The C-terminal domain of DNA gyrase A adopts a DNA-bending β-pinwheel fold

DNA gyrase is unique among enzymes for its ability to actively introduce negative supercoils into DNA. This function is mediated in part by the C-terminal domain of its A subunit (GyrA CTD). Here, we report the crystal structure of this ≈35-kDa domain determined to 1.75-A resolution. The GyrA CTD unexpectedly adopts an unusual fold, which we term a β-pinwheel, that is globally reminiscent of a β-propeller but is built of blades with a previously unobserved topology. A large, conserved basic patch on the outer edge of this domain suggests a likely site for binding and bending DNA; fluorescence resonance energy transfer-based assays show that the GyrA CTD is capable of bending DNA by ≥180° over a 40-bp region. Surprisingly, we find that the CTD of the topoisomerase IV A subunit, which shares limited sequence homology with the GyrA CTD, also bends DNA. Together, these data provide a physical explanation for the ability of DNA gyrase to constrain a positive superhelical DNA wrap, and also suggest that the particular substrate preferences of topoisomerase IV might be dictated in part by the function of this domain.

[1]  J. Wang,et al.  Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine , 1998, Quarterly Reviews of Biophysics.

[2]  JAMES C. Wang,et al.  Cellular roles of DNA topoisomerases: a molecular perspective , 2002, Nature Reviews Molecular Cell Biology.

[3]  N. Cozzarelli,et al.  Preferential relaxation of positively supercoiled DNA by E. coli topoisomerase IV in single-molecule and ensemble measurements. , 2000, Genes & development.

[4]  N. Cozzarelli,et al.  Contrasting Enzymatic Activities of Topoisomerase IV and DNA Gyrase from Escherichia coli* , 1996, The Journal of Biological Chemistry.

[5]  Ying Zhang,et al.  Flexible DNA bending in HU–DNA cocrystal structures , 2003, The EMBO journal.

[6]  N. Cozzarelli,et al.  Contacts between DNA gyrase and its binding site on DNA: features of symmetry and asymmetry revealed by protection from nucleases. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[7]  A. Newton,et al.  Requirement of topoisomerase IV parC and parE genes for cell cycle progression and developmental regulation in Caulobacter crescentus , 1997, Molecular microbiology.

[8]  J. Thompson,et al.  Multiple sequence alignment with Clustal X. , 1998, Trends in biochemical sciences.

[9]  Patrick Forterre,et al.  Phylogenomics of type II DNA topoisomerases , 2003, BioEssays : news and reviews in molecular, cellular and developmental biology.

[10]  N. Grishin,et al.  C‐terminal domain of gyrase A is predicted to have a β‐propeller structure , 2002 .

[11]  K. Marians.  DNA gyrase-catalyzed decatenation of multiply linked DNA dimers. , 1987, The Journal of biological chemistry.

[12]  Anthony Maxwell,et al.  Crystal structure of the breakage–reunion domain of DNA gyrase , 1997, Nature.

[13]  Chung-Cheng Liu,et al.  Type II DNA topoisomerases: Enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break , 1980, Cell.

[14]  A. Maxwell,et al.  A model for the mechanism of strand passage by DNA gyrase. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[16]  L. Klevan,et al.  Deoxyribonucleic acid gyrase-deoxyribonucleic acid complex containing 140 base pairs of deoxyribonucleic acid and an alpha 2 beta 2 protein core. , 1980, Biochemistry.

[17]  G. Charvin,et al.  Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[18]  D. Wigley,et al.  Crystal structure of an N-terminal fragment of the DNA gyrase B protein , 1991, Nature.

[19]  J. Wang,et al.  Recent developments in DNA topoisomerase II structure and mechanism. , 1996, Current opinion in structural biology.

[20]  J. Berger,et al.  Structure of the topoisomerase II ATPase region and its mechanism of inhibition by the chemotherapeutic agent ICRF-187 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Michael D. Stone,et al.  Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Wang,et al.  Supercoiling of the DNA template during transcription. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[23]  M. Gellert,et al.  Structure of the DNA gyrase-DNA complex as revealed by transient electric dichroism. , 1987, Journal of molecular biology.

[24]  D. Waugh,et al.  Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused , 1999, Protein science : a publication of the Protein Society.

[25]  P. Brown,et al.  A sign inversion mechanism for enzymatic supercoiling of DNA. , 1979, Science.

[26]  S. Salzberg,et al.  Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi , 1997, Nature.

[27]  G N Murshudov,et al.  Use of TLS parameters to model anisotropic displacements in macromolecular refinement. , 2001, Acta crystallographica. Section D, Biological crystallography.

[28]  G. Bricogne,et al.  [27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. , 1997, Methods in enzymology.

[29]  M. Paoli,et al.  Protein folds propelled by diversity. , 2001, Progress in biophysics and molecular biology.

[30]  Leroy F. Liu,et al.  DNA-DNA gyrase complex: the wrapping of the DNA duplex outside the enzyme , 1978, Cell.

[31]  N R Cozzarelli,et al.  Effect of supercoiling on the juxtaposition and relative orientation of DNA sites. , 1996, Biophysical journal.

[32]  Tom Alber,et al.  Automated protein crystal structure determination using ELVES. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[33]  M. Gellert,et al.  Site-specific interaction of DNA gyrase with DNA. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Wang,et al.  DNA gyrase and its complexes with DNA: direct observation by electron microscopy , 1985, Cell.

[35]  Massimo Paoli,et al.  Novel sequences propel familiar folds. , 2002, Structure.

[36]  V S Lamzin,et al.  Automated refinement of protein models. , 1993, Acta crystallographica. Section D, Biological crystallography.

[37]  D. Samuels,et al.  Natural synthesis of a DNA‐binding protein from the C‐terminal domain of DNA gyrase A in Borrelia burgdorferi , 1999, The EMBO journal.

[38]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[39]  I. Hickson.  RecQ helicases: caretakers of the genome , 2003, Nature Reviews Cancer.

[40]  K. Kirkegaard,et al.  Mapping the topography of DNA wrapped around gyrase by nucleolytic and chemical probing of complexes of unique DNA sequences , 1981, Cell.

[41]  J. Berger,et al.  Structure and mechanism of DNA topoisomerase II , 1996, Nature.

[42]  N. Cozzarelli,et al.  Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli. , 1995, Genes & development.

[43]  D T Jones,et al.  Protein secondary structure prediction based on position-specific scoring matrices. , 1999, Journal of molecular biology.

[44]  J. Berger,et al.  Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. , 2004, Annual review of biophysics and biomolecular structure.

[45]  Thomas Terwilliger,et al.  SOLVE and RESOLVE: automated structure solution, density modification and model building. , 2004, Journal of synchrotron radiation.

[46]  R. J. Reece,et al.  Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein. , 1991, The Journal of biological chemistry.

[47]  T. Hsieh,et al.  Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites. , 1989, The Journal of biological chemistry.

[48]  R. J. Reece,et al.  The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein. , 1991, Nucleic acids research.

[49]  R F Standaert,et al.  Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. , 1993, Journal of molecular biology.

[50]  J. Wang,et al.  Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[51]  A M Gronenborn,et al.  Minor groove-binding architectural proteins: structure, function, and DNA recognition. , 1998, Annual review of biophysics and biomolecular structure.

[52]  R. J. Reece,et al.  Tryptic fragments of the Escherichia coli DNA gyrase A protein. , 1989, The Journal of biological chemistry.

[53]  Deborah Fass,et al.  Quaternary changes in topoisomerase II may direct orthogonal movement of two DNA strands , 1999, Nature Structural Biology.

[54]  J. Champoux.  DNA topoisomerases: structure, function, and mechanism. , 2001, Annual review of biochemistry.

[55]  A. Khodursky,et al.  Roles of Topoisomerases in Maintaining Steady-state DNA Supercoiling in Escherichia coli * , 2000, The Journal of Biological Chemistry.

[56]  M. Gellert,et al.  DNA gyrase: an enzyme that introduces superhelical turns into DNA. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[57]  R. Clegg.  Fluorescence resonance energy transfer and nucleic acids. , 1992, Methods in enzymology.

[58]  S. Knight,et al.  Disruption of the Borrelia burgdorferi gac Gene, Encoding the Naturally Synthesized GyrA C-Terminal Domain , 2000, Journal of bacteriology.

[59]  K. Marians,et al.  The Interaction of Escherichia coli Topoisomerase IV with DNA (*) , 1995, The Journal of Biological Chemistry.

[60]  G. Orphanides,et al.  Evidence for a conformational change in the DNA gyrase-DNA complex from hydroxyl radical footprinting. , 1994, Nucleic acids research.

[61]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[62]  D. Lilley,et al.  Global structure of three-way DNA junctions with and without additional unpaired bases: a fluorescence resonance energy transfer analysis. , 1997, Biochemistry.