Department of Chemistry , University of Kentucky , 505 Rose Street, Chemistry-Physics Building , Lexington , Kentucky 40506 , United States.
Department of Chemical and Materials Engineering , University of Kentucky , 177 F. Paul Anderson Tower , Lexington , Kentucky 40506 , United States.
J Chem Theory Comput. 2019 Apr 9;15(4):2692-2705. doi: 10.1021/acs.jctc.8b01312. Epub 2019 Mar 13.
Sarcoendoplasmic reticulum Ca-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca) binds SERCA with micromolar affinity, magnesium (Mg) and potassium (K) also compete with Ca binding. However, the molecular bases for these competing ions' influence on the SERCA function and the selectivity of the pump for Ca are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca binding sites via (1) triplicate molecular dynamics (MD) simulations of Mg, Ca, and K-bound SERCA, (2) mean spherical approximation (MSA) theory to score the affinity and selectivity of cation binding to the MD-resolved structures, and (3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that (a) coordination at sites I and II is optimized for Ca and to a lesser extent for Mg and K, as determined by MD-derived cation-amino acid oxygen and bound water configurations, (b) the impaired coordination and high desolvation cost for Mg precludes favorable Mg binding relative to Ca, while K has limited capacity to bind site I, and (c) Mg most likely acts as inhibitor and K as intermediate in SERCA's reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalizes enzyme activity across broad ranges of K, Ca, and Mg concentrations.
肌浆网 Ca-ATP 酶（SERCA）是一种跨膜泵，在将 Ca 转运到肌浆网（SR）中发挥着重要作用。虽然 Ca 与 SERCA 的结合亲和力为微摩尔级，但 Mg 和 K 也与 Ca 竞争结合。然而，这些竞争离子对 SERCA 功能的影响以及泵对 Ca 的选择性的分子基础尚未得到很好的确立。因此，我们使用计算方法通过以下方式解决了在典型的位点 I 和 II Ca 结合位点中阳离子结合的分子决定因素：（1）Mg、Ca 和 K 结合的 SERCA 的三倍分子动力学（MD）模拟，（2）平均球近似（MSA）理论来评分 MD 解析结构中阳离子结合的亲和力和选择性，以及（3）基于 MSA 衍生的亲和力数据的 SERCA 周转率状态模型。我们的主要发现是：（a）通过 MD 衍生的阳离子-氨基酸氧和结合水配置确定，位点 I 和 II 的配位优化用于 Ca，其次是 Mg 和 K，（b）Mg 的配位受损和高去溶剂化成本排除了相对于 Ca 的有利 Mg 结合，而 K 结合位点 I 的能力有限，以及（c）根据 SERCA 周转率的最佳拟合状态模型，Mg 很可能作为抑制剂，K 作为 SERCA 反应循环的中间产物。这些发现为 SERCA 功能提供了一个定量基础，利用了分子尺度的热力学数据，并解释了在广泛的 K、Ca 和 Mg 浓度范围内的酶活性。
