which gave Z-scores above 4.0, most of which were four helix bundles, such as bacterioferritin 22 . However, in these only two of the three Nl helices superimposed simultaneously on two, not always adjacent, bundle helices as a result of a more planar arrangement of helices within Nl. The indications are that the similarities observed reflect the optimum stacking angle of antiparallel helices against one another rather than suggesting a common ancestor for the evolution of these molecules. Domain N2 is made up of residues 110 to 201 and is composed almost entirely of antiparallel β structure, with only one short helical segment from residues 171 to 178, Figure 2 (b,c). The secondary structure has two short three and four stranded antiparallel β pleated sheets interconnected by two stranded β ribbons. For this domain DALI failed to identify any significant homologies to known structures, with a highest Z-score of only 2.1. From the analysis of Harris and Botchan 15 and the present study, the N2 fold appears to be novel. The structure between the Nl and N2 domains (residues 93 to 109) contains two consecutive single turns of helical structure, resulting in a compact and tight turn. It packs closely against elements of both domains and is not a truly independent structural domain. Rather it forms a fulcrum in the L-shape formed by Nl and N2 where it could act as a hinge, allowing the two domains to change their relative conformation in a specific way. Several of the interactions between adjacent regions of chain in the fulcrum are mediated indirectly through H-bonds involving water molecules, suggesting the possibility of flexibility. One of the most striking features of the crystal structure is the association of two E2NT monomers into a tight dimer. The two E2NT monomers pack around the crystallographic 2-fold axis, as shown in Figure 2a. The dimer interface is formed mostly by amino acids from helices α2 and α3 of the Nl domain and by residues 142-144 from the N2 domain. The total buried surface area between the two E2NT is 2 2 n 2026 A° , comparable to the 2444 A° buried between the two E2CT , which are known to form a tight dimer with a K ά of 3-6 x 10 " M ' . In the E2NT dimer interface, each subunit contributes a cluster of seven equivalent residues, invariant or conserved in the 86 known sequences of E2 1 ', with many direct and water-mediated hydrogen bonds and rather few non-polar contacts, Fig. 2. Analysis of the dimer forming surfaces shows that all the direct hydrogen bonds between monomers are made through these seven amino acids. For the invariant Arg37, all possible side-chain hydrogen bonds are made and all are well defined, Figure 2. Three of them are across the dimer interface. One hydrogen bond is critical, from NH2 to the main chain carbonyl oxygen of Leu77. A second hydrogen bond from NH2 is to OG1 of Thr81; in five out of 86 sequences this residue is glutamine, and modelling shows a hydrogen bond is possible to the NE of Arg37. The NH1 of Arg71 H-bonds to the OE1 of residue 80, which is Glu or Gin in all but six variants. At the NE of Arg37 there is an ideal H-bond to water that itself makes another strong H-bond across the dimer interface to the main-chain carbonyl oxygen of residue 142. The role of the invariant Ile73 is the filling of the intersubunit non- polar volume made up of the aliphatic parts of Arg37, Gln76 and of Leu77 - in this case from both monomers. The Leu77 is in a few sequences substituted by valine or isoleucine and in 9 out of 86 known sequences by methionine. Inspection of the structure shows that Leu77 is partially exposed to the solvent and therefore different hydrophobic side chains could be easily accommodated at this site. Another important non-polar side chain is Ala69. Its side chain methyl packs into the surface of the other monomer at van de Waals distance from the main chain of residue 142. The only observed mutation of Ala69 is to Gly, and is easily accommodated. Gln76 is conserved or has homologous substitutions in about 2/3 of E2 sequences; in about 1/4 of the sequences there is methionine or valine at this position 11 . Although hydrophobic substitutions of Gln76 would disrupt the hydrogen bonding to Glu80 across the dimer interface, and to Arg37 from the same subunit, the hydrophobic side chain at residue 76 could instead make a compensating hydrophobic interaction with the adjacent intersubunit hydrophobic pocket formed by Ile73 and Leu77. Modelling of the amino acid variations in the 86 known papillomavirus E2 proteins into the other contacts at the dimer interface shows that they generally can be accommodated (data not shown). The consistency of the hydrogen bonds and van de Waals contacts at the monomer-monomer interface in the various sequences suggests therefore that the E2NT dimer interactions are potentially present in all papillomaviruses. The first experimental evidence for the E2NT dimerisation in the presence of DNA with multiple E2-binding sites was provided by Knight et al in 1991 8 . Their studies showed that intact E2 led to the formation of DNA loops on templates with widely separated E2 binding sites, while a truncated E2, containing the DNA-binding E2CT but missing the N-terminal 161 residues, did not. Such dimerisation is further supported by the observed synergistic transcription activation by a complex of two DNA-bound E2 dimer s 25 . To analyse the functional behaviour of the E2NT dimers further, we measured the dissociation constant by sedimentation equilibrium using analytical ultracentrifugation of recombinant E2NT protein containing the 201 N-terminal amino acids. A value of K - 8.1 ± 4 x 10 '6 M was obtained, indicating medium- strength association. The micromolar range of the E2NT dimer K d is certainly physiologically significant, and compares well with values for other transcription factors which have relatively low dissociation constants, often with the K d values between 1 μM and 20 μM ' . In vivo, the interaction could be enhanced when the two E2NT modules are placed in close proximity. Indeed, E2CT forms dimers which bind to the multiple DNA-binding sites located within the URR of viral DNA with K d of protein:DNA interactions usually in the nanomolar range . Consequently, the local concentration of E2NT, bound to the E2CT via the non-conserved, flexible ~80 amino-acid linker, is effectively increased. E2NT dimer interactions, as seen in the crystal structure, could form either between modules which are already part of a single E2 dimer, formed as a result of E2CT dimerisation interactions and bound to a single E2 binding site on the DNA (Fig. 3 a), or between two preformed E2 dimers located on different E2 binding sites (Fig. 3b). The results of the electron microscopy suggest that the latter dimerisation does occur 8 . Although no direct experimental evidence exists for the former dimerisation, it does also seem possible due to the flexibility of the linker connecting the two modules. We propose that E2 molecules may initially keep their N-terminal modules within their internal dimers, but swap N-terminal modules and cross link to E2 molecules bound to distant DNA binding sites to form active loop structures during transcriptional activation and / or HPV DNA replication (Figure 3d). As discussed below, the effects of mutations on transcriptional transactivation can be explained in terms of the dimer being an essential element in this process. E2 is a regulator of both transcription and viral DNA replication and thus interacts with other viral and host macromolecules in the infected cell. Indication of the possible importance of individual residues in the function comes firstly from the structure, secondly from the extensive set of sequences of the papillomaviral E2's and thirdly from mutagenesis studies on the individual proteins. In the following we make a primary attempt to map the molecule's function onto its structure. The pattern of amino acid conservation for the 86 available papilloma sequences 11 has been analysed using the GCG program suite . The sequences exhibit striking variation, characteristic of some virus families. However, 33 of the total 201 residues in the E2NT construct were totally or highly conserved. Fig. 4a illustrates the distribution of these 33 residues in the dimer. These were categorised into two sets: those with an essentially structural role and those exposed on the surface with a potential for intermolecular interactions. Thirteen residues (Fig. lb) are buried or play a purely structural role within the monomer, they are not expected to be of functional importance and will not be discussed here. A further 12 of these 33 residues stand out as having a structural role in the interface of the Nl and N2 domains. They form three clusters, the first making direct interactions between the two domains (Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vall45) and two separate sets of interactions, one from N2 (Prol06, Lysl 11, Phel68, Tr l34) and the other from Nl (Trp33, Leu94) to the structure connecting them, referred to here as a fulcrum. The first two clusters are shown in Figure 4 b, c and it can be seen that Lysl 11 and Lysl 12 play key roles. Their side chains point in opposite directions to one another and their terminal amino groups are involved in near ideal patterns of hydrogen bonds. The flat surfaces of their extended side chains stack against Tip 134 and Trp92, respectively. This clustering of invariant residues at the interface indicates a functional importance for the relative orientation of Nl and N2. The fulcrum could indeed provide a flexible pivot between the two domains, but there is no direct evidence for this as yet. Finally, while the side chain of Glu90 is held tightly in place by two H-bonds and could have a structural role, its OE2 atom is exposed on the surface and is surrounded by near invariant side-chains, which may thus play a part in interactions with other molecules. Of the remaining eight conserved residues, mutational substitutions of Glu20, GlulOO and Asp 122 30'33 had moderate effects on the transactivation and replication properties of E2, which depended on a particular viral strain. Glu20 lies on the top surface of Nl. Asp 122 lies far away on the distal surface of N2. GlulOO is completely exposed and points into the solvent at the junction of the L between the Nl and N2 domains. The functional role of these amino acids has yet to be clarified. Three conserved amino acids (Arg37, Glu39 and Ile73) have been subjected to point mutation and the effects on the two principal functions of E2, i.e. transactivation and HPV DNA replication have been assessed (reviewed in 4 ,also 31 ' 34 - 35 ). Together with the remaining two conserved amino acids, Glnl2 and Ala69, these residues form two functionally important surfaces (see below). Finally, a number of the mutational results (reviewed in 4 , also 31>34,35 ) correspond to residues that can be assigned to structural roles. Substitution of these residues will lead to substantial conformational changes and a probable inability to fold correctly. This is particularly true for some of the deletion mutants involving the core of the molecule. Knowledge of the structure will allow a more rational choice and design of mutants in the future. The induction of DNA loops by E2NT dimerisation could be important for the construction of the active transcription bubble by targeting DNA-binding transcription factors, bound at distal sites, to the region proximal to the start of transcription (reviewed in 36 ). In support of this, residues Arg37, Ile73 and Gln76 map onto the surface of E2NT involved in dimer formation, and mutations result in considerable disruption of transactivation, while having little effect on replication, 4 - 15 ' 31 . T e structure also shows that Ala69 which points its side chain methyl across the dimer interface, is also critical for transactivation. Mutational substitutions to amino acids with longer side chains should have a knock out effect on E2NT dimer formation and consequently on transactivation. The sites of association with cellular transcription factors AMF-1 (residues 74-134) and TFIIB (134-216) were previously mapped onto the E2NT module (Figure 1) using a series of deletion mutants as well as point mutations 34,35 . These sites were mutually exclusive. In the structure, residues 74-134 include the fulcrum, while residues 134-216 correspond to domain N2. Further biochemical and structural studies can now be planned to characterise these interactions in more detail. Replication of the viral genome is initiated by binding of another viral protein, El, to the origin of DNA replication 4 which is itself flanked by two E2 binding sites, Fig. 3 a. While the function of E2CT dimers is to bind specifically to the DNA sites, E2NT interaction with El enhances the binding of El to this region. Mutational substitutions of Glu39 generally retained transcriptional activation while DNA replication was substantially reduced ' . In the structure, the conserved Glu39 makes every possible hydrogen bond by its side chain carboxyl oxygens, Fig. 4d. One hydrogen bond is to NE2 of Gin 12, which is absolutely conserved in all known sequences of E2. The other three hydrogen bonds are to the water molecules which are part of an intimate net of well-defined water molecules surrounding Glu39 and mediating its interactions with adjacent residues. Interestingly, a number of these protein interactions with water molecules are conserved as they are made to the protein backbone, including carbonyl oxygens of Glnl2, Met36 and Lys68. While mutation of Gin 12 in BPV1 only slightly affected both transactivation and replication, it substantially reduced cooperative origin binding 30,32 . The close positioning of Glnl2 and Glu39 in the three-dimensional structure further enhances the notion that these two resides are involved in interactions with El. The conserved set of interactions at Glnl2/Glu39 suggests that the main chain carbonyl oxygens of Gin 12 and Met36 and the conserved water molecules could be also involved in these interactions. Glnl2/Glu39 are surrounded by Leu8, Ilel5, Met36, Tyr43, Gln57 and Lys68, which are unlikely to contribute into E2/E1 interactions, as these residues are not well conserved in E2 sequences from different papillomaviruses. The Glnl2/Glu39 cluster lies on a side of the Nl domain which is opposite to the side involved in transactivation (and dimerisation), Figure 2c. Notably, the spatial separation of the two functionally important surfaces suggests that E2NT module could be able to interact with El at the same time as it interacts through the dimerisation interface with another E2NT module. The structure reported here for the entire E2 transactivation module, has several implications for understanding of E2 function. It is now possible to map known mutations onto the E2 three-dimensional structure, and to use the knowledge of amino acid conservation and the effects of mutations to assign roles in folding, structure and function to residues. To this end, our results indicate that molecular surfaces involved in transactivation and El -binding are located at opposite sides of the Nl domain of E2NT, suggesting that both surfaces could be accessed simultaneously by other protein factors. In line with these observations, El has been shown to modulate transactivation by directly interacting with E2, leading to repression of transactivation in the presence of excess El . It is not inconceivable that the docking of E2NT dimer with El is sufficient to block further association with other target proteins. The structure shows that the transactivation surface is involved in the formation of the E2NT dimer, which could cross-link E2 molecules bound by their E2CT modules to well-separated DNA sites. Inevitably, such dimerisation would cause DNA to form a loop structure, targeting distally bound transcription factors to regions close to the promoter. While this process has been suggested to be essential for transactivation 36 , the definition of interacting surfaces between E2 and other cellular transcription factors requires a great deal of further study. Our results suggest that the process of DNA loop formation could involve swapping of E2NT modules across E2 dimers bound at separated DNA sites (Fig. 3a-d). The polar components of the monomer-monomer interactions may favour such exchange. Domain swapping is a well-recognised phenomenon that occurs relatively frequently between two individual monomers containing domains connected by a flexible linker 39,40 . E2 is to our knowledge the first example where the swapping event is predicted to occur between dimers. The dimerisation surface of E2 represents a good target for designing anti-viral drugs, since it is essential for viral transcription, there is no homologous human protein and the residues forming the interface are highly conserved among different viral strains. Dynamic interactions between transcription factors play a central role in the regulation of transcription and replication. Dimerisation, heterodimerisation and the monomer-to-dimer transition may play important roles during the control of the papillomavirus life cycle. These processes themselves can be regulated through phosphorylation, proteolysis, interaction with small ligands or changes in their intracellular concentration. It has been suggested that E2 can regulate the switch between early gene expression and viral genome replication during HPV infection 41 . It is possible that dimerisation of E2NT modules plays an essential role during this process. One scenario would be to activate transcription via induction of DNA loop formation at early stages of the viral life cycle. At later stages, when the concentration of expressed E2 proteins within the cell becomes high and comparable with the K d for E2 dimer formation, free E2NT modules could compete for dimerisation with those involved in DNA loop formation and titrate them away, switching off transcription and stimulating replication. It is also possible that other protein factors could be involved in this process, including, for example, El. The invention therefore includes the use of E2NT crystal structure in the design of anti-viral drugs, since it is essential for viral transcription. In the rationalised computational design of drugs using the crystal structure, computational analyses are therefore necessary to determine whether a molecule or the E2NT-binding portion thereof is sufficiently similar to the E2NT structure. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, Mass.) version 3.3, and as described in the accompanying User's Guide, Volume 3 pages. 134-135. The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in Molecular Similarity to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). Atom equivalency within QUANTA is defined by user input and, for the purpose of this invention equivalent atoms may be defined as protein backbone atoms (N, C. alpha., C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses a least squares fitting algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA. For the purpose of one class of embodiments this invention, any set of structure coordinates of a molecule or molecular complex that has a root mean square deviation of conserved residue backbone atoms (N, C. alpha., C, O) of less than 1.5 .ANG. when superimposed-using backbone atoms— on the relevant structure coordinates of E2NT are considered identical. More preferably, the root mean square deviation is less than 1.0 .ANG.. Most preferably, the root mean square deviation is less than 0.5 .ANG.. The term "root mean square deviation" means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the "root mean square deviation" defines the variation in the backbone of a protein from the backbone of E2NT a dimerising portion thereof, for example as defined by the structure coordinates of E2NT described herein. The term "least squares" refers to a method based on the principle that the best estimate of a value is that in which the sum of the squares of the deviations of observed values is a minimum. Materials and Methods Purification and crystallisation. Details of the purification and crystallisation of E2NT have been described previously 16 . Briefly, the ORF encoding the N-terminal 201 residues of HPV- 16 E2 was cloned into the prokaryotic expression plasmid pET15b downstream of the 20- residue His-tag leader sequence; protein was expressed in E. cø// ' BL21(DE3)pLysS and purified using nickel affinity and anion exchange chromatography. Crystals were obtained by hanging drop vapour diffusion with 0.8-1.2M ammonium sulphate, 0.1M triethanolamine pH 8.0-8.3 and 3-5% 2-methyl-2,4-pentanediol. Crystals grew only with very fresh protein preparations and deteriorated in terms of diffraction quality in less than a week. This necessitated freezing and storage of crystals in liquid nitrogen immediately after growth, as discussed above. Structure determination. All data were recorded on cryogenically frozen crystals. A native crystal was frozen for which initial data were recorded to 3.4 A 16 . For the screening of derivatives, crystal stability was even more limiting. Nine crystals were soaked in various heavy atom reagents immediately after growth. The crystals were screened in-house using a MAR research imaging plate on a Rigaku RU200 rotating anode source, by recording 3° of data for each and analysing the fractional isomorphous difference from the native. Three derivatives showed promising differences from the native, in the range of 15-20%) after scaling using SCALEPACK 42 and were stored in liquid nitrogen. The native crystal was transported to EMBL Hamburg where 1.9 A data were measured using synchrotron radiation from beam line XI 1, Table 1. In addition data were recorded at EMBL for the three promising derivatives to about 2.7 A. Two of these derivatives proved useful in phase determination and the structure was solved by multiple isomorphous replacement with anomalous scattering (MIRAS) at 2.7 A. The two derivatives were solved independently using the CCP4 suite 43 from the difference Patterson synthesis and by direct methods as implemented in SHELX 44 . Both contained a single heavy atom site. Phases, calculated using MLPHARE, were enhanced by solvent flattening4 5 using a solvent content of 50 %. The resulting high quality density map was easily interpretable and the initial model was built using QUANTA (Molecular Simulations) for all but four residues of the construct, ignoring the His-tag. The model was completed with REFMAC (resolution 20-1.9 A) using a bulk solvent correction, to an R-factor of 23.3 % (R Free 29.7 % - for 5 % of the data). There are 221 residues in the recombinant protein: the first twenty comprise the His- Tag. The final model contains all but two of the 201 residues of the real protein: residues 125-126 are disordered and lie in a flexible surface loop. Only one residue, HisO, of the His-tag has clear density and an ordered conformation. In addition there are 187 water molecules, which were selected using ARP 46 during the course of refinement. The main statistics of the refined model are shown in Table 2. Analytical ultracentrifugation. Experiments were carried out in an Optima XL-A ultracentrifuge (Beckman-Coultier, CA, USA) using scanning UV optics. During the experiments, the recombinant E2NT was in lOmM TrisHCl pH 8.0, 5mM DTT, 0.2 mM EDTA, 300 mM NaCl. Data were obtained at rotor speeds of 12,000 and 16,000 rpm, and the time to equilibrium was 10-12 hours. All runs were carried out at 293K, and all radial scans were at a wavelength of 280 nm. Dissociation constants were obtained by nonlinear regression using the Beckman ultracentrifuge software. P32059WO Table 1 X-ray data and phasing statistics 31 Table 2 Refinement and model correlation Resolution 1.9 - 10.0 A Number of protein atoms 1622 Number of solvent sites 211 Number of reflections used in refinement 20637 Number of reflections used for Rfree calculation l l l l R-factor + 0.232 Rfree " ' ' 0.305 Average atomic B-factor*, A^ protein atoms 38.0 water molecules 48.5 R.m.s. deviations from ideal geometry (A). Targets in parentheses bond distance 0.013 (0.020) angle distance 0.026 (0.040) chiral volume 0.142 (0.200) t Crystallographic R-factor, R(f ree ) = Σ ||F 0 | - |F C || / ∑ |F 0 | Tabl .e 3 CRYST 54. 680 54 .680 155. 730 90.00 ! 90.00 120.00 : P3121 SCALE1 0.01829 0 .01056 0, .00000 0.00000 SCALE2 0.00000 0 .02112 0, .00000 0.00000 SCALE3 0.00000 0 .00000 0 .00642 0.00000 ATOM 1 N HIS A 0 5. .469 -26, .512 52.262 1, .00 61. .92 ATOM 2 CA HIS A 0 6, .434 -25, .669 51.568 1, .00 61, .84 ATOM 3 C HIS A 0 6, .263 -25 .743 50.051 1, .00 53, .91 ATOM 4 O HIS A 0 6, .089 -24 .713 49.607 1, .00 69, .59 ATOM 5 CB HIS A 0 7, .837 -26, .127 51.965 1, .00 54, .18 ATOM 6 CG HIS A 0 7. .848 -26, .468 53.431 0, .00 99, .00 ATOM 7 ND1 HIS A 0 7, .914 -25, .533 54.412 0, .00 99. .00 ATOM 8 CD2 HIS A 0 7. .732 -27, .728 54.027 0. .00 99. ,00 ATOM 9 CE1 HIS A 0 7. .828 -26, .215 55.570 0. .00 99. .00 ATOM 10 NE2 HIS A 0 7. .723 -27, .531 55.370 0. .00 99, .00 ATOM 11 N MET A 1 6. .663 -26, .896 49.478 1. .00 56. .24 ATOM 12 CA MET A 1 6. ,435 -27, ,076 48.053 1. ,00 56. ,42 ATOM 13 C MET A 1 5. .209 -26. .282 47.619 1. .00 56. ,07 ATOM 14 O MET A 1 5. .293 -25, .299 46.911 1. .00 56. ,51 ATOM 15 CB MET A 1 6, .216 -28, .565 47.788 1, .00 60. .46 ATOM 16 CG MET A 1 6. .856 -29. .020 46.477 0. .00 99. .00 ATOM 17 SD MET A 1 7. .244 -30, .775 46.483 0. .00 99. .00 ATOM 18 CE MET A 1 7, .499 -30, .975 44.711 0, .00 99, .00 ATOM 19 N GLU A 2 4, .035 -26 .755 48.064 1, .00 54, .92 ATOM 20 CA GLU A 2 2, .803 -26, .044 47.744 1, .00 53. .59 ATOM 16 CG MET A 1 6.856 -29.020 46.477 0.00 99.00 ATOM 17 SD MET A 1 7.244 -30 .775 46 .483 0.00 99 .00 ATOM 18 CE MET A 1 7.499 -30 .975 44 .711 0.00 99 .00 ATOM 19 N GLU A 2 4.035 -26 .755 48 .064 1.00 54 .92 ATOM 20 CA GLU A 2 2.803 -26 .044 47 .744 1.00 53 .59 ATOM 21 C GLU A 2 2.870 -24 .570 48 .154 1.00 52 .81 ATOM 22 O GLU A 2 2.555 -23 .664 47 .393 1.00 51 .69 ATOM 23 CB GLU A 2 1.661 -26 .740 48 .482 1.00 56 .88 ATOM 24 CG GLU A 2 2.090 -28 .092 49 .054 0.00 99 .00 ATOM 25 CD GLU A 2 1.019 -28 .610 49 .983 0.00 99 .00 ATOM 26 OE1 GLU A 2 0.454 -27 .819 50 .722 0.00 99 .00 ATOM 27 OE2 GLU A 2 0.761 -29 .811 49 .963 0.00 99 .00 ATOM 28 N THR A 3 3.260 -24 .346 49 .424 1.00 52 .06 ATOM 29 CA THR A 3 3.300 -22 .980 49 .940 1.00 51 .61 ATOM 30 C THR A 3 4.161 -22 .059 49 .070 1.00 50 .30 ATOM 31 O THR A 3 3.731 -21 .006 48 .617 1.00 49 .91 ATOM 32 CB THR A 3 3.858 -23 .023 51 .364 1.00 54 .31 ATOM 33 OG1 THR A 3 2.975 -23 .789 52 .187 1.00 56 .98 ATOM 34 CG2 THR A 3 3.960 -21 .605 51 .935 1.00 55 .18 ATOM 35 N LEU A 4 5.372 -22 .498 48 .717 1.00 50 .11 ATOM 36 CA LEU A 4 6.201 -21 .696 47 .808 1.00 50 .48 ATOM 37 C LEU A 4 5.553 -21 .516 46 .444 1.00 50 .18 ATOM 38 O LEU A 4 5.520 -20 .410 45 .877 1.00 50 .73 ATOM 39 CB LEU A 4 7.603 -22 .286 47 .626 1.00 52 .72 ATOM 40 CG LEU A 4 8.545 -22 .252 48 .826 1.00 56 .58 ATOM 41 CD1 LEU A 4 9.819 -23, .035 48, .583 1.00 55, ,37 ATOM 42 CD2 LEU A 4 8.829 -20, .828 49, .288 1.00 56, .27 ATOM 43 N CYS A 5 5.028 -22, .615 45, .885 1.00 49, ,77 ATOM 44 CA CYS A 5 4.362 -22. .530 44, .587 1.00 48, .93 ATOM 45 C CYS A 5 3.218 -21. .534 44. .589 1.00 48. .27 ATOM 46 O CYS A 5 3.136 -20. .698 43, .682 1.00 47. .03 ATOM 47 CB CYS A 5 3.865 -23. .879 44. .075 1.00 49, ,50 ATOM 48 SG CYS A 5 5.217 -24. .972 43. .626 1.00 50. ,79 ATOM 49 N GLN A 6 2.356 -21. ,627 45. .610 1.00 46. ,85 ATOM 50 CA GLN A 6 1.227 -20. ,718 45. ,675 1.00 47. 22 ATOM 51 C GLN A 6 1.666 -19. ,276 45. ,865 1.00 46. 83 ATOM 52 O GLN A 6 1.050 -18. ,382 45. ,276 1.00 48. 60 ATOM 53 CB GLN A 6 0.272 -21. 079 46. 817 1.00 50. 54 ATOM 54 CG GLN A 6 -0.681 -22. 221 46. 515 1.00 55. 34 ATOM 55 CD GLN A 6 -1.144 -22. 875 47. 806 1.00 59. 63 ATOM 56 OE1 GLN A 6 -1.101 -22. 222 48. 853 1.00 61. 52 ATOM 57 NE2 GLN A 6 -1.482 -24. 156 47. 775 1.00 57. 45 ATOM 58 N ARG A 7 2.628 -19. 035 46. 757 1.00 46. 33 ATOM 59 CA ARG A 7 3.162 -17. 700 46. 938 1.00 46. 38 ATOM 60 C ARG A 7 3.780 -17. 145 45. 650 1.00 44. 90 ATOM 61 O ARG A 7 3.544 -15. 986 45. 355 1.00 45. 29 ATOM 62 CB ARG A 7 4.267 -17. 682 48. 013 1.00 50. 84 ATOM 63 CG ARG A 7 3.690 -17. 869 49. 418 1.00 62. 38 ATOM 64 CD ARG A 7 2.884 -16. 616 49. 765 1.00 71. 41 ATOM 65 NE ARG A 7 3.786 -15. 539 50. 171 1.00 78. 82 ATOM 66 CZ ARG A 7 3.406 -14. 307 50. 474 1.00 83. 50 ATOM 67 NH1 ARG A 7 2.122 -13. 965 50. 423 1.00 85. 24 ATOM 68 NH2 ARG A 7 4.316 -13. 410 50. 841 1.00 85. 59 ATOM 69 N LEU A 8 4.608 -17. 918 44. 984 1.00 45. 22 ATOM 70 CA LEU A 8 5.212 -17. 429 43. 728 1.00 45. 85 ATOM 71 C LEU A 8 4.161 -17. 110 42. 672 1.00 46. 67 ATOM 72 O LEU A 8 4.197 -16. 055 42. 025 1.00 46. 25 ATOM 73 CB LEU A 8 6.185 -18. 477 43. 194 r.oo 41. 52 ATOM 74 CG LEU A 8 6.979 -18. 054 41. 944 1.00 44. 17 ATOM 75 CD1 LEU A 8 7.941 -16. 924 42. 283 1.00 44. 96 ATOM 76 CD2 LEU A 8 7.723 -19. 246 41. 373 1.00 44. 18 ATOM 77 N ASN A 9 3.193 -18. 014 42. 508 1.00 47. 73 ATOM 78 CA ASN A 9 2.065 -17. 825 41. 610 1.00 48. 73 M cri σι σι H H a) -i σι θ (Nj rH O rH O O o < cn ro ^ -O n ro c^ rH rH ^ ^ rH cn cD t^ cy* yi o_i cD oo ^ a) ^ ^ ■ a< ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ r ^ ^ r n ro r o rn ro o ro o ro c M ro ^ ^ r^ ^o (η ^ ^ H ^ ( η ^ ^o aJ H a) lΛ (, l tJl 'J ■ ( <1 ^ u ) ( ) ( η n ( !l O ■ i^ a H flO 'T ( O O H ln ι » l ln σ^ O l^ ln ^- '^ H O O •a , m o r^ o ^ ^o ^ cD O ^D σl (^J ^i ) c^l H ^^l M Φ t cn ω o n H H ιIι N H n H ^ ^D a) rη m « ln (I) H ln r^) m m σι m ^ m co ω m ^^ H H « N ^ N n n al ^ι -^ O l l M rn o H ^ H σl (n ^ n tη Lo i^ σi rH ^ σ^ cN O oo ao ^ ^ co o c^ ^ ^ c D ^ ^ σ cM r^ cj ro ro cN rH iO rH ^ co ^ rH rH i^ m ιoιncooo o«)ιnιη(M'»n ιn ^N H o f ^ in in 001 co r^ n co ' i ^O '^ ri oo co i^ cn i co o ri Ti cD t^ o oo r- rH rH i-H CM CM CM rH rH rH rH rH rH rH rH rH I I I I I I I I I I I I I I I I I I lI lI lI lI I I I I I I I I I I I l l l l o m H oo i H θ c N o o o H i n n iη ^ io c) ^ csj n -sr iD kD ^o r^ H o o o o o o cM o o o cM nn ro i i irno iDcN M H rH rH r CM n o o I I I l l l l I I I σι σι σι c^ cn σι o o o o o o θ rH H rH rH H H <>ι cN N CN CN CN M N M ro n ro c ro rn r ro ^ ^ H ri H H H H H H rl H H H ri H H H H H H H H H H H H H rl H H H H H H H H H H H H H ri H H H H H H H H H H H H H H H H Z z z s z z J J rH ^ ^ hH W w c co w OT Z z z z z s z z ^ w n n cn n M ^ rt; fi; «; >H < H ^ >H < j j J rl J J ^ hi ^ w i» M (a oi co c ffl > ( i ^ -c ac «θ > > > > > u u ϋ u u u o ϋ ϋ o o o ϋ o < «ι; ιi: (i; < ιJ J hi m QQ oi raou BC m o < ffl UDw a KC m o Q D f-c cQ O Q ω N <: CQ o e> α < Q O Q Q < uo υ υo z z υυo u υ u z uυo υw z o υo c cj c o z z αu o υ υ o o z υ α oυ υ u z z υ o o o u υc z j j o u j o α z o σ^ θ H r ^ Ln ^ ι^ cD crι θ H M ro ^ Lθ ^ r^ co σι θ ιH CM rθ ' Lθ ' > [^ oo rι θ rH CM rn iHD r^ co m o H C iη ^r in vD r^ co σi O H rM O ^ in ω i^ co CΛ O H i^ co co co co co co co co co co cfi oi cn cn σi σi σiCΛ CΛ cfi o o o o o o o o o o H H H H rH rH rH rH rH C CM M CM (N0 M M CM rπ r r π ro ro ro r r rη -3' ^ H H H ri H H H H H H H H ri H ri H H rl H H H H H H H H H θ in o o rn o ∞ σi rH oo ^ ro i^ - o si iD o o^ H t^ o cM O O ^ i^ oo in o ^ σi ^ cD oo io cri -o M CN ^ [ ^ σι^ iD ιnr~ iΛ rH tn cn ro crι r iD M ^ oD ir) ^ oo r cα oo ^r o ι ^ crι r rH C -o σ rH σι »^ rH in r ^ -n oo i ^ r r ^ i^ iH ^ Osi i^ M i^ ro ^ iri ^ Ln ^ r ^ ^ n r^ cM ^ i^ σi cn o ^ oo D M rH ^ ri ^ ro UJ Ln r^ r^ r^ lQ ^ -O ^ I^ Cn σi O O H ^ iη C rH CN rO O rO ^ ^ ^ O C rH rH O CN rH rH rH ^ co rπ co co cπ co cn ro co co ro cn ^ ^ ^cn co c co cπ co cπ co m co co co co ωω rH M mo LO LO M H ιn i i r^ r~ [^ [^ r^ αo ∞ ∞ ∞ ∞ ∞ ∞ ∞ ∞ ro σt cn σϊ θ σ σ crι o^ σ σϊ σ σι o o o o o o o o pϊ; pC Λ αi - DS ω c ω m cn w ω n w iϊ S Λ rt Di rt oS rt c- Dj α; !!; □ D D S D D D D Z Z Z Z Z Z Z Z ft ft Oj Oi m ft ft iϋ iU J iiJ dJ lϊ oi Oj Oi rt d! j j j j j j j j tn oi t oi wio oi oi tn oi ϊi c tn M c w ω ω ω ω u i i i K o (j j o u o _3 o u ι <: < rt : ι <: < ι rt: rt; <: : m w w o w w E→ H H O C5 C5 < m o α Q ω ω eζ Da Q Q ia ω N a m o α ω ω ; Λ U Q Q < co o o α <; PQ to m en o o o o o z ϋuo uuz uυz Z U O O O U U O U C C O s υ oo υ υ υ o o z υ c o υ u o z z c o o υ o oo ^; uuouo zuuou N r^ ^ iΛ ia i^ oo σ O rH cπ ^ -o io i^ co cri o H CN ro ^ in io i^ n σϊ O rH CN rn ^ in io t^ oo ^ •^ ^ ■^ ^ ^ •^ ^r Ln -o in in un LO Λ iO -O -n iD iD iβ iD iD iD io iD iO iD i^ i^ i^ i^ ^ r^ i^ r^ i^ i^ co co ∞ H H H H H ri H H ri rl H H rl rl H H H H H ri H H H H rl H H H H H H H ri H H H H H H H H ri H H H H H H H H rl H H H H H H H PI M N N N ATOM 205 OG1 THR A 24 0.042 7.871 34.734 1.00 51.02 ATOM 206 CG2 THR A 24 1 .558 6 .901 33 .094 1 .00 48 .60 ATOM 207 N ASP A 25 1 .124 6 .096 37 .828 1 .00 46 .98 ATOM 208 CA ASP A 25 1 .058 6 .453 39 .234 1 .00 46 .50 ATOM 209 C ASP A 25 2 .193 5 .788 40 .013 1 .00 45 .63 ATOM 210 O ASP A 25 2 .376 4 .578 40 .042 1 .00 44 .95 ATOM 211 CB ASP A 25 -0 .286 6 .024 39 .847 1 .00 53 .36 ATOM 212 CG ASP A 25 -1 .442 6 .789 39 .202 1 .00 62 .10 ATOM 213 OD1 ASP A 25 -1 .605 7 .997 39 .498 1 .00 64 .72 ATOM 214 OD2 ASP A 25 -2 .185 6 .192 38 .392 1 .00 62 .72 ATOM 215 N LEU A 26 3 .000 6 .633 40 .614 1 .00 45 .40 ATOM 216 CA LEU A 26 4 .167 6 .207 41 .381 1 .00 45 .37 ATOM 217 C LEU A 26 3 .834 5 .157 42 .418 1 .00 45 .85 ATOM 218 O LEU A 26 4 .563 4 .170 42 .565 1 .00 46 .38 ATOM 219 CB LEU A 26 4 .763 7 .483 41 .982 1 .00 44 .87 ATOM 220 CG LEU A 26 6 .056 7 .341 42 .783 1 .00 44 .69 ATOM 221 CD1 LEU A 26 7 .128 6 .688 41 .931 1 .00 40 .20 ATOM 222 CD2 LEU A 26 6 .529 8 .703 43 .267 1 .00 46 .93 ATOM 223 N ARG A 27 2 .741 5 .281 43 .178 1 .00 45 .28 ATOM 224 CA ARG A 27 2 .266 4 .241 44 .065 1 .00 45 .19 ATOM 225 C ARG A 27 2 .251 2 .841 43 .483 1 .00 44 .47 ATOM 226 O ARG A 27 2 .610 1, .886 44, .187 1 .00 44 .56 ATOM 227 CB ARG A 27 0 .852 4 .494 4 .607 1 .00 51 .04 ATOM 228 CG ARG A 27 0 .713 5, .531 45, .690 1 .00 61 .27 ATOM 229 CD ARG A 27 -0 .715 6 .081 45, .714 1 .00 66 .89 ATOM 230 NE ARG A 27 -0 .927 6 .984 46 .839 1 .00 75 .54 ATOM 231 CZ ARG A 27 -2 .083 7, .555 47, .170 1 .00 79 .36 ATOM 232 NH1 ARG A 27 ■ 3, .184 7, .331 46, .456 1 .00 80, .70 ATOM 233 NH2 ARG A 27 • 2, .152 8, .359 48, .228 1, .00 79, .78 ATOM 234 N ASP A 28 1, .771 2. .632 42. .255 1 .00 43, ,06 ATOM 235 CA ASP A 28 1, .785 1, .318 41, .648 1, .00 41. .77 ATOM 236 C ASP A 28 3, ,195 0. .797 41. ,385 1, ,00 41, ,18 ATOM 237 O ASP A 28 3. .408 -0, .409 41. ,402 1, .00 40. ,08 ATOM 238 CB ASP A 28 1. ,014 1. .325 40. ,303 1, ,00 44. ,91 ATOM 239 CG ASP A 28 • 0. .450 1. ,655 40. ,560 1, ,00 53. ,31 ATOM 240 OD1 ASP A 28 ■ 1, ,014 2. .560 39. ,920 1, ,00 53, ,29 ATOM 241 OD2 ASP A 28 •1. .022 0. ,983 41. ,446 1, .00 52, ,80 ATOM 242 N HIS A 29 4. .132 1. ,700 41. 062 1. ,00 40. ,87 ATOM 243 CA HIS A 29 5. ,510 1. 269 40. 787 1. ,00 40. ,10 ATOM 244 C HIS A 29 6. ,208 0. .795 42. 073 1, ,00 39, ,08 ATOM 245 O HIS A 29 6. ,987 -0. 143 42. 045 1. ,00 38. 12 ATOM 246 CB HIS A 29 6. 246 2. 473 40. 166 1. ,00 40. 60 ATOM 247 CG HIS A 29 5. ,590 2. 806 38. 837 1. .00 42. ,13 ATOM 248 ND1 HIS A 29 5. 069 1. 810 38. 042 1. ,00 42. 28 ATOM 249 CD2 HIS A 29 5. 373 3. 980 38. 192 1. ,00 44. 10 ATOM 250 CE1 HIS A 29 4. 552 2. 348 36. 943 1. 00 43. 73 ATOM 251 NE2 HIS A 29 4. 738 3. 656 37. 014 1. 00 42. 95 ATOM 252 N ILE A 30 5. 896 1. 454 43. 152 1. 00 39. 44 ATOM 253 CA ILE A 30 6. 339 0. 990 44. 501 1. 00 39. 95 ATOM 254 C ILE A 30 5. 899 -0. 426 44. 746 1. 00 40. 09 ATOM 255 O ILE A 30 6. 658 -1. 303 45. 181 1. 00 41. 16 ATOM 256 CB ILE A 30 5. 843 1. 991 45. 550 1. 00 40. 65 ATOM 257 CGI ILE A 30 6. 563 3. 321 45. 334 1. 00 40. 86 ATOM 258 CG2 ILE A 30 6. 125 1. 537 47. 004 1. 00 41. 39 ATOM 259 CD1 ILE A 30 6. 060 4. 498 46. 138 1. 00 42. 24 ATOM 260 N ASP A 31 4. 631 -0. 764 44. 485 1. 00 41. 09 ATOM 261 CA ASP A 31 4. 082 -2. 089 44. 758 1. 00 40. 37 ATOM 262 C ASP A 31 4. 718 -3. 130 43. 856 1. 00 40. 59 ATOM 263 O ASP A 31 4. 965 -4. 277 44. 244 1. 00 40. 70 ATOM 264 CB ASP A 31 2. 566 -2. 080 44. 459 1. 00 42. 53 ATOM 265 CG ASP A 31 1. 886 -3. 379 44. 801 1. 00 44. 66 ATOM 266 OD1 ASP A 31 1. 799 -4. 311 43. 991 1. 00 46. 03 ATOM 267 OD2 ASP A 31 1. 495 -3. 517 45. 987 1. 00 53. 28 co IT) M H CN M O n co an^ co ^ ifl r rO M' ^ rO H n σ> co o o -o io iD io r- r^ co oo o io ^? n r ^ ^ i ) Lo _ r M ro r rH o o^ cx5 ' ' iD iD u> r- co ri O rH rH CM in oi co ci H i'i H m in Mn 'S' i' O i o HD i ai n co co -n oo r^ o ri o r^ o co o co rH CM n co r- co m N 'T σi ^ in co nJ o o H Λ H ro rM e ro -» m CM ro CM m ro ■» in n n i n M M' fi r 'i' iO r- r~ Mn ω m τ I I I I I I I I I I I I I I I I I I I I I I I I I I I I i io ^ σi i o ^ in in H Λ M o ro ^ o ^ co ^ M ^ ^ σi in H i o o r^ o o ιn ^r iD rι o m r~ ω o r~ cfι co ^ ιn r- iD ι ι iN U! H r σι o co ιn o rM in o «! ■^ r rH Lθ iθ ιn r^ o ^ r^ o cM ^ ao ^ -n -n cβ , ^ CM r^ r o crι i ^< Lθ rι co cM iD ano ^ in r~ H rO "» o ) ro N i- ) ( ci [^ co ro rM co o cM ^ cM ir) r- ro «) N r- σno o (θ M^ r o rM co >j (θ co H H θ θ ιn N in m r N N in o M H θno N co o ι o m u3 σι cN i N in -ι ιn m u3 rι co ^ ιn o o ω oi ι^ cM θ ιn rθ H >D ' * ω uo rn i N co fι θ H θ n θ ri θ H H N H rNi N ri CN CN CN CN CN CNI N N CN CN CN CO C^ C^ m r C^ rO r C^ CO r C CO po ro i^ on ro m ro ro r m ro rn c r r oo ro ro ro ro r^ oi cϊJ oi ctf oϊ ffS i-S i^ c-ϊ pS c-i ai aj i-j i-j ai H i > >H >H H ; i >H >H >H >H H e^ c a; pi cn p-; ps c c tf h ^ h B h H H ^ ^ B h h B t< fH h B B H H h H H J hi H t j j H j -; ι ι; ϊ! ϊ; ι; ι x ϊ ϊ 2 _; -; _; s 2 s ∑ rt < <; < fl; rt; <; <: CM rH CN rH < (ΣHJ D Q W H N 3C <; ωuQawωωisiNa Λ; cquaω <c „ PQ rj a a ω . „ω en o a ω ; Q O Q W N K zυuouuooocjoozυυo oυuuzoυυυυzc cjoc oocjzzuυooozc υz Z U U O U O OI U Z U U O U U U Z U Z co σι θ H M ro <^ Lθ i r^ co σι θ H C r ^ ιn iD r^ cD n o rH M <η ^ Ln ιo r^ co cn o rH M r^ i iD r^ r^ r^ r^ r~ r^ r^ ι^ r~ ι^ co co co co co oo co co co co σi rι σι cn n σ\ σι σι o^ n o o o o o CN CN CN CN CN CN C CN CN CN CN CM CN C\1 CN C\] CN CN CN C\J CN CN CN CN CN CN CN CN CN CN CN CN C^ *0 0000000000000000000000000000 00000000 00 00000000 000000 000000 0000 0 © ; ι< fit; ; < fiC ; fiC ; fiC rt; fit; rt; fiC fiC fit; fiC ι fiC ι fiC fiC ι< ; c < ; rt; fiC rt; fi fi( fi^ O ATOM 331 NH2 ARG A 37 12.948 -5.572 52.719 1.00 32.86 ATOM 332 N LEU A 38 9 .920 -9 .544 46 .841 1 .00 33 .92 ATOM 333 CA LEU A 38 9 .330 -10 .768 47 .372 1 .00 34 .82 ATOM 334 C LEU A 38 9 .592 -11 .985 46 .532 1 .00 36 .08 i ATOM 335 O LEU A 38 9 .879 -13 .050 47 .066 1 .00 35 .88 ATOM 336 CB LEU A 38 7 .806 -10 .549 47 .542 1 .00 37 .00 ATOM 337 CG LEU A 38 7 .048 -11 .843 47 .862 1 .00 39 .41 ATOM 338 CD1 LEU A 38 7 .338 -12 .303 49 .271 1 .00 36 .17 ATOM 339 CD2 LEU A 38 5 .544 -11 .698 47 .633 1 .00 42 .91 10 ) ATOM 340 N GLU A 39 9 .532 -11 .873 45 .176 1 .00 36 .01 ATOM 341 CA GLU A 39 9 .903 -12 .982 44 .328 1 .00 35 .41 ATOM 342 C GLU A 39 11 .310 -13 .492 44 .650 1 .00 35 .95 ATOM 343 O GLU A 39 11 .524 -14 .706 44 .610 1 .00 34 .71 ATOM 344 CB GLU A 39 9 .826 -12 .621 42 .814 1 .00 33 .83 15i ATOM 345 CG GLU A 39 9 .999 -13 .858 41 .944 1 .00 35 .55 ATOM 346 CD GLU A 39 10 .153 -13 .499 40 .467 1 .00 44 .56 ATOM 347 OE1 GLU A 39 11 .229 -12 .997 40 .106 1 .00 42 .84 ATOM 348 OE2 GLU A 39 9 .219 -13 .700 39 .690 1 .00 42 .80 ATOM 349 N CYS A 40 12 .280 -12 .600 44 .916 1 .00 35 .37 20 I ATOM 350 CA CYS A 40 13 .616 -13 .054 45 .262 1 .00 35 .02 ATOM 351 C CYS A 40 13 .603 -13. .852 46 .574 1 .00 35 .78 ATOM 352 O CYS A 40 14 .329 -14, .842 46 .621 1 .00 35 .14 ATOM 353 CB CYS A 40 14 .587 -11 .879 45 .434 1 .00 34 .19 ATOM 354 SG CYS A 40 14 .743 -10 .845 43 .945 1 .00 35 .07 25 ' ATOM 355 N ALA A 41 12 .796 -13, .419 47, .540 1 .00 36 .59 ATOM 356 CA ALA A 41 12 .772 -14 .160 48, .820 1 .00 38 .13 ATOM 357 C ALA A 41 12, .191 -15, .553 48, .590 1 .00 37 .52 ATOM 358 O ALA A 41 12, ,659 -16, .527 49, ,200 1, .00 38, .32 ATOM 359 CB ALA A 41 11. .955 -13. .380 49, .827 1, .00 36, .08 30 1 ATOM 360 N ILE A 42 11, .221 -15, .674 47, .663 1, .00 37, .54 ATOM 361 CA ILE A 42 10, .629 -16, .995 47. .397 1, .00 36. .18 ATOM 362 C ILE A 42 11, ,626 -17. .922 46, ,753 1. .00 36, .44 ATOM 363 O ILE A 42 11, ,856 -19. .069 47, ,133 1, ,00 35, .31 ATOM 364 CB ILE A 42 9, ,325 -16. ,907 46. .581 1, .00 36, ,30 35 ATOM 365 CGI ILE A 42 8. ,225 -16. ,165 47. .345 1, .00 38, .51 ATOM 366 CG2 ILE A 42 8. ,865 -18, ,282 46, ,108 1. .00 38, ,23 ATOM 367 CD1 ILE A 42 7. .114 -15, ,700 46. ,390 1, .00 41, ,57 ATOM 368 N TYR A 43 12. ,321 -17. ,436 45, ,707 1, ,00 35. ,53 ATOM 369 CA TYR A 43 13. 341 -18. ,254 45. ,060 1. ,00 36. ,20 40 1 ATOM 370 C TYR A 43 14, ,479 -18. ,536 46. ,047 1, .00 36. ,21 ATOM 371 O TYR A 43 15. 091 -19. 597 45. ,993 1. ,00 36. ,69 ATOM 372 CB TYR A 43 13. 884 -17. 474 43. 838 1. 00 36. ,61 ATOM 373 CG TYR A 43 13. 065 -17. 637 42. 572 1. ,00 38. ,55 ATOM 374 CD1 TYR A 43 12. 717 -16. 512 41. 820 1. 00 39. 59 5 ATOM 375 CD2 TYR A 43 12. 644 -18. 871 42. 116 1. 00 39. 53 ATOM 376 CE1 TYR A 43 11. 998 -16. 626 40. 646 1. 00 41. 22 ATOM 377 CE2 TYR A 43 11. 943 -19. 006 40. 918 1. 00 40. 40 ATOM 378 CZ TYR A 43 11. 604 -17. 884 40. 210 1. 00 40. 78 ATOM 379 OH TYR A 43 10. 847 -17. 954 39. 081 1. 00 41. 19 0 ATOM 380 N TYR A 44 14. 794 -17. 563 46. 906 1. 00 35. 43 ATOM 381 CA TYR A 44 15. 933 -17. 815 47. 811 1. 00 37. 45 ATOM 382 C TYR A 44 15. 547 -19. 008 48. 716 1. 00 37. 65 ATOM 383 O TYR A 44 16. 329 -19. 945 48. 876 1. 00 38. 00 ATOM 384 CB TYR A 44 16. 205 -16. 555 48. 635 1. 00 38. 20 5 ATOM 385 CG TYR A 44 17. 445 -16. 670 49. 503 1. 00 40. 09 ATOM 386 CD1 TYR A 44 17. 398 -17. 286 50. 756 1. 00 41. 30 ATOM 387 CD2 TYR A 44 18. 663 -16. 206 49. 041 1. 00 40. 77 ATOM 388 CE1 TYR A 44 18. 569 -17. 412 51. 492 1. 00 42. 23 ATOM 389 CE2 TYR A 44 19. 833 -16. 312 49. 776 1. 00 42. 80 0 ATOM 390 CZ TYR A 44 19. 746 -16. 907 51. 023 1. 00 43. 39 ATOM 391 OH TYR A 44 20. 863 -17. 049 51. 798 1. 00 45. 51 ATOM 392 N LYS A 45 14. 334 -18. 982 49. 224 1. 00 38. 11 ATOM 393 CA LYS A 45 13. 891 -20. 078 50. 118 1. 00 40. 98 ATOM 394 C LYS A 45 13.832 -21.403 49.387 1.00 41.49 ATOM 395 O LYS A 45 14 .315 -22 .472 49 .789 1 .00 42 .02 ATOM 396 CB LYS A 45 12 .537 -19 .754 50 .750 1 .00 40 .25 ATOM 397 CG LYS A 45 11 .968 -20 .894 51 .614 1 .00 48 .19 ATOM 398 CD LYS A 45 12 .824 -21 .269 52 .813 1 .00 51 .93 ATOM 399 CE LYS A 45 12 .671 -20 .308 53 .983 1 .00 59 .68 ATOM 400 NZ LYS A 45 13 .979 -20 .145 54 .698 1 .00 61 .34 ATOM 401 N ALA A 46 13 .307 -21 .357 48 .139 1 .00 42 .39 ATOM 402 CA ALA A 46 13 .230 -22 .586 47 .356 1 .00 41 .88 ATOM 403 C ALA A 46 14 .613 -23 .167 47 .179 1 .00 41 .96 ATOM 404 O ALA A 46 14 .828 -24 .368 47 .347 1 .00 42 .06 ATOM 405 CB ALA A 46 12 .561 -22 .294 46 .004 1 .00 45 .41 ATOM 406 N ARG A 47 15 .605 -22 .341 46 .839 1 .00 42 .18 ATOM 407 CA ARG A 47 16 .967 -22 .806 46 .649 1 .00 42 .89 ATOM 408 C ARG A 47 17 .567 -23 .364 47 .949 1 .00 44 .97 ATOM 409 O ARG A 47 18 .270 -24 .377 47 .899 1 .00 44 .85 ATOM 410 CB ARG A 47 17 .873 -21 .700 46 .134 1 .00 39 .95 ATOM 411 CG ARG A 47 19 .278 -22 .115 45 .751 1 .00 44 .08 ATOM 412 CD ARG A 47 19 .323 -23 .087 44 .564 1 .00 51 .87 ATOM 413 NE ARG A 47 20 .701 -23 .450 44 .306 1 .00 57 .92 ATOM 414 CZ ARG A 47 21 .372 -24 .042 43 .351 1 .00 62 .27 ATOM 415 NH1 ARG A 47 20 .819 -24 .506 42 .243 1 .00 60 .85 ATOM 416 NH2 ARG A 47 22 .696 -24 .175 43 .516 1 .00 66 .76 ATOM 417 N GLU A 48 17. .287 -22 .673 49 .051 1 .00 45 .14 ATOM 418 CA GLU A 48 17, .783 -23 .090 50, .364 1 .00 48 .18 ATOM 419 C GLU A 48 17 .266 -2 .500 50 .681 1 .00 48 .66 ATOM 420 O GLU A 48 18, .009 -25, .362 51, .122 1 .00 49 .01 ATOM 421 CB GLU A 48 17, .202 -22, .208 51, ,472 1 .00 50 .97 ATOM 422 CG GLU A 48 17, .987 -21. .039 51, .955 1 .00 59 .37 ATOM 423 CD GLU A 48 17. .911 -20. .689 53, .432 1, .00 58, .91 ATOM 424 OE1 GLU A 48 16, ,891 -20, .144 53, .912 1, .00 64, .65 ATOM 425 OE2 GLU A 48 18, ,904 -20, .949 54, .156 1, .00 64, .43 ATOM 426 N MET A 49 16. ,000 -24. ,730 50. ,361 1, ,00 49. .75 ATOM 427 CA MET A 49 15. .358 -26. .017 50, .510 1, .00 50. .42 ATOM 428 C MET A 49 15. ,738 -27. ,106 49. ,543 1. .00 50. .69 ATOM 429 O MET A 49 15. 197 -28. ,218 49. ,620 1. ,00 51. ,52 ATOM 430 CB MET A 49 13. 840 -25. ,835 50. ,513 1. .00 53. .09 ATOM 431 CG MET A 49 13. 351 -25. 048 51. ,719 1. ,00 55. ,67 ATOM 432 SD MET A 49 11. 617 -24. 613 51. ,508 1. ,00 64. ,13 ATOM 433 CE MET A 49 10. 922 -24. 927 53. 123 1. ,00 64. 34 ATOM 434 N GLY A 50 16. 616 -26. 852 48. 592 1. 00 50. 09 ATOM 435 CA GLY A 50 17. 159 -27. 787 47. 664 1. ,00 48. 51 ATOM 436 C GLY A 50 16. 332 -27. 949 46. 384 1. 00 48. 34 ATOM 437 O GLY A 50 16. 603 -28. 920 45. 679 1. 00 47. 16 ATOM 438 N PHE A 51 15. 383 -27. 074 46. 084 1. 00 48. 32 ATOM 439 CA PHE A 51 14. 628 -27. 205 44. 837 1. 00 48. 99 ATOM 440 C PHE A 51 15. 442 -26. 616 43. 675 1. 00 49. 10 ATOM 441 O PHE A 51 16. 187 -25. 659 43. 884 1. 00 48. 66 ATOM 442 CB PHE A 51 13. 266 -26. 527 44. 904 1. 00 54. 66 ATOM 443 CG PHE A 51 12. 370 -27. 030 46. 005 1. 00 62. 30 ATOM 444 CD1 PHE A 51 12. 237 -28. 382 46. 261 1. 00 66. 64 ATOM 445 CD2 PHE A 51 11. 648 -26. 142 46. 784 1. 00 64. 64 ATOM 446 CE1 PHE A 51 11. 418 -28. 837 47. 281 1. 00 67. 20 ATOM 447 CE2 PHE A 51 10. 813 -26. 584 47. 788 1. 00 65. 80 ATOM 448 CZ PHE A 51 10. 708 -27. 937 48. 047 1. 00 65. 88 ATOM 449 N LYS A 52 15. 455 -27. 331 42. 554 1. 00 49. 49 ATOM 450 CA LYS A 52 16. 204 -26. 877 41. 377 1. 00 48. 93 ATOM 451 C LYS A 52 15. 254 -26. 192 40. 393 1. 00 47. 94 ATOM 452 O LYS A 52 15. 644 -25. 414 39. 516 1. 00 46. 65 ATOM 453 CB LYS A 52 16. 905 -28. 073 40. 739 1. 00 54. 00 ATOM 454 CG LYS A 52 17. 964 -28. 697 41. 640 1. 00 61. 04 ATOM 455 CD LYS A 52 18. 931 -29. 536 40. 811 1. 00 64. 27 ATOM 456 CE LYS A 52 19 . 717 -30 . 537 41 . 711 0 . 00 99 . 00 ATOM 457 NZ LYS A 52 20.300 -31.674 41.001 0.00 99.00 ATOM 458 N HIS A 53 13 .968 -26 .480 40 .589 1 .00 46 .17 ATOM 459 CA HIS A 53 12 .924 -25 .816 39 .834 1 .00 45 .58 ATOM 460 C HIS A 53 11 .697 -25 .685 40 .724 1 .00 44 .64 5 ATOM 461 O HIS A 53 11 .566 -26 .465 41 .649 1 .00 44 .95 ATOM 462 CB HIS A 53 12 .568 -26 .542 38 .531 1 .00 44 .28 ATOM 463 CG HIS A 53 11 .635 -27 .700 38 .758 1 .00 46 .45 ATOM 464 ND1 HIS A 53 10 .277 -27 .525 38 .978 1 .00 45 .21 ATOM 465 CD2 HIS A 53 11 .868 -29 .026 38 .829 1 .00 45 .27 10 ) ATOM 466 CE1 HIS A 53 9 .709 -28 .698 39 .163 1 .00 45 .18 ATOM 467 NE2 HIS A 53 10 .655 -29 .629 39 .076 1 .00 49 .44 ATOM 468 N ILE A 54 10 .895 -24 .693 40 .468 1 .00 43 .90 ATOM 469 CA ILE A 54 9 .598 -24 .513 41 .100 1 .00 44 .62 ATOM 470 C ILE A 54 8 .589 -24 .354 39 .976 1 .00 43 .53 15 ) ATOM 471 O ILE A 54 8 .704 -23 .454 39 .123 1 .00 44 .50 ATOM 472 CB ILE A 54 9 .627 -23 .258 41 .996 1 .00 51 .01 ATOM 473 CGI ILE A 54 8 .222 -22 .740 42 .337 1 .00 51 .18 ATOM 474 CG2 ILE A 54 10 .420 -22 .138 41 .313 1 .00 54 .51 ATOM 475 CD1 ILE A 54 8 .293 -22 .022 43 .690 1 .00 55 .08 20 ) ATOM 476 N ASN A 55 7 .679 -25 .327 39 .853 1 .00 42 .57 ATOM 477 CA ASN A 55 6 .719 -25 .332 38 .748 1 .00 41 .98 ATOM 478 C ASN A 55 7 .391 -25 .383 37 .389 1 .00 41 .70 ATOM 479 O ASN A 55 6 .958 -24 .848 36 .368 1 .00 41 .10 ATOM 480 CB ASN A 55 5 .639 -24, .266 38 .828 1 .00 41 .18 25 > ATOM 481 CG ASN A 55 4 .611 -24 .596 39 .915 1, .00 45 .37 ATOM 482 OD1 ASN A 55 4, .481 -25, .766 40, .285 1, .00 42 .95 ATOM 483 ND2 ASN A 55 3 .908 -23, .589 40 .393 1, .00 49 .59 ATOM 484 N HIS A 56 8, .508 -26, .115 37, .343 1, .00 42, .09 ATOM 485 CA HIS A 56 9, .304 -26, .336 36. .155 1, .00 41, .80 30 ) ATOM 486 C HIS A 56 9, ,996 -25, .085 35, .651 1. .00 42, .37 ATOM 487 O HIS A 56 10. ,579 -25. .115 34, .573 1, ,00 41, .39 ATOM 488 CB HIS A 56 8. .509 -27. .081 35. .059 1, .00 38, .13 ATOM 489 CG HIS A 56 8. ,140 -28. .417 35. ,639 1. .00 39. .93 ATOM 490 ND1 HIS A 56 8. ,997 -29. .476 35, ,683 1. .00 40. .64 35i ATOM 491 CD2 HIS A 56 7. .000 -28. .811 36. .253 1, .00 40. ,69 ATOM 492 CE1 HIS A 56 8. ,401 -30. ,496 36. ,292 1. .00 46. ,16 ATOM 493 NE2 HIS A 56 7, ,192 -30, ,108 36. ,648 1. ,00 44, ,07 ATOM 494 N GLN A 57 10. ,115 -24. ,074 36. ,492 1. ,00 42. ,01 ATOM 495 CA GLN A 57 10. ,867 -22, ,845 36, ,209 1. .00 43. ,31 40 1 ATOM 496 C GLN A 57 12. .178 -22. ,922 37. ,011 1. ,00 42. ,73 ATOM 497 O GLN A 57 12. ,135 -23. ,367 38. .159 1. ,00 42. ,65 ATOM 498 CB GLN A 57 10. ,017 -21. 730 36. ,771 1. 00 44. ,03 ATOM 499 CG GLN A 57 10. 191 -20. 286 36. 476 1. 00 54. 62 ATOM 500 CD GLN A 57 8. 889 -19. 530 36. 723 1. 00 54. 38 5 ATOM 501 OE1 GLN A 57 8. 881 -18. 332 36. 990 1. 00 54. 35 ATOM 502 NE2 GLN A 57 7. 774 -20. 248 36. 641 1. 00 57. 38 ATOM 503 N VAL A 58 13. 308 -22. 526 36. 435 1. 00 41. 67 ATOM 504 CA VAL A 58 14. 592 -22. 695 37. 122 1. 00 41. 06 ATOM 505 C VAL A 58 14. 654 -21. 870 38. 395 1. 00 39. 90 0 ' ATOM 506 O VAL A 58 14. 141 -20. 751 38. 425 1. 00 40. 26 ATOM 507 CB VAL A 58 15. 766 -22. 343 36. 168 1. 00 41. 94 ATOM 508 CGI VAL A 58 15. 770 -20. 860 35. 859 1. 00 41. 59 ATOM 509 CG2 VAL A 58 17. 085 -22. 872 36. 676 1. 00 43. 75 ATOM 510 N VAL A 59 15. 193 -22. 451 39. 461 1. 00 40. 08 5 ATOM 511 CA VAL A 59 15. 414 -21. 673 40. 713 1. 00 40. 24 ATOM 512 C VAL A 59 16. 878 -21. 205 40. 635 1. 00 39. 98 ATOM 513 O VAL A 59 17. 761 -22. 042 40. 559 1. 00 41. 27 ATOM 514 CB VAL A 59 15. 250 -22. 591 41. 945 1. 00 40. 48 ATOM 515 CGI VAL A 59 15. 437 -21. 111 43. 243 1. 00 44. 12 0 ATOM 516 CG2 VAL A 59 13. 830 -23. 161 42. 037 1. 00 39. 62 ATOM 517 N PRO A 60 17. 121 -19. 911 40. 686 1. 00 40. 13 ATOM 518 CA PRO A 60 18. 466 -19. 380 40. 576 1. 00 40. 49 ATOM 519 C PRO A 60 19. 312 -19. 737 41. 806 1. 00 40. 57 co ID o o o rn m m o ωro O CN -sf lO coι in r- rocM CN O O O rH CN COCO CN cn Co r LO O o O o i ^ r^ co mmm ^ fi mo co ^ iD -i coσi om ifi Ln ^ ro inin ^ ro rs in ^ in ^ ^ N H inioro H o σi i π ro ^ ^ o m ^ N H rl H H H H H H H M H H H H H H C H H H H H H H H rl H H rl H H H ri H ri H ri H H I rH rH r-i rH r-t rH I I I ^- I I I I I I I i-H rH rH rH rH I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I H H O O M N IN r Ol CO Mfl r~ O O CN CN CM CM CN CN CN CN rH rH rH rH rH rH rH rH O O O O rH rH r rH iH rH rH CN CN CN CN CN vJ CN rO r ro r ^ ^ ^ '^ '^ ^ '^ tn i in i^ I^ I > D IΩ ID V^ D V~3 1^ V£> I IO ID ID I ID I£> I IO ID I I IO I I£> ID ID ID I^ IO I£> I^ ^ I ID I ID ^ U3 1 ID ID *£>.*& lO lJD l lQ lO lO lD l lO fiC fiC fiC <! ι^ ι fiC fiC fiC < £ζ fiC <; : rt: fiC ι< cC ; fiC fiC cC fiC fit; ι r^ ι ι^ o o o o pc; cύ os pd oi & c ncnn n nonetoco v C ςC C - -C -C D -c -c w w ω ω ω ω ω ω j ^ H i^ fiC fiC κ < fiC ω ω ω ω ω ω H H H >H >H >H >H H >H u 0j Hi En E-> En H En ιa; ιa; > > > > > > > o) co n !n oι ιn ( H i ι 4 ri i iH t 4 Hl ι 4 fiC c ι< fiC fit; fit; ι- : ι ^ rH CM rH CN rH CM rH CM pa j Q fit; m u u a; m u α Q K, PQ CQ D CJ SC mo £, OQ O Q W N fit; CQ O Q Q fiC ra o Q ω N o o oo z o ooo oo z o υ o o o υ o -s u o o c z o o o o o υ z o oooo z o ooo o o z s C 000 CJ 02 2 U 0 o o o u υ z O H C ro rH c CM Cj cM CM L in in L ui Λ Ln Lθ -θ L ooooooooσoooooooooooo ooooooooσooooooooooσo CM H ^r Lθ - " ι iD iD i^ iD 3 ι _o ^ r^ r^ cD '^ r m H H θ ι σι r r L Ln «j , iD r~ r^ oo σ^ σ^ ^ ^ LO O to ^ L ^ r^ ^ '^ O '^ '^ Ni o m ω iO M^ co CΛ o r- oo ^* ^J 1 ^ , *^ ^* ^* ^* ^3* ^* ^* ^ϊ 1 ^* ^* ^J* ^J* ^ 1 ^3* ^* ^3* CO CO ^^ ^* ^T 1 ^f ^* ^J* ^I 1 ^ji ^3^ ^3* ^J* ^3* ^3 ^^* *3* ^T* ^ * ^* ^3* ^3* ^3 ^ * ^^ ^3* ^T* ^ ^ * l' co o r L in σi in co ^ iD CM ^ i^ cD ^ ^ cn i^ o N CNi r^ r^ oo ^ co r^ o ^ co rH O i^ r^ Ni rH in co i^ co r^ rH r-~ co CM r— Λ ^ o o H ri H fl o i^ ω H CΛ ω m ^ ^ ^ o H ^ H Oi in H iβ m o ui in ^ o m H in N fO ω i^ tM Oi o H ro o H m H o r^ N ^ n o N ro o i^ -»^r-oιn o ^ H Hn o ^ ω ιn m ιnω ^ O H ^ ιn m H σ « ) m ιB ιη ^ ^f l ^ n r1 r ^ (N ^ N H co o H fn ι N ^c cι co o r<^ O H ι riO "ϊ ^O ln co ιn LO ID H r~ CO σ> co r^ ID en LO ^r r in iD iD in ir) ^T C CM '^Γ LO L I ^I* o o CM I I I I I I I I I I I I I I I I I I I I I I I I I I θ ^ Cn o CN I^ CO O -^ O rH CS1 ^ CN lO CQ O^ lΩ m θ rθ Cn oO '^ C^ rH rH ^ rH l cθ < Cn ^ o^ ιn cτι c < N r~ m o o m tn c r m cD σι o r ^ o ^ o N N ^ Lθ rH ^ Ln r iD sj i i ι iD N Co iD x3 j' θ rH c^ r co Lθ ιn cn rH rH co o ιo iΛ r~ [~ ro r r^ rH CM Lθ L^ r~ io r~ io iD co c co o O rH CN rH co co iD iD co cn θ rH cn < ) r r roio io m -iii ) ir)i->vD w r~^ i co cno noiO'3 , ro (M ro ^ ^iO N H ri H HH HH HH rl H H H H H H M HNHH H H HHH H ri oιm fl CΛm o oo oooo o H ri H H H H H H HN NN N Mn rorn nn oιη c ' ^ « ^ ^ ^ ^ ^ f inιnιn ιnιn ιnιn ι ιo^ iD iou)io «) ϊ)ifl r- ιDωωωι^ ^ r~^ ^^ r~ ^ ^ r»r^ ^^r~ ^ r-^ι^^ ι-- r~r~^ r~ r~^ ^r~ ^ ^ ^- ^ ^- r~ ^ ^r~^ r^^ ^r-r~r- ^- ^^ ^ ^ ^^ ^ ^ rtj ι-ι; et; fiC fii; fit; «< eC ι< ι^ fiC fiC ι< fi fii! κC fiC C fiC fiC fiC fi fiC fi fi<; fit; fiC fit; fi^ ZZZZZZZZZ-3-3 «: co a tjQ Q e; mu Q ω w c oα cC CQ cj α fi CQ O Q ω H fiC BODQ e PQ Q ω ω ct; zoooo z υ cj oo υ oυ z u o o υ υ o o z z o υ o υ z υ o o o cj o υ z υ υ o o uυ o o UUOUUUU z o o oo o o o z z o r ^ Lθ iD [^ oo rι σ rH CM r ^ L iD r^ cD θ θ rH CM r ^ ιn u3 r^ cx3 σι θ rH \i r ' in ιo r~ D θ^ θ rH c^ OT ∞ oo ∞ ∞ cD ∞ c o cri crϊ cri cri cn o^ cri σi O O O O O O O O o o L L θ L L Ln LO O L U0 ιn ιO - ι > n LO tn lO lD lD M0 lD lD ^ lD i > l^ l lO l l^ lD ^ ^ ι ) lD oooooooooooooo oooo oooo ooooooooooooooooooooooooooooo oo oooooo oooo H H EH .H EH EH EH EH H EH EH H EH EH EH EH H EH H H H H EH EH .H H EH H EH H EH .H .H H .H EH EH EH H EH EH EH .H © o cn rM ro Λ i' Λo ι r i oooooooooo oooooooooσ co m r iD r^ coc^ oo H i LOiOL '^'^ ro ^ ^ L in cM M rH r^ cxj co cn cn c^ooo DCD r^ ra∞ c^ ^ ^ ^ lO l l ^ ^ ^ ^ ^^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ L lO lO ^ ^ ^ ^ ^ ^ ^ ^ ^ I , ^ ^ ^ ^ ^ , <J 1 ^J' ^r ^l , '3 , ^ ^ , ^J , ^' <3 , 1 ^ ^ ^ 1 ^P LO Ln i r rH lD lD l^ C\l CM rH ∞ CD r~ r Cn cn θ rH CO lθ L OO rH '^ <^ CM rH r _O rH r M ^ O^ CO ^ ro cN -o iΩ U3 θ io cn r cN H - θ Ln ιn '^ '^ cN CN θ OT co r^ r *3< ιo ιn co ιn ∞ [^ rH CM r^ L ^ cn rH c o^ rH rH D r o ^ o^ co m co r^ o^ cM r^ rH cri r^ LO ^ ^ ^' ^ co r^ co oo rH ■ ^ LO CN r^ H H ^ ιn ^ ^ -l ^ ω -^ ιn ln ιD ^ rn N » ιn ιn ^ ^ ^ ^^ N C H ^ m m o co o o Λ o o H C^ι Λ o o fl cn o H co o m o co m co co o H N π I _D o o r^ in o r f r~ r~ r^ r~ r~ ι^ ∞ ∞ ∞ ∞ coco ∞ crι crι θ crι c crι y> c o o o o o o r~ r^ r^ r~ [^ r~ ι^ t^ r^ r~ r^ r^ [^ r^ [^ r^ r^ c^ r^ r~ r^ co coco co ao co co oo cD co co co cD ∞ x) ∞ eC fiC f< ι< fiC fid fiC < <ι; fiC <C κC fiC fiC fiC fiC < ^ fiC ^ < s fiC < fiC fil! fiC fiC <^ D D S D O D IU IIi CS J ei i liJ S D r D D D D > r3 n z3 r3 £> o$ & oi tt a: c£ e6 ω a ω u ω u tt c6 o6 cχ pl eύ pi eύ & ei cύ &, oύ z z z z z z wωωwωwκMwκffiK3sωwωωωωωc-i ej O O J J O O EH EH EH EH EH EH EH H H H rH rH EH EH EH EH H EH EH EH EH EH EH EH ; et; fit; si; fit; fit; CM CM rH CM CQ C5 Q Q fit; CQ CJ CJ fiC CQ CJ Q Q fiC CQ C Q ω w 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HHHHHHHHHHHHHHHriHHH l£> l0 0 ^ O 0D ∞ C0 rH CN CN lD ιn c ^ ^ ιn c o] C^ O O [^ O ^ CM C^ M lD ι 00 C0 l£> lD C C l^ ^ r^ CTl rH M CM CN LO O lD O CO CO θ en ^ ^ r c rH ^ H n θ C l lD ^ 3 CO rH C C ^ ^ CO ^ < l CN rH ιn cn iD CN ιn eM ^ i^ o co in oo cM CM c in cM rH ^ c t^ cD iD i c^ iD r^ r^ co co r~ o iD io co ^ in c r~ i CN H C^ rH CM rH O O CM ^ \l in CTl C000 CD r~ in ^ ^ C0 CM n r^ r^ lD in CD O CD in ^ ^ in r M C0 rH C0 rH M rH ^ ^ ^ ■ ^ ^ ^ ^ ^ ^ ^ co co co co o c c c c o c ro c c co r ^ c o r co co c r co r co c rH cn oo t^ O rπ r^ iD iD ^ ∞ CN cn cn in ^ c CN rH r^ i^ cN CN c rH c r^ c cN c rH rH r^ r^ c^ c^ rH t^ LO ∞ rH CO Cn ^ CM O O CD CD CO t^ rH CO Cn rH CN CN CM in O rH Cri CN CO rH rO lD CO rH CO CO rH r^ o o cM iD O in co i o i o oo c t^ iD αs in cM r^ rH r^ H rH c ro ^ t^ H i cn cM iD io e c cN CM ^ rH i^ o cD in e^ M H o e o^ cn c co cD O O^ co r~ o cri O rH CM CM r r~ LO LO L <a' LO c ^i" ^t' M ^ L vD ιn r~ r-- co cn cM θ o oo O CM rH CO CN O O o en cn o rH rH O rH CM CM CM CM rH rH rH rH rH rH CM rH rH rH CM rH CM CM CM CM CM rH rH rH rH rH rH rH rH rH rH ■-H rH rH rH rH rH rH rH rH rH rH rH rH ∞ lO CD CO I^ CN O rH ∞ CO ∞ CTl Cn rH O l ^ in ro CN CTl C CD C^ rH ^ ∞ in ∞ r^ ^ t^ ^ co LO O rH CM r^ o rH c in t^ '^ cD in LO O H i cN cn co cn o t^ iΩ rH C r^ cn cn o r^ iD ^ ι^ ι^ r~ iD ^ c rH CN rH o co cN [^ ιn i cθ rH i£> oo cn r ro σϊ L iD r^ m co ι^ c oo t^ cn c σ c cn cn cΛ rH o θ H θ θ o cn cn c c en cD co oo o o cn o rH rH < i H CM rH CM CM cα cD ∞ c^ CM CM CN CM CM CM CN CO CM CM CN CN CO CO CO CO CO CO CO CM CM CM CM CN CM CM Cvl CO CO CM CO rO r CO CO C^ o o o o oo i-ti icij fiC fiC fi fiC fi i^ fiC fiC fiC fiC fiC fiC fi i^ fiC fi fiC fiC fiC fiC fiC fiC fiC fi fiCi fi fi^ >π ω ω ω H wω ω ω cD cj cDCD C Cjo o ϋ cD ct; ^ c^ eel cd (ϊi ctioj ii Bj ω PB H ω w ω up ω H^ ι-3 z z z z z z Lj yl 3 ^ ^ ^ ^ Ll l CLl, ol a & (£ ι% cύ (£ cύ Pύ cύ x x . . ι .τ! 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ι^ fid eC fiC cC fit; fiC fit! fi fi fi fi C! < < <C fiC fit! fiC fi fiC fiC! fit! fiC fiC κC fit! fiC fit! fiC fi^ j jjtnw tnωc tβtΛ t»nw rt < <; >H H ι < ι >< ι H z z z z z z zz z J hi ^ J j HH u ωω ω ω ω ω Cώ J J h- r^ l-H I-H M rH J cC fiC fiC fit: rf H H H H H H H H H H j ^ J J J h J ιJ j μi j i J h J J l q j μi j μi J l l ; (i; l ι< (i; j μi μ| i i J J H CD CD CD O CD CD CD CD CD > > > > > > > S a S S ffi B S B X B [J [J U O O U lJ O U (3 U U U (J) U U U > > > > > > > H H H H H H H CM CM CM CM _α cc co D Q ω ω fiC Q CO CO fit; CQ CO Q α ω ω fit; CQ CQ to Q ω ω fi CQ CD CD cC CQ CD CD u z cj υo υu oo o z CJ J υ u U CJ z o o o CJ CJ Z o CJ z z CJ CJ O CJ z o O O z CJ J o z CJ υ o J o o o z z CJ CJ O O O CJ Z CJ CJ O CJ CJ CJ ∞ c o rH CN c ^ ιn ιo c^ oo c o rH CN co ^ ι <-3 r^ ∞ c o rH CN c ^ Ln ι^ r^ ∞ en o r^ CM C c o c c co c o o ι^ o ^ -^ ^ ^ ^ ^' '^ "τ ^ ^ ι Ln ι ιn L Lθ L ι ι Ln iD iD iD in in in tn LO in in in in in in in in in -o in in in in LO LO in in in -O Lo in in in in L^ IΛ « C! 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fi <C κC fid fi fi fi fid fiC fid fiC fi fi fi fid fi^ ES ES ES ES ES ES ES ES S ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES S ES ES ES S o o o o o o o o o o o o o o o oo o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o E oSoES EoS SoEoSoESoESoESoES EoSoES EoS EoSoES EoS EoS EoSoES EoS EoS EoS EoSoES SoSoEoS EoS EoS EoS EoS EoSoES SoSoSooooooooooooooooooooooooooooo m ID r~ oo oi r~- r- r~ c~ r- t — i — i — r^ t — ^ r^ t — t — ■ r~ r- r- Γ~ r~ 2222222222222 ooooooooooooo 90 *o IΛ © cM O^ o iD '^ c rH oo rH CΛ H in i^ '^ LO cn ^ ^r rH L iD co '^ -^ cM O Ln rH i i rO Ln ^ r^ M iXϊ r^ c rH C CN iD r~ ^r ι co u3 ro ∞ ιn '^ ι ι cM CD iD eM c cD t^ rH o θ rH θ rH r^ ι- r^ © © o^ ^ι H N c^l H M π^lπn rn ^ ιn ^ ^ l ι ^ ^^D ιn u^u) ^ιDιo ^ιnω^ o ω ιnω ι ιncΛOl ^ m o o o H ωo H (MH H O pa L lβ O lO lO lD β lO lβ O lO <-3 lD <-3 lO lβ lD lO l_3 O O lO lD lO lO lD lO <-3 lO O O H ooooooooooooooooooooooooooooooooooooooooooooooooooooσ ooooooooooooooσoooooooooooooooooooooooooooooooooooooo U α. rH rH rH rH rH CN cn c O Oi iD mm TOΓ- o I rH CM CN rH rH en ^r c ^f in ^r o e o in rH iD c co io co rH o cM rH -=f , oo eM co ^r CM en co co in r~ -a' r- cn o co cM cn o oooooi co in io ^ MO CM CM CN CO CM T rH CM rH C CM rH CM CN CM rH CO CO CO ^r CO CM I I I CM CM CM rH rH CM CM CM CO CM rH CM CN ^r CN I O rH CM C ^ L iD i^ co cn o rH vi c ^ i iD i^ co en o rH C co ^ in iD i^ co en o rH CN o ^ i-O iD r- CD c o n ro o ron roro r rn π^ ^ ^ ^ ^ <f ^ ^ * ^ mm m m m m<DΦiDiD iD ioΦiD i iD [^ r- r^ c^^ r^ r~ ι^ ^ ^ ∞ro rH rH rH rH rH rH rH rH rH rH rH rH rH M rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH rH M rH rH rH M ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES S ES ES ES ES ES ES ES ES ES ES ES ES S fi fi fiC fid fi fi fid « fid fid cd fi fid fi fi i fiC fiC fid fi fid fi fi fid i :: fid fi fid < fi c fid fi fid fid fid fid fi fid fid fi cd fid fi fid fi fi fi fid fid fid ES ES ES ES ES ES ES ES ES S ES ES ES ES ES ES ES ES ES ES ES ES ES S S ES ES ES S DS ES ES E5 ES ES E5 ES ES ES ES ES ES ES ES ES E5 ES ES E5 ES ES ES ES o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o ES £ E5 E5 ES E5 E5 Es Es Es £ Es ES ES ES Es Es E5 E5 E5 E5 !5 S 5 S S g S ES S E5 E5 ooooooooooooooooooooooooooooooooooooooooooooooooooooo rH rH rH rH 2 fi fid fid fid fid fid References 1. zur Hausen, H. 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Claims: Claims 1. A crystallised molecular complex of an E2 N-terminal module (E2NT) dimer protein or homologue thereof, comprising residues vital for transcriptional and replicational activities of said protein. 2. An E2NT dimer protein according to Claim 1 wherein the residues lie on opposite sides of an N-terminal domain. 3. An E2NT dimer protein according to either preceding claim wherein the residues comprise a plurality of residue clusters associated with a structural role at an interface between Nl and N2 terminal domains of respective monomers within the dimer. 4. An E2NT dimer according to Claim 3 comprising three clusters. 5. An E2NT dimer according to either of Claims 3 or 4 wherein a first cluster of vital residues is associated with interactions between Nl and N2 domains and comprises any one or more of the following residues Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vall45. 6. An E2NT dimer according to any one of Claims 3-5 wherein a second cluster of residues is associated with Nl interactions and comprises either or both of residues Trp33 and Leu94. 7. An E2NT dimer according to any one of Claims 3-6 wherein a third cluster of residues is associated with N2 interactions and comprises any one or more of the following residues Prol06, Lysl 11, Phel68, Trpl34. 8. An E2NT dimer according to any preceding claim further comprising residues associated with transactivation and/or replication properties of E2. 9. An E2NT dimer according to Claim 8 wherein the residues comprise any one or more of the following residues Glu20, GlulOO, Asp 122, Arg37, Glu39, Ile73, Glnl2 and Ala69. 10. Use of a crystallised molecular complex of an E2 N-terminal module (E2NT) dimer protein according to any preceding claim or homologue thereof in mapping mutations onto an E2 three-dimensional structure so as to identify areas of amino acid conservation and the effect of mutations on folding of the E2 protein. 11. Use according to Claim 10 in rationalised antiviral drug design. 12. An in vitro method for identifying and/or selecting a candidate therapeutic agent, the method comprising determining interaction of a E2 N-terminal module (E2NT) dimer in a sample by contacting said sample with said candidate therapeutic agent and measuring DNA loop formation in E2. 13. Use of the method according to Claim 12 in identifying and/or selecting an antiviral candidate therapeutic agent. 14. Use according to Claim 13 wherein identification/selection of the candidate therapeutic agent depends on its ability to interfere with or block interactions of E2NT so as to interfere or block viral and/or cellular transcription factors. 15. Use of an E2NT dimerisation inhibitor for the preparation of a medicament for treatment of conditions that arise as a result of HPV infection. 16. Use according to Claim 15 for the treatment of warts, proliferative skin lesions and/or cervical cancer. 17. A method of monitoring the efficacy of an antiviral therapy in a patient receiving a medicament for the treatment of an HPV infection comprising taking a sample from said patient and measuring E2NT interactions and/or DNA loop formation. 18. Use of a dimerisation surface of an crystallised molecular complex of an E2 N-terminal module (E2NT) dimer protein or homologue thereof according to any one of Claims 1-9 as a target site for interaction with putative antiviral agents and/or for measuring efficacy of said agents. 19. A method for identifying and/or selecting a candidate therapeutic agent, comprising applying rationalised drug design to a crystal structure obtainable by crystallising E2NT, cryogenically freezing the crystals and generating the crystal structure using X-ray diffraction. 20. A method of claim 19, wherein the method by which the E2NT crystal structure is obtainable comprises crystallisation using hanging-drop vapour diffusion. 21. A method of claim 19 or claim 20 wherein the method by which E2NT crystal structure is obtainable comprises X-ray diffraction using uranium acetate and gold cyanide E2NT derivatives and refining with data extending to 1.9 A spacing. 22. A method of any of claims 19 to 21, wherein the crystal structure comprises the portions of amino acids Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vall45, Prol06, Lysl 11, Phel68, Trpl34, Trp33 and Leu94. 23. A method of any of claims 19 to 22, wherein the rationalised drug design comprises designing drugs which interact with the dimerisation surface of E2NT. 24. A computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises or a three-dimensional representation of a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5A, wherein said computer comprises: (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vall45, Prol06, Lysl 11, Phel68, Trpl34, Trp33 and Leu94 according to Table 3; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central-processing unit coupled to said working memory and to said machine- readable data storage medium for processing said machine readable data into said three-dimensional representation; and (d) a display coupled to said central-processing unit for displaying said three- dimensional representation. 25. The computer according to claim 24, wherein said three-dimensional representation is of a molecule or molecular complex is defined by the set of structure coordinates according to Table 3, or wherein said three-dimensional representation is of a homologue of said molecule or molecular complex, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. 26. A computer for determining at least a portion of the structure coordinates corresponding to an X-ray diffraction pattern of a molecule or molecular complex, wherein said computer comprises: (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structural coordinates according to Table 3; (b) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises an X-ray diffraction pattern of said molecule or molecular complex; (c) a working memory for storing instructions for processing said machine-readable data of (a) and (b); (d) a central-processing unit coupled to said working memory and to said machine- readable data storage medium of (a) and (b) for performing a Fourier transform of the machine readable data of (a) and for processing said machine readable data of (b) into structure coordinates; and (e) a display coupled to said central-processing unit for displaying said structure coordinates of said molecule or molecular complex. 27. A crystallised molecule or molecular complex comprising a dimerisation surface defined by structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vall45, Prol06, Lysl 11, Phel68, Trpl34, Trp33 and Leu94 according to Table 3 or a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. 28. The crystallized molecule or molecular complex according to claim 27, wherein said molecule or molecular complex is defined by the set of structure coordinates according to Table 3, or a homologue thereof, wherein said homologue has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. 29. A machine-readable data storage medium, comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three- dimensional representation of a molecule or molecular complex comprising a dimerisation surface defined by structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vail 45, Prol06, Lysl 11, Phel68, Trpl34, Trp33 and Leu94 according to Table 3, or a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. 30. The machine-readable data storage medium according to claim 7, wherein said molecule or molecular complex is defined by the set of structure coordinates according to Table 3, or a homologue of said molecule or molecular complex, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A. 31. A machine-readable data storage medium comprising a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, wherein: said first set of data comprises a Fourier transform of at least a portion of the structural coordinates according to Table 3; and said second set of data comprises an x-ray diffraction pattern of a molecule or molecular complex. 32. A method for evaluating the ability of a chemical entity to associate with a molecule or molecular complex according to claim 27 or claim 28 comprising the steps of: a. employing computational means to perform a fitting operation between the chemical entity and a dimerisation surface of the molecule or molecular complex; and b. analysing the results of said fitting operation to quantify the association between the chemical entity and the dimerisation surface. 33. A drug or therapeutic agent identified, assessed or selected using a crystallised molecular complex of an E2NT protein or its crystal structure or using a complex of any of claims 1 to 9, a method of claim 12, a use of any of claims 13, claim 14 or 18. a method of any of claims 20 to24 or 32 or a product of any of claims 25 to 31.

Inventors: ANTSON ALFRED, MAITLAND NORMAN, ANTSON, ALFRED, MAITLAND, NORMAN
Applicants: ANTSON ALFRED, MAITLAND NORMAN, UNIV YORK, ANTSON, ALFRED, MAITLAND, NORMAN, THE UNIVERSITY OF YORK
Application year: 2000
Main classification: C07K14
IPC codes: A61K38, C07K14