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 either H(+), Li(+), or Na(+), but prefers the coupling with Na(+). Previously, we determined the structures of the inward- and outward-facing conformation of MelB(St), as well as the molecular recognition for galactoside and Na(+). However, the molecular mechanisms for H(+)- and Na(+)-coupled symport still remain poorly understood. We have solved two x-ray crystal structures of MelB(St) 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 previously. We determined the energetic contributions of three major Na(+)-binding residues in cation selectivity for Na(+) and H(+) by the free energy simulations. 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 with poor activities at higher ΔpH and better activities at reversal ΔpH was observed, supporting that the membrane potential is 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.