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A Wonacott

Publications and source records attributed to A Wonacott.

16 recordsLinked to original sources
PubMed2000

Crystal structure of the ffh and EF-G binding sites in the conserved domain IV of Escherichia coli 4.5S RNA.

BACKGROUND: Bacterial signal recognition particle (SRP), consisting of 4.5S RNA and Ffh protein, plays an essential role in targeting signal-peptide-containing proteins to the secretory apparatus in the cell membrane. The 4.5S RNA increases the affinity of Ffh for signal peptides and is essential for the interaction between SRP and its receptor, protein FtsY. The 4.5S RNA also interacts with elongation factor G (EF-G) in the ribosome and this interaction is required for efficient translation. RESULTS: We have determined by multiple anomalous dispersion (MAD) with Lu(3+) the 2.7 A crystal structure of a 4.5S RNA fragment containing binding sites for both Ffh and EF-G. This fragment consists of three helices connected by a symmetric and an asymmetric internal loop. In contrast to NMR-derived structures reported previously, the symmetric loop is entirely constituted by non-canonical base pairs. These pairs continuously stack and project unusual sets of hydrogen-bond donors and acceptors into the shallow minor groove. The structure can therefore be regarded as two double helical rods hinged by the asymmetric loop that protrudes from one strand. CONCLUSIONS: Based on our crystal structure and results of chemical protection experiments reported previously, we predicted that Ffh binds to the minor groove of the symmetric loop. An identical decanucleotide sequence is found in the EF-G binding sites of both 4.5S RNA and 23S rRNA. The decanucleotide structure in the 4.5S RNA and the ribosomal protein L11-RNA complex crystals suggests how 4.5S RNA and 23S rRNA might interact with EF-G and function in translating ribosomes.

Bacterial Proteins↗
PubMed1999

Crystal structures of thrombin complexed to a novel series of synthetic inhibitors containing a 5,5-trans-lactone template.

The binding modes of four active site-directed, acylating inhibitors of human alpha-thrombin have been determined using X-ray crystallography. These inhibitors (GR157368, GR166081, GR167088, and GR179849) are representatives of a series utilizing a novel 5, 5-trans-lactone template to specifically acylate Ser195 of thrombin, resulting in an acyl complex. In each case the crystal structure of the complex reveals a binding mode which is consistent with the formation of a covalent bond between the ring-opened lactone of the inhibitor and residue Ser195. Improvements in potency and selectivity of these inhibitors for thrombin are rationalized on the basis of the observed protein/inhibitor interactions identified in these complexes. Occupation of the thrombin S2 and S3 pockets is shown to be directly correlated with improved binding and a degree of selectivity. The binding mode of GR179849 to thrombin is compared with the thrombin/PPACK complex [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471] as this represents the archetypal binding mode for a thrombin inhibitor. This series of crystal structures is the first to be reported of synthetic, nonpeptidic acylating inhibitors bound to thrombin and provides details of the molecular recognition features that resulted in nanomolar potency.

Binding Sites↗
PubMed1998

X-ray and spectrophotometric studies of the binding of proflavin to the S1 specificity pocket of human alpha-thrombin.

Proflavin can be used to study the interactions of inhibitors and substrates with thrombin by monitoring the changes in the visible absorption spectrum that occur on dye displacement. We have used microspectrophotometric methods to investigate the binding of proflavin to crystals of an alpha-thrombin-hirugen complex and have determined the structure by X-ray crystallography. The proflavin molecule binds in the S1 pocket of the enzyme with one of the amino groups hydrogen bonded to the carboxylate of Asp-189 while the protonated ring nitrogen is hydrogen bonded to the carbonyl of Gly-219. This result indicates that the proflavin displacement assay can be used to specifically monitor the binding of inhibitors to the S1 pocket.

Absorption↗
PubMed1996

Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin.

BACKGROUND: UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), catalyses the first committed step of bacterial cell wall biosynthesis and is a target for the antibiotic fosfomycin. The only other known enolpyruvyl transferase is 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme involved in the shikimic acid pathway and the target for the herbicide glyphosate. Inhibitors of enolpyruvyl transferases are of biotechnological interest as MurA and EPSP synthase are found exclusively in plants and microbes. RESULTS: The crystal structure of Escherichia coli MurA complexed with UDP-N-acetylglucosamine (UDP-GlcNAc) and fosfomycin has been determined at 1.8 A resolution. The structure consists of two domains with the active site located between them. The domains have a very similar secondary structure, and the overall protein architecture is similar to that of EPSP synthase. The fosfomycin molecule is covalently bound to the cysteine residue Cys115, whereas UDP-GlcNAc makes several hydrogen-bonding interactions with residues from both domains. CONCLUSIONS: The present structure reveals the mode of binding of the natural substrate UDP-GlcNAc and of the drug fosfomycin, and provides information on the residues involved in catalysis. These results should aid the design of inhibitors which would interfere with enzyme-catalyzed reactions in the early stage of the bacterial cell wall biosynthesis. Furthermore, the crystal structure of MurA provides a model for predicting active-site residues in EPSP synthase that may be involved in catalysis and substrate binding.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1996

The x-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 angstrom resolution.

Phosphomannose isomerase (PMI) catalyses the reversible isomerization of fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P). Absence of PMI activity in yeasts causes cell lysis and thus the enzyme is a potential target for inhibition and may be a route to antifungal drugs. The 1.7 A crystal structure of PMI from Candida albicans shows that the enzyme has three distinct domains. The active site lies in the central domain, contains a single essential zinc atom, and forms a deep, open cavity of suitable dimensions to contain M6P or F6P The central domain is flanked by a helical domain on one side and a jelly-roll like domain on the other.

Binding Sites↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies of human RANTES.

The chemotactic cytokine RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted) is a potent chemoattractant and activator of a number of leukocytes, with a molecular mass of 8 kDa. Crystals of this protein have been grown from 100 mM sodium acetate buffer (pH 4.6) containing 200 mM magnesium acetate, with 20% (w/v) PEG 4000 and 6% (v/v) glycerol. The crystals grow as thick rods, which diffract to at least 1.8 A resolution on a rotating anode X-ray source. The crystals belong to space group p2(1)2(1)2(1) with unit cell dimensions a = 95.14 A, b = 57.58 A and c = 24.01 A with alpha = beta = gamma = 90 degrees. The asymmetric unit contains two molecules of the RANTES monomer, with a VM of 2.0 A(3)/Da.

Chemokine CCL5↗
PubMed1994

X-ray crystallographic studies of a series of penicillin-derived asymmetric inhibitors of HIV-1 protease.

In the development of a treatment for AIDS, the HIV-1 protease has been identified as a good target enzyme for inhibitor design. We previously reported a series of dimeric penicillin-derived C2-symmetric HIV-1 protease inhibitors [Humber, D., et al. (1993) J. Med. Chem. 36, 3120-3128]. In an attempt to reduce the size and optimize the binding of these C2-symmetric inhibitors, molecular modeling studies led to a novel series of monomeric penicillin-derived inhibitors of HIV-1 protease. The binding modes of these monomeric inhibitors have been characterized by X-ray crystallographic and NMR studies. Crystal structures of HIV-1 protease complexed to three inhibitors (GR123976, GR126045, and GR137615) from this series identify the molecular details of the interactions. The binding of GR123976 (IC50 = 2.3 microM) exhibits good hydrophobic contacts but few electrostatic interactions. A strategy of structure-based design and chemical synthesis led to the elaboration of GR123976 to optimize interactions with the protein. Crystallographic analysis of HIV-1 protease complexed to GR126045 and GR137615 identified these interactions with the catalytic aspartates and the protein binding pockets. The crystal structures of the three complexes confirm the presence of the major interactions modeled in order to optimize potency and reveal details of the molecular recognition by HIV-1 protease of this novel series of nonpeptidic inhibitors.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray analysis of Candida albicans phosphomannose isomerase.

Crystals of recombinant phosphomannose isomerase from Candida albicans have been obtained in a form suitable for X-ray diffraction analysis. The enzyme plays a key role in the biosynthesis of the mannan component of the fungal cell wall. It crystallizes in monoclinic space group C2, with cell dimensions a = 124.9 A, b = 52.9 A, c = 85.9 A and beta = 127.4 degrees. The crystals diffract to Bragg spacings beyond 1.7 A, native data have been collected to 2.4 A and a search for heavy-metal derivatives is in progress. The asymmetric unit contains one molecule of the enzyme (M(r) approximately 49,000) with a Vm of 2.3 A3/Da.

Candida albicans↗
PubMed1993

A series of penicillin-derived C2-symmetric inhibitors of HIV-1 proteinase: structural and modeling studies.

The binding modes of a series of penicillin-derived C2 symmetric dimer inhibitors of HIV-1 proteinase were investigated by NMR, protein crystallography, and molecular modeling. The compounds were found to bind in a symmetrical fashion, tracing and S-shaped course through the active site, with good hydrophobic interactions in the S1/S1' and S2/S2' pockets and hydrogen bonding of inhibitor amide groups. Interactions with the catalytic aspartates appeared poor and the protein conformation was very similar to that seen in complexes with peptidomimetics, in spite of the major differences in ligand structure.

Amino Acid Sequence↗
PubMed1993

Determinants of coenzyme specificity in glyceraldehyde-3-phosphate dehydrogenase: role of the acidic residue in the fingerprint region of the nucleotide binding fold.

On the basis of the three-dimensional structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of sequence comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, a series of mutants of GAPDH from Bacillus stearothermophilus have been constructed. The results deduced from kinetic and binding studies suggest that the absence of activity of the wild-type GAPDH with NADP as a cofactor is the consequence of at least three factors: (1) steric hindrance, (2) electrostatic repulsion between the charged carboxyl group of Asp32 and the 2'PO4, and (3) structural determinants at the subunit interface of the tetramer. The best value for kcat/KM and KD for NADP was observed for the D32A-L187A-P188S mutant. This triple mutation leads to a switch in favor of NADP specificity but with a kcat/KM ratio 50- and 80-fold less than that observed for the wild type with NAD and for the chloroplast GAPDH with NADP, respectively. Substituting the invariant chloroplastic Thr33-Gly34-Gly35 for the B. stearothermophilus Leu33-Thr34-Asp35 residues on the double mutant Ala187-Ser188 does not improve significantly the affinity for NADP while substituting Ala32 for Asp32 on the double mutant does. Clearly, other subtle adjustments in the adenosine subsite are needed to reconcile the presence of the carboxylate group of Asp32 and the 2'-phosphate of NADP. Kinetic studies indicate a change of the rate-limiting step for the mutants. This could be the consequence of an incomplete apo-holo transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis.

By combining our knowledge of the crystal structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the sequence of the photosynthetic NADP-dependent GAPDH of the chloroplast, two particular amino acid residues were predicted as the principal determinants of differing coenzyme specificity. By use of site-directed mutagenesis, the amino acids Leu 187 and Pro 188 of GAPDH from Bacillus stearothermophilus have been replaced with Ala 187 and Ser 188, which occur in the sequence from the chloroplast enzyme. The resulting mutant was shown to be catalytically active not only with its natural coenzyme NAD but also with NADP, thus confirming the initial hypothesis. This approach has not only enabled us to alter the coenzyme specificity by minimal amino acid changes but also revealed factors that control the relative affinity of the enzyme for NAD and NADP.

Amino Acid Sequence↗
PubMed1990

The nicotinamide subsite of glyceraldehyde-3-phosphate dehydrogenase studied by site-directed mutagenesis.

Directed mutagenesis has been used to study the nicotinamide subsite of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Residue Asn313 is involved together with the carboxyamide moiety of the nicotinamide ring in a complex network of hydrogen bonding interactions which fix the position of the pyridinium ring of NAD to which hydride transfer occurs at the C-4 position in the catalytic reaction. The asparagine side-chain has been replaced by that of the Thr and Ala residues and results in mutants with very similar properties. Both mutants show much weaker binding of NAD and lower catalytic efficiency. The mutant Asn313----Thr still exhibits strict B-stereospecificity in hydride transfer and retains the property of negative co-operativity in NAD binding. These experiments strongly suggest that the mutant enzyme undergoes the apo----holo sub-unit structural transition associated with coenzyme binding but that the nicotinamide ring is no longer as rigidly held in its pocket as in the wild type enzyme. The results shed light on the details of the molecular interactions which are responsible for negative co-operativity in this enzyme.

Apoenzymes↗
PubMed1989

Role of the histidine 176 residue in glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis.

The catalytically essential amino acid, histidine 176, in the active site of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been replaced with an asparagine residue by site-directed mutagenesis. The role of histidine 176 as a chemical activator, enhancing the reactivity of the thiol group of cysteine 149, has been demonstrated, with iodoacetamide as a probe. The esterolytic properties of GAPDH, illustrated by the hydrolysis of p-nitrophenyl acetate, have been also studied. The kinetic results favor a role for histidine 176 not only as a chemical activator of cysteine 149 but also as a hydrogen donor facilitating the formation of tetrahedral intermediates. These results support the hypothesis that histidine 176 plays a similar role during the oxidative phosphorylation of glyceraldehyde 3-phosphate.

Binding Sites↗
PubMed1989

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148.

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase, Ser148----Ala, was produced by oligonucleotide-directed mutagenesis. The study of the catalytic properties of this mutant has shown that this mutation significantly affects the Michaelis constant of inorganic phosphate and to a lesser extent that of 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate. This result is consistent with model-building studies which show that, for the phosphorylation step of catalysis, inorganic phosphate must bind to the anion recognition site designated Pi with the C(3) phosphate of the acyl-enzyme intermediate in the alternative anion site Ps. Studies of the enantiomeric specificity using D- and L-glyceraldehyde as substrates show that the hydroxyl group of Ser148, combined with the presence of the C(3) phosphate of the substrate, enhances stereospecificity as well as catalysis. However, the stereospecific effect cannot be a consequence of the direct interaction of Ser148 with the C(2)-hydroxyl of the substrate. The changed Km for glyceraldehyde-3-phosphate suggests that the initial step of hemithioacetal formation may take place with its C(3) phosphate bound in the Pi site. This supports the molecular mechanism proposed by Moody (1984). Therefore, catalysis could be enhanced through interactions of the serine hydroxyl group not only with inorganic phosphate but also with the C(3) phosphate of glyceraldehyde-3-phosphate.

Binding Sites↗
PubMed1988

Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase.

Oligonucleotide-directed mutagenesis was employed to produce mutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Escherichia coli and Bacillus stearothermophilus. Three different mutants proteins--His176----Asn, Cys149----Ser, Cys149----Gly--were isolated from one or both of the enzymes. The study of the properties of these mutants has shown that Cys149 is clearly responsible for the information of a charge-transfer transition, named the Racker band, observed during the NAD+ binding to apoGAPDH. This result excludes a similarity between the Racker band and the charge-transfer transition observed following the alkylation of GAPDH by 3-chloroacetyl pyridine-adenine dinucleotide.

Binding Sites↗