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André Aubry

Publications and source records attributed to André Aubry.

At least 19 recordsLinked to original sources
PubMed2006

1,3,5-Triazepane-2,6-diones as structurally diverse and conformationally constrained dipeptide mimetics: identification of malaria liver stage inhibitors from a small pilot library.

The development of the 1,3,5-triazepane-2,6-dione system as a novel, conformationally restricted, and readily accessible class of dipeptidomimetics is reported. The synthesis of the densely functionalized 1,3,5-triazepane-2,6-dione skeleton was achieved in only four steps from a variety of simple linear dipeptide precursors. To extend the practical value of 1,3,5-triazepane-2,6-diones, a general polymer-assisted solution-phase synthesis approach amenable to library production in a multiparallel format was developed. The conformational preferences of the 1,3,5-triazepane-2,6-dione skeleton were investigated in detail by NMR spectroscopy and X-ray diffraction. The ring exhibits a characteristic folded conformation which was compared to that of related dipeptide-derived scaffolds including the more planar 2,5-diketopiperazine (DKP). Molecular and structural diversity was increased further through post-cyclization appending operations at urea nitrogens. Preliminary biological screens of a small collection of 1,3,5-triazepane-2,6-diones revealed inhibitors of the underexplored malaria liver stage and suggest strong potential for this dipeptide-derived scaffold to interfere with and to modulate biological pathways.

Combinatorial Chemistry Techniques↗
PubMed2006

The first crystal structure of a thioacylenzyme intermediate in the ALDH family: new coenzyme conformation and relevance to catalysis.

Crystal structures of several members of the nonphosphorylating CoA-independent aldehyde dehydrogenase (ALDH) family have shown that the peculiar binding mode of the cofactor to the Rossmann fold results in a conformational flexibility for the nicotinamide moiety of the cofactor. This has been hypothesized to constitute an essential feature of the catalytic mechanism because the conformation of the cofactor required for the acylation step is not appropriate for the deacylation step. In the present study, the structure of a reaction intermediate of the E268A-glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, obtained by soaking the crystals of the enzyme/NADP complex with the natural substrate, is reported. The substrate is bound covalently in the four monomers and presents the geometric characteristics expected for a thioacylenzyme intermediate. Control experiments assessed that reduction of the coenzyme has occurred within the crystal. The structure reveals that reduction of the cofactor upon acylation leads to an extensive motion of the nicotinamide moiety with a flip of the reduced pyridinium ring away from the active site without significant changes of the protein structure. This event positions the reduced nicotinamide moiety in a pocket that likely constitutes the exit door for NADPH. Arguments are provided that the structure reported here constitutes a reasonable picture of the first thioacylenzyme intermediate characterized thus far in the ALDH family and that the position of the reduced nicotinamide moiety observed in GAPN is the one suitable for the deacylation step within all of the nonphosphorylating CoA-independent ALDH family.

Acylation↗
PubMed2006

The X-ray structure of the N-terminal domain of PILB from Neisseria meningitidis reveals a thioredoxin-fold.

The secreted form of the PilB protein was recently shown to be bound to the outer membrane of Neisseria gonorrhoeae and proposed to be involved in survival of the pathogen to the host's oxidative burst. PilB is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase (Msr) A and B activities respectively, i.e. the ability to reduce specifically the S and the R enantiomers of the sulfoxide function of the methionine sulfoxides, which are easily formed upon oxidation of methionine residues. The N-terminal domain of PilB (Dom1(PILB)) of N.meningitidis, which possesses a CXXC motif, was recently shown to recycle the oxidized forms of the PilB Msr domains in vitro, as the Escherichia coli thioredoxin (Trx) 1 does. The X-ray structure of Dom1(PILB) of N.meningitidis determined here shows a Trx-fold, in agreement with the biochemical properties of Dom1(PILB). However, substantial structural differences with E.coli Trx1 exist. Dom1(PILB) displays more structural homologies with the periplasmic disulfide oxidoreductases involved in cytochrome maturation pathways in bacteria. The active site of the reduced form of Dom1(PILB) reveals a high level of stabilization of the N-terminal catalytic cysteine residue and a hydrophobic environment of the C-terminal recycling cysteine in the CXXC motif, consistent with the pK(app) values measured for Cys67 (<6) and Cys70 (9.3), respectively. Compared to cytochrome maturation disulfide oxidoreductases and to Trx1, one edge of the active site is covered by four additional residues (99)FLHE(102). The putative role of the resulting protuberance is discussed in relation to the disulfide reductase properties of Dom1(PILB).

Amino Acid Sequence↗
PubMed2005

Crystal structure and solution NMR dynamics of a D (type II) peroxiredoxin glutaredoxin and thioredoxin dependent: a new insight into the peroxiredoxin oligomerism.

Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol-containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research. We present here a combined X-ray and nuclear magnetic resonance (NMR) study of a plant Prx that belongs to the D-Prx (type II) subfamily. The Populus trichocarpa Prx is the first Prx shown to be regenerated in vitro by both the glutaredoxin and thioredoxin systems. The crystal structure and solution NMR provide evidence that the reduced protein is a specific noncovalent homodimer both in the crystal and in solution. The dimer interface is roughly perpendicular to the plane of the central beta sheet and differs from the interface of A- and B-Prx dimers, where proteins associate in the plane parallel to the beta sheet. The homodimer interface involves residues strongly conserved in the D (type II) Prxs, suggesting that all Prxs of this family can homodimerize. The study provides a new insight into the Prx oligomerism and the basis for protein-protein and enzyme-substrate interaction studies by NMR.

Amino Acid Sequence↗
PubMed2005

The three-dimensional structures of peptide methionine sulfoxide reductases: current knowledge and open questions.

Methionine sulfoxides are easily formed in proteins exposed to reactive oxidative species commonly present in cells. Their reduction back to methionine residues is catalyzed by peptide methionine sulfoxide reductases. Although grouped in a unique family with respect to their biological function, these enzymes are divided in two classes named MsrA and MsrB, depending on the sulfoxide enantiomer of the substrate they reduce. This specificity-based classification differentiates enzymes which display no sequence homology. Several three-dimensional structures of peptide methionine sulfoxide reductases have been determined, so that members of both classes are known to date. These crystal structures are reviewed in this paper. The folds and active sites of MsrAs and MsrBs are discussed in the light of the methionine sulfoxide reductase sequence diversity.

Amino Acid Sequence↗
PubMed2004

Synthesis and structure of symmetrical bicyclic hexapeptides bridged by metathesis reaction.

[structure: see text] Bicyclic hexapeptides 1a-c were synthesized via an intramolecular ring-closing metathesis reaction on solid phase followed by an N- to C-terminal cyclization in solution. Structural elucidation showed that these compounds assumed a C2-symmetrical structure with two beta-turns. The trans-ethylene plane was found to occupy two positions in rapid interconversion. One of the bicyclic hexapeptides crystallized with five water molecules, which made an arch above the ethylene group.

Cyclization↗
PubMed2004

Expression, purification, crystallization and preliminary X-ray diffraction data of methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis.

Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Suitable crystals for X-ray diffraction experiments were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 195.2, b = 192.5, c = 83.5 A, and contain one tetramer per asymmetric unit. X-ray diffraction data were collected to 2.5 A resolution using a synchrotron-radiation source. The crystal structure was solved by the molecular-replacement method.

Aldehyde Oxidoreductases↗
PubMed2004

tert-Butyl (6S)-4-hydroxy-6-isobutyl-2-oxo-1,2,5,6-tetrahydropyridine-1-carboxylate and tert-butyl (4R,6S)-4-hydroxy-6-isobutyl-2-oxopiperidine-1-carboxylate.

X-ray studies reveal that tert-butyl (6S)-6-isobutyl-2,4-dioxopiperidine-1-carboxylate occurs in the 4-enol form, viz. tert-butyl (6S)-4-hydroxy-6-isobutyl-2-oxo-1,2,5,6-tetrahydropyridine-1-carboxylate, C14H23NO4, when crystals are grown from a mixture of dichloromethane and pentane, and has an axial orientation of the isobutyl side chain at the 6-position of the piperidine ring. Reduction of the keto functionality leads predominantly to the corresponding beta-hydroxylated delta-lactam, tert-butyl (4R,6S)-4-hydroxy-6-isobutyl-2-oxopiperidine-1-carboxylate, C14H25NO4, with a cis configuration of the 4-hydroxy and 6-isobutyl groups. The two compounds show similar molecular packing driven by strong O-H...O=C hydrogen bonds, leading to infinite chains in the crystal structure.

Journal Article↗
PubMed2004

(6S)-6-Isobutylpiperidine-2,4-dione and (4R,6S)/(4S,6S)-4-hydroxy-6-isobutylpiperidin-2-one.

The crystal structure of (6S)-6-isobutylpiperidine-2,4-dione, C9H15NO2, shows that the keto tautomer is favoured in the solid state. The reduction of the keto functionality leads to the corresponding 4-hydroxy-6-isobutylpiperidin-2-one, C9H17NO2, with an 84:16 cis/trans ratio, containing the 4R,6S and 4S,6S isomers; the ratio of the two isomers was determined by NMR analysis of the reaction mixture. Crystals obtained from the mixture of both isomers have been studied and shown to contain the isomers in a 86:14 ratio. Hence, both X-ray and NMR analyses show that crystallization does not select the major diastereomer formed by the reduction. In both crystal structures, the two independent molecules dimerize through an R2(2)(8) hydrogen-bond motif between adjacent amide groups.

Journal Article↗
PubMed2003

Purification, crystallization and preliminary X-ray diffraction data of L7Ae sRNP core protein from Pyrococcus abyssii.

The L7Ae sRNP core protein from Pyrococcus abyssii was crystallized using the sitting-drop vapour-diffusion method. Crystals were obtained in the presence of MgCl(2), PEG 2000 MME and acetate buffer at pH 4.0. A native data set has been collected at 2.9 A resolution using a rotating-anode generator at room temperature. Crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 70.7, b = 112.9, c = 34.8 A. There are two monomers of MW 14 200 Da per asymmetric unit and the packing density V(M) is 2.45 A(3) Da(-1). A molecular-replacement analysis gave solutions for the rotation and translation functions.

Archaeal Proteins↗
PubMed2003

The functional glycosyltransferase signature sequence of the human beta 1,3-glucuronosyltransferase is a XDD motif.

The human beta 1,3-glucuronosyltransferase I (GlcAT-I) is the key enzyme responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide of proteoglycans (GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta 1-O-serine). We have investigated the role of aspartate residues Asp194-Asp195-Asp196 corresponding to the glycosyltransferase DXD signature motif, in GlcAT-I function by UDP binding experiments, kinetic analyses, and site-directed mutagenesis. We presented the first evidence that Mn2+ is not only essential for GlcAT-I activity but is also required for cosubstrate binding. In agreement, kinetic studies were consistent with a metal-activated enzyme model whereby activation probably occurs via binding of a Mn2+.UDP-GlcA complex to the enzyme. Mutational analysis showed that the Asp194-Asp195-Asp196 motif is a major element of the UDP/Mn2+ binding site. Furthermore, determination of the individual role of each aspartate showed that substitution of Asp195 as well as Asp196 to alanine strongly impaired GlcAT-I activity, whereas Asp194 replacement produced only a moderate alteration of the enzyme activity. These findings along with molecular modeling and three-dimensional structure comparison of the GlcAT-I catalytic center with that of the Bacillus subtilis glycosyltransferase SpsA provided evidence that the interactions of Asp195 with the ribose moiety of UDP and of Asp196 with the metal cation Mn2+ were crucial for GlcAT-I function. Altogether, these results indicated that, similarly to the SpsA enzyme, the nucleotide binding site of GlcAT-I contains a XDD motif rather than a DXD motif.

Amino Acid Motifs↗
PubMed2003

Crystallization and preliminary X-ray studies of the glutaredoxin from poplar in complex with glutathione.

A monocysteinic mutant of poplar glutaredoxin (C30S) has been overproduced and purified. The protein has been crystallized in complex with glutathione using the hanging-drop vapour-diffusion technique in the presence of PEG 4000 as a precipitating agent. A native data set was collected at 1.55 A resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.7, b = 49.1, c = 104.8 A. Isomorphous crystals of a selenomethionine derivative were grown under the same conditions. Three data sets were collected at 1.73 A using the FIP synchrotron beamline at the ESRF. The positions of the Se atoms were determined and model rebuilding and refinement are in progress.

Cloning, Molecular↗
PubMed2003

Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate.

The crystal structure of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus was solved in complex with its cofactor, NAD, and its physiological substrate, D-glyceraldehyde 3-phosphate (D-G3P). To isolate a stable ternary complex, the nucleophilic residue of the active site, Cys(149), was substituted with alanine or serine. The C149A and C149S GAPDH ternary complexes were obtained by soaking the crystals of the corresponding binary complexes (enzyme.NAD) in a solution containing G3P. The structures of the two binary and the two ternary complexes are presented. The D-G3P adopts the same conformation in the two ternary complexes. It is bound in a non-covalent way, in the free aldehyde form, its C-3 phosphate group being positioned in the P(s) site and not in the P(i) site. Its C-1 carbonyl oxygen points toward the essential His(176), which supports the role proposed for this residue along the two steps of the catalytic pathway. Arguments are provided that the structures reported here are representative of a productive enzyme.NAD.D-G3P complex in the ground state (Michaelis complex).

Amino Acid Substitution↗
PubMed2002

Comparative analysis of thermoadaptation within the archaeal glyceraldehyde-3-phosphate dehydrogenases from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus.

To gain insight into the molecular determinants of thermoadaptation within the family of archaeal glyceraldehyde-3-phosphate dehydrogenases (GAPDH), a homology-based 3-D model of the mesophilic GAPDH from Methanobacterium bryantii was built and compared with the crystal structure of the thermophilic GAPDH from Methanothermus fervidus. The homotetrameric model of the holoenzyme was initially assembled from identical subunits completed with NADP molecules. The structure was then refined by energy minimization and simulated-annealing procedures. PROCHECK and the 3-D profile method were used to appraise the model reliability. Striking molecular features underlying the difference in stability between the enzymes were deduced from their structural comparison. First, both the increase in hydrophobic contacts and the decrease in accessibility to the protein core were shown to discriminate in favor of the thermophilic enzyme. Besides, but to a lesser degree, the number of ion pairs involved in cooperative clusters appeared to correlate with thermostability. Finally, the decreased stability of the mesophilic enzyme was also predicted to proceed from both the lack of charge-dipole interactions within alpha-helices and the enhanced entropy of unfolding due to an increase in chain flexibility. Thus, archaeal GAPDHs appear to be governed by thermoadaptation rules that differ in some aspects from those previously observed within their eubacterial counterparts.

Adaptation, Physiological↗
PubMed2002

Disulphide dimerised peptide creates a crystal contact in an anti-peptide antibody.

Although many Fab derived from human and murine antibodies have been crystallized, several such fragments, including anti-peptide Fab 4 x 11, remain problematic for crystallisation. Diffracting crystals have been obtained for this Fab only in complex with a cysteine containing peptide CGGIRGERA. The IRGERA portion contained in the C-terminus of histone H3 is recognised by the monoclonal antibody and the CGG portion is an essential spacer. Crystallisation experiments and seeding studies show that disulphide bond formation is essential for obtaining crystals. Data has been collected to 2.7 A resolution, and the structure solved by molecular replacement. The asymmetric unit contains two antibodies whose binding sites are face-to-face in an non-crystallographic approximate two-fold rotational axis. We find two Fab in the asymmetric unit with the two antigen combining sites facing each other. This result was unexpected since it has been common practice to avoid the cysteine-containing peptides because of heterogeneity, since the peptide solution is likely to contain both dimeric and monomeric peptides. This result suggests that the cysteine containing peptides used in immunisation for coupling them to a carrier protein could also be used to screen for crystals. Structural data obtained for the same peptides as those used in the immunisation is valuable to evaluate to what extent the linker to the carrier protein may have contributed to the shaping of the antibody binding site. The introduction of exposed cysteines on the surface of proteins by site directed mutagenesis could help resolve difficult crystallisation cases. Should these not form a disulphide bridge in the crystal but nonetheless crystallize, the free cysteine could be used to make a heavy atom derivative for isomorphous replacement.

Amino Acid Sequence↗
PubMed2002

Crystallization and preliminary X-ray diffraction studies of the peptide methionine sulfoxide reductase B domain of Neisseria meningitidis PILB.

Peptide methionine sulfoxide reductases (Msr) are ubiquitous enzymes that catalyse the reduction of free and protein-bound methionine sulfoxide back to methionine via sulfenic acid chemistry. Two classes of Msrs, MsrA and MsrB, have been described. The fact that the two Msrs display opposite stereoselectivities and have no sequence identity suggests that there is no structural similarity between the two classes. No three-dimensional structure of a MsrB is known. In the present report, the MsrB subdomain of Neisseria meningitidis PILB was used to grow orthorhombic crystals by the hanging-drop vapour-diffusion technique. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.0, b = 118.6, c = 138.5 A. Crystals of selenomethionine-substituted MsrB were grown under the same conditions in order to use the MAD method for structure determination. Three diffraction data sets at 1.8 A resolution were collected. The positions of the Se atoms were determined and should result in a full structure determination.

Crystallography, X-Ray↗
PubMed2002

Crystallization and preliminary X-ray data of a bifunctional peroxiredoxin from poplar.

Two variants (wild type and V152C mutant) of a bifunctional poplar peroxiredoxin have been overexpressed in Escherichia coli cells. The two recombinant enzymes were purified and crystallized using the hanging-drop vapour-diffusion technique. Data sets were collected to 1.62 and 2.48 A resolution using X-ray synchrotron-source radiation from two crystal forms of wild-type peroxiredoxin which belonged to the monoclinic space group P2(1) (with unit-cell parameters a = 59.26, b = 68.80, c = 75.71 A, beta = 93.45 degrees ) and to the orthorhombic space group P2(1)2(1)2 (with unit-cell parameters a = 64.70, b = 130.73, c = 35.59 A), respectively. Data were also collected to 2.17 A resolution using a home X-ray source from a V152C peroxiredoxin crystal which belongs to the triclinic space group (P1), with unit-cell parameters a = 36.65, b = 41.53, c = 58.06 A, alpha = 70.52, beta = 93.45, gamma = 64.31 degrees. Phases have been obtained using molecular replacement with the structure of human peroxiredoxin V (PDB code 1hd2) as a search model. Refinement of the structures is in progress.

Cloning, Molecular↗