Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure

Three trypsin inhibitors (TIs), from the seeds of the squash Momordica coch inchinensis (MCo), have been isolated and purified using gel filtration, io n exchange chromatography, and reverse-phase HPLC. Their sequences could be determined only after proteolytic cleavages. In the case of MCoTI-I and -I I, it was shown that their polypeptide backbones are cyclic, a structure th at has never been described in squash TIs. They contain 34 amino acid resid ues with 3 disulfide bridges and measured molecular masses of 3453.0 and 34 80.7, respectively. They are the largest known macrocyclic peptides contain ing disulfide bridges. Their sequences show strong homology to other squash TIs, suggesting a similar three-dimensional structure and an analogous mec hanism of action. A model of MCoTI-II was constructed by analogy to the cry stal structure of the complex between bovine trypsin and CMTI-I, indicating that the linker connecting the two termini is flexible and does not impose significant geometrical constraints. This flexibility allows an Asp-Gly pe ptide bond rearrangement to occur in this region, giving rise to two isofor ms of MCoTI-II. Although the importance of cyclization is not clear, it mig ht confer increased stability and resistance to proteolysis. A minor specie s, MCoTI-III, was also characterized as containing 30 amino acid residues w ith a molecular mass of 3379.6. This component possesses a linear backbone with a blocked N-terminus. MCoTIs represent interesting candidates for drug design, either by changing their specificity of inhibition or by using the ir structure as natural scaffolds bearing new binding activities.