Crystallographic and Kinetic Evidence of Allostery in a Trypsin-like Protease

摘要

Protein allostery is based on the existence of multiple conformations innequilibrium linked to distinct functional properties. Although evidence of allosteric transitionsnis relatively easy to identify by functional studies, structural detection of a pre-existingnequilibrium between alternative conformations remains challenging even for textbooknexamples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombinnexists in equilibrium between two conformations where the active site is either collapsed (E*)nor accessible to substrate (E). However, structural demonstration that the two conformationsnexist in the same enzyme construct free of ligands has remained elusive. Here we reportnthe crystal structure of the thrombin mutant N143P in the E form, which complements thenrecently reported structure in the E* form, and both the E and E* forms of the thrombinnmutant Y225P. The side chain of W215 moves 10.9 Å between the two forms, causing andisplacement of 6.6 Å of the entire 215u0001217 segment into the active site that in turn opens orncloses access to the primary specificity pocket. Rapid kinetic measurements of p-aminobenzamidinenbinding to the active site confirm the existence of the E*u0001E equilibrium in solution for wild-type and the mutantsnN143P and Y225P. These findings provide unequivocal proof of the allosteric nature of thrombin and lend strong support to thenrecent proposal that the E*u0001E equilibrium is a key property of the trypsin fold.