Institute for Biocomplexity and Informatics and Department of Biological Sciences, University of Calgary, 2500 University Drive, BI558, Calgary, Alberta, Canada T2N 1N4.
Biochemistry. 2011 Mar 22;50(11):1848-56. doi: 10.1021/bi101454f. Epub 2011 Feb 15.
Recent progress in crystallographic studies of sodium-coupled secondary transporters has revealed striking similarities in the structural organization of ion and solute binding. Previous reports suggested that the Na2 sodium binding site in the neurotransmitter sodium symporter (NSS) leucine transporter (LeuT) is conserved across sodium/proton coupled secondary transporters of many distantly related families. This site is implicated in the conformational dynamics controlled by the binding and release of both translocated solute and ion(s) through a mechanism that largely remains unknown. In this study, we used extensive equilibrium molecular dynamics simulations, potential of mean force (PMF) computations, and quasi-harmonic analysis of the LeuT transporter with and without sodium ion bound at the Na2 site to delineate the role of this site in the conformational dynamics of the protein. PMF computations show that in presence of the sodium ion in Na2 the conserved T354 residue is locked into a single rotameric state in contrast to two degenerate states available in the absence of ion in Na2. Molecular dynamics (MD) simulations suggest the formation of a stable water wire from the cytoplasm to the Na2 site in the occluded state. It is plausible that local hydration plays an important role in transport cycle facilitating release of the ion from Na2. An unbinding of the ion from the Na2 site leads to a tightening of the extracellular thin gates and a destabilization of the intracellular thin gate and thus may promote an unbinding of the cotransported substrate. The study lends additional support to the hypothesis that one of the main drivers in the transport cycle of Na-coupled secondary transporters is the binding of the Na2 ion that controls dynamical equilibrium between an inward-facing to an outward-facing conformation.
最近，对钠离子偶联辅助转运体的晶体结构研究取得了进展，揭示了离子和溶质结合的结构组织具有惊人的相似性。之前的报告表明，神经递质钠协同转运体（NSS）亮氨酸转运体（LeuT）中的 Na2 钠离子结合位点在许多亲缘关系较远的家族的钠离子/质子偶联辅助转运体中是保守的。该位点涉及通过一种机制控制构象动力学，该机制在很大程度上仍然未知，这种机制涉及到被转运的溶质和离子的结合和释放的构象动力学。在这项研究中，我们使用了广泛的平衡分子动力学模拟、平均力势（PMF）计算以及有和没有钠离子结合在 Na2 位点的 LeuT 转运体的准谐分析，以描绘该位点在蛋白质构象动力学中的作用。PMF 计算表明，在 Na2 中有钠离子的情况下，保守的 T354 残基被锁定在单一旋转构象状态，而在没有离子的情况下则有两种简并状态。分子动力学（MD）模拟表明，在闭塞状态下，从细胞质到 Na2 位点形成了一条稳定的水线。局部水合作用在促进离子从 Na2 释放的转运循环中可能起着重要作用。离子从 Na2 位点的脱附导致细胞外细门的收紧和细胞内细门的不稳定性，从而可能促进共转运底物的脱附。该研究进一步支持了这样一种假设，即钠离子偶联辅助转运体的转运循环的主要驱动力之一是 Na2 离子的结合，它控制着内向到外向构象的动态平衡。
