Characterizing Conformational Ensemble and Free Energy Landscape of ABC Exporters using a Novel System-Specific Sampling Approach

Abstract

During their transport cycle, ATP-binding cassette (ABC) exporters undergo large-scale conformational changes between inward-facing (IF) and outward-facing (OF) states. Using a novel approach based on designing system-specific reaction coordinates and employing nonequilibrium simulations, we have recently characterized the IF-OF conformational transition pathway of MsbA, a bacterial ABC exporter whose structure has been solved in multiple functional states including an OF conformation and two IF conformations (termed IF-closed and IF-open based on the extent to which the cytoplasmic side is open). Due to low resolution of MsbA crystal structures in IF conformation, only Cα atoms have been reported; however our unique system-specific sampling strategy results in a stable all-atom model of MsbA in the IF conformation in the presence of explicit membrane and water. In order to more accurately characterize the conformational ensemble of nucleotide-free apo MsbA, we also employ a free energy calculation technique based on a combination of umbrella sampling and replica exchange methods, using a reaction coordinate defined based on the orientation of transmembrane helices. The results indicate that the IF-closed conformation is associated with a relatively deep minimum and the IF-open-like conformations are also thermally accessible. However, the deepest free energy basin is associated with an IF conformation, more open than IF-closed and less open than IF-open, resembling the crystal structures of P-glycoprotein (a MsbA homolog) which are obtained at higher resolutions. The overall picture emerging from the reconstructed free energies is that MsbA is fairly flexible in its resting state in the absence of nucleotides and substrates. The approach proposed here provides a framework to study large-scale conformational changes of membrane transporters whose computational investigation at an atomic resolution may not be currently feasible using conventional methods.