PDF(Pubmed)
Abstract：
Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.
摘要：
肠沙门氏菌血清型鼠伤寒二糖通透酶（MelBSt）是主要的促进者超家族（MFS）转运蛋白的原型，在人类健康和疾病中起着重要作用。MelBSt催化半乳糖苷与Na的共生，Li+,或H+，但更喜欢与Na+偶联。以前,我们确定了MelBSt的向内和向外构象的结构以及半乳糖苷和Na的分子识别。然而,H+和Na+偶联共生的分子机制仍然知之甚少。在这项研究中,我们解析了MelBSt的两个X射线晶体结构，阳离子结合位点突变体D59C处于未结合的apo状态，D55C处于配体结合状态，和两个结构显示向外的构象几乎相同的公布。我们通过自由能模拟确定了三个主要的Na结合残基对Na和H的选择的能量贡献。转运试验表明，D55C突变体将MelBSt转化为仅H偶联的转运体，以及自由能扰动计算，Asp59被确认为MelBSt的唯一质子化位点。出乎意料的是，H偶联的蜜二糖转运在较大的大体积ΔpH下表现出较差的活性，在反向ΔpH下表现出更好的活性，支持跨膜静电定位质子和相关膜电位的新理论，作为由MelBSt介导的H+耦合对称的主要驱动力。生物能学，和自由能模拟，证明了MelBSt阳离子结合袋中主要结合残基的不同作用。
