Characterizing the Pathway, Free Energy, and the Effect of Substrate Binding on Structural Transition of MFS Transporter GlpT between Inward- and Outward-Facing States

Abstract

Glycerol-3-phosphate transporter (GlpT) is an MFS secondary transporter whose structure has been only solved in the apo inward-facing (IF) state. Despite extensive experimental and computational studies, the structure of the outward-facing (OF) and occluded states, an accurate description of the IF-OF transition pathway, and the associated energetics are missing. We have studied the IF-OF structural transition of GlpT using a novel approach based on a combination of several state-of-the-art sampling techniques such as nonequilibrium driven MD, umbrella sampling, and Hamiltonian replica exchange MD. Biasing the system along a set of system-specific holonomic coordinates allowed us to adequately sample the relevant regions of the phase space around the transition path in the presence of explicit solvent and membrane. The free energy profiles of the IF-OF transition were reconstructed for both the apo and the substrate-bound GlpT, using a novel implementation of the generalized weighted histogram analysis method (WHAM). Our results indicate that the known IF conformation is indeed the global minimum in the free energy profile of apo GlpT; however, there exists another low free energy minimum associated with an OF conformation with a free energy barrier of only 10 kcal/mol between the two states. More interestingly, the binding of a substrate not only lowers this barrier but also stabilizes at least two intermediate/occluded states that were not observed in the apo GlpT simulations. Our results not only verify the “alternating-access” mechanism but also provide a detailed description of the IF-OF transition mechanism of GlpT at an atomic level, establishing a quantitative basis for the study of MFS transporters and introducing a novel approach to study the large-scale transitions in membrane transporters.