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• W1560750175 abstract "Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-Å crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) β-sheet domain (amino acids 1–90) and a predominantly α-helical carboxyl-terminal (C) domain (amino acids 91–346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an “open” conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix. Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-Å crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) β-sheet domain (amino acids 1–90) and a predominantly α-helical carboxyl-terminal (C) domain (amino acids 91–346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an “open” conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix. Topoisomerases I and II are involved in virtually all DNA transactions and are the targets of clinically effective anti-cancer and anti-infective drugs (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2218) Google Scholar, 2Corbett K.D. Berger J.M. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 95-118Crossref PubMed Scopus (351) Google Scholar). Topoisomerases exploit a tyrosine nucleophile to attack the phosphodiester backbone, yielding a covalent enzyme-DNA adduct on one side of the resulting break that permits the passage of strand(s) through the break. Type I enzymes operate by cleaving one DNA strand and passing another strand through the nick; type II enzymes cleave both DNA strands and allow passage of a duplex segment through the double strand break. Closure of the break by reversal of the cleavage step results in a change to the topology of DNA, with no net effect on its chemical structure. Type I topoisomerases are subclassified as type IA or type IB enzymes, depending on whether they form a 5′- or 3′-phosphotyrosyl adduct, respectively. Type IA enzymes are distributed widely in the bacterial, archaeal, and eukaryal domains of life. Type IB enzymes are found in eukarya, eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2218) Google Scholar, 2Corbett K.D. Berger J.M. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 95-118Crossref PubMed Scopus (351) Google Scholar, 3Shuman S. Biochim. Biophys. Acta. 1998; 1400: 321-337Crossref PubMed Scopus (93) Google Scholar, 4Zhang H. Barcelo J.M. Lee B. Kohlhagen G. Zimonjic D.B. Popescu N.C. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10608-10613Crossref PubMed Scopus (172) Google Scholar, 5Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar, 6Benarroch D. Claverie J.M. Raoult D. Shuman S. J. Virol. 2006; 80: 314-321Crossref PubMed Scopus (41) Google Scholar). Eukaryotic nuclear type IB enzymes are large monomeric polypeptides, typically >90 kDa, whereas the viral and bacterial type IB polypeptides are much smaller, typically ∼33–36 kDa. Despite their differences in size, the poxvirus and nuclear type IB enzymes have a common core tertiary structure and catalytic mechanism (7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (785) Google Scholar), which is shared with the tyrosine recombinase family (9Guo F. Gopaul D.N. van Duyne G.D. Nature. 1997; 389: 40-46Crossref PubMed Scopus (486) Google Scholar, 10Hickman A.B. Waninger S. Scocca J.J. Dyda F. Cell. 1997; 89: 227-237Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Kwon H.J. Tirumalai R. Landy A. Ellenberger T. Science. 1997; 276: 126-131Crossref PubMed Scopus (182) Google Scholar, 12Subramanya H.S. Arciszewska L.K. Baker R.A. Bird L.E. Sherratt D.J. Wigley D.B. EMBO J. 1997; 16: 5178-5187Crossref PubMed Scopus (178) Google Scholar), thereby suggesting a common ancestry for type IB topoisomerases and tyrosine recombinases (7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (785) Google Scholar). The active sites of poxvirus and nuclear type IB topoisomerases consist of five conserved functional groups, e.g. Arg-130, Lys-167, Arg-220, His-265, and Tyr-274 in vaccinia virus topoisomerase IB (vaccinia TopIB), which execute the cleavage and religation transesterification steps of the catalytic cycle (13Cheng C. Wang L.K. Sekiguchi J. Shuman S. J. Biol. Chem. 1997; 272: 8263-8269Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 14Petersen B.O. Shuman S. J. Biol. Chem. 1997; 272: 3891-3896Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 15Wittschieben J. Shuman S. Nucleic Acids Res. 1997; 25: 3001-3008Crossref PubMed Scopus (73) Google Scholar, 16Tian L. Claeboe C.D. Hecht S.M. Shuman S. Mol. Cell. 2003; 12: 199-208Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 17Yang Z. Champoux J.J. J. Biol. Chem. 2001; 276: 677-685Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 18Interthal H. Quigley P.M. Hol W.G. Champoux J.J. J. Biol. Chem. 2004; 279: 2984-2992Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 19Krogh B.O. Shuman S. Mol. Cell. 2000; 5: 1035-1041Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 20Tian L. Claeboe C.D. Hecht S.M. Shuman S. Structure (Lond.). 2005; 13: 513-520Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 21Krogh B.O. Shuman S. J. Biol. Chem. 2002; 277: 5711-5714Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Vaccinia TopIB is composed of two domains separated by a flexible protease-sensitive linker (22Sharma A. Hanai R. Mondragón A. Structure (Lond.). 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 23Sekiguchi J. Shuman S. J. Biol. Chem. 1995; 270: 11636-11645Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The 234-aa 3The abbreviations used are: aa, amino acid(s); DraTopIB, D. radiodurans topoisomerase IB; MES, 4-morpholineethanesulfonic acid; r.m.s.d., root mean square deviation. carboxyl-terminal (C) domain contains the active site and catalyzes DNA transesterification and supercoil relaxation but has reduced affinity for DNA compared with the full-length enzyme (24Cheng C. Shuman S. J. Biol. Chem. 1998; 273: 11589-11595Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The 80-aa amino-terminal (N) domain interacts with DNA in the major groove (22Sharma A. Hanai R. Mondragón A. Structure (Lond.). 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 25Sekiguchi J. Shuman S. EMBO J. 1996; 15: 3448-3457Crossref PubMed Scopus (46) Google Scholar, 26Sekiguchi J. Shuman S. Nucleic Acids Res. 1997; 25: 3649-3656Crossref PubMed Scopus (20) Google Scholar). The atomic structures of the individual domains of vaccinia TopIB have been determined by x-ray crystallography (7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 22Sharma A. Hanai R. Mondragón A. Structure (Lond.). 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Whereas the fold and active site of the predominantly α-helical catalytic domain are conserved in nuclear type IB topoisomerases (nuclear TopIB) and the tyrosine recombinases (7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), the amino-terminal domain, which consists primarily of β-strands (22Sharma A. Hanai R. Mondragón A. Structure (Lond.). 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), has no counterpart in tyrosine recombinases, although it is structurally homologous to part of the much larger amino-terminal domain of nuclear TopIB (27Redinbo M.R. Champoux J.J. Hol W.G. Curr. Opin. Struct. Biol. 1999; 9: 29-36Crossref PubMed Scopus (57) Google Scholar). Bacterial type IB topoisomerases were discovered recently (5Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar) and are similar to poxvirus type IB topoisomerases with respect to their size, primary structures, and domain organization (Fig. 1). Mutational analyses indicate that the transesterification mechanism and active site constituents of bacterial TopIB adhere closely to those of vaccinia TopIB (5Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar). A major difference between the viral and bacterial enzymes is their cleavage site specificity. Whereas poxvirus and mimivirus TopIB transesterify at a pentapyrimidine cleavage site 5′-(C/T)CCTT↓ in duplex DNA (6Benarroch D. Claverie J.M. Raoult D. Shuman S. J. Virol. 2006; 80: 314-321Crossref PubMed Scopus (41) Google Scholar, 28Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar), the same element cannot be cleaved by Deinococcus radiodurans topoisomerase IB (DraTopIB), the only member of the bacterial TopIB clade that has been characterized to date (5Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar). Nuclear TopIB is also unable to transesterify at the poxvirus cleavage site (29Morham S.G. Shuman S. J. Biol. Chem. 1992; 267: 15984-15992Abstract Full Text PDF PubMed Google Scholar). It has been proposed that contacts of the poxvirus TopIB with DNA trigger assembly of a competent active site by recruitment of several of the catalytic residues that are either disordered or out of position in the free enzyme (7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 30Krogh B.O. Shuman S. J. Biol. Chem. 2001; 276: 36091-36099Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 31Tian L. Claeboe C.D. Hecht S.M. Shuman S. Structure (Lond.). 2004; 12: 31-40Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 32Tian L. Sayer J.M. Jerina D.M. Shuman S. J. Biol. Chem. 2004; 279: 39718-39726Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Structural studies of tyrosine recombinases underscore the theme that the active site might not be preassembled in the free enzyme prior to productive DNA binding (11Kwon H.J. Tirumalai R. Landy A. Ellenberger T. Science. 1997; 276: 126-131Crossref PubMed Scopus (182) Google Scholar, 12Subramanya H.S. Arciszewska L.K. Baker R.A. Bird L.E. Sherratt D.J. Wigley D.B. EMBO J. 1997; 16: 5178-5187Crossref PubMed Scopus (178) Google Scholar). A fuller understanding of target site recognition by type IB enzymes and the conformational changes that accompany DNA binding will depend on capturing the structures of a single enzyme in free and DNA-bound states or on structural comparisons of enzymes with different site specificity in the same functional states along the reaction pathway. These goals have been elusive. The structures of a free full-length poxvirus or nuclear TopIB remain unsolved, perhaps because domain motions about the linker are an impediment to crystallization. Whereas the human TopIB has been crystallized bound to DNA (8Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (785) Google Scholar), no DNA cocrystal has been obtained for the vaccinia enzyme. Here we report the structure of the intact D. radiodurans topoisomerase IB, a prototypal bacterial type IB topoisomerase. The structure verifies the predicted similarity between viral, nuclear, and bacterial type IB enzymes. Important findings are: (i) the active site of DraTopIB is substantially preassembled and seemingly poised for transesterification and (ii) the relative orientation of the catalytic and amino-terminal domains in the apoenzyme clearly needs to change to engage duplex DNA in the circumferential binding mode used by viral and nuclear enzymes (7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (785) Google Scholar, 33Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Abstract Full Text PDF PubMed Google Scholar). A comparison with the human TopIB-DNA cocrystal structure suggests how viral and bacterial TopIB enzymes might bind circumferentially to DNA. DraTopIB Purification and Crystallization—DraTopIB was produced and purified as described previously (5Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar). Selenomethionine-substituted protein was produced using the methionine pathway inhibition method (34Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1091) Google Scholar). For crystallization, the protein was further purified by hydrophobic (Tosoh Biosep; TSK-Gel Phenyl-5PW) and gel filtration chromatography (Pharmacia Corporation; Sephacryl S-100). Purified protein was dialyzed into 50 mm Tris-HCl (pH 7.5), 0.5 m NaCl, 1 mm EDTA, and 1 mm dithiothreitol, and concentrated to 5 mg/ml. Initial crystallization trials, using 96-well crystallization plates set up with a Hydra-II crystallization robot, gave small plate-like crystals in a variety of polyethylene glycol conditions at 10 °C, which were refined further. Refined conditions yielded plate-like crystals in 12% polyethylene glycol 3350 (w/v), 0.1 m MES (pH 6.5), 0.2 m ammonium chloride using the hanging drop vapor diffusion method. Typical crystals were .4×.4×.05 mm. Prior to data collection, DraTopIB crystals were transferred to a solution containing 25% glycerol in addition to the mother liquor in several steps (5% glycerol increments/step and soaking for 2 min/step), harvested using a rayon crystal-mounting loop, and flash-cooled in liquid nitrogen. Throughout the transfer, the crystals were kept at 4 °C. Structure Determination and Refinement—All data were collected from a single crystal at 100 K using synchrotron radiation at the DuPont-Northwestern-Dow Collaborative Access Team Synchrotron Research Center at the Advanced Photon Source. Diffraction data were measured at a wavelength corresponding to an energy value of ∼35 eV above the theoretical absorption edge of selenium (E = 12,693 eV, λ = 0.9768 Å). To minimize radiation damage, the crystal was translated halfway through data collection. In addition, a low resolution data set was collected from the same crystal after the high resolution data had been measured. All data were processed using XDS (35Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3243) Google Scholar) and scaled using SCALA software programs (36Collaborative Computational Project 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). DraTopIB crystals belong to space group P21 with unit cell dimensions a = 38.09, b = 64.97, and c = 76.62 Å, β = 91.78° and have one molecule in the asymmetric unit. Data collection statistics are listed in Table 1.TABLE 1Data collection and refinement statisticsSe-MetData collectionDetector type/sourceMAR-CCD/APSWavelength (Å)0.9768Resolution (Å)1.75Measured reflections227,246Unique reflections36,231Completeness (%)aNumbers in parentheses represent values in the highest resolution shell96.4 (96.4)Rsym (%)a,Numbers in parentheses represent values in the highest resolution shellbRsym = Σ|I – 〈I 〉|/ΣI, where I = observed intensity, and 〈I 〉 = average intensity obtained from multiple measurements4.4 (16.3)Rmeas (%)a,Numbers in parentheses represent values in the highest resolution shellcRmeas as defined by Ref. 665.1 (21.3)Phasing (27–1.75 Å)Phasing power (acentric)dPhasing power = r.m.s.d. (|Fh|/E), where |Fh| = heavy atom structure factor amplitude and E = residual lack of closure error1.546Rcullis (acentric)eRcullis = Σ||Fh(obs)| – |Fh(calc)||/Σ|Fh(obs)|, where |Fh(obs)| = observed heavy atom structure factor amplitude and |Fh(calc)| = calculated heavy atom structure factor amplitude0.685Figure of merit (acentric/centric)0.812/0.685RefinementResolution (Å)27.5–1.75 (1.8–1.75)Number of reflections; working set/test set34,412/1,819 (2,287/117)R-factorfR-factor = Σ||Fo| – |Fc||/Σ|Fo|, where |Fo| = observed structure factor amplitude and |Fc| = calculated structure factor amplitude19.7 (23.3)RfreegRfree, R-factor based on 5% of the data excluded from refinement23.2 (32.6)Protein atoms2,507Water molecules321Other atoms1r.m.s.d.Bond lengths (Å)0.009Bond angles (°)1.06Average B-factor (Å2)hNumbers in parentheses represent r.m.s.d.Main chain34.3 (3.9)Side chain35.9 (4.7)Solvent41.6 (11.8)a Numbers in parentheses represent values in the highest resolution shellb Rsym = Σ|I – 〈I 〉|/ΣI, where I = observed intensity, and 〈I 〉 = average intensity obtained from multiple measurementsc Rmeas as defined by Ref. 66Diederichs K. Karplus P.A. Nat. Struct. Biol. 1997; 4: 269-275Crossref PubMed Scopus (789) Google Scholard Phasing power = r.m.s.d. (|Fh|/E), where |Fh| = heavy atom structure factor amplitude and E = residual lack of closure errore Rcullis = Σ||Fh(obs)| – |Fh(calc)||/Σ|Fh(obs)|, where |Fh(obs)| = observed heavy atom structure factor amplitude and |Fh(calc)| = calculated heavy atom structure factor amplitudef R-factor = Σ||Fo| – |Fc||/Σ|Fo|, where |Fo| = observed structure factor amplitude and |Fc| = calculated structure factor amplitudeg Rfree, R-factor based on 5% of the data excluded from refinementh Numbers in parentheses represent r.m.s.d. Open table in a new tab Three of five possible selenium sites were identified using the Shake and Bake program (37Weeks C.M. DeTitta G.T. Hauptman H.A. Thuman P. Miller R. Acta Crystallogr. Sect. A. 1994; 50: 210-220Crossref PubMed Scopus (134) Google Scholar, 38DeTitta G.T. Weeks C.M. Thuman P. Miller R. Hauptman H.A. Acta Crystallogr. Sect. A. 1994; 50: 203-210Crossref PubMed Scopus (87) Google Scholar). An additional site was identified after refinement of the heavy atom model using the SHARP program (39delaFortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). The missing fifth site corresponds to the disordered amino-terminal methionine. The four selenium sites were refined and phases to 1.75 Å were calculated using SHARP. Phases were improved by density modification using Solomon (40Abrahams J.P. Leslie A.G. Acta Crystallogr. Sect. D. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar) and DM programs (41Cowtan K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, Vol. 31. Daresbury Laboratory, Daresbury, UK1994: 34-38Google Scholar) as implemented in SHARP. The resulting electron density map clearly showed elements of protein secondary structure. Experimental density for the active site region is shown in Fig. 2. The high quality of the experimental map enabled the use of ARP/warp programs (42Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar) to trace automatically 253 of 346 amino acids in the polypeptide chain mainly corresponding to the C domain, which is better ordered. Additional model building was carried out in the program O (43Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The model was refined with the REFMAC5 program (44Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) using data to 1.75 Å resolution. The program ARP was used to place water molecules into peaks >1.5σ in a 2Fo - Fc difference Fourier map and within hydrogen bonding distances. No electron density was seen for several loops in the N domain and one loop in the C domain. As part of the refinement, the translation/liberation/screw parameters were refined for each domain. The final model contained residues 2–16, 20–24, 39–138, and 149–337 plus 321 water molecules and 1 unidentified atom. The model also included 12 amino acids showing alternative side chain conformations. All residues were found within the most favored or allowed regions in the Ramachandran plot. Refinement statistics are listed in Table 1. Figures were made with the programs MOLSCRIPT (45Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), RASTER3D (46Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D. 1994; D50: 869-873Crossref Scopus (2859) Google Scholar), and PYMOL (47DeLano W.L. The PyMOL User's Manual. DeLano Scientific, San Carlos, CA2002Google Scholar). Coordinates and structure factors for DraTopIB have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under the accession code 2F4Q. Overview of the DraTopIB Structure—The structure of DraTopIB was determined by Single Anomalous Dispersion phasing as described under “Experimental Procedures.” The final model, containing one DraTopIB protomer in the asymmetric unit, was refined at 1.75 Å resolution (Rfree = 23.2% and r = 19.7%; Table 1). DraTopIB is composed of an N domain spanning aa 1–90 and a C domain from aa 91–346. Most of the amino acids in the C domain were clearly visible in the electron density map (e.g. see Fig. 2), except for a disordered loop from residues 139–148. The density in the N domain was of poorer quality, with several disordered loops and missing side chains, especially near the amino terminus. The N domain (Fig. 1, red) comprises three α helices plus a five strand antiparallel β-sheet in a tertiary structure similar to that of the N domain of vaccinia TopIB (22Sharma A. Hanai R. Mondragón A. Structure (Lond.). 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The first helix, α0N, which has no equivalent in vaccinia TopIB, packs against the C domain. The structure of the C domain, which contains the active site, is similar to the catalytic domain of vaccinia TopIB (7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). It can be described as a two-lobed fold. Lobe 1 (Fig. 1, cyan) comprises residues 115–233 and contains four helices plus two β-sheets; one sheet is formed by three anti-parallel strands (β1-β3) and the second by two anti-parallel strands (β4-β5). Lobe 2 (Fig. 1, blue) comprises eight α helices (α1 and α6–10) formed by residues 91–113 and 234–346. The last nine amino acids of the protein are part of lobe 2 but are not visible in the crystal structure. The two lobes make extensive interactions, and the active site residues are located at the interface between the lobes. Similarity to Viral TopIB—A search of the Protein Data Bank using the DALI server (48Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3566) Google Scholar) and the Structural Classification of Proteins data base (49Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5610) Google Scholar) using secondary structure matching (50Krissinel E. Henrick K. Acta Crystallogr. Sect. D. 2004; 60: 2256-2268Crossref PubMed Scopus (3189) Google Scholar) confirmed that the C domain of DraTopIB is structurally similar to the catalytic domains of vaccinia TopIB, human TopIB, and several tyrosine recombinases, thereby supporting the proposed common ancestry of the TopIB and tyrosine recombinase enzymes (5Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar, 7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). The search also highlighted the similarity of the N domains of DraTopIB and vaccinia TopIB; otherwise, no significant similarities to any other protein or class of proteins were uncovered. The N domains of DraTopIB and vaccinia TopIB are superimposable with an r.m.s.d. of 1.05 Å for 42 Cα atoms; the C domains superposed with an r.m.s.d. of 3.0 Å for 132 Cα atoms (Fig. 3A). Considered individually, lobes 1 and 2 of DraTopIB superposed on lobes 1 and 2 of vaccinia TopIB with r.m.s.d. values of 2.05 Å and 2.35 Å for 82 and 52 Cα atoms, respectively. The secondary structure elements of lobe 1 aligned well, except for the presence of a surface β-sheet inserted between helices α4 and α5 of DraTopIB (aa 204–216) (Fig. 1). A disordered surface loop in DraTopIB (aa 139–148) corresponds closely to a disordered segment in vaccinia TopIB (aa 129–136), with an important difference being that there is well defined electron density for the first essential arginine of the catalytic pentad (Arg-137) in DraTopIB immediately preceding the start of the disordered loop, whereas the equivalent Arg-130 side chain of vaccinia TopIB is part of the missing segment (Fig. 1). Lobe 2 of vaccinia and DraTopIB differ significantly with respect to their secondary structures flanking the tyrosine nucleophile of the active site. Whereas Tyr-274 of vaccinia TopIB is located within a long continuous helix (helix α8 from aa 269–283), the corresponding region of DraTopIB is broken into two shorter helices (α8′ and α8″), such that the Tyr-289 nucleophile is now situated in the loop connecting the short helices (Figs. 1 and 3C). This has the effect of changing dramatically the position of the catalytic tyrosine, as discussed under “A Preassembled Active Site in DraTopIB.” The structures of vaccinia and DraTopIB also differ in the positioning of the α9 helices distal to the catalytic tyrosine (Fig. 1), although the final α10 helix does occupy an equivalent position in both proteins. Similarity to Nuclear TopIB and Insights into Circumferential Binding—Fig. 3B shows a superposition of the N and C domains of DraTopIB on the structure of human nuclear TopIB in its complex with DNA (8Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (785) Google Scholar). Nuclear TopIB is a much larger protein than vaccinia or DraTopIB and contains multiple structural components that have no counterpart in the viral and bacterial proteins. Nuclear TopIB forms a C-shaped clamp around duplex DNA. The two ends of the C-clamp consist of loops (the so-called ”lips“) that meet in a noncovalent “kiss” to envelop the target site (Fig. 3B). A subset of the β-strands of the N domain of DraTopIB that comprises a DNA-binding surface in vaccinia TopIB (24Cheng C. Shuman S. J. Biol. Chem. 1998; 273: 11589-11595Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 25Sekiguchi J. Shuman S. EMBO J. 1996; 15: 3448-3457Crossref PubMed Scopus (46) Google Scholar, 51Koster D.A. Cr" @default.
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• W1560750175 title "Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA" @default.
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