Abstract
The Major Facilitator Superfamily (MFS) transporters are an essential class of secondary active transporters involved in various physiological and pathological processes. The melibiose permease (MelB), which catalyzes the stoichiometric symport of the disaccharide melibiose and monovalent cations (e.g., Na+, H+, or Li+), is a key model for understanding the cation-coupled symport mechanisms. Extensive experimental data has established that positive cooperativity between the cargo melibiose and the coupling cation is central to the symport mechanism. However, the structural and energetic origins of this cooperativity remain unclear at the atomistic level for MelB and most other coupled transporters. Here, leveraging recently resolved structures in inward- and outward-facing conformations, we employed the string method and replica-exchange umbrella sampling simulation techniques to comprehensively map the all-atom free energy landscapes of the Na+-coupled melibiose translocation across the MelB in Salmonella enterica serovar Typhimurium (MelBSt), in comparison with the facilitated melibiose transport in a uniporter mutant. The simulation results unravel asymmetrical free energy profiles of melibiose translocation, which is tightly coupled to protein conformational changes in both the N- and C-terminal domains. Notably, the cytoplasmic release of the melibiose induces the simultaneous opening of an inner gate, resulting in a high-energy state of the system. Periplasmic sugar binding and cytoplasmic melibiose released are dynamically coupled with changes in the internal gating elements along the translocation pathway. The outward-facing sugar-bound state is thermodynamically most stable, while the occluded state is a transient state. The binding of Na+ facilitates melibiose translocation by increasing the melibiose-binding affinity and decreasing the overall free energy barrier and change. The cooperative binding of the two substrates results from the allosteric coupling between their binding sites instead of direct electrostatic interaction. These findings add substantial new atomic-level details into how Na+ binding facilitates melibiose translocation and deepen the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵3 Leading Contact
Minor modifications to the abstract and discussions to highlight the major findings and significance of the results.