Allosteric effects of the coupling cation in melibiose transporter MelB

The major facilitator superfamily (MFS) transporters play significant roles in human health and disease. Salmonella enterica serovar Typhimurium melibiose permease (MelB_St) catalyzes the symport of galactosides with Na⁺, H⁺, or Li⁺ and is a prototype of MFS transporters. We published the structures of MelB_St in both inward- and outward-facing conformations, bound to galactoside or Na⁺, and proposed that positive cooperativity of the co-transported solutes is crucial for the symport mechanism. Here, we elucidated the underlying mechanisms by analyzing MelB_St dynamics and the effects of melibiose, Na⁺, or both using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We also refined the determinants of sugar recognition by solving the crystal structures of a uniporter D59C MelB_St complexed with melibiose and other sugars, and by identifying a critical water molecule involved in sugar recognition. Our integrated studies, combining structures, HDX-MS, and molecular dynamics simulations, support the conclusion that sugar-binding affinity is directly correlated with protein dynamics. Na⁺ acts as an allosteric activator, reducing the flexibility of dynamic residues in the sugar-binding site and in the cytoplasmic gating salt-bridge network, thereby increasing sugar-binding affinity. This study provides a molecular-level framework of the symport mechanism that could serve as a general model for cation-coupled symporters.