Michel Marraud

Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor

Summary: The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti‐AChR antibody response is polyclonal. However, a small extracellular region of the AChR a‐subunit, the main immunogenic region (MIR), seems to be a major target for anti‐AChR antibodies. A major loop containing overlapping epitopes for several anti‐MIR monoclonal antibodies (mAbs) lies within residues α67–76 at the extreme synaptic end of each a‐subunit; however, anti‐MIR mAbs are functionally and structurally quite heterogeneous. Anti‐MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplas‐mic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine‐binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti‐AChR antibodies cross‐reactive with noti‐AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR, especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in‐depth understanding, and for possible specific immunotherapy, of MG.

Characterization of the methionine sulfoxide reductase activities of PILB, a probable virulence factor from Neisseria meningitidis

PILB has been described as being involved in the virulence of bacteria of Neisseria genus. The PILB protein is composed of three subdomains. In the present study, the central subdomain (PILB-MsrA), the C terminus subdomain (PILB-MsrB), and the fused subdomain (PILB-MsrA/MsrB) of N. meningitidiswere produced as folded entities. The central subdomain shows a methionine sulfoxide reductase A (MsrA) activity, whereas PILB-MsrB displays a methionine sulfoxide reductase B (MsrB) activity. The catalytic mechanism of PILB-MsrB can be divided into two steps: 1) an attack of the Cys-494 on the sulfur atom of the sulfoxide substrate, leading to formation of a sulfenic acid intermediate and release of 1 mol of methionine/mol of enzyme and 2) a regeneration of Cys-494 via formation of an intradisulfide bond with Cys-439 followed by reduction with thioredoxin. The study also shows that 1) MsrA and MsrB display opposite stereoselectivities toward the sulfoxide function; 2) the active sites of both Msrs, particularly MsrB, are rather adapted for binding protein-bound MetSO more efficiently than free MetSO; 3) the carbon Cα is not a determining factor for efficient binding to both Msrs; and 4) the presence of the sulfoxide function is a prerequisite for binding to Msrs. The fact that the two Msrs exhibit opposite stereoselectivities argues for a structure of the active site of MsrBs different from that of MsrAs. This is further supported by the absence of sequence homology between the two Msrs in particular around the cysteine that is involved in formation of the sulfenic acid derivative. The fact that the catalytic mechanism takes place through formation of a sulfenic acid intermediate for both Msrs supports the idea that sulfenic acid chemistry is a general feature in the reduction of sulfoxides by thiols.

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.

Helix-forming oligoureas: Temperature-dependent nmr, structure determination, and circular dichroism of a nonamer with functionalized side chains

To further investigate the degree of structural homology between γ-peptides A and N,N′-linked oligoureas B, we prepared oligourea nonamer 2 containing Ala, Val, Leu, Phe, Tyr and Lys side chains. Oligomer 2 was synthesized on solid support from activated monomers, i.e., from enantiomerically pure succinimidyl {2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl}carbamates 3a–f that are further substituted at C(2) of the ethyl moiety. These precursors were conveniently prepared from N-Fmoc-protected β3-amino acids with corresponding side chains. Detailed NMR studies (DQF-COSY, TOCSY, and ROESY) in (D5)pyridine revealed that 2 adopts a regular (P)-2.5 helical secondary structure very similar to that previously determined for oligourea heptamer 1 and closely related to the (P)-2.614 helix of γ-peptides. Temperature-dependent NMR further demonstrated the conformational homogeneity and remarkable stability of the structure of 2 in pyridine. The CD spectrum of 2 (0.2 mM) was recorded in MeOH with the aim to gain more information about the conformation of oligoureas. In contrast to 2.6-helical γ-peptides, which display only a weak or no Cotton effect, oligourea 2 exhibits an intense positive Cotton effect at ca. 203 nm ([Θ]=+373000 deg cm2 dmol−1) that decreases only slowly upon increasing the temperature.

Solution Structure of a Retro-inverso Peptide Analogue Mimicking the Foot-and-Mouth Disease Virus Major Antigenic Site STRUCTURAL BASIS FOR ITS ANTIGENIC CROSS-REACTIVITY WITH THE PARENT PEPTIDE

The antigenic activity of a 19-mer peptide corresponding to the major antigenic region of foot-and-mouth disease virus and its retro-enantiomeric analogue was found to be completely abolished when they were tested in a biosensor system in trifluoroethanol. This suggests that the folding pattern, which is α-helix in trifluoroethanol (confirmed by CD measurement), does not correspond to the biologically relevant conformation(s) recognized by antibodies. The NMR structures of both peptides were thus determined in aqueous solution. These studies showed that the two peptides exhibit similar folding features, particularly in their C termini. This may explain in part the cross-reactive properties of the two peptides in aqueous solution. However, the retro-inverso analogue appears to be more rigid than the parent peptide and contains five atypical β-turns. This feature may explain why retro-inverso foot-and-mouth disease virus peptides are often better recognized than the parent peptide by anti-virion antibodies.

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.

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.

A γ-turn induced by a highly constrained cyclopropane analogue of phenylalanine (c3diPhe) in the solid state

Abstract In order to evaluate the possible influence of the side chain orientation on the backbone conformation we have synthesized the model dipeptides tBuCO- l -Pro-c3diPhe-NHiPr, where c3diPhe represents (2S,3S)- and (2R,3R)-1-amino-2,3-diphenylcyclopropanecarboxylic acid, two cyclopropane analogues of phenylalanine. In the solid state, the (2S,3S)c3diPhe-containing compound adopts a classical βII-turn disposition. In contrast, the dipeptide incorporating the (2R,3R) enantiomer exhibits an open βII-turn structure that lacks the usual i+3 to i hydrogen bond, together with a γ-turn centred at the c3diPhe residue.

Efficient access to all four stereoisomers of phenylalanine cyclopropane analogues by chiral HPLC

Bonded polysaccharide-derived chiral stationary phases were found to be useful for the preparation of the four stereoisomers of the cyclopropane analogue of phenylalanine (c(3)Phe) as well as for the direct determination of the enantiomeric purity of c(3)Phe derivatives by HPLC. Three chiral stationary phases, consisting of cellulose and amylose derivatives chemically bonded on allylsilica gel, were tested. The mixed 10-undecenoate/3, 5-dimethylphenylcarbamate of cellulose, 10-undecenoate/3, 5-dimethylphenylcarbamate of amylose and 10-undecenoate/p-methylbenzoate of cellulose were the starting polysaccharide derivatives for CSP-1, CSP-2, and CSP-3, respectively. Using mixtures of n-hexane/chloroform/2-propanol as mobile phase on a semi-preparative column (150 mm x 20 mm ID) containing CSP-2, we separated about 1.7 g of racemic cis-methyl 1-tert-butoxycarbonylamino-2-phenylcyclopropanecarboxylate (cis-6) and 1.2 g of racemic trans-methyl-1-tert-butoxycarbonylamino-2-phenylcycloprop-anecarboxyl ate (trans-6) by successive injections. Copyright 1999 Wiley-Liss, Inc.