André Aubry

Crystal structure of the Escherichia coli peptide methionine sulphoxide reductase at 1.9 A resolution.

BACKGROUND Peptide methionine sulphoxide reductases catalyze the reduction of oxidized methionine residues in proteins. They are implicated in the defense of organisms against oxidative stress and in the regulation of processes involving peptide methionine oxidation/reduction. These enzymes are found in numerous organisms, from bacteria to mammals and plants. Their primary structure shows no significant similarity to any other known protein. RESULTS The X-ray structure of the peptide methionine sulphoxide reductase from Escherichia coli was determined at 3 A resolution by the multiple wavelength anomalous dispersion method for the selenomethionine-substituted enzyme, and it was refined to 1.9 A resolution for the native enzyme. The 23 kDa protein is folded into an alpha/beta roll and contains a large proportion of coils. Among the three cysteine residues involved in the catalytic mechanism, Cys-51 is positioned at the N terminus of an alpha helix, in a solvent-exposed area composed of highly conserved amino acids. The two others, Cys-198 and Cys-206, are located in the C-terminal coil. CONCLUSIONS Sequence alignments show that the overall fold of the peptide methionine sulphoxide reductase from E. coli is likely to be conserved in many species. The characteristics observed in the Cys-51 environment are in agreement with the expected accessibility of the active site of an enzyme that reduces methionine sulphoxides in various proteins. Cys-51 could be activated by the influence of an alpha helix dipole. The involvement of the two other cysteine residues in the catalytic mechanism requires a movement of the C-terminal coil. Several conserved amino acids and water molecules are discussed as potential participants in the reaction.

Transferability of Multipole Charge-Density Parameters: Application to Very High Resolution Oligopeptide and Protein Structures

Crystallography at sub-atomic resolution permits the observation and measurement of the non-spherical character of the electron density (parameterized as multipoles) and of the atomic charges. This fine description of the electron density can be extended to structures of lower resolution by applying the notion of transferability of the charge and multipole parameters. A database of such parameters has been built from charge-density analysis of several peptide crystals. The aim of this study is to assess for which X-ray structures the application of transferability is physically meaningful. The charge-density multipole parameters have been transferred and the X-ray structure of a 310 helix octapeptide Ac-Aib2-L-Lys(Bz)-Aib2-L-Lys(Bz)-Aib2-NHMe refined subsequently, for which diffraction data have been collected to a resolution of 0.82 A at a cryogenic temperature of 100 K. The multipoles transfer resulted in a significant improvement of the crystallographic residual factors wR and wR free. The accumulation of electrons in the covalent bonds and oxygen lone pairs is clearly visible in the deformation electron-density maps at its expected value. The refinement of the charges for nine different atom types led to an additional improvement of the R factor and the refined charges are in good agreement with those of the AMBER molecular modelling dictionary. The use of scattering factors calculated from average results of charge-density work gives a negligible shift of the atomic coordinates in the octapeptide but induces a significant change in the temperature factors (DeltaB approximately 0.4 A2). Under the spherical atom approximation, the temperature factors are biased as they partly model the deformation electron density. The transfer of the multipoles thus improves the physical meaning of the thermal-displacement parameters. The contribution to the diffraction of the different components of the electron density has also been analyzed. This analysis indicates that the electron-density peaks are well defined in the dynamic deformation maps when the thermal motion of the atoms is moderate (B typically lower than 4 A2). In this case, a non-truncated Fourier synthesis of the deformation density requires that the diffraction data are available to a resolution better than 0.9 A.

Thioredoxins and related proteins in photosynthetic organisms: molecular basis for thiol dependent regulation

Thioredoxins are small molecular weight disulfide oxidoreductases specialized in the reduction of disulfide bonds on other proteins. Generally, the enzymes which are selectively and reversibly reduced by these proteins oscillate between an oxidized and inactive conformation and a reduced and active conformation. Thioredoxin constitutes the archetype of a family of protein disulfide oxidoreductases which comprises glutaredoxin and protein disulfide isomerase. Thioredoxin and glutaredoxin serve many roles in the cell, including the redox regulation of target enzymes and transcription factors. They can also serve as hydrogen donors to peroxiredoxins, recently discovered heme free peroxidases, the function of which is to get rid of hydroperoxides in the cell. This review describes the molecular basis for the functioning and interaction between these enzymes in photosynthetic organisms.

Crystal structures of peptides and modified peptides

The X-ray diffraction experiments on peptides and related molecules which have been carried out in Western Europe, except Italy, in the last eight years are reviewed. The crystal structures of some bioactive peptides such as Leu-enkephalin (a neurotransmitter), cyclosporin A (an immunomodulator in both the free and protein-bound state), balhimycin (an antibiotic) and octreotide (a somatostatin analogue) are briefly presented. Crystallized N- and C-protected model peptides have given an insight into the folding tendency and folding modes depending on the peptide sequences. The crystal structures of various pseudopeptide molecules reveal how the three-dimensional structure of peptide analogues can be modulated by substituting non-peptide groups for the peptide bond. A few examples of structural mimetics of the beta- and gamma-turns, and of templates for alpha-helix induction are also presented.

Hydrazino and N-Amino peptides. Chemical and structural aspects

Abstract The regioselective acylation of the nitrogens in hydrazino acetic acid has been studied to obtain the hydrazide and N-amino amide peptidomimetic groups Their conformational influence on the β-turn structure has also been considered.

Crystal growth and structure of La2(C2O4)3 9.5H2O

Abstract Single crystals of La 2 (C 2 O 4 ) 3 9.5H 2 O have been grown in a silica gel by “in situ” generation of oxalic acid. Crystal structure analysis has been carried out in order to complete the results given by Ollendorff and Weigel in a preliminary report (Inorg. Nucl. Chem. Letters 5, 263 (1969)). The value of 9.5 water molecules was that indicated by both thermogravimetry and X-ray diffraction.

The couple Pro/AzaPro : A means of β-turn formation control synthesis and conformation of two AzaPro-containing dipeptides

Abstract On the basis of two synthesized AzaPro-containing dipeptides, the conformational influence of the azaproline residue (a nitrogen atom is substituted for the Pro-CH α ) on the β-turn occurrence was rested according to its relative position in the azadipeptide sequence.

A Structural Analysis of the Catalytic Mechanism of Methionine Sulfoxide Reductase A from Neisseria meningitidis

The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product-methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.

Folding Types of Dipeptides Containing the Diastereoisomeric Cyclopropanic Analogues of Phenylalanine

Abstract In order to consider the possible influence of the orientation of a side chain on the peptide backbone, we have studied the molecular structure of four t BuCO-Pro-c 3 Phe-NHMe model dipeptides, where c 3 Phe denotes each of the four diastereoisomeric 2,3-methanophenylalanines, by using IR and proton NMR experiments. All four derivatives are β-folded, but the folding type depends on the stereochemistry of the cyclopropane moiety.

Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans (GAPN) belongs to the aldehyde dehydrogenase (ALDH) family, which catalyzes the irreversible oxidation of a wide variety of aldehydes into acidic compounds via a two-step mechanism: first, the acylation step involves the formation of a covalent ternary complex ALDH-cofactor-substrate, followed by the oxidoreduction process which yields a thioacyl intermediate and reduced cofactor and second, the rate-limiting deacylation step. Structural and molecular factors involved in the chemical mechanism of GAPN have recently been examined. Specifically, evidence was put forward for the chemical activation of catalytic Cys-302 upon cofactor binding to the enzyme, through a local conformational rearrangement involving the cofactor and Glu-268. In addition, the invariant residue Glu-268 was shown to play an essential role in the activation of the water molecule in the deacylation step. For E268A/Q mutant GAPNs, nucleophilic compounds like hydrazine and hydroxylamine were shown to bind and act as substrates in this step. Further studies were focused at understanding the factors responsible for the stabilization and chemical activation of the covalent intermediates, using X-ray crystallography, site-directed mutagenesis, kinetic and physico-chemical approaches. The results support the involvement of an oxyanion site including the side-chain of Asn-169. Finally, given the strict substrate-specificity of GAPN compared to other ALDHs with wide substrate specificity, one has also initiated the characterization of the G3P binding properties of GAPN. These results will be presented and discussed from the point of view of the evolution of the catalytic mechanisms of ALDH.