Tr-Noe and MD studies of Leishmania gp63 SRYD-containing sequences bound to anti-SRYD monoclonal antibody

Summary The I²⁵⁰ ASRYDQL²⁵⁷ synthetic octapeptide of theLeishmania major surface glycoprotein gp63, which efficiently inhibits parasite attachment to the macrophage receptors and mimics antigenically and functionally the RGDS sequence of fibronectin, was studied by 2D TR-NOESY in the presence of an anti-SRYD monoclonal antibody (mAbSRYD) that recognizes both SRYD-containing peptides and the cognate protein on intact parasites. Molecular modeling was performed using distance constraints obtained from TR-NOEs. The bound structure was compared with that of the free peptide in DMSO solution and with the crystal structure of the RYD fragment of the OPG2 Fab, an antireceptor antibody that mimics an RGD cell adhesion site.     

Conformational and antigenic properties of SRYD-containing peptide analogues of Leishmania gp63 adhesion site

Summary The antigenicity and conformational properties of the Ser-Arg-Tyr-Asp (SRYD) segment (252–255) of the major surface glycoprotein ofLeishmania, gp63, which plays a key role in the parasite-macrophage attachement, are presented. It was found that the antibody recognition, using anti-IASRYDQL antibodies, of the SRYD-containing analogues, Ac-SRYD-NH₂ (1), ANIASRYD-NH₂ (2), Ac-SRYD (3), SRYD (4) and ANIASRYD (5), is rather similar. The structure of the SRYD moiety in analogues 1 and 2 is characterized by the presence of a type I -turn, stabilized by the formation of a hydrogen bonding between the C-terminaltrans-carboxamide proton and the Arg-CO and an ionic bridge between arginine and aspartic acid side chains, while the conformation of compounds 3, 4 and 5 is stabilized by an ionic bridge between the arginine side chain and the C-terminal carboxylate group. A common structural motif involving the arginine side chain in an ionic interaction is identified in all the SRYD analogues, which may explain the observed similarities in the antibody recognition of the reported peptides.     

NMR study of thymulin, a lymphocyte differentiating thymic nonapeptide. Conformational states of free peptide in solution

The nonapeptide less than Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (formerly called serum thymic factor) is a factor produced by the thymic epithelium, which needs a zinc ion to express its immunoregulatory properties. We report here on 1H and 13C NMR investigation of the conformational properties of the free peptide in aqueous medium and in dimethyl sulfoxide-d6 solution by a combination of homo- and heteronuclear one- and two-dimensional experiments. The various resonances have been assigned in a straightforward manner on the basis of 1H,1H COSY spectroscopy for the recognition of the proton spin systems; two-dimensional NOESY spectra with the correlation peaks across amide bonds and for the amino acid sequence assignment; amide bonds and for the amino acid sequence assignment; 13C,1H COSY experiments using selective polarization transfer from 1H- to 13C-nucleus via the 13C,1H long-range couplings for the attribution of the carboxyl and carbonyl groups; and 13C,1H COSY experiments with selective polarization transfer via the 13C,1H direct couplings for the assignment of all the aliphatic carbons. Other experiments such as pH-dependent chemical shifts, combined use of multiple and selective proton-decoupled 1H and 13C NMR spectra, the temperature and the concentration dependence of the proton shifts of the amide resonances, the solvent dependences of peptide carbonyl carbon resonances, and comparison of the spectra with three different analogues were performed. In aqueous solution, the data are compatible with the assumption of a highly mobile dynamic equilibrium among different conformations, whereas in dimethyl sulfoxide-d6, a more rigid structure is found involving three internal hydrogen bonds. These observations provide an insight into the conformational tendencies of this peptidic hormone in two different media.     

A solid-phase synthesis of three aza-, iminoaza- and reduced aza-peptides from the same precursor

Using the Boc-strategy, a step-by-step synthesis on the PAM solid supportof three aza-, iminoaza- and reduced aza-peptide homologues is described.From the same hydrazinocarbonyl peptide-PAM precursor, the coupling ofeither a Boc-amino acid or a Boc-amino aldehyde gives rise to an aza-peptideor an iminoaza-peptide containing theC-CO-NH-N-CO-NH-C orC-CH=N-N-CO-NH-C surrogate of the peptide motif, respectively. In situreduction of the latter by NaBH₃CN leads to a reducedaza-peptide containing theC-CH₂-NH-N-CO-NH-C moiety. The key step synthesis of thehydrazinocarbonyl peptide-PAM precursor is carried out by coupling on thegrowing peptide chain the N-Boc-aza-amino acid chloride obtained by theaction of triphosgene on the corresponding N-Boc-hydrazine. Thesemodifications have been introduced in position 1-2 of the YLGYLEQLLRbenzodiazepine-like decapeptide     

Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.     

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin)

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.     

Solution structure and backbone dynamics of the reduced form and an oxidized form of E. coli methionine sulfoxide reductase A (MsrA): structural insight of the MsrA catalytic cycle

Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (MetSO)-containing proteins, back to methionine (Met). MsrAs are stereospecific for the S epimer whereas MsrBs reduce the R epimer of MetSO. Although structurally unrelated, the Msrs characterized so far display a similar catalytic mechanism with formation of a sulfenic intermediate on the catalytic cysteine and a concomitant release of Met, followed by formation of at least one intramolecular disulfide bond (between the catalytic and a recycling cysteine), which is then reduced by thioredoxin. In the case of the MsrA from Escherichia coli, two disulfide bonds are formed, i.e. first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and the second recycling Cys206. Three crystal structures including E. coli and Mycobacterium tuberculosis MsrAs, which, for the latter, possesses only the unique recycling Cys198, have been solved so far. In these structures, the distances between the cysteine residues involved in the catalytic mechanism are too large to allow formation of the intramolecular disulfide bonds. Here structural and dynamical NMR studies of the reduced wild-type and the oxidized (Cys51-Cys198) forms of C86S/C206S MsrA from E. coli have been carried out. The mapping of MetSO substrate-bound C51A MsrA has also been performed. The data support (1) a conformational switch occurring subsequently to sulfenic acid formation and/or Met release that would be a prerequisite to form the Cys51-Cys198 bond and, (2) a high mobility of the C-terminal part of the Cys51-Cys198 oxidized form that would favor formation of the second Cys198-Cys206 disulfide bond.     

Structure of antibody-bound peptides and retro-inverso analogues. A transferred nuclear Overhauser effect spectroscopy and molecular dynamics approach

The three-dimensional structures of the two L-peptides, H-CGGIRGERA-OH, called L(A), and H-CGGIRGERG-OH, called L(G), corresponding or close to the IRGERA sequence present in the C-terminal region (residues 130-135) of histone H3, and their retro-inverso analogues HO-mAreGriGGC-NH2, called RI(mA), and HO-mGreGriGGC-NH2, called RI(mG), have been studied by two-dimensional 1H NMR and molecular dynamics calculations in association with a monoclonal antibody generated against L(A). At 25 degrees C, the affinity constants of the monoclonal antibody with respect to RI(mA) and RI(mG) were 75- and 270-fold higher than those measured with the homologous L(A) and L(G) peptides, respectively. Due to the spontaneous epimerization of the mA malonic residue, RI(mA) gave rise to two sets of resonances. With regard to the NH amide region, one set was similar to that for RI(mG) while the second was similar to those for the parent L-peptides L(A) and L(G). The antibody-bound conformations of the two couples of L- and retro-inverso peptides have been analyzed using molecular modeling calculations based on the transferred NOE interproton distances. Folded structures appeared in both cases with a type II' beta-turn in the parent GGIR sequence and a type I' beta-turn in the retro-inverso reGr sequence.     

Isomerization of the Xaa-Pro peptide bond induced by ionic interactions of arginine

Inclusion of Arg or Pro residues in proteins and peptides has been proved to play an essential role in biochemical functions through ionic interactions, conformational transitions, and formation of turns as well. In this study we present the conformational properties of the Ac-Arg-Ala-Pro (1), Ac-Arg-Ala-Pro-NH₂ (2), Ac-Arg-Pro-Asp-NH₂ (3), and Ac-Arg-Pro-Asp (4) tripeptides, using ¹H-nmr spectroscopy and molecular dynamics. These peptides were modeled with the aim of studying the role of the Arg-guanidinium to carboxylate ionic interactions on the Xaa-Pro peptide bond isomerization. It was found with 1 and 4 that arginine preferentially interacts with the C-terminal carboxylate group, even though the β-carboxylate is also accessible. This tendency of the Arg moiety was found to induce the cis disposition of the Ala-Pro peptide bond in 1. It was also confirmed that the Arg…Asp side chain-side chain ionic interaction in 3 plays a key role in backbone folding and structural stabilization through a type I β-turn. The nmr pattern for 3 showed a remarkable similarity with that for various Arg-Tyr-Asp containing peptides, a sequence that is crucial for the adhesion properties of the Leishmania gp63 glycoprotein. © 1996 John Wiley & Sons, Inc.