Allosteric effects of the coupling cation in melibiose transporter MelB.

Abstract

The major facilitator superfamily (MFS) transporters play significant roles in human health and disease. <i>Salmonella enterica</i> 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.