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In a previous study, severe acute respiratory syndrome coronavirus (SARS-CoV) was cultured in the presence of bananin, an effective adamantane-related molecule with antiviral activity. In the present study, we show that all bananin-resistant variants exhibit mutations in helicase and membrane protein, although no evidence of bananin interference on their mutual interaction has been found. A structural analysis on protein sequence mutations found in SARS-CoV bananin-resistant variants was performed. The S259/L mutation of SARS-CoV helicase is always found in all the identified bananin-resistant variants, suggesting a primary role of this mutation site for bananin activity. From a structural analysis of SARS-CoV predicted helicase structure, S259 is found in a hydrophilic surface pocket, far from the enzyme active sites and outside the helicase dimer interface. The S/L substitution causes a pocket volume reduction that weakens the interaction between bananin and SARS-CoV mutated helicase, suggesting a possible mechanism for bananin antiviral activity.

A linear decapeptide, HGASYQDLGH, was synthesized and used as a model to evaluate the effect of nickel addition upon non-covalent backbone cyclization. The NMR data, obtained for the peptide in the presence of the metal ion, support the existence of predominant folded structures in solution, where the two His residues are maintained close to each other. These results suggest that insertion of even a single His residue at each peptide terminus can be used efficiently to reduce peptide flexibility without any backbone modification. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd.

We have developed a novel competitive method to select from a phage display library a single chain Fv which is able to mimic the alpha-bungarotoxin binding site of the muscle nicotinic receptor. The single chain Fv was selected from a large synthetic library using alpha-bungarotoxin-coated magnetic beads. Toxin-bound phages were then eluted by competition with affinity purified nicotinic receptor. Recognition of the toxin by the anti-alpha-bungarotoxin single chain Fv was very similar to that of the receptor, such as indicated by the epitope mapping of alpha-bungarotoxin through overlapping synthetic peptides. Moreover, several positively charged residues located in the toxin second loop and in the C-terminal region were found to be critical, to a similar extent, for toxin recognition by the single chain Fv and the receptor. However, although the anti-alpha-bungarotoxin single chain Fv seems to mimic the toxin binding site of the nicotinic receptor, it does not bind other nicotinic agonists or antagonists. Our results suggest that competitive selection of anti-ligand antibody phages can allow the production of receptor-mimicking molecules directly and exclusively targeted at one specific ligand. Since physiologically and pharmacologically different ligands can produce opposite effects on receptor functions, such selective ligand decoys can have important therapeutic applications.

A synthetic tridecapeptide, corresponding to the 30242 fragment of the S1 subunit of pertussis toxin,has been structurally characterised by using NMR spectroscopy. The molecule corresponds to a T-cellepitope of the bacterial toxin which has been extensively analysed with the alanine scanning approach tocheck the relevance of each residue for the biological activity of the peptide. Five of these Ala-substitutedanalogs have also been spectroscopically studied. In the experimental conditions used, different extentsof helicity were found for the six peptides in a way which cannot be related to their capabilities of ofbinding to major histocompatibility complex (MHC) class II and inducing T-cell proliferation. Backboneflexibility around helical transient conformations seems to constitute the structural intermediate step be-tween the structure of the corresponding sequence within the parental protein and in the MHC class IIcomplex. A model of the latter complex, which accounts for the different biological activities of theanalogs, is proposed.Keywords: structure; NMR; T-cell epitope; pertussis toxin; major-histocompatibility-complex class II.The design of new generations of antibacterial drugs and how sequence changes could induce modulations on molecularsynthetic vaccines depends on the understanding, at the atomic structure and antigenic and immunogenic functions.level, of the fine mechanisms of the various recognition pro- We discuss here the results of the NMR structural investiga-cesses occurring in the immune system. In several cases detoxi- tion on the natural sequence of that fragment, DNVLDHLTGR-fying bacterial toxins has proved to be a safe strategy to produce SCQ, and on analogs bearing an Ala substitution in positions 5,vaccines through chemical and/or molecular genetics approaches 7, 9, 10 and 12. These five mutants were selected as representa-[1, 2]. Convenient alternatives to develop new vaccines and tive of distinct immunological activity classes, both for MHCdrugs could be found by observing the mimicry or the favour- class II binding and for T-cell proliferation [4]. The present com-able interference of the T-cell-mediated immune response. parative analysis of the available functional data and conforma-T-cell epitopes are short linear peptides which can be easily tional results could yield a rational basis for the design of newsynthesised and handled and are thus good candidates for im- vaccines and drugs.mune response assays either as activators or repressors. Thestructure/function characteristics of some of those which interactwith the class II major histocompatibility complex (MHC) are MATERIALS AND METHODSfocused in the present report.The fragment 30242 from the S1 subunit of pertussis toxin The six peptides, DNVLDHLTGRSCQ plus five Ala substi-binds to the MHC class II DR1 molecule and stimulates T-cell tuted analogs in positions 5, 7, 9, 10 and 12, henceforth calledproliferation [3]. The same binding and stimulation capabilities respectively per0, per5, per7, per9, per10 and per12, were syn-have been investigated by the alanine scanning procedure, i.e. thesised by a solid-phase method, as previously reported [4].for each analog of the parent sequence obtained by systematic All the