Outward- to Inward-Facing Transition of MsbA Transporter: A Mechanistic Picture at Atomic Resolution

Abstract

MsbA is a structurally well-characterized, bacterial ATP-binding cassette (ABC) exporter. It undergoes a large-scale conformational change between the inward-facing (IF) and the outward-facing (OF) states during its transport cycle. Despite extensive experimental and computational studies on MsbA and its homologs, a unified mechanistic picture describing the OF-IF conformational transition of ABC exporters is still missing. In order to study this transition at an atomic level, we have used a novel approach based on a nonequilibrium driven scheme and performed an extensive set of molecular dynamics simulations that sample the OF-to-IF reaction-path ensemble of the apo MsbA in the presence of explicit solvent and membrane. using several distinct system-specific biasing protocols, we were able to steer the system along a large number of different paths in a low-dimensional holonomic coordinate space. Nonequilibrium work relations were employed to interpret the results and to determine the optimal transition path. Our results provide clear evidence that the opening of the cytoplasmic gate in the apo MsbA is prohibitively disfavored when the extracellular gate is open, an observation consistent with the “alternating-access mechanism”. More interestingly, we observe that the closure of the extracellular gate does not result in a stable IF conformation unless the nucleotide-binding domains (NBDs) undergo a “twisting” motion that involves a drastic change in their relative orientation. We thus propose a “door-knob mechanism” for the OF-to-IF transition of MsbA that asserts a crucial role for the “twisting” of NBDs in the opening of the cytoplasmic gate. More generally, our results call into question the simplistic models of NBD dissociation/dimerization that ignore the important role of the relative orientation between the NBDs in promoting the OF-IF conformational transitions of ABC exporters.