What question did this study set out to answer?

This research aims to unveil the molecular mechanisms behind cooperativity in MelB St during substrate transport.

February 21, 2026

BPS2026 – Molecular basis of the cooperativity between the primary substrate-binding affinity and conformational dynamics in the melibiose permease MelBSt

Key Points

This research aims to unveil the molecular mechanisms behind cooperativity in MelB St during substrate transport.
Utilized hydrogen-deuterium exchange mass spectrometry (HDX-MS) for protein dynamics analysis
Investigated conformational changes of MelB St with melibiose and Na +
Analyzed structural and functional data to understand symport mechanisms
Positive cooperativity between melibiose and Na + was identified as integral to MelB St function
MelB St dynamics show concerted movements correlating inward- and outward-facing states
Enhanced sugar-binding affinity occurs through allosteric modulation linked to cation-binding activities

Abstract

The major facilitator superfamily (MFS) transporters play important roles in human health and disease. Salmonella enterica serovar Typhimurium melibiose permease (MelB St ), which catalyzes the stoichiometric symport of galactosides with Na + , H + , or Li + , serves as a prototype of this important transporter superfamily. Structures of MelB St in both inward- and outward-facing conformations without substrate (apo) or with galactosides or Na + have been determined. Positive cooperativity between the two co-transported solutes has been proposed to be integral to the symport mechanism of this symporter; however, the molecular mechanism is still not fully understood. In this study, MelB St protein dynamics and the effects of melibiose and/or Na + were investigated using the label-free hydrogen-deuterium exchange mass spectrometry (HDX-MS). Our integrated analyses of the HDX-MS data sets with structural and functional results showed that the MelB St dynamics involve a similar concerted movement of conformational changes between outward- and inward-facing states. The overlapping peptides covering the Na + -binding pocket and nearby sugar-binding residues were highly protected, while the peptides covering the sugar-binding residues farther from the cation-binding site exhibited significant deuteration. Notably, the dynamics of those sugar-binding residues and the cytoplasmic salt-bridge network that is responsible for opening and closing the cytoplasmic cavity belong to the same interconnected dynamic group, and all cooperatively respond to melibiose or Na + binding, especially when both are bound. The results indicate that MelB St structural motions, conformational transition, and sugar-binding affinity are coordinated and function together. Na + binding to the rigid cation-binding pocket restricts the dynamics of remote sugar-binding residues via suppressing the dynamics of the cytoplasmic salt-bridge network, thereby increasing sugar-binding affinity allosterically. Our research has yielded valuable insights into the mechanisms of allostery-based symport.

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