Structural Features and Energetics of the Periplasmic Entrance Opening of the Outer Membrane Channel TolC Revealed by Molecular Dynamics Simulation and Markov State Model Analysis.

TolC is a channel protein responsible for substrate translocation across the outer membrane, and it is also a part of the tripartite multidrug efflux pumps in Gram-negative bacteria. The crystal structure of TolC shows that the periplasmic entrance is tightly closed in the resting state, while substrate translocation definitely requires the entrance to open. How the occluded periplasmic entrance opens to allow passage of substrates remains elusive. In this work, we constructed a Markov state model from swarms of all-atom molecular dynamics (MD) simulation trajectories, which delineates the energetics of the conformational changes of TolC. Opening of the periplasmic entrance results in a monotonic increase in free energy and is accompanied by disruption of interprotomer interactions, whereas the intraprotomer interactions remain intact. Multi-ion potential of mean force (PMF) profiles for Na and Cl permeation along the channel have been calculated, and the cation/anion permeability ratio derived from which are in good agreement with electrophysiological experiments. These results not only deepen our understanding of conformational dynamics of isolated TolC but also provide valuable vision of its functioning state in tripartite efflux pumps.