Crystal structure of a replication protein encoded by a plasmid isolated from a patient with multiple sclerosis

Abstract

The present invention relates to crystalline forms of replication proteins encoded by plasmids isolated from multiple sclerosis patients, crystal structural information obtained therefrom, methods for preparing such crystalline forms, their use for identifying and / or designing inhibitors of said replication proteins, and methods for identifying, optimizing and designing compounds that should have the ability to interact with replication proteins or the ability to inhibit replication proteins.

Description

[0001] The present invention relates to crystalline forms of replication proteins encoded by plasmids isolated from multiple sclerosis patients, crystal structural information obtained therefrom, methods for preparing such crystalline forms, their use for identifying and / or designing inhibitors of said replication proteins, and methods for identifying, optimizing and designing compounds that should have the ability to interact with replication proteins or to inhibit the ability of replication proteins. Background Art

[0002] Beef and milk consumption is considered a risk factor for the development of degenerative and malignant diseases in humans, such as colon and breast cancer ( 1-3 Indeed, epidemiological data suggest an association between these cancers and the ingestion of cattle products derived from aurochs, a species of cattle from Eurasia ( 4-7 ). Beef and milk factor (BMMF) is a circular, single-stranded episomal DNA (<3 kb) that has been detected in beef and milk products. BMMF is thought to play a role in human malignant and degenerative diseases. BMMF encodes a replication initiator protein (Rep) that is actively transcribed and translated in human cells. Therefore, BMMF may represent a possible etiologic factor for such diseases ( 8-10 ). However, BMMF have also been isolated from patients with multiple sclerosis and studies have suggested that these are possible infectious agents of the disease ( 10-13 ).

[0003] Typically, BMMFs encode an autonomous plasmid trans-acting replication initiator protein, called "Rep". Rep binds to the origin of replication on the DNA (called on) and in most cases, includes a set of repeated DNA elements (called repeatons), which are present in most BMMFs ( 5 The replication of various plasmids (including circular Rep-encoding single-stranded (CRESS) DNA viruses) also requires the binding of Rep to specific DNA sequences ( 14 In prokaryotes, Rep plays a central role in maintaining plasmid copy number, as reported for the F plasmid in Escherichia coli ( 15 This regulation is also crucial for the replication of plasmid-derived, phage-like, or virus-like DNA genomes ( 16 ). Rep is essential for the replication of multidrug-resistant bacteria in humans ( 17 ) and studies have shown that Rep plays a role in transmissible amyloid disease ( 18-20 ).

[0004] The X-ray crystal structure of Rep has been well characterized, and the structural basis for autonomous replication has been described ( 24-27). Rep consists of two winged helix domains (called WH1 and WH2), which are essentially fused N-terminal and C-terminal proteins. Rep switches between monomeric and dimeric forms, depending on specific functions and binding to DNA ( 28 These oligomeric forms are complemented by large structural changes involving both domains. The structural transition requires refolding and / or displacement of certain α-helices and β-strands on Rep ( 26 ). In the dimeric form, Rep functions as a repressor, with WH2 binding to each operator DNA repeat and WH1 acting to form the dimerization interface. In the monomeric form, Rep functions as a replication initiator, with WH1 undergoing a large structural shift, i.e., dimer disassembly, thus allowing WH1 to bind to one repeat end and WH2 to bind to the opposite repeat end.

[0005] Recently, episomal circular DNA was isolated from brain samples of patients with multiple sclerosis (isolate MSBI1.176, accession number LK931491.1) ( 11 The MSBI1.176 Rep exhibits 98% amino acid identity with the Rep encoded by Sphinx-1.76 (GenBank ADR65123.1 and HQ444404.1), which was isolated from cultures and brain preparations of pathogens associated with transmissible encephalopathy ( 21 In addition, there are indications that the Rep encoded by Sphinx.1.76 is detected in neural cells (GT1-cell line) and brain samples of mouse CNS, hamster CNS and human glioblastoma based on Sphinx-1.76-specific antibodies. 22 Serology based on the MSBI1.176 Rep antigen showed a positive immune response to healthy human blood donors and indicated a possible pre-exposure to these pathogens ( 23 ). Therefore, it is becoming increasingly important to decipher the functions of BMMF in human malignant and degenerative diseases.

[0006] Purpose of the Invention

[0007] Having a crystal structure of the MSBI1.176Rep protein would be very helpful for deciphering the function of BMMF in human malignant and degenerative diseases, which has been unsuccessful so far. Such a crystal structure would also help to identify and / or design inhibitors of the replication protein, as well as methods for identifying, optimizing and designing compounds that have the ability to interact with the replication protein or inhibit the replication protein.

[0008] Therefore, the object of the present invention is to provide the crystal structure coordinates of MSBI1.176 Rep protein, a method for obtaining a crystal form of MSBI1.176 Rep protein, and a method for designing, identifying and optimizing compounds as MSBI1.176 Rep protein inhibitors based on the crystal structure and crystal structure coordinates. DETAILED DESCRIPTION

[0009] The objects of the invention are achieved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are apparent from the dependent claims, the description, the drawings and the examples of the present application.

[0010] Therefore, the present invention relates to a crystalline form of the MSBI1.176 Rep protein as characterized in claim 1. Preferred embodiments are subject matter of the dependent claims.

[0011] MSBI1 Rep-derived full-length replication protein

[0012] GenBank: CDS63398.1

[0013] (Encoded in the replication competent episomal DNA MSBI1.176)

[0014] Embl accession number DNA sequence: LK931491.1

[0015] Source: Human brain with multiple sclerosis

[0016] >MSBI1.176 putative replication initiation protein

[0017] MSDLIVKDNALMNASYNLALVEQRLILLAIIEARETGKGINANDPLTVHASSYINQFNVERHTAYQALKDACKDLFARQFSYQEKRERGRINITSRWVSQIGYMDDTATVEIIFAPAVVPLITRLEEQFTQYDIEQISGLSSAYAVRMYELLICWRSTGKTP IIELDEFRKRIGVLDTEYTRTDDNLKMRVIELALKQINEHTDITASYEQHKKGRVITGFSFKFKHKKQNSDKTPKNSDSSPRIVKHSQIPTNIVKQPENAKMSDLEHRASRVTGEIMRNRLSDRFKQGDESAIDMMKRIQSEIITDAIADQWESKLEEFGVVF

[0018] During the experiments leading to the present invention, the X-ray crystal structure of the MSBI1.176 WH1 domain (residues 2-133) was solved. The asymmetric unit consists of a WH1 dimer, i.e., two protomers (called A and B). The electron density of most proteins is well resolved. However, due to the lack of discernible electron density, residues 36-39 could not be matched to the B protomer, although the electron density was discernible in the other protomers. The WH1 structure includes five α-helices (α1-α5) and five β-strands (β1-β5) in each protomer ( Figure 1 ). The A and B protomers are closely related However, a small structural shift was observed at the β2-β3 hairpin, suggesting some flexibility in this region. Importantly, at improved resolution compared to previous structures ( 24-27 ), effectively adding water molecules to this Rep structure.

[0019] The data collection and refinement statistics for the MSBI1.176 WH1 protein structure are summarized in Table 1:

[0020] Table 1. Data collection and refinement statistics for the MSBI1.176 WH1 protein structure.

[0021]

[0022]

[0023] a The values ​​in parentheses are for the highest resolution shell.

[0024] Database searches for closely related structures and sequences revealed that MSBI1.176 WH1 has exceptionally low amino acid identities of 28% and 17% with Pseudomonas syringae RepA WH1 (dRepA, PDB ID 1HKQ) and Escherichia coli RepE fusion WH1-2 (RepE54, 1REP), respectively. 24 , 25 ). Similar to dRepA, MSBI1.176 WH1 is also folded into a replication-inert dimer, while RepE54 (WH1-2 construct) was crystallized as a monomer starting form. Superposition of MSBI1.176 WH1 and dRepA WH1 shows that the two domains are structurally similar Both have the typical five α-helices and five β-strands ( Figure 2Several structural similarities and differences were observed between the two Reps. The MSBI1.176 and dRepA dimer interface involves five β-strands (β1-β5-β4-β3-β2) anchored by a similar number of backbone binding interactions, albeit not at the same residues ( Figure 3 A). The results suggest that the dimer interface features in different Rep isolates may be functionally relevant. The inventors observed that water molecules bind at the dimer interface ( Figure 3 B) However, how these water molecules stabilize the dimer interface and / or are displaced upon binding DNA and changing conformation is still unclear.

[0025] The inventors also observed that the MSBI1.176 WH1 region including α1-α2-α5 is similar in orientation to dRepA, with a typical α1-α2 bend thus forming a V-shaped structure ( Figure 3 C). This region of dRepA that forms the linker to WH2 also contains a hydrophobic seven-fold pocket that typically contains a number of leucine residues (e.g., dRepA: Leu12, Leu19, and Leu26; RepE: Leu24, Leu31, and Leu39). The MSBI1.176 WH1 hydrophobic pocket also includes or consists of three leucine residues, i.e., Leu11, Leu18, and / or Leu25, which are similar in position to those in dRepA. Not surprisingly, there are no water molecules in the MSBI1.176 WH1 hydrophobic pocket ( Figure 3 B).

[0026] Overall, many MSBI1.176 WH1 structural features are conserved relative to other known dimeric Rep structures ( 24-27 ). However, the movement of the loops as well as different α-helices and β-strands has been observed in different structures. When compared to the β2-β3-hairpin of dRepA, the β2-β3-hairpin in the case of MSBI1.176 is shifted by approx. ( Figure 4 In the case of RepE54, this equivalent β2-β3-hairpin (residues 97-110) was not added to the structure due to missing electron density ( 20 , 24 This suggests that the β2-β3-hairpin of RepE54 is flexible and that this flexibility may play a role by destabilizing the antiparallel β2-β3-hairpin and blocking dimerization ( 20 , 24 ). However, the β2-β3-hairpin of MSBI1.176 WH1 is clearly fixed by direct main-chain interactions, unlike the dRepA structure ( Figure 3A). In addition, we believe that water-mediated interactions at the dimer interface may also add further stability to the hairpin ( Figure 3 B).

[0027] Previous modeling analysis of the dRepA domain suggested that six basic residues (dRepA numbering: Lys74, Arg81, Arg91, Arg93, Lys62, and Arg78) on α2, β2, β3, and adjacent loops may be located after the minor groove of the DNA backbone ( 24 ). In the MSBI1.176 WH1 structure, six basic residues were also found in this region. These are thought to represent DNA interaction sites that include or consist of: Lys69, Lys73 (located in α2), Lys85 (β2), Arg90 (β3), Arg78 and / or Arg96 (on the adjacent loop). In DNA interaction sites, positively charged amino acids are preferred to cause electrostatic interactions with the negative charges of DNA. Although for MSBI1.176 WH1 Lys73 A / B链 ,Lys85 A / B链 and Arg90 A链 Electron density for the side chains is weak; two of these residues (Arg78 and Arg96) are located at equivalent dRepA positions and are thought to interact with the DNA molecule ( 24 ). Although MSBI1 WH1 has a three amino acid insertion in the β2 strand that extends the sheet, the function of the β2-β3 sheet orientation of MSBI1.176 WH1 is not obvious. Presumably, this insertion shifts Lys85 (β2) and Arg90 (β3) of MSBI1.176 WH1 elegantly on the β2-β3 sheet when compared to the equivalent dRepA residues Arg81 and Arg91. From another perspective, the β2-β3 sheet of MSBI1.176 is quite oriented, while the β2-β3 hairpin of dRepA hooks in the opposite direction ( Figure 2 ).

[0028] The elucidation of the MSBI1.176 WH1 structure is a critical step toward better understanding how structural features may vary between Rep proteins in different groups of BMMF DNA. The finding that this MSBI1.176 WH1 protein isolated from patients with multiple sclerosis is very similar to the prokaryotic Rep structure is of great significance. Overall, this new structural information supports the development and design of new drug targets that can inhibit the oligomeric properties of Rep. According to the present invention, X-ray crystallography was used to determine the Rep WH1 domain encoded on BMMF (MSBI1.176) isolated from multiple sclerosis human brain samples to a resolution of 1.5 Å. Compared to other Rep structures, MSBI1.176WH1 has a remarkably similar overall structure despite having low (28%) amino acid identity. MSBI1.176 WH1 contains elements shared with other Reps, including five α-helices, five β-strands, and a hydrophobic pocket. Interestingly, the β2-β3-hairpin of MSBI1.176 WH1 is shifted by approximately 1% when compared to other Reps. This region is known to interact with DNA, and the amino acid insertion in the MSBI1.176 WH1 hairpin shifts the positively charged DNA binding residues further along the β2-β3 sheet. The present data also show that water molecules additionally stabilize the α-helices and β-strands in the protein. Overall, these new findings support that the MSBI1.176 Rep may have comparable roles and functions to other known Reps from different sources.

[0029] Furthermore, the present invention enables the establishment of methods for identifying inhibitors of MSBI1.176, as well as methods for preparing crystal forms of MSBI1.176 Rep protein and their crystal structure information. Data enables rational drug design based on the use of such structural data.

[0030] The present invention also provides the possibility of identifying and / or designing inhibitors of the MSBI 1.176Rep protein, as well as crystalline forms of the MSBI 1.176Rep protein, crystal structure information obtained from the crystalline forms, methods for preparing such crystalline forms, their use in identifying and / or designing inhibitors of the activity of the MSBI 1.176Rep protein, and the diagnostic and / or pharmaceutical uses of these inhibitors of the MSBI 1.176Rep protein identified by these methods.

[0031] The term "crystal form" or "crystal structure" (which are used interchangeably) refers to the crystal form of the MSBI1.176 Rep protein with or without surfactant and / or nucleic acid (especially: DNA) bound to the MSBI1.176 Rep protein.

[0032] As used herein, the term "unit cell" refers to the smallest repeating unit that can generate a crystal using only a translation operation. The unit cell is the smallest unit of volume, which contains all the structural and symmetric information of the crystal, and can reproduce the figure in all spaces by translation. The structural information is therefore the figure (atom) plus all the surrounding space, and the symmetric information means mirror, slide, axis and inversion center. Translation refers to the movement of the unit cell edge length along the edge of the unit cell. The asymmetric unit is the smallest unit that can be rotated and translated to generate a unit cell using only the symmetric operations allowed by crystallographic symmetry. The asymmetric unit can be a molecule or a subunit of a multimeric protein, but it can also be more than one. Therefore, the asymmetric unit is the smallest unit of volume that contains all the structural information and can reproduce the unit cell by using symmetric operations. The shape of the unit cell is constrained by the symmetric elements that aggregate the group. For space groups, seven lattice types may be: triclinic, monoclinic, orthorhombic, tetragonal, hexagonal, rhombic, and cubic. In crystallography, space groups are also called crystallographic groups or Fedorov groups. The edges of the unit cell define a set of unit vector axes a, b and c, which have the dimensions of the unit cell as their lengths a, b and c, respectively. These vectors need not be at right angles, and the angles between two axes are noted: α between the bc axes, β between the ac axes, and γ between the ab axes. Different shapes emerge depending on the restrictions placed on the lengths of the three edges (a, b and c) and the values ​​of the three angles (α, β and γ).

[0033] In another preferred embodiment of the present invention, the crystal form of the MSBI1.176 Rep protein comprises or further comprises a hydrophobic pocket, wherein the hydrophobic pocket comprises amino acids Leu11, Leu18 and Leu25.

[0034] Potential inhibitors of the MSBI1.176 Rep protein may bind to the DNA interaction site in order to reduce the activity of the Rep protein.Therefore, methods for designing, identifying and / or optimizing compounds that may have the ability to bind to the MSBI1.176 Rep protein may be based on crystal structure coordinates.

[0035] MSBI1.176 Potential inhibitors of Rep protein can bind to the Rep oligomerization interface and modulate structure-based functions and protein activity / localization / stability / modification.

[0036] Potential inhibitors of the MSBI1.176 Rep protein could bind to yet unknown protein interaction interfaces, modulating structure-based functions and protein activity / localization / stability / modification.

[0037] Potential inhibitors of MSBI1.176 Rep proteins may bind to structurally related prokaryotic Rep (eg, from Acinetobacter baumannii species) to reduce the activity of such Rep proteins from different sources based on steric hindrance of DNA binding, oligomerization, and / or additional protein interaction interfaces.

[0038] The crystalline form of the MSBI1.176 Rep protein as disclosed herein can be obtained by the following crystallization method. Therefore, the present invention relates to a method for crystallizing the MSBI1.176 Rep protein, which comprises the steps of:

[0039] (a) preparing a solution of the recombinantly produced MSBI1.176 WH1 domain in a crystallization reagent, the crystallization reagent preferably comprising at least one inorganic salt and at least one precipitating agent;

[0040] (b) Crystallization of the MSBI1.176 WH1 domain by vapor diffusion.

[0041] At least one inorganic salt is used in an amount between 0.01 mM and 2 M, preferably between 0.1 mM and 1 M, more preferably between 0.1 M and 0.5 M, more preferably about 0.2 M. If more than one inorganic salt is used, the concentrations mentioned above refer to the concentration of all the inorganic salts together and not the concentration of each single salt used in the inorganic salt mixture.

[0042] Preferably, the inorganic salt is selected from the group comprising or consisting of ammonium chloride, ammonium sulfate, ammonium acetate, ammonium fluoride, ammonium bromide, ammonium iodide, ammonium nitride, calcium chloride, calcium acetate, magnesium acetate, magnesium formate, magnesium nitrate, potassium acetate, potassium bromide, potassium fluoride, potassium chloride, potassium iodide, potassium nitrate, sodium acetate, sodium hydroxide, sodium bromide, sodium fluoride, sodium iodide, sodium nitrate, sodium sulfate, sodium chloride, zinc chloride, zinc sulfate, zinc acetate. The preferred inorganic salt is magnesium acetate.

[0043] In theory, at least one precipitation agent competes with protein solute for water, thus causing the supersaturation of protein. Crystals are usually only grown by supersaturated state, and therefore they can be grown by precipitate. Salt, polymer and organic solvent are suitable precipitation agents, with the mixture of precipitation agent or precipitation agent relative to the gross weight of buffered precipitation solution, use with the following amount: by weight 5% to by weight 50%, preferably by weight 10% to by weight 40% and more preferably by weight 15% to 35%, and most preferably by weight 20% to by weight 30%.

[0044] The precipitant used in the buffered precipitation solution of step (b) may preferably be selected from the group comprising or consisting of 2-methyl-2,4-pentanediol, glycerol, polyethylene glycol (PEG), pentaerythritol propoxylate, pentaerythritol ethoxylate, sodium polyacrylate, hexylene glycol, isopropanol, ethanol, tert-butyl alcohol, dioxane, ethyleneimine polymer, etylene glycol, propylene glycol, polyacrylic acid, polyvinyl pyrrolidone, 2-ethoxyethanol or a mixture thereof. PEG is most preferred as the precipitant, preferably having a molecular weight in the range of PEG 200 to PEG 20,000, more preferably having a molecular weight in the range of PEG 1,000 to PEG 18,000, and even more preferably having a molecular weight in the range of PEG 3,000 to PEG 15,000. PEG is a very preferred precipitant, which is preferably used in an amount of 20-30% by weight of the buffered precipitation solution.

[0045] In the most preferred practice, the crystallization buffer comprises 0.2 M magnesium acetate and 20% PEG3350.

[0046] In a preferred embodiment, crystallization is performed using a hanging drop or sitting drop method. The "hanging drop vapor diffusion" technique is the most popular method for crystallizing macromolecules. The principle of vapor diffusion is direct. A droplet consisting of a mixture of sample and crystallization reagent is brought into equilibrium with a liquid reservoir vapor of the reagent. Typically, the droplet contains a lower reagent concentration than the reservoir. In order to reach equilibrium, water vapor leaves the droplet and eventually enters the reservoir. As water leaves the droplet, an increase in the sample in the direction of supersaturation occurs. As water leaves the droplet and enters the reservoir, both sample and reagent concentrations increase. Equilibrium is reached when the reagent concentration in the droplet is approximately the same as the reagent concentration in the reservoir.

[0047] A further important aspect of the present invention relates to the use of a crystalline form of the MSBI1.176 Rep protein for identifying, optimizing and / or designing a computer prediction model for an inhibitor of the MSBI1.176 Rep protein. Knowing the exact position of the amino acid atoms in the DNA interaction site provides the following possibilities: designing a suitable inhibitor, identifying a suitable inhibitor, such as from a compound library, or optimizing a known suitable inhibitor by increasing the inhibitory potential. Suitable inhibitors can be designed, identified and optimized using standard computer-based methods and software programs well known in the art. Various commercially available software programs can be used to analyze and compare data in a computer-based system. Those skilled in the art will readily recognize which algorithms or execution software packages available for computer analysis can be utilized or adapted for use in a computer-based system. A target structural motif or a target motif refers to any reasonably selected sequence or combination of sequences, wherein one or more sequences are selected based on a three-dimensional configuration or an electron density map formed by folding the target motif. There are various target motifs known in the art. Various structural formats for input and output modes can be used to input and output information in a computer-based system of the present invention. Various comparison methods can be used to compare the target sequence or target motif to the data storage means to identify structural motifs or interpret electron density maps derived in part from atomic coordinates / X-ray diffraction data. Those skilled in the art will readily appreciate any of the publicly available computer modeling programs that can be used. Suitable software that can be used to view, analyze, design and / or model proteins includes Alchemy TM 、LabVision TM 、Sybyl TM 、Molcadd TM 、Leapfrog TM 、Matchmaker20 TM 、Genefold TM and Sitel TM (acquired from Tripos Inc., St. Louis, MO), Quanta TM 、MacroModel TM and GRASP TM 、Univision TM 、Chem 3D TM and Protein Expert TM .

[0048] Therefore, in a further aspect, the present invention relates to a method for designing, identifying and optimizing inhibitors of the MSBI1.176 Rep protein by using a crystal form and structural coordinates or at least one DNA interaction site associated with the crystal form to design, identify and optimize inhibitors with the aid of a computer-based method or software program.

[0049] Atomic coordinates / X-ray diffraction data can be used to create a physical model that can be used to design a molecular model of a compound subsequently, and the compound should have the ability or characteristic of inhibiting and / or interacting with the following: a determined DNA interaction site or other structural or functional domains or subdomains, such as a pocket near a hydrophobic pocket and / or a DNA interaction site. Alternatively, the atomic coordinates / X-ray diffraction data of the complex can be represented as atomic model output data on a computer-readable medium, which can be used for a computer modeling system to calculate different molecules that are expected to inhibit and / or interact with the following: a determined site, or other structural or functional domains or subdomains. For example, computer analysis of data allows the three-dimensional interaction of MSBI1.176 Rep protein and compound to be calculated to determine that the compound is bound to specific one or more domains or one or more subdomains, or to change the configuration of specific one or more domains or one or more subdomains. The compound determined by the analysis of the physical or computer model can then be synthesized and tested for biological activity in an appropriate manner.

[0050] If the inhibitor is identified from a compound library or if a known inhibitor is optimized by theoretical chemical modification, it is desirable to test the actual compound in order to determine the inhibitory effect and continue the optimization process. Therefore, preferably the above method for identifying and / or optimizing a compound further comprises the following steps:

[0051] (a) obtaining identified or optimized compounds; and

[0052] (b) contacting the identified or optimized compound with MSBI1.176 Rep protein to determine the inhibitory effect on MSBI1.176 Rep protein.

[0053] It is not necessary to use all of the structural coordinates listed in Table 1. Thus, only the structural coordinates of the hydrophobic pocket and / or the DNA interaction site may be used.

[0054] In this application, X-ray crystallography was used to determine the structure of the Rep WH1 domain encoded on BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample to a resolution of Although MSBI1.176 WH1 has low (28%) amino acid identity compared to other Rep structures, its overall structure is significantly similar. MSBI1.176 contains elements shared with other Reps, including five α-helices, five β-strands, and a hydrophobic pocket. These new findings suggest that MSBI1.176Rep may have similar roles and functions to other known Reps from different sources. Rep is important for the replication of plasmid or self-sufficient episomal nucleic acids in different hosts. Therefore, it has been considered that such proteins and DNA encoding Rep may be associated with disease. Therefore, detailed structural and functional characterization of Rep is needed. The Rep described in this study is encoded by human bioactive beef and milk factor MSBI1.176 isolated from patients with multiple sclerosis. Specific serum antibodies were found in a group of healthy human blood bank donors, indicating that the general population is exposed to such pathogens. The discovery that this MSBI1.176-encoded RepWH1 protein is very similar to the Rep structure of prokaryotes may have important consequences and point to possible disease-related adaptations of these pathogens to humans. This new structural information may aid in the development and design of therapeutic / preventive drugs that can inhibit these Reps from different sources.

[0055] The following abbreviations are used for typical and altered amino acids referred to herein.

[0056] Amino Acids

[0057] Ala Alanine

[0058] Arg Arginine

[0059] Asn Asparagine

[0060] Asp Aspartic acid (Aspartate)

[0061] Cys Cysteine

[0062] Gin Glutamine

[0063] Glu Glutamate (glutamate)

[0064] Glycine

[0065] His

[0066] Ile Isoleucine

[0067] Leu Leucine

[0068] Lys

[0069] Met Methionine

[0070] Phe Phenylalanine

[0071] Proline

[0072] Ser Serine

[0073] Thr Threonine

[0074] Trp Tryptophan

[0075] Tyr Tyrosine

[0076] Val Sequence Listing <110> German Public Interest Foundation for Cancer Research (Deutsches Krebsforschungszentrum) <120> Crystal structure of a replication protein encoded by a plasmid isolated from a patient with multiple sclerosis <130> K3469PCT <150> EP18206532.6 <151> 2018-11-15 <160> 2 <170> BiSSAP 1.3.6 <210> 1 <211> 324 <212> PRT <213> Sphinx <220> <223> MSBI1.76 protein <400> 1 Met Ser Asp Leu Ile Val Lys Asp Asn Ala Leu Met Asn Ala Ser Tyr 1 5 10 15 Asn Leu Ala Leu Val Glu Gln Arg Leu Ile Leu Leu Ala Ile Ile Glu 20 25 30 Ala Arg Glu Thr Gly Lys Gly Ile Asn Ala Asn Asp Pro Leu Thr Val 35 40 45 His Ala Ser Ser Tyr Ile Asn Gln Phe Asn Val Glu Arg His Thr Ala 50 55 60 Tyr Gln Ala Leu Lys Asp Ala Cys Lys Asp Leu Phe Ala Arg Gln Phe 65 70 75 80 Ser Tyr Gln Glu Lys Arg Glu Arg Gly Arg Ile Asn Ile Thr Ser Arg 85 90 95 Trp Val Ser Gln Ile Gly Tyr Met Asp Asp Thr Ala Thr Val Glu Ile 100 105 110 Ile Phe Ala Pro Ala Val Val Pro Leu Ile Thr Arg Leu Glu Glu Gln 115 120 125 Phe Thr Gln Tyr Asp Ile Glu Gln Ile Ser Gly Leu Ser Ser Ala Tyr 130 135 140 Ala Val Arg Met Tyr Glu Leu Leu Ile Cys Trp Arg Ser Thr Gly Lys 145 150 155 160 Thr Pro Ile Ile Glu Leu Asp Glu Phe Arg Lys Arg Ile Gly Val Leu 165 170 175 Asp Thr Glu Tyr Thr Arg Thr Asp Asn Leu Lys Met Arg Val Ile Glu 180 185 190 Leu Ala Leu Lys Gln Ile Asn Glu His Thr Asp Ile Thr Ala Ser Tyr 195 200 205 Glu Gln His Lys Lys Gly Arg Val Ile Thr Gly Phe Ser Phe Lys Phe 210 215 220 Lys His Lys Lys Gln Asn Ser Asp Lys Thr Pro Lys Asn Ser Asp Ser 225 230 235 240 Ser Pro Arg Ile Val Lys His Ser Gln Ile Pro Thr Asn Ile Val Lys 245 250 255 Gln Pro Glu Asn Ala Lys Met Ser Asp Leu Glu His Arg Ala Ser Arg 260 265 270 Val Thr Gly Glu Ile Met Arg Asn Arg Leu Ser Asp Arg Phe Lys Gln 275 280 285 Gly Asp Glu Ser Ala Ile Asp Met Met Lys Arg Ile Gln Ser Glu Ile 290 295 300 Ile Thr Asp Ala Ile Ala Asp Gln Trp Glu Ser Lys Leu Glu Glu Phe 305 310 315 320 Gly Val Val Phe <210> 2 <211> 131 <212> PRT <213> Sphinx <220> <223> RepA domain <400> 2 Met Val Asp Asn Lys Val Thr Gln Ser Asn Lys Leu Ile Glu Ser Ser 1 5 10 15 His Thr Leu Thr Leu Asn Glu Lys Arg Leu Val Leu Cys Ala Ala Ser 20 25 30 Leu Ile Asp Ser Arg Lys Pro Leu Pro Lys Asp Gly Tyr Leu Thr Ile 35 40 45 Arg Ala Asp Thr Phe Ala Glu Val Phe Gly Ile Asp Val Lys His Ala 50 55 60 Tyr Ala Ala Leu Asp Asp Ala Ala Thr Lys Leu Phe Asn Arg Asp Ile 65 70 75 80 Arg Arg Tyr Val Lys Gly Lys Val Val Glu Arg Met Arg Trp Val Phe 85 90 95 His Val Lys Tyr Arg Glu Gly Gly Cys Val Glu Leu Gly Phe Ser Pro 100 105 110 Thr Ile Ile Pro His Leu Thr Met Leu His Lys Glu Phe Thr Ser Tyr 115 120 125 Gln Leu Lys 130 BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 .X-ray crystal structure of MSBI1.176 WH1 dimer.

[0078] The MSBI1.176 WH1 protomer A chain is colored cyan and the B chain is orange. One protomer includes five α-helices (α1-α5) and five β-strands (β1-β5). The dimer interface involves two chains (β4-β3).

[0079] Figure 2 .Structural comparison with the closely matching prokaryotic RepA WH1.

[0080] MSBI1.176 WH1 and RepA WH1 have 28% amino acid identity. Superposition with RepA (grey) shows that the two WH1 dimers are highly similar, with Structural differences were observed in the extended loops, notably the loops connecting α2 and β1 and β2 and β3.

[0081] Figure 3 .MSBI1.176 WH1 has structural similarity to MSBI1.176 WH1.

[0082] (A) Five β-sheets (β1-β5-β4-β3-β2) are shown for backbone interactions in MSBI1.176 WH1 (cyan and orange) and RepAWHI (grey). The β-strands are held in place by numerous backbone hydrogen bonds (dashed lines), similar to RepA, including the dimer interface (β4-β3).

[0083] (B) The region containing α1-α2-α5 is oriented similarly to that in RepA. This region creates a V-shaped structure and α5 is the linker region of the WH2 domain. The hydrophobic pocket also contains three leucine residues, namely Leu11, Leu18 and Leu25, which are similarly positioned in RepA (data not shown).

[0084] Figure 4 Superposition of the β2-β3 hairpin of .MSBI1.176 WH1 on the prokaryotic Rep proteins with PDB codes 1hkq (RepA) and 2z9o (RepE).

[0085] When compared to the equivalent RepA hairpin, the β2-β3 hairpin of MSBI1.176 WH1 was shifted by ca. The β2-β3 hairpin of MSBI1.176 WH1 interacts with the β2-β3 strand of ... Figure 3 a) and water-mediated interactions. Figure 3 (A) The β2-β3 hairpin twist differs between the MSBI1.176 and RepA WH1 structures. The RepE β2-β3 hairpin is located between these two WH1 β2-β3 hairpins.

[0086] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that function well in the practice of the present invention, and therefore can be considered to construct preferred modes of its practice. In view of this description, modifications and alternative embodiments of various aspects of the present invention will be apparent to those skilled in the art.

[0087] Therefore, this specification should be interpreted as illustrative only and is intended to teach those skilled in the art the general method of implementing the invention. It should be understood that the inventive forms shown and described herein are considered examples of implementation. Elements and materials can be substituted for those illustrated and described herein, parts and processes can be interchanged, and certain features of the invention can be utilized independently, all of which will be apparent to those skilled in the art after having the benefit of the description of the invention.

[0088] Example

[0089] Example 1: Method for obtaining MSBI1 WH1 crystals

[0090] MSBI1.176 DNA (LK931491.1) was isolated from brain samples of patients with multiple sclerosis ( 11 As previously described for the protruding domain of human norovirus ( 29 ), the MSBI1 WH1 domain (residues 1-135) was expressed in E. coli and purified. Briefly, the codon-optimized WH1 was cloned into the modified expression vector pMal-c2X (Geneart) and transformed into BL21 cells for protein expression. The transformed cells were grown in LB medium supplemented with 100 μg / ml ampicillin at 37°C for 4 hours. The cells were cultured at 22°C at OD 600 Expression was induced by IPTG (0.75mM) at 0.7 for 18h. The cells were collected by centrifugation at 6000rpm for 15min and broken by sonication on ice. The fusion MBSM protein with His tag was first purified from a Ni column (Qiagen), dialyzed in gel filtration buffer (GFB: 25mM Tris-HCl and 300mM NaCl) with 10mM imidazole, and digested with HRV-3C protease (Novagen) at 4°C overnight. The cleaved MSBI1 WH1 was then applied to the Ni column again to separate and collect the cleaved protein, and dialyzed in GFB at 4°C overnight. The MSBI1 WH1 protein was further purified by size exclusion chromatography, concentrated to 5mg / ml and stored in GFB at 4°C. MSBI1 WH1 crystals were grown at 18°C ​​for ~6-10 days in a 1:1 mixture of protein sample and mother liquor (0.2M magnesium acetate and 20% PEG3350) using the hanging drop vapor diffusion method. Prior to data collection, MSBI1 WH1 crystals were transferred to a cryoprotectant containing a mother solution of 40% PEG3350 and subsequently snap frozen in liquid nitrogen.

[0091] Example 2: X-ray diffraction

[0092] X-ray diffraction data of the MSBI1 WH1 domain were collected at beamlines ID23-1 and ID30B at the European Synchrotron Radiation Facility (ESRF). For single-wavelength anomalous diffraction (S-SAD) experiments using native sulfur, X-ray diffraction was performed on beamline ID23-1 equipped with a Dectris pixel array detector PILATUS-6M. Diffraction data from seven crystals were collected. The X-ray beam size at the sample position was 50 μm, and the size of the crystal was approximately 70x70x200 μm 3 In order to reduce the impact of radiation damage, a spiral data collection strategy was adopted. A local data set was collected for initial phase extension and at ID30B A second local data set was collected for structure refinement. The optimal experimental parameters for data collection were obtained using the MxCube software ( 31 BEST( 30 ) designed. A single native dataset was processed with XDS while multiple datasets of S-SAD were processed with XDS and then merged using XSCALE ( 32 ).

[0093] Some datasets were collected using S-SAD for further processing ( 33 ). Use as in HKL2MAP( 34 ) was used for the S-SAD phasing procedure using the SHELXC / D / E pipeline implemented in . One thousand trials were performed in SHELXC to determine the substructure. resolution and is truncated to SHELXD correctly identified all 24 sulfur sites with anomalous signals. Four hundred and fifteen residues were automatically built by SHELXE, which produced interpretable plots for further processing. Finally, based on the first S-SAD native dataset collected, ARP-wARP was subsequently used for automatic model building ( 35 ). Used in COOT( 36 ) and PHENIX( 37 The structure was refined using the second highest resolution native dataset in multiple rounds of manual model building in ). The structure was verified using Molprobity and Procheck. Interactions were analyzed using Accelrys Discovery Studio (version 4.1), and hydrogen bond distances were Figures and protein contact potentials were generated using PyMOL. Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with the accession code 6H24.

[0094] Example 3: Modeling of DNA binding

[0095] Previous modeling analysis of the RepA domain indicated that six basic residues (RepA numbering: Lys74, Arg81, Arg91, Arg93, Lys62, and Arg78) on α2, β2, β3, and the adjacent loops may be located after the minor groove of the DNA backbone (24). In the MSBI1.176 WH1 structure, six basic residues were also found in this region, namely Lys69, Lys73 (both located on α4), Lys85 (β2), Arg90 (β3), Arg78, and Arg96 (both on the adjacent loops). Although MSBI1.176 WH1 Lys73 A / B链 ,Lys85 A / B链 and Arg90 A链 Electron density for the side chains is weak, and two of these residues (Arg78 and Arg96) are located in equivalent RepA positions and are thought to interact with the DNA molecule (24). Although MSBI1.176 WH1 has a three amino acid insertion on the β2 strand that extends the sheet, the function of the β2-β3 sheet orientation of MSBI1.176 WH1 is not obvious. Presumably, this insertion shifts Lys85 (β2) and Arg90 (β3) of MSBI1.176 WH1 elegantly on the β2-β3 sheet when compared to the equivalent RepA residues Arg81 and Arg91. From another perspective, the β2-β3 sheet of MSBI1.176 is quite saturated, while the β2-β3 hairpin of RepA hooks in the opposite direction.

[0096]

[0097] The above amino acid sequence alignment of MSBI1.176 (LK931491.1) and RepA (PDB ID 1HKQ) was generated using ClustalW (Genetyx). Secondary structural elements are shown and determined using the crystal structure. Identical and homologous residues are highlighted in the filled background and box, respectively. Presumably, the DNA molecule will interact along the dimer interface and possibly with the six basic residues in this region, namely Lys69 (α4), Lys73 (α4), Arg78, Lys85 (β2), Arg90 (β3) and Arg96. The basic amino acid residues of RepA that are thought to be involved in DNA interaction are indicated by inverted triangles. Mark and mark the equivalent of MSBI1.176 WH1 with a triangle (▲).

[0098] References

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Claims

1. Crystals of the MSBI1.176 Rep protein WH1 domain, characterized by: The crystals have a crystal structure having (a) a space group of P21 and (b) unit cell dimensions of a = 32.38 Å ± 1-2 Å, b = 77.77 Å ± 1-2 Å, and c = 47.68 Å ± 1-2 Å, α = 90°, β = 90.66°, and γ = 90°; wherein, The crystal comprises a hydrophobic pocket including Leu11, Leu18 and / or Leu25 as defined in SEQ ID. NO: 1; and The crystals comprise a DNA interaction site defined in SEQ ID. NO: 1 including residues Lys69, Lys73, Lys85, Arg90, Arg78 and / or Arg96.

2. A method for producing a crystal of the MSBI1.176 Rep protein WH1 domain or a crystallizable fragment thereof according to claim 1, the method comprising the following steps: (a) preparing a solution of the WH1 domain of the recombinantly produced MSBI1.176 Rep protein in a crystallization reagent, (b) The WH1 domain of the MSBI1.176 Rep protein was crystallized by vapor diffusion.

3. The method according to claim 2, wherein: The crystallization reagent contains 0.2 M magnesium acetate and 20% PEG3350.

4. The method according to claim 2, wherein: The vapor diffusion is a hanging drop vapor diffusion method.

5. A method for screening inhibitors of MSBI1.176 Rep protein, the method comprising the following steps: (a) providing a solution of the MSBI1.176 Rep protein WH1 domain or a crystallizable fragment thereof, (b) contacting at least one candidate compound with the MSBI1.176 Rep protein WH1 domain in the solution, (c) preparing a crystal of the MSBI1.176 Rep protein WH1 domain according to claim 1, and (d) identifying candidate binding compounds for the MSBI1.176 Rep protein, wherein the binding of the candidate compounds to the DNA interaction site as defined in claim 1 is determined.

6. Use of the crystal of the WH1 domain of the MSBI1.176 Rep protein according to claim 1 in obtaining atomic spatial relationship data.

7. The use according to claim 6, which is used for screening, identifying, designing or optimizing drugs that bind to the MSBI1.176 Rep protein.

8. Use of the crystal of the WH1 domain of the MSBI1.176 Rep protein according to claim 1 in computer screening of the ability of candidate compounds to bind to the MSBI1.176 Rep protein.

9. The use according to claim 8, wherein the candidate compound has the ability to bind to the DNA interaction site defined in claim 1.

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