The Bowman-Birk inhibitor reactive site loop sequence represents an independent structural β-hairpin motif

Abstract

We have determined the NMR structure in aqueous solution of a disulphide-cyclised 11-residue peptide that forms a stable β-hairpin, incorporating a type VIb β-turn. The structure is found to be extremely well ordered for a short peptide, with the 30 lowest energy simulated annealing structures having an average pairwise r.m.s. deviation of only 0.36 Å over the backbone. All but three side-chains adopt distinct conformations, allowing a detailed analysis of their involvement in cross-strand interactions. The peptide sequence analysed originates from a previously reported study, which identified potent inhibitors of human leukocyte elastase from screening a combinatorial peptide library based on the short protein β-sheet segment that forms the reactive site loop of Bowman-Birk inhibitors. A detailed comparison of the peptide’s solution structure with the corresponding region in the whole protein structure reveals a very good correspondence not only for the backbone (r.m.s. deviation ≈0.7 Å) but also for the side-chains. This isolated β-hairpin retains the biologically active “canonical conformation” typical of small serine proteinase inhibitor proteins, which explains why it retains inhibitory activity. Since the structural integrity is sequence-inherent and does not depend upon the presence of the remaining protein, this β-hairpin represents an independent structural motif and so provides a useful model of this type of protein architecture and its relation to biological function. The relationship between the conformation of this β-hairpin and its biological activity is discussed.