Biomedical subjects

S P Wood

Publications and source records attributed to S P Wood.

At least 19 recordsLinked to original sources

PubMed2006

High resolution structure of BipD: an invasion protein associated with the type III secretion system of Burkholderia pseudomallei.

Burkoldheria pseudomallei is a Gram-negative bacterium that possesses a protein secretion system similar to those found in Salmonella and Shigella. Recent work has indicated that the protein encoded by the BipD gene of B. pseudomallei is an important secreted virulence factor. BipD is similar in sequence to IpaD from Shigella and SipD from Salmonella and is therefore likely to be a translocator protein in the type-III secretion system of B. pseudomallei. The crystal structure of BipD has been solved at a resolution of 2.1 A revealing the detailed tertiary fold of the molecule. The overall structure is appreciably extended and consists of a bundle of antiparallel alpha-helical segments with two small beta-sheet regions. The longest helices of the molecule form a four-helix bundle and most of the remaining secondary structure elements (three helices and two three-stranded beta-sheets) are formed by the region linking the last two helices of the four-helix bundle. The structure suggests that the biologically active form of the molecule may be a dimer formed by contacts involving the C-terminal alpha-helix, which is the most strongly conserved part of the protein. Comparison of the structure of BipD with immunological and other data for IpaD indicates that the C-terminal alpha-helix is also involved in contacts with other proteins that form the translocon.

Amino Acid Sequence↗

PubMed2006

Crystallization and preliminary X-ray diffraction analysis of BipD, a virulence factor from Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis, possesses a protein-secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to secrete virulence-associated proteins into target cells of the host organism. The BipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and most likely functionally analogous to IpaD from Shigella and SipD from Salmonella. Thus, the BipD protein is likely to be a component of a type III protein-secretion system (TTSS) in B. pseudomallei. Proteins in the same class as BipD, such as IpaD and SipD, are thought to act as extracellular chaperones to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and might even link the translocon pore with the secretion needle. There is evidence that the translocator proteins also bind an integrin which stimulates actin-mediated insertion of the bacterium into the host-cell membrane. Native BipD has been crystallized in a monoclinic crystal form that diffracts X-rays to 2.5 angstroms resolution. BipD protein which incorporates selenomethionine (SeMet-BipD) has also been expressed and forms crystals which diffract to a higher resolution of 2.1 angstroms.

Bacterial Proteins↗

PubMed2006

Structure of the neuronal protein calexcitin suggests a mode of interaction in signalling pathways of learning and memory.

The three-dimensional structure of the neuronal calcium-sensor protein calexcitin from Loligo pealei has been determined by X-ray analysis at a resolution of 1.8A. Calexcitin is up-regulated following Pavlovian conditioning and has been shown to regulate potassium channels and the ryanodine receptor. Thus, calexcitin is implicated in neuronal excitation and plasticity. The overall structure is predominantly helical and compact with a pronounced hydrophobic core between the N and C-terminal domains of the molecule. The structure consists of four EF-hand motifs although only the first three EF hands are involved in binding calcium ions; the C-terminal EF-hand lacks the amino acids required for calcium binding. The overall structure is quite similar to that of the sarcoplasmic calcium-binding protein from Amphioxus although the sequence identity is very low at 31%. The structure shows that the two amino acids of calexcitin phosphorylated by protein kinase C are close to the domain interface in three dimensions and thus phosphorylation is likely to regulate the opening of the domains that is probably required for binding to target proteins. There is evidence that calexcitin is a GTPase and the residues, which have been implicated by mutagenesis in its GTPase activity, are in a short but highly conserved region of 3(10) helix close to the C terminus. This helix resides in a large loop that is partly sandwiched between the N and C-terminal domains suggesting that GTP binding may also require or may cause domain opening. The structure possesses a pronounced electropositive crevice in the vicinity of the 3(10) helix, that might provide an initial docking site for the triphosphate group of GTP. These findings elucidate a number of the reported functions of calexcitin with implications for neuronal signalling.

Amino Acid Sequence↗

PubMed2005

Structure of Chlorobium vibrioforme 5-aminolaevulinic acid dehydratase complexed with a diacid inhibitor.

The structure of Chlorobium vibrioforme 5-aminolaevulinic acid dehydratase (ALAD) complexed with the irreversible inhibitor 4,7-dioxosebacic acid has been solved. The inhibitor binds by forming Schiff-base linkages with lysines 200 and 253 at the active site. The structure reported here provides a definition of the interactions made by both of the substrate molecules (A-side and P-side substrates) with the C. vibrioforme ALAD and is compared and contrasted with structures of the same inhibitor bound to Escherichia coli and yeast ALAD. The structure suggests why 4,7-dioxosebacic acid is a better inhibitor of the zinc-dependent ALADs than of the zinc-independent ALADs.

Binding Sites↗

PubMed2005

Crystallization and preliminary X-ray diffraction analysis of calexcitin from Loligo pealei: a neuronal protein implicated in learning and memory.

The neuronal protein calexcitin from the long-finned squid Loligo pealei has been expressed in Escherichia coli and purified to homogeneity. Calexcitin is a 22 kDa calcium-binding protein that becomes up-regulated in invertebrates following Pavlovian conditioning and is likely to be involved in signal transduction events associated with learning and memory. Recombinant squid calexcitin has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P2(1)2(1)2(1). The unit-cell parameters of a = 46.6, b = 69.2, c = 134.8 A suggest that the crystals contain two monomers per asymmetric unit and have a solvent content of 49%. This crystal form diffracts X-rays to at least 1.8 A resolution and yields data of high quality using synchrotron radiation.

Calcium↗

PubMed2005

Structure of yeast 5-aminolaevulinic acid dehydratase complexed with the inhibitor 5-hydroxylaevulinic acid.

The X-ray structure of the enzyme 5-aminolaevulinic acid dehydratase (ALAD) from yeast complexed with the competitive inhibitor 5-hydroxylaevulinic acid has been determined at a resolution of 1.9 A. The structure shows that the inhibitor is bound by a Schiff-base link to one of the invariant active-site lysine residues (Lys263). The inhibitor appears to bind in two well defined conformations and the interactions made by it suggest that it is a very close analogue of the substrate 5-aminolaevulinic acid (ALA).

Aminolevulinic Acid↗

PubMed2004

The atomic resolution structure of methanol dehydrogenase from Methylobacterium extorquens.

The crystal structure of methanol dehydrogenase (MDH) from Methylobacterium extorquens has been refined without stereochemical restraints at a resolution of 1.2 A. The high-resolution data have defined the conformation of the tricyclic pyrroloquinoline quinone (PQQ) cofactor ring as entirely planar. The detailed definition of the active-site geometry has shown many features that are similar to the quinohaemo-protein alcohol dehydrogenases from Comamonas testosteroni and Pseudomonas putida, both of which possess MDH-like and cytochrome c-like domains. Conserved features between the two types of PQQ-containing enzyme suggest a common pathway for electron transfer between MDH and its physiological electron acceptor cytochrome cL. A pathway for proton transfer from the active site to the bulk solvent is also suggested.

Alcohol Dehydrogenase↗

PubMed2004

The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism.

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 A resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the alpha/beta hydrolase fold family, comprising eight beta-strands interconnected by loops and helices. The 2.8 A resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.

Amino Acid Sequence↗

PubMed2004

Crystallization and X-ray analysis of the Y75N mutant of Mucor pusillus pepsin complexed with inhibitor.

Y75N mutant Mucor pusillus pepsin has been overexpressed in yeast, purified and cocrystallized with the iodine-containing human renin inhibitor CP-113972 [(2R,3S]-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S-methyl-cysteinyl)amino-4]-cyclohexyl-2-hydroxybutanoate] for X-ray crystallography. Tetragonal complex crystals with space group P4(3)2(1)2 were produced by the hanging-drop vapour-diffusion method and diffracted to 3.0 A. The crystals exhibited unit-cell parameters a = b = 182.5, c = 99.1 A and contained four molecules in the asymmetric unit. A 96% complete data set was collected at 298 K using Cu Kalpha X-rays from a rotating-anode generator. Solution of the crystal structure of Y75N mutant M. pusillus pepsin is under way by molecular replacement using the molecular coordinates of wild-type M. pusillus pepsin as a model.

Butyrates↗

PubMed2003

The structure of endothiapepsin complexed with the gem-diol inhibitor PD-135,040 at 1.37 A.

The crystal structure of endothiapepsin complexed with the gem-diol inhibitor PD-135,040 has been anisotropically refined to a resolution of 1.37 A. The structure of this inhibitor complex is in agreement with previous structures of endothiapepsin gem-diol inhibitor complexes that have been used to develop proposed catalytic mechanisms. However, the increase in resolution over previous structures confirms the presence of a number of short hydrogen bonds within the active site that are likely to play an important role in the catalytic mechanism. The presence of low-barrier hydrogen bonds was indicated in a previous one-dimensional H NMR spectrum.

Aspartic Acid Endopeptidases↗

PubMed2003

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Amino Acid Sequence↗

PubMed2002

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism.

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.

Aspartic Acid Endopeptidases↗

PubMed2002

Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis.

The normal plasma protein serum amyloid P component (SAP) binds to fibrils in all types of amyloid deposits, and contributes to the pathogenesis of amyloidosis. In order to intervene in this process we have developed a drug, R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid, that is a competitive inhibitor of SAP binding to amyloid fibrils. This palindromic compound also crosslinks and dimerizes SAP molecules, leading to their very rapid clearance by the liver, and thus produces a marked depletion of circulating human SAP. This mechanism of drug action potently removes SAP from human amyloid deposits in the tissues and may provide a new therapeutic approach to both systemic amyloidosis and diseases associated with local amyloid, including Alzheimer's disease and type 2 diabetes.

Amyloidosis↗

PubMed2001

A neutron Laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism.

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing nonhydrolyzable analogues of the scissile peptide bond. However, the positions of protons on the catalytic aspartates and the ligand in these complexes have not been determined with certainty. Thus, our objective was to locate crucial protons at the active site of an inhibitor complex since this will have major implications for a detailed understanding of the mechanism of action. We have demonstrated that high-resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1 A to an R-factor of 23.5% and an R(free) of 27.4%. This work represents the largest protein structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exchanged their hydrogen atoms with D2O in the mother liquor. The majority of residues resisting exchange are buried within core beta-sheet regions of the molecule. The neutron maps confirm that the protein has a number of buried ionized carboxylate groups which are likely to give the molecule a net negative charge even at very low pH, thereby accounting for its low pI. The functional groups at the catalytic center have clearly undergone H-D exchange despite being buried by the inhibitor occupying the active site cleft. Most importantly, the data provide convincing evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. This has an important bearing on mechanistic proposals for this class of proteinase.

Aspartic Acid↗

PubMed2001

The x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors.

The structures of 5-aminolaevulinic acid dehydratase (ALAD) complexed with substrate (5-aminolaevulinic acid) and three inhibitors: laevulinic acid, succinylacetone and 4-keto-5-aminolaevulinic acid, have been solved at high resolution. The ligands all bind by forming a covalent link with Lys263 at the active site. The structures define the interactions made by one of the two substrate moieties that bind to the enzyme during catalysis. All of the inhibitors induce a significant ordering of the flap covering the active site. Succinylacetone appears to be unique by inducing a number of conformational changes in loops covering the active site, which may be important for understanding the co-operative properties of ALAD enzymes. Succinylacetone is produced in large amounts by patients suffering from the hereditary disease type I tyrosinaemia and its potent inhibition of ALAD also has implications for the pathology of this disease. The most intriguing result is that obtained with 4-keto-5-amino-hexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263. It appears that we have defined the structure of an intermediate of Schiff base formation that the substrate forms upon binding to the P-site of the enzyme.

Aminolevulinic Acid↗

PubMed2001

Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L).

Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 A resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome c(L), which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.

Alcohol Oxidoreductases↗

PubMed2001

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors.

The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.

Catalytic Domain↗

PubMed2000

Molecular chaperone properties of serum amyloid P component.

The selective binding of serum amyloid P component (SAP) to proteins in the pathological amyloid cross-beta fold suggests a possible chaperone role. Here we show that human SAP enhances the refolding yield of denatured lactate dehydrogenase and protects against enzyme inactivation during agitation of dilute solutions. These effects are independent of calcium ions and are not inhibited by compounds that block the amyloid recognition site on the B face of SAP, implicating the A face and/or the edges of the SAP pentamer. We discuss the possibility that the chaperone property of SAP, or its failure, may contribute to the pathogenesis of amyloidosis.

Binding Sites↗