Abstract
Salmonella enterica serovar Typhimurium melibiose permease (MelB(St)) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelB(St) catalyzed the symport of galactosides with Na(+), Li(+), or H(+) but prefers the coupling with Na(+). Previously, we determined the structures of the inward- and outward-facing conformation of MelB(St) and the molecular recognition for galactoside and Na(+). However, the molecular mechanisms for H(+)- and Na(+)-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelB(St), the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na(+)-binding residues for the selection of Na(+) and H(+) by free energy simulations. Transport assays showed that the D55C mutant converted MelB(St) to a solely H(+)-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelB(St). Unexpectedly, the H(+)-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H(+)-coupled symport mediated by MelB(St). This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelB(St).