Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins

The "knottin" fold is a stable cysteine-rich scaffold, in which one disulfi de crosses the macrocycle made by two other disulfides and the connecting b ackbone segments. This scaffold is found in several protein families with n o evolutionary relationships. In the past few years, several homologous pep tides from the Rubiaceae and Violaceae families were shown to define a new structural family based on macrocyclic knottin fold. We recently isolated f rom Momordica Cochinchinensis seeds the first known macrocyclic squash tryp sin inhibitors. These compounds are the first members of a new family of cy clic knottins, In this paper, we present NMR structural studies of one of t hem, MCoTI-II, and of a P-Asp rearranged form, MCoTI-IIb. Both compounds di splay similar and well-defined conformations. These cyclic squash inhibitor s share a similar conformation with noncyclic squash inhibitors such as CPT I-II, and it is postulated that the main effect of the cyclization is a red uced sensitivity to exo-proteases. On the contrary, clear differences were detected with the three-dimensional structures of other known cyclic knotti ns, i.e., kalata B1 or circulin A. The two-disulfide cystine-stabilized P-s heet motif [Heitz et al, (1999) Biochemistry 38, 10615-10625] is conserved in the two families, whereas in the C-to-N linker, one disulfide bridge and one loop are differently located. The molecular surface of MCoTI-II is alm ost entirely charged in contrast to circulin A that displays a well-marked amphiphilic character. These differences might explain why the isolated mac rocyclic squash inhibitors from M. cochinchinensis display no significant a ntibacterial activity, whereas circulins and kalata B1 do.