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D B Wigley

Publications and source records attributed to D B Wigley.

At least 55 records · Page 3Linked to original sources
PubMed1996

Crystal structure of a DExx box DNA helicase.

There are a wide variety of helicases that unwind helical DNA and RNA substrates. The twelve helicases that have been identified in Escherichia coli play a role in almost all cellular processes involving nucleic acids. We have solved the crystal structure of a monomeric form of a DNA helicase from Bacillus stearothermophilus, alone and in a complex with ADP, at 2.5 and 2.9 A resolution, respectively. The enzyme comprises two domains with a deep cleft running between them. The ATP-binding site, which is situated at the bottom of this cleft, is formed by motifs that are conserved across the superfamily of related helicases. Unexpected structural homology with the DNA recombination protein, RecA, suggests how ATP binding and hydrolysis may drive conformational changes of the enzyme during catalysis, and implies that there is a common mechanism for all helicases.

Adenosine Diphosphate↗
PubMed1996

Crystallization and preliminary crystallographic analysis of the DNA gyrase B protein from B. stearothermophilus.

DNA gyrase B (GyrB) from B. stearothermophilus has been crystallized in the presence of the non-hydrolyzable ATP analogue, 5'-adenylyl-beta-gamma-imidodiphosphate (ADPNP), by the dialysis method. A complete native data set to 3.7 A has been collected from crystals which belonged to the cubic space group I23 with unit-cell dimension a = 250.6 A. Self-rotation function analysis indicates the position of a molecular twofold axis. Low-resolution data sets of a thimerosal and a selenomethionine derivative have also been analysed. The heavy-atom positions are consistent with one dimer in the asymmetric unit.

Journal Article↗
PubMed1996

Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.

Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA.

Adenosine Monophosphate↗
PubMed1996

Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7.

The crystal structure of the ATP-dependent DNA ligase from bacteriophage T7 has been solved at 2.6 A resolution. The protein comprises two domains with a deep cleft running between them. The structure of a complex with ATP reveals that the nucleotide binding pocket is situated on the larger N-terminal domain, at the base of the cleft between the two domains of the enzyme. Comparison of the overall domain structure with that of DNA methyltransferases, coupled with other evidence, suggests that DNA also binds in this cleft. Since this structure is the first of the nucleotidyltransferase superfamily, which includes the eukaryotic mRNA capping enzymes, the relationship between the structure of DNA ligase and that of other nucleotidyltransferases is also discussed.

Adenosine Triphosphate↗
PubMed1996

Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization.

The bacteriophage T7 DNA ligase gene was amplified using polymerase chain reaction-based methods and cloned into a T7 promoter-based expression vector. The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of protein per liter of bacterial culture. An initial physical and biochemical characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt-ended DNA fragments. Inhibition of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated. The enzyme has been crystallized from methoxypolyethylene glycol. The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 A. The unit cell dimensions are a = 66.1 A, b = 87.6 A, and c = 78.6 A, with one monomer in the asymmetric unit (Vm = 2.77 A3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized.

Bacteriophage T7↗
PubMed1996

Preliminary crystallographic analysis of the ParE subunit of Escherichia coli topoisomerase IV.

The ParE subunit of Escherichia coli topoisomerase IV has been crystallized in the presence of the non-hydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP). The crystals are of the orthorhombic space group, P2(1)2(1)2(1), with unit-cell dimensions a = 92.6, b = 119.1, c = 135.3 A. Data have been collected to 3.5 A resolution from frozen native crystals. Self-rotation function analysis of these data indicate the position of a molecular twofold axis. Higher resolution native data are being collected and a derivative search is underway.

Journal Article↗
PubMed1996

The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography.

This study describes the first crystal structures of a complex between a DNA topoisomerase and a drug. We present the structures of a 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein in complexes with two different inhibitors of the ATPase activity of DNA gyrase, namely the coumarin antibiotic, novobiocin, and GR122222X, a member of the cyclothialidine family. These structures are compared with the crystal structure of the complex with an ATP analogue, adenylyl-beta-gamma-imidodiphosphate (ADPNP). The likely mechanism, by which mutant gyrase B proteins become resistant to inhibition by novobiocin are discussed in light of these comparisons. The three ligands are quite dissimilar in chemical structure and bind to the protein in very different ways, but their binding is competitive because of a small degree of overlap of their binding sites. These crystal structures consequently describe a chemically well characterized ligand binding surface and provide useful information to assist in the design of novel ligands.

Adenylyl Imidodiphosphate↗
PubMed1996

A wasp head with a relaxing bite.

The crystal structure of a 92 kDa fragment of the yeast type II topoisomerase reveals a toroidal structure with a large central cavity that is likely to be involved in the translocation of a DNA duplex during catalysis.

Catalysis↗
PubMed1996

Enzymatic ketonization of 2-hydroxymuconate: specificity and mechanism investigated by the crystal structures of two isomerases.

5-Carboxymethyl-2-hydroxymuconate isomerase (CHMI) and 4-oxalocrotonate tautomerase (4-OT) are enzymes that catalyze the isomerization of unsaturated ketones. They share a common enzyme mechanism, although they show a preference for different substrates. There is no apparent sequence homology between the enzymes. To investigate the molecular mechanism and the basis for their substrate specificity, we have determined the crystal structures of the two enzymes at high resolution. 4-OT is hexameric, with the subunits arranged with 32 symmetry. CHMI is trimeric and has extensive contacts between subunits, which include secondary structural elements. The central core of the CHMI monomer has a fold similar to a 4-OT dimer, but the secondary structural elements that form the subunit contacts around the 3-fold axis are different in the two enzymes. The region of greatest similarity between the two enzymes is a large pocket that is proposed to be the active site. The enzymes appear to operate via a "one-base" mechanism, and the possible role of residues in this pocket is discussed in view of this idea. Finally, the molecular basis for substrate specificity in the two enzymes is discussed.

Binding Sites↗
PubMed1996

Molecular mechanisms of drug inhibition of DNA gyrase.

DNA gyrase, an enzyme unique to prokaryotes, has been implicated in almost all processes that involve DNA. Although efficient inhibitors of this protein have been known for more than 20 years, none of them have enjoyed prolonged pharmaceutical success. It is only recently that the mechanisms of inhibition for some of these classes of drugs have been established unequivocally by X-ray crystallography. It is hoped that this detailed structural information will assist the design of novel, effective inhibitors of DNA gyrase.

4-Quinolones↗
PubMed1995

Structure and mechanism of DNA topoisomerases.

DNA topoisomerases are ubiquitous enzymes that control the level of supercoiling of DNA in cells. There are several classes, each with distinct properties, which are briefly discussed in this review. High-resolution X-ray crystallographic structures have been obtained for fragments of two classes of these enzymes, which when combined with biochemical data, reveal a great deal about the gymnastics that the enzymes undergo during catalysis and provide fascinating snapshots of their mechanisms. These mechanisms are discussed in detail. Finally, the first structure of a topoisomerase in a complex with an antibiotic was recently solved. This structure is briefly discussed with regard to the biochemical activity of the compound.

DNA Topoisomerases, Type I↗
PubMed1994

The third IgG-binding domain from streptococcal protein G. An analysis by X-ray crystallography of the structure alone and in a complex with Fab.

Protein G is a cell surface-associated protein from Streptococcus that binds to IgG with high affinity. We have determined the X-ray crystal structures of the third IgG-binding domain (domain III) alone to a resolution of 1.1 A (final R-factor of 19.3%), and in complex with an Fab fragment to 2.6 A (final R-factor of 16.8%). The structure of domain III is similar to the lower-resolution crystal structures of protein G domains determined previously by other investigators, but shows some minor differences when compared with the equivalent NMR structures. Domain III binds to the immunoglobulin by formation of an antiparallel interaction between the second beta-strand in domain III and the last beta-strand in the CH 1 domain. There is also a minor site of interaction between the C-terminal end of the alpha-helix in protein G and the first beta-strand in the CH 1 domain. Previous studies by NMR on the interactions between protein G and IgG have concluded that different portions of the protein G domain are involved in binding to the Fab and Fc portions. The results presented here support these findings and permit a detailed analysis of the recognition of Fab by protein G; formation of the complex buries a large water-accessible area, of a magnitude comparable with that found in antibody/antigen interactions. The majority of hydrogen bonds between the two proteins involve main-chain atoms from the CH 1 domain. The CH 1 domain residues that are in contact with protein G are shown to be highly conserved in alignments of mouse and human gamma chain amino acid sequences. We conclude that the binding site for protein G on Fab is relatively invariant across different species and gamma chain subclasses, providing an explanation for the widespread recognition of Fab fragments from mouse and human antibodies by protein G. The solution of the crystal structures of domain III alone and bound to Fab has demonstrated that there is no major structural change apparent in either protein on formation of the complex.

Amino Acid Sequence↗
PubMed1994

Preliminary crystallographic analysis of 4-oxalocrotonate tautomerase reveals the oligomeric structure of the enzyme.

Crystals of recombinant 4-oxalocrotonate tautomerase from Pseudomonas sp. strain CF600 have been obtained in a form suitable for X-ray analysis. The enzyme is a highly efficient catalyst and is unusual in that it consists of subunits of only 62 amino acids. It crystallises in the triclinic space group, P1, with unit cell dimensions a = 39.6 A, b = 51.5 A, c = 51.6 A, alpha = 60.0 degrees, beta = 81.4 degrees, gamma = 69.6 degrees. The crystals diffract to beyond 1.9 A resolution and are stable to irradiation with X-rays. Preliminary crystallographic data are not consistent with the previously suggested pentameric structure, but indicate that the complex is in fact a hexamer with 32 symmetry.

Computer Simulation↗
PubMed1994

Crystallization of inhibitor complexes of an N-terminal 24 kDa fragment of the DNA gyrase B protein.

A 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein has been crystallized in the presence of novobiocin. One crystal form has been obtained that is orthorhombic, P2(1)2(1)2(1), with unit cell dimensions a = 40.3 A, b = 47.7 A, c = 111.9 A. The asymmetric unit of this crystal form contains one molecule (Vm = 2.24 A3/Da). Complete native data have been collected to 2.5 A resolution. This same protein fragment has also been crystallized in the presence of GR122222X, an inhibitor that is structurally related to cyclothialidine. These crystals also exhibit P2(1)2(1)2(1) symmetry but have unit cell dimensions of a = 68.8 A, b = 68.6 A, c = 48.6 A. The Vm value of this crystal form is 2.39 A3/Da, assuming one molecule in the asymmetric unit, and native data have been collected to 2.0 A resolution. Molecular replacement studies of both complexes are underway.

Binding Sites↗
PubMed1994

Allosteric activation in Bacillus stearothermophilus lactate dehydrogenase investigated by an X-ray crystallographic analysis of a mutant designed to prevent tetramerization of the enzyme.

The crystal structure of a mutant Bacillus stearothermophilus lactate dehydrogenase, into which an additional loop has been engineered in order to prevent tetramerization of the enzyme, has been solved and refined at 2.4 A. The minimal repeat unit in the crystal is a dimer and the tetramer cannot be generated by any of the crystallographic symmetry operations in P2(1). The loop protrudes out into the solvent, stabilized by a good hydrogen bonding arrangement, and clearly sterically hinders tetramer formation. This is the first structure of B. stearothermophilus lactate dehydrogenase (bsLDH) in which the allosteric activator fructose, 1,6-bisphosphate (FBP) is not present. To investigate the mechanism of allosteric activation in this enzyme we have compared the structure with a ternary complex of B. stearothermophilus lactate dehydrogenase. Many of our observations confirm those reported from a comparison of FBP-bound ternary bsLDH complex with an FBP free LDH from another bacterial source, Bifidobacterium longum. Our results suggest that quaternary structural alterations may have less influence on the mechanism than previously reported. The differences in the quaternary structural behaviour of these two enzymes is discussed.

Allosteric Regulation↗
PubMed1994

Three-dimensional model of Escherichia coli gyrase B subunit crystallized in two-dimensions on novobiocin-linked phospholipid films.

Two-dimensional crystals of the Escherichia coli DNA gyrase B subunit were obtained upon specific interactions with novobiocin linked phospholipid films. A three-dimensional surface model of the protein was generated by analysing images of tilted negatively stained crystals. The structure showed, at 2.5 to 3.0 nm resolution, two elongated arms organised as a V-shaped protein: the bottom of the V contains the novobiocin binding site, and the extremities of the arms mediate protein-protein interactions between the two monomers in the unit cell. Image analysis of frozen hydrated two-dimensional crystals resulted in a 1.0 nm resolution projection map that shows structural elements not revealed with negative staining. Electron microscopic structural data were compared with the crystallographic structure of the 43 kDa N-terminal fragment of the B subunit complexed with a non hydrolysable ATP analogue.

Binding Sites↗