Abstract
The bacterial mechanosensitive channel, MscL, opens in response to elevated membrane tension during osmotic shock. Some mutations, like L17A and V21A, can reduce the activation tension threshold, thus offering an approach to study the mechanism of MscL gating. We employed all-atom molecular dynamics to simulate the L17A, V21A double mutant of MscL under a tension of 30 mN/m. Under these conditions, the closed state initially adopts a funnel-like conformation. Subsequently, five chains of MscL undergo sequential transitions into asymmetric states (S1, S2, etc.). Within its "open" fragment, the S1 state is similar to the expanded state of Methanosarcina acetivorans MscL and has a conductance 10 times lower than the open state. We applied committor analysis and a nonlinear regression model to construct a reaction coordinate for the transition between the closed and the S1 state as a linear combination of interatomic distances and contacts. The main contributions to the reaction coordinate are (1) the disruption of the "cytoplasmic" contact sites between the considered chain and two adjacent chains, (2) the delipidation of the lipid-binding pocket, formed by the I82, V86, and V22 residues, and (3) pulling the two neighboring chains apart via the tension sensors. The free energy profile along the reaction coordinate was calculated using the umbrella sampling approach. The S1 state is approximately 5 kJ/mol more favorable than the closed state under tension. The height of the free energy barrier for the transition toward the S1 state is approximately 10 kJ/mol, which is in reasonable agreement with the corresponding average transition time, estimated to be 133 ± 13 ns. The results and approach can be employed to elucidate the wild-type protein gating mechanism.