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M C Wilce

Publications and source records attributed to M C Wilce.

At least 19 recordsLinked to original sources
PubMed2000

Macromolecular crystallography as a tool for investigating drug, enzyme and receptor interactions.

1. Protein crystallography is an essential tool for the discovery and investigation of pharmacological interactions at the molecular level. It allows investigators to directly visualize the three-dimensional structures of proteins, including enzymes, receptors and hormones. 2. Increasingly, knowledge of these interactions is being used in the drug-discovery process. This is popularly called structure-based drug design. The desired drug could be an enzyme inhibitor or an agonist that mimics endogenous transmitters or hormones. 3. Once the 3-D structure of a pharmacologically relevant target is known, computational processes can be used to search databases of compounds to identify ones that may interact strongly with the target. Lead compounds can be improved using the 3-D structure of the complex of the lead compound and its biological target. 4. The present review describes the processes involved in the determination of a structure by means of protein crystallography and the use of structures in the drug-discovery process. A number of successful examples of structure-based drug design are described. The limitations of the techniques are discussed.

Animals↗
PubMed1999

The structure of plastocyanin from the cyanobacterium Phormidium laminosum.

The crystal structure of the 'blue' copper protein plastocyanin from the cyanobacterium Phormidium laminosum has been solved and refined using 2.8 A X--ray data. P. laminosum plastocyanin crystallizes in space group P43212 with unit-cell dimensions a = 86.57, c = 91.47 A and with three protein molecules per asymmetric unit. The final residual R is 19.9%. The structure was solved using molecular replacement with a search model based on the crystal structure of a close homologue, Anabaena variabilis plastocyanin (66% sequence identity). The molecule of P. laminosum plastocyanin has 105 amino-acid residues. The single Cu atom is coordinated by the same residues - two histidines, a cysteine and a methionine - as in other plastocyanins. In the crystal structure, the three molecules of the asymmetric unit are related by a non-crystallographic threefold axis. A Zn atom lies between each pair of neighbouring molecules in this ensemble, being coordinated by a surface histidine residue of one molecule and by two aspartates of the other.

Amino Acid Sequence↗
PubMed1999

Rubredoxin from Clostridium pasteurianum. Structures of G10A, G43A and G10VG43A mutant proteins. Mutation of conserved glycine 10 to valine causes the 9-10 peptide link to invert.

The four cysteine ligands which coordinate the Fe atom in the electron-transfer protein rubredoxin lie on loops of the polypeptide which form approximate local twofold symmetry. The cysteine ligands in the protein from Clostridium pasteurianum lie at positions 6, 9, 39 and 42. Two glycine residues adjacent to the cysteine ligands at positions 10 and 43 are conserved in all rubredoxins, consistent with the proposal that a beta-carbon substituent at these positions would eclipse adjacent peptide carbonyl groups [Adman et al. (1975). Proc. Natl Acad. Sci. USA, 72, 4854-4858]. X-ray crystal structures of the three mutant proteins G10A, G43A and G10VG43A are reported. The crystal structures of the single-site mutations are isomorphous with the native protein, space group R3; unit-cell parameters are a = 64.3, c = 32.9 A for G10A and a = 64.4, c = 32.8 A for G43A. The crystals of the double mutant, G10VG43A, were in space group P43212, unit-cell parameters a = 61.9, c = 80.5 A, with two molecules per asymmetric unit. The observed structural perturbations support the hypothesis that mutation of the conserved glycine residues would introduce strain into the polypeptide. In particular, in the G10VG43A protein substitution of valine at Gly10 causes the 9-10 peptide link to invert, relieving steric interaction between Cys9 O and Val10 Cbeta. This dramatic change in conformation is accompanied by the loss of the 10N-HcO6 hydrogen bond, part of the chelate loop Thr5-Tyr11. The new conformation allows retention of the 11N-HcS9 hydrogen bond, but converts it from a type II to a type I hydrogen bond. This occurs at the cost of a less tightly packed structure. The structural insights allow rationalization of 1H NMR data reported previously for the 113CdII-substituted proteins and of the negative shifts observed in the FeIII/FeII mid-point potentials upon mutation.

Alanine↗
PubMed1999

Structure and function of the bacterial mechanosensitive channel of large conductance.

Mechanosensation in bacteria involves transducing membrane stress into an electrochemical response. In Escherichia coli and other bacteria, this function is carried out by a number of proteins including MscL, the mechanosensitive channel of large conductance. MscL is the best characterized of all mechanosensitive channels. It has been the subject of numerous structural and functional investigations. The explosion in experimental data on MscL recently culminated in the solution of the three-dimensional structure of the MscL homologue from Mycobacterium tuberculosis. In this review, much of these data are united and interpreted in terms of the newly published M. tuberculosis MscL crystal structure.

Amino Acid Sequence↗
PubMed1998

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli.

The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-A resolution, for a dipeptide-inhibited complex at 2.3-A resolution, and for a low-pH inactive form at 2.7-A resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.

Aminopeptidases↗
PubMed1997

Crystal structures of the copper-containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone.

The crystal structures of the copper enzyme phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter globiformis (AGAO) have been determined and refined for three forms of the enzyme: the holoenzyme in its active form (at 2.2 A resolution), the holoenzyme in an inactive form (at 2.8 A resolution), and the apoenzyme (at 2.2 A resolution). The holoenzyme has a topaquinone (TPQ) cofactor formed from the apoenzyme by the post-translational modification of a tyrosine residue in the presence of Cu2+. Significant differences between the three forms of AGAO are limited to the active site. The polypeptide fold is closely similar to those of the amine oxidases from Escherichia coli [Parsons, M. R., et al. (1995) Structure 3, 1171-1184] and pea seedlings [Kumar, V., et al. (1996) Structure 4, 943-955]. In the active form of holo-AGAO, the active-site Cu atom is coordinated by three His residues and two water molecules in an approximately square-pyramidal arrangement. In the inactive form, the Cu atom is coordinated by the same three His residues and by the phenolic oxygen of the TPQ, the geometry being quasi-trigonal-pyramidal. There is evidence of disorder in the crystals of both forms of holo-AGAO. As a result, only the position of the aromatic group of the TPQ cofactor, but not its orientation about the Cbeta-Cgamma bond, is determined unequivocally. In apo-AGAO, electron density consistent with an unmodified Tyr occurs at a position close to that of the TPQ in the inactive holo-AGAO. This observation has implications for the biogenesis of TPQ. Two features which have not been described previously in amine oxidase structures are a channel from the molecular surface to the active site and a solvent-filled cavity at the major interface between the two subunits of the dimer.

Amine Oxidase (Copper-Containing)↗
PubMed1997

Crystallization, structural determination and analysis of a novel parasite vaccine candidate: Fasciola hepatica glutathione S-transferase.

Glutathione S-transferases (GSTs) represent the major class of detoxifying enzymes from parasitic helminths. As a result, they are candidates for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cattle against fasciolosis. This helminth contains at least seven GST isoforms, of which four have been cloned. The cloned isoforms (Fh51, Fh47, Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. Crystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4122, with one monomer in the asymmetric unit, which corresponds to a very high solvent content of approximately 75%. The physiological dimer is generated via a crystallographic 2-fold rotation. The three-dimensional structure of Fh47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: an N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees interdomain rotation in comparison to Sj26, where the equivalent residue is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh47 crystal structure, reveal critical differences in the residues lining the xenobiotic binding site, particularly at residue positions 9, 106 and 204. In addition, differences amongst the isoforms in the non-substrate binding site were noted, which may explain the observed differential binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed regions of the molecule.

Amino Acid Sequence↗
PubMed1997

Homology model for the human GSTT2 Theta class glutathione transferase.

A tertiary model of the human GSTT2 Theta class glutathione transferase is presented based on the recently solved crystal structure of a related thetalike isoenzyme from Lucilia cuprina. Although the N-terminal domains are quite homologous, the C-terminal domains share less than about 20% identity. The model is used to consolidate the role of Ser 11 in the active site of the enzyme as well as to identify other residues and mechanisms of likely catalytic importance. The T2 subfamily of theta class enzymes have been shown to inactivate reactive sulfate esters arising from arylmethanols. A possible reaction pathway involving the conjugation of glutathione with one such sulfate ester, 1-menaphthyl-sulfate, is described. It is also proposed that the C-terminal region of the enzyme plays an important role in allowing substrate access to the active site.

Amino Acid Sequence↗
PubMed1996

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND: Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS: The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS: There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Amine Oxidase (Copper-Containing)↗
PubMed1996

A structurally derived consensus pattern for theta class glutathione transferases.

We have recently determined the first crystal structure of a theta class glutathione transferase. We have aligned the amino acid sequences of members of the family using the crystal structure as a guide. The alignment has revealed a consensus pattern of residues that first identifies a protein as belonging to the glutathione transferase superfamily, and second is able to distinguish theta class members from other classes of glutathione transferases. The consensus residues unique to the theta class are found to cluster mostly on the hydrophilic surface and flanking loops of helix 2, a region found to be structurally diverse amongst crystal structures of the different glutathione transferase classes. When the consensus pattern was scanned against sequence databases, a number of matches were made with proteins not formally identified as glutathione transferases. Some of these matches indicated that several stress-related proteins belong to the theta class GST family.

Amino Acid Sequence↗
PubMed1995

Evidence for an essential serine residue in the active site of the Theta class glutathione transferases.

A consistent feature of the Alpha-, Mu- and Pi-class glutathione transferases (GSTs) is the presence near the N-terminus of a tyrosine residue that contributes to the activation of glutathione. While this residue appears to be conserved in many Theta-class GSTs, its absence in some suggested that the Theta-class GSTs may have a significantly different structure or catalytic mechanism. The elucidation of the crystal structure of the Theta-class GST from the Australian sheep blowfly, Lucilia cuprina, has indicated that a serine residue rather than a tyrosine residue can form a hydrogen bond with the glutathionyl sulphur atom. The present studies show that mutation of Ser-9 to alanine substantially inactivates the L. cuprina GST, confirming its importance in the reaction mechanism. As this serine is conserved in all Theta-class enzymes reported so far, it seems that an active-site serine is a significant factor that distinguishes the Theta-class GSTs from members of the Alpha-, Mu- and Pi-class isoenzymes.

Amino Acid Sequence↗
PubMed1995

Crystal structure of a theta-class glutathione transferase.

Glutathione S-transferases (GSTs) are a family of enzymes involved in the cellular detoxification of xenotoxins. Cytosolic GSTs have been grouped into four evolutionary classes for which there are representative crystal structures of three of them. Here we report the first crystal structure of a theta-class GST. So far, all available GST crystal structures suggest that a strictly conserved tyrosine near the N-terminus plays a critical role in the reaction mechanism and such a role has been convincingly demonstrated by site-directed mutagenesis. Surprisingly, the equivalent residue in the theta-class structure is not in the active site, but its role appears to have been replaced by either a nearby serine or by another tyrosine residue located in the C-terminal domain of the enzyme.

Amino Acid Sequence↗
PubMed1994

Insights into autoregulation from the crystal structure of twitchin kinase.

Many protein kinases are self-regulated by an intrasteric mechanism where part of the enzyme's structure directly inhibits the active site. This inhibitory structure is called a pseudosubstrate and specific regulators are required to remove it from the active site to allow substrates access. Removal of the pseudosubstrate sequence from members of the myosin light-chain kinase subfamily, including twitchin kinase, activates them but it is not known whether the pseudosubstrate sequence binds to the active site. Native twitchin is a 753K protein (6,839 residues) located in muscle A-bands of the nematode Caenorhabditis elegans and because of its size has not been easy to study. We have determined the crystal structure, refined to 2.8 A resolution, of a recombinant fragment (residues 5,890 to 6,262) of twitchin kinase that contains the catalytic core and a 60 residue carboxy-terminal tail. The C-terminal tail extends through the active site, wedged between the small and large lobes of the structure and making extensive contacts with the catalytic core which accounts for autoinhibition and provides direct support for the intrasteric mechanism of protein kinase regulation.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies of a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina.

Crystals of a glutathione S-transferase from the Australian sheep blowfly Lucilia cuprina have been grown from ammonium sulphate by the hanging drop vapour diffusion method. Successful crystallization required the presence of the inhibitor S-hexylglutathione. The crystals belong to the tetragonal space group P4(1)22 (or P4(3)22) with cell dimensions of a = b = 88.1 A and c = 66.9 A. They contain one monomer in the asymmetric unit and diffract beyond 2.8 A resolution.

Animals↗
PubMed1994

Crystallization and preliminary X-ray analysis of the auto-inhibited twitchin kinase.

An auto-inhibited fragment of twitchin kinase (residues 5890 to 6262) has been crystallized by vapor diffusion techniques using polyethylene glycol 4000 as the precipitant at pH 7.25 to 7.5 at 4 degrees C. We have found that MgSO4 and glycerol were essential for large crystal growth. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit cell dimensions of a = 144.1 A, b = 168.3 A and c = 60.6 A. They are suitable for X-ray analysis and diffract to a resolution of at least 2.8 A.

Animals↗
PubMed1994

Vaccination of sheep against Fasciola hepatica with glutathione S-transferase. Identification and mapping of antibody epitopes on a three-dimensional model of the antigen.

The glutathione S-transferases (FhGST) of the liver fluke Fasciola hepatica have been identified as novel vaccine candidates that protect sheep against a fluke infection. With the use of overlapping peptides covering the predicted amino acid sequences of four FhGST cDNAs, we have defined the linear epitopes recognized by polyclonal antibody from sheep vaccinated with FhGST. Dominant and minor epitopes were found to be present on all four of the sequences although some epitopes were shown to be specific to particular FhGST. A high percentage of the FhGST peptides were found to be antigenic although considerable variability in response to the peptides was observed among the animals. This analysis was extended to the IgG1 and IgG2 response at the peptide level. Based on the recently solved crystal structure of the rat mu-class GST 3-3, a three-dimensional model of one of the FhGST sequences was generated that allowed the predicted spatial localization of defined epitopes. Most epitopes were localized on regions of high flexibility and accessibility. A comparison of epitopes on FhGST with the B cell epitopes on Sm28, a 28-kDa GST from Schistosoma mansoni, has found few similarities. There was no correlation between an antibody response to linear peptide epitopes and the level of protection induced in sheep by vaccination with FhGST.

Amino Acid Sequence↗
PubMed1993

Conformational states of human placental glutathione transferase as probed by limited proteolysis.

Limited proteolysis experiments have been carried out on human placental glutathione transferase in its different forms. The reduced enzyme, as well as the oxidized form and that inactivated with cystamine were all sensitive to 10% (w/w) trypsin, under nondenaturing conditions. The proteolytic cleavage was accompanied by a concomitant loss of enzymatic activity. On the contrary, the presence of glutathione or glutathione conjugates strongly protected the reduced enzyme against inactivation and from the proteolytic attack. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only the peptide bond between Lys 44 and Ala 45 was cleaved. Since Lys 44 has been demonstrated to be involved in the glutathione binding, it is suggested that the region surrounding this amino acid residue (alpha B helix) could be more exposed to the solvent, in the absence of glutathione. Crystallographic data also indicated that this region is flexible, supporting the idea that it may be involved in the observed conformational change upon glutathione binding.

Alanine↗