High-resolution structures of three new trypsin-squash-inhibitor complexes: a detailed comparison with other trypsins and their complexes

An anionic trypsin from Atlantic salmon and bovine trypsin have been comple xed with the squash-feed inhibitors, CMTI-I (Cucurbita maxima trypsin inhib itor I, P1 Arg) and CPTI-II (Cucurbita pepo trypsin inhibitor II, P1 Lys). The crystal structures of three such complexes have been determined to 1.5- 1.8 Angstrom resolution and refined to crystallographic R factors ranging f rom 17.6 to 19.3%. The two anionic salmon-trypsin complexes (ST-CPTI and ST -CMTI) and the bovine-trypsin complex (BT-CPTI) have been compared to other trypsin-inhibitor complexes by means of general structure and primary and secondary binding features. In all three new structures, the primary bindin g residue of the inhibitor binds to trypsin in the classical manner, but wi th small differences in the primary and secondary binding patterns. Lysine in CPTI-II binds deeper in the specificity Docket of bovine trypsin than ly sine in other known lysine-bovine-trypsin complexes, and anionic salmon try psin lacks some of the secondary binding interactions found in the complexe s formed between squash inhibitors and bovine trypsin. The ST-CMTI complex was formed from the reactive-site-cleaved form of the inhibitor. However, w ell defined electron density was observed for the P1-P1' peptide bond, toge ther with a hydrogen-bonding pattern virtually identical to those of all se rine-protease-protein-inhibitor complexes. indicating a resynthesis of the scissile bond.