Liang Ruibin, Guan Lan
bioRxiv. 2024 Sep 14:2024.08.21.608993. doi: 10.1101/2024.08.21.608993.
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.
主要易化子超家族（MFS）转运蛋白是一类重要的次级主动转运蛋白，参与各种生理和病理过程。蜜二糖通透酶（MelB）催化二糖蜜二糖与单价阳离子（如Na⁺、H⁺或Li⁺）的化学计量同向转运，是理解阳离子偶联同向转运机制的关键模型。大量实验数据表明，底物蜜二糖与偶联阳离子之间的正协同作用是同向转运机制的核心。然而，对于MelB和大多数其他偶联转运蛋白而言，这种协同作用在原子水平上的结构和能量起源仍不清楚。在此，利用最近解析的向内和向外构象结构，我们采用弦方法和副本交换伞形采样模拟技术，全面绘制了鼠伤寒沙门氏菌（MelBSt）中Na⁺偶联的蜜二糖跨MelB转运的全原子自由能景观，并与单向转运突变体中蜜二糖的易化转运进行了比较。模拟结果揭示了蜜二糖转运的不对称自由能分布，其与N端和C端结构域中的蛋白质构象变化紧密耦合。值得注意的是，蜜二糖的胞质释放诱导了内部闸门的同时打开，导致系统处于高能状态。周质糖结合和胞质蜜二糖释放与沿转运途径的内部闸门元件变化动态耦合。向外的糖结合状态在热力学上最稳定，而封闭状态是一个过渡状态。Na⁺的结合通过增加蜜二糖结合亲和力、降低整体自由能势垒和变化来促进蜜二糖转运。两种底物的协同结合源于它们结合位点之间的变构偶联，而非直接的静电相互作用。这些发现为Na⁺结合如何促进蜜二糖转运增添了大量新的原子水平细节，并加深了对阳离子偶联转运蛋白同向转运机制分子基础的基本理解。
