Compromise and accommodation in ecotin, a dimeric macromolecular inhibitorof serine proteases

Ecotin is a dimeric serine protease inhibitor from Escherichia coli which b inds proteases to form a hetero-tetramer with three distinct interfaces: an ecotin-ecotin dimer interface, a larger primary ecotin-protease interface, and a smaller secondary ecotin-protease interface. The contributions of th ese interfaces to binding and inhibition are unequal. To investigate the co ntribution and adaptability of each interface, we have solved the structure of two mutant ecotin-trypsin complexes and compared them to the structure of the previously determined wild-type ecotin-trypsin complex. Wild-type ec otin has an affinity of 1 nM for trypsin, while the optimized mutant, ecoti n Y69F, D70P, which was found using phage display technologies, inhibits ra t trypsin with a K-i value of 0.08 nM. Ecotin 67-70A, M84R which has four a lanine substitutions in the ecotin-trypsin secondary binding site, along wi th the M84R mutation at the primary site, has a K-i value against rat tryps in of 0.2 nM. The structure of the ecotin Y69F, D70P-trypsin complex shows minor structural changes in the ecotin-trypsin tetramer. The structure of t he ecotin 67-70A, M84R mutant bound to trypsin shows large deviations in th e tertiary and quaternary structure of the complex. The trypsin structure s hows no significant changes, but the conformation of several loop regions o f ecotin are altered, resulting in the secondary site releasing its hold on trypsin. The structure of several regions previously considered to be rigi d is also significantly modified. The inherent flexibility of ecotin allows it to accommodate these mutations and still maintain tight binding through the compromises of the protein-protein interfaces in the ecotin-trypsin te tramer. A comparison with two recently described ecotin-like genes from oth er bacteria suggests that these structural and functional features are cons erved in otherwise distant bacterial Lineages. (C) 2000 Academic Press.