Abstract
The equilibrium between active E and inactive E* forms of thrombin is assumed to be governed by the allosteric binding of a Na+ ion. Here we use molecular dynamics simulations and Markov state models to sample transitions between active and inactive states. With these calculations we are able to compare thermodynamic and kinetic properties depending on the presence of Na+. For the first time, we directly observe sodium-induced conformational changes in long-timescale computer simulations. Thereby, we are able to explain the resulting change in activity. We observe a stabilization of the active form in presence of Na+ and a shift towards the inactive form in Na+-free simulations. We identify key structural features to quantify and monitor this conformational shift. These include the accessibility of the S1 pocket and the reorientation of W215, of R221a and of the Na+ loop. The structural characteristics exhibit dynamics at various timescales: Conformational changes in the Na+ binding loop constitute the slowest observed movement. Depending on its orientation, it induces conformational shifts in the nearby substrate binding site. Only after this shift, residue W215 is able to move freely, allowing thrombin to adopt a binding-competent conformation.
Highlights
The equilibrium between active E and inactive E* forms of thrombin is assumed to be governed by the allosteric binding of a Na+ ion
To understand the mechanism of the transition itself and to gain information about the timescales of the conformational changes involved in the transition, we performed and analysed molecular dynamics (MD) simulations
Classical MD simulations of 1 μs length started from the E and the E* form extend the conformational space covered by X-ray structures
Summary
The equilibrium between active E and inactive E* forms of thrombin is assumed to be governed by the allosteric binding of a Na+ ion. We use molecular dynamics simulations and Markov state models to sample transitions between active and inactive states With these calculations we are able to compare thermodynamic and kinetic properties depending on the presence of Na+. We observe a stabilization of the active form in presence of Na+ and a shift towards the inactive form in Na+-free simulations. It induces conformational shifts in the nearby substrate binding site. After this shift, residue W215 is able to move freely, allowing thrombin to adopt a binding-competent conformation. The presence or absence of Na+ in the X-ray structure models is often unreliable as Na+ ions and water molecules have an equal number of electrons and are difficult to distinguish[11]
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