Distinct roles of the major binding residues in the cation-binding pocket of MelB
Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt 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 MelBSt, 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 MelBSt 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 MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport with poor activities at higher {Delta}pH and better activities at reversal {Delta}pH was observed, supporting that the membrane potential is the primary driving force for the H+-coupled symport mediated by MelBSt. 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.