Distinct roles of the major binding residues in the cation-binding pocket of MelB

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.