Allosteric Networks in Thrombin

Abstract

Serine proteases are found ubiquitously in both eukaryotes and prokaryotes however the dynamic motions of this largest peptidase family remains unknown. All serine proteases have a double β-barrel core surrounded by connecting loops and helices, but compared to the prototypical serine protease, chymotrypsin, thrombin has more extended loops that are thought to impart greater specificity. We analyzed apo-thrombin and active site-bound (PPACK-thrombin) using a combination of MD, accelerated MD and NMR. Community analysis of the MD ensembles identified groups of residues linked through correlated motions and physical contacts. AMD simulations, calibrated on measured residual dipolar couplings, revealed correlated loop motions connecting the active site with distal allosteric regulator binding sites. The backbone dynamics profile (from ps to ms motions) of thrombin was determined in both states using R1, R2, 15N-{1H}NOEs, TROSY Hahn-Echo, and relaxation dispersion experiments. The apo thrombin backbone is highly dynamic and shows motions across multiple time scales. The various surface loops move on different time scales. The substrate-binding loops showed intermediate time scale motions that are quenched upon PPACK binding. TROSY Hahn-echo and relaxation dispersion experiments reveal a large number of residues throughout the protein undergoing temporally correlated µs-ms motions. These include the Na+-binding loop and the β-strand connecting exosite 1 and the active site, both of which are implicated in allosteric coupling of effector binding sites with the active site. The results show a network of slowly exchanging residues extends through the entire apo-thrombin molecule.