Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA.
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA.
J Biol Chem. 2021 Sep;297(3):101040. doi: 10.1016/j.jbc.2021.101040. Epub 2021 Aug 2.
Ryanodine receptor type 1 (RyR1) releases Ca ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca-binding site to sensitize RyR1 to Ca. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca. However, enhanced RyR1 activation occurred in the presence of Ca, AMPPCP, and Caf. In the absence of Ca, Na inhibited [H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca, Na is predicted to inhibit RyR1 by interacting with the Ca-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca-binding site, and conversely, Ca binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca-, ATP-, and Caf-binding sites.
肌质网 Ryanodine 受体 1 型（RyR1）从骨骼肌细胞的肌质网释放 Ca 离子，从而引发肌肉收缩。多种内源性和外源性效应物调节 RyR1，如 ATP、Ca、咖啡因（Caf）和 Ryanodine。冷冻电镜确定了三个共激活剂 Ca、ATP 和 Caf 的结合位点。然而，这些激活剂的核心调节和协同作用的机制仍有待确定。在这里，我们使用 [H]Ryanodine 配体结合测定和分子动力学模拟来检验以下假设，即 ATP-和 Caf-结合位点与 Ca-结合位点相互作用，使 RyR1 对 Ca 敏感。我们报告说，非水解型 ATP 类似物磷酸甲硫氨酸磷酸腺苷酯（AMPPCP）或 Caf 可在 Ca 存在或不存在的情况下激活 RyR1。然而，在 Ca、AMPPCP 和 Caf 存在的情况下，增强了 RyR1 的激活。在没有 Ca 的情况下，Na 抑制 [H]Ryanodine 结合，但不损害 AMPPCP 和 Caf 对 RyR1 的激活。计算分析表明，Ca、ATP 和 Caf 结合位点通过与激活核心的羧基末端结构域和其他结构域相互作用来调节 RyR1 蛋白的稳定性。在没有 Ca 的情况下，当存在 ATP 和 Caf 时，Na 预计通过与 Ca 结合位点相互作用来抑制 RyR1。我们的数据表明，ATP 和 Caf 结合改变了 Ca 结合位点的构象，反之亦然，Ca 结合改变了 ATP-和 Caf-结合位点的构象。我们得出结论，Ca、ATP 和 Caf 通过涉及 Ca、ATP 和 Caf 结合位点的变构相互作用网络来调节 RyR1。
