The role of ecotin dimerization in protease inhibition

Ecotin is a homodimeric protein from Escherichia coli that inhibits many se rine proteases of the chymotrypsin fold, often with little effect from the character or extent of enzyme substrate specificity. This pan-specificity o f inhibition is believed to derive from formation of a heterotetrameric com plex with target proteases involving three types of interface: the dimeriza tion interface, a primary substrate-like interaction, and a smaller seconda ry interaction between the partner ecotin subunit and the protease. A monom eric ecotin variant (mEcotin) and a single-chain ecotin dimer (scEcotin) we re constructed to study the effect of a network of protein interactions on binding affinity and the role of dimerization in broad inhibitor specificit y, mEcotin was produced by inserting a beta -turn into the C-terminal arm, which normally exchanges with the other subunit. While the dimerization con stant (K-dim) of wild-type (WT) ecotin was found to be picomolar by subunit exchange experiments using FRET and by association kinetics, mEcotin was m onomeric up to 1 mM as judged by gel filtration and analytical centrifugati on. A crystal structure of uncomplexed mEcotin to 2.0 Angstrom resolution v erifies the design, showing a monomeric protein in which the C-terminal arm folds back onto itself to form a beta -barrel structure nearly identical t o its dimeric counterpart. The kinetic rate constants and equilibrium disso ciation constants for monomeric and dimeric ecotin Variants were determined with both trypsin and chymotrypsin. The effect of the secondary binding si te on affinity was found to vary inversely with the strength of the interac tion at the primary site. This compensatory effect yields a nonadditivity o f up to 5 kcal/mol and can be explained in terms of the optimization of bin ding orientation. Such a mechanism of adaptability allows femtomolar affini ties for two proteases with very different specificities. (C) 2001 Academic Press.