Structure and allostery in major facilitator superfamily symport mechanisms

The solute carriers of the major facilitator superfamily (MFS) play significant roles in human health and disease. The melibiose transporter of Salmonella enterica serovar Typhimurium (MelB_St) catalyzes the stoichiometric symport of galactosides with Na⁺, H⁺ or Li⁺ and is a well studied prototype of MFS transporters. With a large body of data from extensive functional analyses using multiple transport and binding assays, structural characterizations of multiple states by X-ray crystallography and cryoEM single-particle analysis, and dynamic elucidations using hydrogen-deuterium exchange mass spectrometry (HDX-MS), the molecular recognition of the primary substrate galactoside and its coupling cation Na⁺, H⁺ or Li⁺ has been elucidated at the molecular level. Cooperative binding of both solutes is recognized as the core symport mechanism that supports the transporter's functions more effectively in a sugar-scarce environment. Structural and dynamic studies show that conformational dynamics significantly influence sugar binding but have minimal effect on Na⁺. Na⁺ acts as an allosteric activator, increasing sugar affinity by stabilizing the inner barrier and constraining conformational flexibility. All data are consistent with the previously constructed stepped-binding kinetic model for melibiose symport with Na⁺; in addition, MFS uniport and antiport mechanisms are discussed.