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. 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 superfamily. We have published the structures of the inward- and outward-facing conformations of MelB St with galactoside or Na + bound, determined the binding thermodynamic cycle, and proposed that positive cooperativity between the two co-transported solutes plays a key role in the symport mechanism of MelB St . The molecular basis for this core mechanism remains unclear. In this study, we determined the molecular basis for this core symport mechanism through analyzing the structural dynamics of MelB St and effects induced by melibiose, Na + , or both 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 a uniporter D59C MelB St bound to melibiose and other sugars, and identified a critical water molecule as part of sugar recognition. Our integrated studies from structure, HDX-MS, and molecular dynamics simulations support the conclusion that sugar-binding affinity is directly correlated with protein dynamics. The binding of the coupling cation at a remote site functions as an allosteric activator to restrain the conformational flexibility of dynamic residues in the sugar-binding site and in the cytoplasmic gating salt-bridge network, thereby increasing sugar-binding affinity allosterically. This study provides a molecular-level schematic of the fundamental symport mechanism via positive cooperativity, which may serve as a general mechanism for cation-coupled symporters.