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• W1966668240 abstract "DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped β-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (–) DNA supercoiling. DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped β-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (–) DNA supercoiling. DNA topoisomerases solve the topological problems that arise in the course of normal DNA metabolism. Among the numerous functions of these essential enzymes are resolution of DNA catenation during replication and cell division and relaxation of DNA (+) and (–) supercoiling 1For our purposes, the term “supercoil” is restricted to topological changes that alter the linking number in a topologically linked DNA. The term “superhelix” refers to a physical superstructure of duplex DNA that may include both changes in writhe and twist from the relaxed state. “Solenoidal” is used as previously defined, and is interchangeable with “toroidal” (52Cozzarelli N.R. Boles T.C. White J.H. Cozzarelli N.R. Wang J.C. DNA Topology and Its Biological Effects. Cold Spring Harbor Laboratory Press, Plainview, NY1990: 139-184Google Scholar, 53Crick F.H. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2639-2643Crossref PubMed Scopus (242) Google Scholar). 1For our purposes, the term “supercoil” is restricted to topological changes that alter the linking number in a topologically linked DNA. The term “superhelix” refers to a physical superstructure of duplex DNA that may include both changes in writhe and twist from the relaxed state. “Solenoidal” is used as previously defined, and is interchangeable with “toroidal” (52Cozzarelli N.R. Boles T.C. White J.H. Cozzarelli N.R. Wang J.C. DNA Topology and Its Biological Effects. Cold Spring Harbor Laboratory Press, Plainview, NY1990: 139-184Google Scholar, 53Crick F.H. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2639-2643Crossref PubMed Scopus (242) Google Scholar). resulting from transcriptional and replicative unwinding of the genome (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2070) Google Scholar). In addition to relieving torsional stress, some topoisomerases are also capable of introducing supercoils (2Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2181) Google Scholar). In particular, bacterial DNA gyrase catalyzes the ATP-dependent introduction of (–) supercoils (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2070) Google Scholar). This activity, unique to gyrase among all topoisomerases, helps maintain prokaryotic genomes at a (–) superhelical density, which in turn is thought to lower the energy barrier for unwinding DNA during transcription and replication (3Espeli O. Marians K.J. Mol. Microbiol. 2004; 52: 925-931Crossref PubMed Scopus (79) Google Scholar, 4Zechiedrich E.L. Khodursky A.B. Bachellier S. Schneider R. Chen D. Lilley D.M.J. Cozzarelli N.R. J. Biol. Chem. 2000; 275: 8103-8113Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Gyrase, similar to all members of the type II topoisomerase family (Topo IIs), 2The abbreviations used are: Topo, DNA topoisomerase; GyrA, gyrase A; NTD, N-terminal domain; CTD, C-terminal domain; βME, 2-mercaptoethanol; r.m.s.d., root mean square deviation; β-CA loop, the loop containing residues 555–576. 2The abbreviations used are: Topo, DNA topoisomerase; GyrA, gyrase A; NTD, N-terminal domain; CTD, C-terminal domain; βME, 2-mercaptoethanol; r.m.s.d., root mean square deviation; β-CA loop, the loop containing residues 555–576. catalyzes the ATP-dependent passage of one DNA duplex (T-segment) through another (G-segment), a process that requires transient introduction of a double-stranded DNA break (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2070) Google Scholar). Intramolecular passage of a T-segment results in relaxation or induction of supercoils, whereas intermolecular passage results in decatenation. Most Topo IIs perform both decatenation and relaxation; yet, interestingly, gyrase exhibits an overriding preference to perform intramolecular and unidirectional ((+) to (–) node) transfer of a T-segment through a contiguous G-segment. Gyrase is an A2B2 tetramer comprising GyrA and GyrB subunits. ATPase activity resides in the N-terminal domain of GyrB (GyrB-NTD); the core cleavage-reunion complex is made up of the C-terminal domain of GyrB (GyrB-CTD) (5Noble C.G. Maxwell A. J. Mol. Biol. 2002; 318: 361-371Crossref PubMed Scopus (87) Google Scholar) plus the N-terminal domain of GyrA (GyrA-NTD) (2Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2181) Google Scholar, 6Hockings S.C. Maxwell A. J. Mol. Biol. 2002; 318: 351-359Crossref PubMed Scopus (17) Google Scholar). Both of the hallmark functional features unique to gyrase, its unidirectional supercoiling activity and strong preference for compact intramolecular T-segment transfer, have been attributed to the C-terminal domain of GyrA (GyrA-CTD) (7Kampranis S.C. Maxwell A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14416-14421Crossref PubMed Scopus (129) Google Scholar). This domain is conserved among gyrases and also members of the Topo IV family, in which it is known as ParC-CTD. Eukaryotic Topo IIs possess a CTD, but it appears to be completely different in structure and function from the prokaryotic CTDs (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2070) Google Scholar, 8Caron P.R. Watt P. Wang J.C. Mol. Cell. Biol. 1994; 14: 3197-3207Crossref PubMed Scopus (83) Google Scholar, 9Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (99) Google Scholar, 10Austin C.A. Marsh K.L. BioEssays. 1998; 20: 215-226Crossref PubMed Scopus (230) Google Scholar). Selective removal of the GyrA-CTD from Escherichia coli gyrase results in loss of the ability to introduce (–) supercoils in relaxed and negatively supercoiled DNA, a gain of the ability to relax (–) supercoils, and a 30-fold increase in decatenation activity (7Kampranis S.C. Maxwell A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14416-14421Crossref PubMed Scopus (129) Google Scholar); addition of the GyrA-CTD in trans results in restoration of the capacity to introduce (–) supercoils (11Reece R.J. Maxwell A. J. Biol. Chem. 1991; 266: 3540-3546Abstract Full Text PDF PubMed Google Scholar, 12Critchlow S.E. Maxwell A. Biochemistry. 1996; 35: 7387-7393Crossref PubMed Scopus (82) Google Scholar). Plasmid nicking/religation experiments indicate that the E. coli GyrA-CTD and full-length gyrase (in the absence of ATP) both bind DNA such that (+) superhelicity is introduced, whereas GyrB + GyrA-NTD does not alter global DNA topology upon binding (7Kampranis S.C. Maxwell A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14416-14421Crossref PubMed Scopus (129) Google Scholar, 13Liu L.F. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2098-2102Crossref PubMed Scopus (151) Google Scholar, 14Kampranis S.C. Bates A.D. Maxwell A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8414-8419Crossref PubMed Scopus (126) Google Scholar, 15Reece R.J. Maxwell A. Nucleic Acids Res. 1991; 19: 1399-1405Crossref PubMed Scopus (127) Google Scholar). Furthermore, E. coli gyrase preferentially binds and processes (+) supercoiled DNA (16Bates A.D. O'Dea M.H. Gellert M. Biochemistry. 1996; 35: 1408-1416Crossref PubMed Scopus (53) Google Scholar), whereas GyrB + GyrA-NTD has no such preference (14Kampranis S.C. Bates A.D. Maxwell A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8414-8419Crossref PubMed Scopus (126) Google Scholar). These data have been interpreted to suggest that DNA wraps around gyrase in a manner dependent upon the presence of the GyrA-CTD, producing (+) superhelicity, and that this wrapping plays a crucial role in controlling the directionality of supercoil induction. Additional support for the notion that the full E. coli gyrase wraps a large continuous stretch of DNA has been provided by transient electric dichroism (17Rau D.C. Gellert M. Thoma F. Maxwell A. J. Mol. Biol. 1987; 193: 555-569Crossref PubMed Scopus (53) Google Scholar) and atomic force microscopy experiments (18Heddle J.G. Mitelheiser S. Maxwell A. Thomson N.H. J. Mol. Biol. 2004; 337: 597-610Crossref PubMed Scopus (63) Google Scholar); footprinting experiments have shown that gyrase protects ∼128 bp of DNA, nearly all of which shows a cleavage periodicity typical for DNA bound to a surface (19Kirkegaard K. Wang J.C. Cell. 1981; 23: 721-729Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 20Morrison A. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1416-1420Crossref PubMed Scopus (100) Google Scholar, 21Orphanides G. Maxwell A. Nucleic Acids Res. 1994; 22: 1567-1575Crossref PubMed Scopus (79) Google Scholar, 22Liu L.F. Wang J.C. Cell. 1978; 15: 979-984Abstract Full Text PDF PubMed Scopus (133) Google Scholar), whereas eukaryotic Topo IIs, which lack any counterpart to the GyrA-CTD, protect only ∼35 bp (23Lee M.P. Sander M. Hsieh T. J. Biol. Chem. 1989; 264: 21779-21787Abstract Full Text PDF PubMed Google Scholar, 24Thomsen B. Bendixen C. Lund K. Andersen A.H. Sorensen B.S. Westergaard O. J. Mol. Biol. 1990; 215: 237-244Crossref PubMed Scopus (55) Google Scholar). Together, these experiments imply a unique role for the GyrA-CTD in DNA wrapping and T-segment presentation by gyrase. Type IV topoisomerases (Topo IVs), the other type II subclass containing a GyrA-like CTD, are unlike gyrases in that they are unable to introduce (–) supercoils in relaxed and negatively supercoiled DNA. Furthermore, Topo IV has a much higher decatenation activity than gyrase (25Ullsperger C. Cozzarelli N.R. J. Biol. Chem. 1996; 271: 31549-31555Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Notwithstanding these differences, both classes show an overriding preference to process (+) nodes (26Stone M.D. Bryant Z. Crisona N.J. Smith S.B. Vologodskii A. Bustamante C. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8654-8659Crossref PubMed Scopus (148) Google Scholar, 27Charvin G. Bensimon D. Croquette V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9820-9825Crossref PubMed Scopus (119) Google Scholar). This (+) node preference is absent in all type II topoisomerases not belonging to the gyrase or Topo IV subclasses; these other Topo IIs relax and decatenate (+) and (-) nodes at similar rates (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2070) Google Scholar). Although less biochemical data are available on the ParC-CTD than the GyrA-CTD, it is reasonable to infer that the ParC-CTD also gives rise directly to the preference of Topo IV to process (+) nodes (28Hsieh T.J. Farh L. Huang W.M. Chan N.L. J. Biol. Chem. 2004; 279: 55587-55593Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Representative structures of the GyrA-NTD (29Morais Cabral J.H. Jackson A.P. Smith C.V. Shikotra N. Maxwell A. Liddington R.C. Nature. 1997; 388: 903-906Crossref PubMed Scopus (399) Google Scholar) and the GyrB-ATPase domain from E. coli gyrase are known (30Berger J.M. Biochim. Biophys. Acta. 1998; 1400: 3-18Crossref PubMed Scopus (105) Google Scholar, 31Wigley D.B. Davies G.J. Dodson E.J. Maxwell A. Dodson G. Nature. 1991; 351: 624-629Crossref PubMed Scopus (486) Google Scholar), but the structure of the E. coli GyrA-CTD has not been available until now. The first and only structure of a GyrA-CTD, derived from the spirochete Borrelia burgdorferi, was recently published and revealed an intriguing new fold, designated a β-pinwheel (32Corbett K.D. Shultzaberger R.K. Berger J.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7293-7298Crossref PubMed Scopus (128) Google Scholar). Here we report the structure of the E. coli GyrA-CTD (Fig. 1A) along with Topo I relaxation assays. Together these data form the basis of a model for how the directional bias of supercoil induction by E. coli gyrase is imparted by its C-terminal domain. The E. coli model is distinct from that proposed for B. burgdorferi, as supported by significant structural differences and divergent biochemical properties of these two GyrA-CTDs. Such differences reveal an unexpected richness of diversity in the mechanisms of DNA strand management by gyrase. Protein Preparation—Plasmids encoding E. coli GyrA-CTD (532–853 or 532–841) and B. burgdorferi GyrA-CTD (501–810) were cloned from E. coli K12 or B. burgdorferi (American Type Culture Collection 35210D), respectively, into pET30a (Novagen) with tobacco etch virus protease-cleavable N-terminal His6 tags and fully sequenced. Proteins were overexpressed in E. coli BL21(DE3)-pLysS or Rosetta2(DE3)-pLysS cells at 37 °C. Lysis was accomplished by sonication in 50 mm sodium phosphate, pH 8.0, 500 mm NaCl, 15 mm imidazole, 10 mm βME, and 1 mm phenylmethylsulfonyl fluoride. Both proteins were purified by nickel-nitrilotriacetic acid-agarose (Qiagen). The E. coli GyrA-CTD was further purified by a Hi-Trap heparin column (Amersham Biosciences) and eluted with 50 mm sodium phosphate, pH 8.0, 5 mm βME, 1 mm EDTA, 10% glycerol, and 300–2000 mm NaCl. The B. burgdorferi and E. coli GyrA-CTDs for Topo I relaxation assays were finally purified by gel filtration (Superdex 200 (Amersham Biosciences) in 20 mm HEPES, pH 7.5, 500 mm NaCl, 10% glycerol, 5 mm βME, and 1 mm EDTA. Structure Determination—Crystals (∼300 × 200 × 100 μm) of the E. coli GyrA-CTD (532–853), with an N-terminal His6 tag, were grown by hanging drop vapor equilibration in Nextal plates as follows: 5 μl of 10–15 mg/ml protein solution (10–20 mm sodium phosphate, pH 7.4–7.8, 650 mm NaCl, 5 mm βME, and 1 mm EDTA) were mixed with 5 μl of well solution (100 mm Tris·HCl, pH 7.6–8.2, and 1–4% polyethylene glycol 1500) and equilibrated at room temperature overnight against 1 ml of well solution. Extensive additive screening, removal of the N-terminal His6 tag, and a smaller protein construct comprising residues 532–841 failed to improve the crystal quality. Crystals were cryoprotected by the stepwise addition of ethylene glycol and glycerol (to final concentrations of 20 and 8%, respectively) to a polyethylene glycol 1500-fortified mother liquor. The selenomethionine derivative of the CTD was prepared in the same strain used for the native protein production (33Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1086) Google Scholar). In addition, 10 mm dithiothreitol supplanted βME as the reducing agent throughout the SeMet protein purification and crystallization. The native x-ray diffraction dataset (dmin = 2.6 Å) was collected at the Macromolecular Diffraction at the Cornell High Energy Synchrotron Source (MacCHESS) A1 beamline, and a multiwavelength anomalous dispersion dataset (dmin = 3.0 Å) was obtained at the National Synchrotron Light Source X-4 station at 100 K. Reflection data were integrated and scaled with the HKL2000 program (34Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38523) Google Scholar). Real-space locations of all twelve of the expected selenomethionines in the asymmetric unit were placed using SOLVE (35Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (433) Google Scholar). The initial model was built, using Quanta 2000 (Accelrys, San Diego, CA), into a density-modified map generated using the DM module of the Collaborative Computational Project 4 suite (36Project Collaborative Computational Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19747) Google Scholar, 37Cowtan K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar). This model was subjected to rounds of simulated annealing, energy minimization, individual B-factor refinement in crystallography NMR software (version 1.1) against the native dataset interspersed with manual rebuilding to produce a model with final values of Rwork = 22.7% and Rfree = 26.7%. Noncrystallographic symmetry was not employed during the refinement. Electron density was weak or absent for a significant number of side chains: protomers A and B had 2102 and 2091 atoms built with 32 and 46 side-chains partially disordered (110 and 175 atoms not modeled), respectively. The figures were created with Ribbons 3.22 and GRASP1.2 software (the GRASP surface was created with a model in which the disordered parts of side chains with β-carbon density were modeled to most appropriately reflect surface charge distributions) (38Petrey D. Honig B. Methods Enzymol. 2003; 374: 492-509Crossref PubMed Scopus (198) Google Scholar, 39Carson M. J. Appl. Crystallogr. 1991; 24: 958-961Crossref Scopus (783) Google Scholar). PROCHECK and SFCHECK were used to inform model building and validate the final model (40Laskowski R.J. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-290Crossref Google Scholar, 41Vaguine A.A. Richelle J. Wodak S.J. Acta Crystallogr. 1999; D55: 191-205Crossref Scopus (858) Google Scholar). Topo IB Relaxation Assays—Negatively supercoiled pBR322 was prepared using a GigaKit (Qiagen) (14Kampranis S.C. Bates A.D. Maxwell A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8414-8419Crossref PubMed Scopus (126) Google Scholar, 42Musgrave D.R. Sandman K.M. Reeve J.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10397-10401Crossref PubMed Scopus (96) Google Scholar). The pBR322-resuspended pellet was dialyzed against 20 mm Tris, pH 8.0, 1 mm EDTA and concentrated on a 30 K polyethersulfone membrane (Vivaspin) to 0.85 μg/ml. To obtain undamaged pBR322, it is essential to never freeze the DNA. The reaction buffer included 35 mm Tris, pH 7.4, 50 mm NaCl, 1.85 mm MgCl2, 5 mm spermidine, 100 μg/ml bovine serum albumin, 9 μg/ml tRNA, 6.5% glycerol, 5.5 mm βME, 1 mm ATP, 2 mm HEPES, 0.1 mm EDTA, 300 nm pBR322, and 0.6–6.0 μm GyrA-CTD (E. coli (532–841) or B. burgdorferi; the results are identical with or without His6 tags). T4 DNA ligase (100 units, New England Biolabs) was added to each reaction. After 10 min, DNA Topo IB from vaccinia virus (10 units, Epicentre) was added to a final volume of 10 μl, and the reactions were incubated for 4–6 h at 37 °C. Phenol:chloroform:isoamyl alcohol (25: 24:1) buffered to pH 8.0 was used to extract the protein from the reactions, followed by ethanol precipitation. The DNA was resuspended in 20 mm Tris, pH 8.0, 1 mm EDTA. Gels with ∼0.5 μg of DNA/lane (1.1% agarose in 1× TPE (Tris phosphate, EDTA) or 1× TAE (Tris acetate, EDTA)) were run at 4 V/cm for 10 min and then 1.0 V/cm for 19–21 h. After ethidium bromide staining, the topoisomer bands were quantitated in ImageQuant TL (Amersham Biosciences) and plotted in Kaleidagraph (Synergy Software), where they were fit to gaussian curves. Structure Analysis—Crystals of the E. coli GyrA-CTD (residues 531–853 plus an N-terminal hexahistidine tag) produced diffraction data to a limiting resolution of 2.6 Å (supplemental Table I). Experimental phases were determined by multiwavelength anomalous dispersion phasing using selenomethionine-labeled protein crystals and then applied to a native data set. The final model includes two protomers/asymmetric unit, each comprising residues 535–841, with a large disordered loop from 555 to 576 in protomer A and 563 to 575 in protomer B (Fig. 1C). This disordered loop, hereafter referred to as the β-CA loop, contains a known trypsin cleavage site plus the “GyrA box,” an indel used to distinguish GyrA from ParC replete with positively charged residues (43Reece R.J. Maxwell A. J. Biol. Chem. 1989; 264: 19648-19653Abstract Full Text PDF PubMed Google Scholar, 44Ward D. Newton A. Mol. Microbiol. 1997; 26: 897-910Crossref PubMed Scopus (66) Google Scholar). The version of the GyrA-CTD that yielded the highest quality diffraction data contained an R562C mutation in the β-CA loop, but the loop was similarly disordered in the 2.9-Å structure of the non-mutated protein (data not shown); because the two proteins were indistinguishable structurally, we used the R562C protein for complete refinement and structural interpretation. We note that the dissociation constant of the GyrA-CTD for DNA is unaffected by the presence of the R562C mutation (data not shown). The two protomers possess nearly identical overall structures and can be superimposed with a Cα r.m.s.d. of 1.2 Å. Significant structural differences between the two are localized entirely to flexible elements located at the crystal packing interface, namely the ordering of a small stretch of the β-CA loop (residues 555–562) in protomer B into a short α-helix and the minor adjustment of a nearby loop (residues 608–617) (Fig. 1D, wedges). Upon removal of these parts from the superposition, the r.m.s.d. drops to 0.7 Å. Because the short helix in protomer B appears to be a crystal packing artifact, we have used protomer A for all figures, except Fig. 1, C and D. The crystal structure of the E. coli GyrA-NTD (29Morais Cabral J.H. Jackson A.P. Smith C.V. Shikotra N. Maxwell A. Liddington R.C. Nature. 1997; 388: 903-906Crossref PubMed Scopus (399) Google Scholar) was ordered through residue 522, thus only 12 amino acids are missing between the structures of the two domains of GyrA (Fig. 2A). The overall fold of the E. coli GyrA-CTD is similar to that recently described for the structure of the B. burgdorferi GyrA-CTD (32Corbett K.D. Shultzaberger R.K. Berger J.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7293-7298Crossref PubMed Scopus (128) Google Scholar), with both bearing a superficial similarity to the well known β-propeller (45Jawad Z. Paoli M. Structure (Camb.). 2002; 10: 447-454Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Similar to β-propeller proteins, the GyrA-CTDs contain serially repeated subdomains known as “blades,” each of which is composed of a four-stranded antiparallel β-sheet (Fig. 1, A and B). Closer inspection, however, reveals that the topology of the blades in the GyrA-CTDs is completely different from that in β-propeller proteins; this distinction prompted Corbett et al. (32Corbett K.D. Shultzaberger R.K. Berger J.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7293-7298Crossref PubMed Scopus (128) Google Scholar) to describe the GyrA-CTD fold as a β-pinwheel. Whereas the β-strands that make up the blades in a β-propeller reside on a contiguous stretch of polypeptide sequence, the β-pinwheel is a more interdigitated structure, with the polypeptide chain meandering back and forth between adjacent blades (Fig. 1, A and B). Although each blade in the β-pinwheel fold of the E. coli GyrA-CTD is nearly identical to its counterpart in the B. burgdorferi GyrA-CTD β-pinwheel (average r.m.s.d. = 1.5 Å), the spatial arrangement differs significantly; while the blades of the B. burgdorferi GyrA-CTD form a toroidal β-pinwheel, the blades of the E. coli GyrA-CTD are arranged in an ascending spiral around a screw axis (Fig. 3). If the B. burgdorferi GyrA-CTD has the appearance of a flat washer, then the E. coli GyrA-CTD has the appearance of a lock washer. Consequently, a global superposition of the B. burgdorferi and E. coli proteins yields an r.m.s.d. of 4.59 Å (5.45 Å for protomer B), in marked contrast to the excellent correspondence of blade-to-blade superpositions. The significant divergence in overall shape can clearly be observed in a Cα superposition using the N-terminal blade of each structure (Fig. 3). To gain a more quantitative estimate of the difference in helicity of the two proteins, we computed a screw axis (Fig. 3, blue rod) and measured the displacement of the center of mass for blades 1 and 6 (Fig. 3, spheres) along the axis. Although the B. burgdorferi β-pinwheel has almost no axial displacement (0.04 Å) from blade 1 to 6, the E. coli β-pinwheel exhibits pronounced axial displacement (11.03 Å). Consonant with the axial displacement of the propeller blades, the interface between blades 1 and 6 is much more extensive in the B. burgdorferi GyrA-CTD structure than in the E. coli structure (2,150 Å2 of solvent-inaccessible surface versus 466 Å2, respectively). In the B. burgdorferi structure, a significant fraction of this interface is contributed by the GyrA box on the β-CA loop (991 Å2), whereas the whole loop is disordered in the E. coli GyrA-CTD. The Bacillus stearothermophilus ParC-CTD (Topo IV) structure, reported while this work was under review, also has a β-pinwheel fold with the blades arranged in an ascending spiral (28Hsieh T.J. Farh L. Huang W.M. Chan N.L. J. Biol. Chem. 2004; 279: 55587-55593Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar); the E. coli GyrA-CTD and the B. stearothermophilus ParC-CTD superimposed with an overall r.m.s.d. of 1.5 Å (Fig. 3). The most notable structural difference between these two domains is the presence of α-helices in the β-CA loop and at the C terminus of the ParC-CTD. The latter packs against strand A of blade 1, resulting in a much more extensive interaction between blades 1 and 6 (1086 Å2) than in the E. coli GyrA-CTD structure (466 Å2). It is formally possible that the differences in overall fold between these three structures result from crystal packing. Two independent lines of evidence suggest this is not likely. Protomers A and B of the E. coli GyrA-CTD asymmetric unit are virtually identical in fold, despite packing differently within the unit cell (Fig. 1, C and D, and supplemental Fig. 3). Further, the B. stearothermophilus ParC-CTD, which also shares this spiral fold, packs in yet another form (space group P32) (supplemental Fig. 3) (28Hsieh T.J. Farh L. Huang W.M. Chan N.L. J. Biol. Chem. 2004; 279: 55587-55593Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). To gain insight into the charge density and distribution on the surface of the E. coli GyrA-CTD, we calculated its surface electrostatic potential (Fig. 2B). Strikingly, a positively charged strip extends along the midriff of the protein, following the right-handed superhelical rise of the β-pinwheel. When DNA is modeled into the structure so as to maximize its electrostatic interactions with this positive strip, the right-handed superhelical rise in the positive strip imparts a corresponding superhelical rise in the bound DNA (Fig. 2B). Interestingly, although the B. burgdorferi structure also contains a positive strip (32Corbett K.D. Shultzaberger R.K. Berger J.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7293-7298Crossref PubMed Scopus (128) Google Scholar), it lacks a spiral sense and hence is not expected to impart any superhelical rise upon its bound DNA. In either case, most of the predicted DNA contacts can be attributed to the preponderance of arginines and lysines on the long loops connecting the 310 helix of one propeller blade with the outermost β-strand in its neighbor (Figs. 1A and 4). It is conceivable and perhaps even likely that these basic loops undergo some local conformational adjustment upon DNA binding but unlikely that they change so drastically as to alter the overall course of the positive strip. The right-handed helical arrangement of blades from N to C terminus suggests a model for DNA wrapping, whereby binding along the midriff of the protein surface imparts right-handed superhelicity in the DNA (Fig. 2B). Topo I Readout of Wrapping—To compare changes in DNA topology upon binding of the E. coli or the B. burgdorferi GyrA-CTDs, Topoisomerase IB was used to relax pBR322 plasmids in the presence of either CTD (Fig. 5) (14Kampranis S.C. Bates A.D. Maxwell A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8414-8419Crossref PubMed Scopus (126) Google Scholar, 42Musgrave D.R. Sandman K.M. Reeve J.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10397-10401Crossref PubMed Scopus (96) Google Scholar). Compensatory torsional force introduced by local deformation of DNA topology by a given protein binding event is relaxed by the Topo IB. When the proteins are extracted from the DNA, the sum of local deformations is observable as a global topological change. A similar experiment (plasmid nicking-religa" @default.
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• W1966668240 title "A Superhelical Spiral in the Escherichia coli DNA Gyrase A C-terminal Domain Imparts Unidirectional Supercoiling Bias" @default.
• W1966668240 cites W1539796472 @default.
• W1966668240 cites W1542411948 @default.
• W1966668240 cites W1544840202 @default.
• W1966668240 cites W1575065817 @default.
• W1966668240 cites W1608247033 @default.
• W1966668240 cites W1933777541 @default.
• W1966668240 cites W1968709898 @default.
• W1966668240 cites W1970623512 @default.
• W1966668240 cites W1972550219 @default.
• W1966668240 cites W1973079886 @default.
• W1966668240 cites W1973167563 @default.
• W1966668240 cites W1973665766 @default.
• W1966668240 cites W1976621352 @default.
• W1966668240 cites W1986191025 @default.
• W1966668240 cites W1989346952 @default.
• W1966668240 cites W1993353309 @default.
• W1966668240 cites W1994155152 @default.
• W1966668240 cites W2001641653 @default.
• W1966668240 cites W2006942426 @default.
• W1966668240 cites W2007848866 @default.
• W1966668240 cites W2008364078 @default.
• W1966668240 cites W2010590744 @default.
• W1966668240 cites W2012879549 @default.
• W1966668240 cites W2026326554 @default.
• W1966668240 cites W2042446618 @default.
• W1966668240 cites W2050137671 @default.
• W1966668240 cites W2053658925 @default.
• W1966668240 cites W2054857925 @default.
• W1966668240 cites W2060268336 @default.
• W1966668240 cites W2061111901 @default.
• W1966668240 cites W2063629343 @default.
• W1966668240 cites W2072413382 @default.
• W1966668240 cites W2083272025 @default.
• W1966668240 cites W2089865676 @default.
• W1966668240 cites W2091619525 @default.
• W1966668240 cites W2092052699 @default.
• W1966668240 cites W2101685360 @default.
• W1966668240 cites W2109778555 @default.
• W1966668240 cites W2123374199 @default.
• W1966668240 cites W2131977436 @default.
• W1966668240 cites W2132600317 @default.
• W1966668240 cites W2135469082 @default.
• W1966668240 cites W2139477659 @default.
• W1966668240 cites W2162861161 @default.
• W1966668240 cites W2163169974 @default.
• W1966668240 cites W2171267013 @default.
• W1966668240 cites W2218064154 @default.
• W1966668240 cites W4236530034 @default.
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