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), which catalyzes the symport of galactosides with Na⁺, H⁺, or Li⁺, is a prototype of this important transporter superfamilies. We have published the structures of the inward- and outward-facing conformations of MelB_St with galactoside or Na⁺ bound, and determined the binding thermodynamic cycle. We have proposed that positive cooperativity between the two co-transported solutes plays a key role in the symport mechanism of MelB_St; however, the molecular basis for this core mechanism remains unclear. In this study, we investigated the structural dynamics induced by melibiose, Na⁺, or both on MelB_St using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We also refined the specific determinants for the sugar recognition in both protein and galactoside molecules by solving the crystal structures of D59C MelB_St bound to melibiose and two other sugars that contain different numbers of sugar units, and identified a critical water molecule as part of the specific determinants from a α-NPG-bound structure. Our integrated structural and HDX-MS analyses support the notion that the binding of the coupling cation at a remote site stabilizes those dynamic sidechains in the sugar-binding pocket, leading to a high-affinity state. This study provides the molecular basis for the essential symport mechanism through positive cooperativity, which may serve as a general mechanism for most cation-coupled symporters.