Sodium-induced population shift drives activation of thrombin

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.

Figure 1

Structural comparison between active and inactive forms of thrombin. The active E form (depicted 3LU9, green) and the inactive E* form (depicted 3BEI, red) mainly differ in the bottom of the non-prime site and the Na⁺ binding loop. The detailed view shows the loop chosen for further analyses (V213–T229).

Figure 2

MSMs of thrombin without Na⁺ and with Na⁺ ions. (a) The free energy surface projected on the TICA space shows the shift of the populations depending on the presence of Na⁺. The position of the X-ray structures of the E form (3LU9) and the E* form (3BEI), from which the TMD simulations were started, are marked. (b) Calculated probability of E-like and E*-like states and mean first passage times (mfpt) for the transitions between the states. Brighter colors denote the confidence interval of the probabilities based on the uncertainties of the Bayesian MSMs, for errors of the mfpts please refer to the Supplementary Tables 1 and 2. (c) Comparison between three representative structures of the metastable states, E and E* (white), and an X-ray structure of the respective form (green and red).

Figure 3

Distributions of internal distances in the E and the E* state, based on the MSMs without and with Na⁺. The frames are weighted according to the probabilities calculated from the MSMs, so that the combined area under both curves totals to 1. The distribution of (a) φ torsion of D221 (PhiD), (b) distance between W215 and the catalytic triad (WCT), (c) the distance between R221a and the catalytic triad (RCT) and (d) the distance between G193 and G216 (GG) in the E state (green) and the E* state (red) are displayed. The left column shows the results for the simulations done without Na⁺, the right column the results for simulation with added Na⁺. The panels above the distributions show the values for the features in X-ray structures.

Figure 4

ψ entropies along the backbone of thrombin. The ψ entropies are a measurement for the localized flexibility. They are calculated for the structural ensemble of the E and E* metastable states from the MSMs without and with Na⁺.