Long-timescale molecular dynamics simulations elucidate the dynamics and kinetics of exposure of the hydrophobic patch in troponin C

Abstract

Troponin (Tn) is an important regulatory protein in the thin-filament complex of cardiomyocytes. Calcium binding to the troponin C (TnC) subunit causes a change in its dynamics that leads to the transient opening of a hydrophobic patch on TnC's surface, to which a helix of another subunit, troponin I (TnI), binds. This process initiates contraction, making it an important target for studies investigating the detailed molecular processes that underlie contraction. Here we use microsecond-timescale Anton molecular dynamics simulations to investigate the dynamics and kinetics of the opening transition of the TnC hydrophobic patch. Free-energy differences for opening are calculated for wild-type Ca(2+)-bound TnC (∼8 kcal/mol), V44Q Ca(2+)-bound TnC (3.2 kcal/mol), E40A Ca(2+)-bound TnC (∼12 kcal/mol), and wild-type apo TnC (∼20 kcal/mol). These results suggest that the mutations have a profound impact on the frequency with which the hydrophobic patch presents to TnI. In addition, these simulations corroborate that cardiac wild-type TnC does not open on timescales relevant to contraction without calcium being bound.

Figure 1

Interhelical angles over the course of the simulations for (A) the Anton wild-type Ca²⁺-bound TnC simulation, (B) the Anton V44Q Ca²⁺-bound TnC simulation, (C) the Anton E40A Ca²⁺-bound TnC simulation, and (D) the Anton wild-type apo TnC simulation. At the top of the figure is a ribbon representation of TnC in its closed and open conformations. The helices and the A/B interhelical angle are labeled.

Figure 2

Surface representation of a closed and an open conformation from the Anton V44Q Ca²⁺-bound TnC simulation. (A) TnC in its closed conformation at the beginning of the trajectory. The interhelical A/B angle is 135.1°. (B) TnC in its open conformation, after ∼1.9 μs of simulation time. The interhelical A/B angle is 80.9°. Helices A and B are labeled for clarity.

Figure 3

Dependence of computed free-energy differences between open and closed states on the cutoff angle. Values for the Anton wild-type Ca²⁺-bound TnC simulation, the Anton V44Q Ca²⁺-bound TnC simulation, the Anton E40A Ca²⁺-bound TnC simulation, and the Anton wild-type apo TnC simulation are shown. Free energies are calculated based on the Boltzmann distribution of states. The widely accepted open-closed cutoff criterion of 90° is marked by a vertical black line.

Figure 4

Dependence of average simulation time between opening events on the cutoff angle. Values for the Anton wild-type Ca²⁺-bound TnC simulation, the Anton V44Q Ca²⁺-bound TnC simulation, the Anton E40A Ca²⁺-bound TnC simulation, and the Anton wild-type apo TnC simulation are shown. The widely accepted open-closed cutoff criterion of 90° is marked by a vertical black line.