Das Avisek, Rui Huan, Nakamoto Robert, Roux Benoît
Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57(th) Street, Chicago,IL 60637, USA.
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, PO Box 800886, 480Ray C. Hunt Drive, Charlottesville, VA 22908, USA.
J Mol Biol. 2017 Mar 10;429(5):647-666. doi: 10.1016/j.jmb.2017.01.007. Epub 2017 Jan 16.
Ion pumps are integral membrane proteins responsible for transporting ions against concentration gradients across biological membranes. Sarco/endoplasmic reticulum Ca-ATPase (SERCA), a member of the P-type ATPases family, transports two calcium ions per hydrolyzed ATP molecule via an "alternating-access" mechanism. High-resolution crystallographic structures provide invaluable insight on the structural mechanism of the ion pumping process. However, to understand the molecular details of how ATP hydrolysis is coupled to calcium transport, it is necessary to gain knowledge about the conformational transition pathways connecting the crystallographically resolved conformations. Large-scale transitions in SERCA occur at time-scales beyond the current reach of unbiased molecular dynamics simulations. Here, we overcome this challenge by employing the string method, which represents a transition pathway as a chainofstates linking two conformational endpoints. Using a multiscale methodology, we have determined all-atom transition pathways for three main conformational transitions responsible for the alternating-access mechanism. The present pathways provide a clear chronology and ordering of the key events underlying the active transport of calcium ions by SERCA. Important conclusions are that the conformational transition that leads to occlusion with bound ATP and calcium is highly concerted and cooperative, the phosphorylation of Asp351 causes areorganization of the cytoplasmic domains that subsequently drives the opening of the luminal gate, and thereclosing of luminal gate induces a shift in the cytoplasmic domains that subsequently enables the dephosphorylation of Asp351-P. Formation of transient residue-residue contacts along the conformational transitions predicted by the computations provide an experimental route to test the general validity of the computational pathways.
离子泵是整合膜蛋白，负责逆浓度梯度跨生物膜转运离子。肌浆网/内质网钙ATP酶（SERCA）是P型ATP酶家族的成员，通过“交替访问”机制，每水解一个ATP分子转运两个钙离子。高分辨率晶体结构为离子泵浦过程的结构机制提供了宝贵的见解。然而，要了解ATP水解如何与钙转运偶联的分子细节，有必要了解连接晶体解析构象的构象转变途径。SERCA中的大规模转变发生的时间尺度超出了目前无偏分子动力学模拟的范围。在这里，我们通过采用弦方法克服了这一挑战，该方法将转变途径表示为连接两个构象端点的一系列状态。使用多尺度方法，我们确定了负责交替访问机制的三个主要构象转变的全原子转变途径。目前的途径为SERCA主动转运钙离子的关键事件提供了清晰的时间顺序和排序。重要的结论是，导致结合ATP和钙时发生堵塞的构象转变是高度协同的，Asp351的磷酸化导致细胞质结构域的重组，随后驱动腔门打开，腔门的重新关闭导致细胞质结构域的移位，随后使Asp351-P去磷酸化。沿着计算预测的构象转变形成瞬时残基-残基接触，为测试计算途径的普遍有效性提供了一条实验途径。
