PDF(Pubmed)
Abstract：
Previous cryo-electron micrographs suggested that the skeletal muscle Ca2+ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca2+ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293 cells for [3H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca2+/Mg2+-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca2+-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca2+/Mg2+-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca2+-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca2+/Mg2+-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.
摘要：
先前的低温电子显微照片表明，骨骼肌Ca2释放通道，RyR1受EF手Ca2+结合结构域和胞质环(S2-S3环)之间的复杂相互作用调节。然而,这些相互作用的精确分子细节和相互作用的功能后果仍然难以捉摸。这里,我们采用分子动力学模拟来探索EF手-S2-S3环界面内参与氢键相互作用的特定氨基酸对。我们的模拟揭示了两个关键的相互作用：（1）K4101（EF手）与D4730（S2-S3环路）和（2）E4075，Q4078和D4079（EF手）与R4736（S2-S3环路）。为了探究这些相互作用的功能意义，我们构建了突变体RyR1cDNA，并在HEK293细胞中表达它们，用于[3H]ryanodine结合测定。我们的结果表明EF手的突变，特别是K4101E和K4101M，导致对Ca2/Mg2依赖性抑制的亲和力降低。有趣的是,K4101E突变增加了Ca2+依赖性激活的亲和力。相反，S2-S3环中的突变，D4730K和D4730N,没有显着改变Ca2/Mg2依赖性抑制的亲和力。我们之前的发现与骨骼疾病相关的RyR1突变，R4736Q和R4736W,Ca2+依赖性抑制受损，与目前的结果一致。在硅诱变分析中，与我们的功能数据一致，表明突变后氢键模式改变。一起来看,我们的研究结果强调了EF手-S2-S3环相互作用在Ca2+/Mg2+依赖性RyR1抑制中的关键作用,并提供了针对该结构域相互作用治疗骨骼肌病的潜在治疗策略的见解.
