Correlated Motions and Residual Frustration in Thrombin

Abstract

Thrombin is the central protease in the cascade of blood coagulation proteases. The structure of thrombin consists of a double β-barrel core surrounded by connecting loops and helices. Compared to chymotrypsin, thrombin has more extended loops that are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity. Previous experiments showed thermodynamic coupling between ligand binding at the active site and distal exosites. We present a combined approach of molecular dynamics (MD), accelerated molecular dynamics (AMD), and analysis of the residual local frustration of apo-thrombin and active-site-bound (PPACK-thrombin). Community analysis of the MD ensembles identified changes upon active site occupation in groups of residues linked through correlated motions and physical contacts. AMD simulations, calibrated on measured residual dipolar couplings, reveal that upon active site ligation, correlated loop motions are quenched, but new ones connecting the active site with distal sites where allosteric regulators bind emerge. Residual local frustration analysis reveals a striking correlation between frustrated contacts and regions undergoing slow time scale dynamics. The results elucidate a motional network that probably evolved through retention of frustrated contacts to provide facile conversion between ensembles of states.

Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold

“…scales, consistent with recent NMR measurements (14) and molecular dynamics calculations (84,85): a slow time scale (45 ms) for the interconversion of the E* and E ensembles detected by stopped-flow measurements, and a fast time scale, perhaps spectroscopically silent, for the interconversion of conformers within each ensemble. The rapid equilibrium within the E* ensemble likely involves different degrees of occlusion of the active site, all of them incompatible with substrate binding.…”

NMR reveals a dynamic allosteric pathway in thrombin

“…These regions are all interesting for different reasons. The 170 s loop, which is highly frustrated9, may be exchanging between an inactive conformation and an active conformation. Substrate-binding (mimicked by PPACK) may select the more stable, proteolytically active conformation.…”

“…We recently completed computational studies which revealed temporal fluctuations of the apo-thrombin ground-state that are largely uncorrelated8. Binding of TM to the anion-binding exosite 1 (ABE1) of thrombin increased the collective correlated motions and this effect extended from the TM binding site into the active site loops, a result that was also seen upon active site occupation89. These computational studies suggest that effector and/or substrate binding alters the conformational ensemble of thrombin to affect distal functional regions.…”

Computational approaches to mapping allosteric pathways

“…Fuglestad and colleagues [36••] used a number of methods to characterize thrombin allosteric networks. They find that different methods correlate to fluctuations occurring on differing timescales.…”