The refined 2.0 8, X-ray crystal structure of the complex formed between bovine /3-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima) Received 7 November 1988The stoichiometric complex formed between bovine j?-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0 -2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or B-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-31-201, Cys-101-221 and Cys-161-281. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypep tidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-21 (P4) -Glu-91 (P4') which contains the reactive site bond Arg-51 -Be-61 and is in a conformation observed also for other serine proteinase inhibitors.

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An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.