Department of Chemistry, University of California, Davis, California 95616, United States.
Department of Molecular and Cellular Biology, University of California, Davis, California 95616, United States.
Biochemistry. 2021 Apr 13;60(14):1088-1096. doi: 10.1021/acs.biochem.1c00152. Epub 2021 Mar 23.
Calmodulin (CaM) regulates the activity of a Ca channel known as the cardiac ryanodine receptor (RyR2), which facilitates the release of Ca from the sarcoplasmic reticulum during excitation-contraction coupling in cardiomyocytes. Mutations that disrupt this CaM-dependent channel inactivation result in cardiac arrhythmias. RyR2 contains three different CaM binding sites: CaMBD1 (residues 1940-1965), CaMBD2 (residues 3580-3611), and CaMBD3 (residues 4246-4275). Here, we report a crystal structure of Ca-bound CaM bound to RyR2 CaMBD3. The structure reveals Ca bound to the four EF-hands of CaM as well as a fifth Ca bound to CaM in the interdomain linker region involving Asp81 and Glu85. The CaM mutant E85A abolishes the binding of the fifth Ca and weakens the binding of CaMBD3 to Ca-bound CaM. Thus, the binding of the fifth Ca is important for stabilizing the complex in solution and is not a crystalline artifact. The CaMBD3 peptide in the complex adopts an α-helix (between Phe4246 and Val4271) that interacts with both lobes of CaM. Hydrophobic residues in the CaMBD3 helix (Leu4255 and Leu4259) form intermolecular contacts with the CaM N-lobe, and the CaMBD3 mutations (L4255A and L4259A) each weaken the binding of CaM to RyR2. Aromatic residues on the opposite side of the CaMBD3 helix (Phe4246 and Tyr4250) interact with the CaM C-lobe, but the mutants (F4246A and Y4250A) have no detectable effect on CaM binding in solution. We suggest that the binding of CaM to CaMBD3 and the binding of a fifth Ca to CaM may contribute to the regulation of RyR2 channel function.
钙调蛋白（CaM）调节一种称为心脏兰尼碱受体（RyR2）的 Ca 通道的活性，该通道在心肌细胞兴奋-收缩偶联过程中促进 Ca 从肌浆网释放。破坏这种 CaM 依赖性通道失活的突变会导致心律失常。RyR2 包含三个不同的 CaM 结合位点：CaMBD1（残基 1940-1965）、CaMBD2（残基 3580-3611）和 CaMBD3（残基 4246-4275）。在这里，我们报告了一个结合 Ca 的 CaM 与 RyR2 CaMBD3 结合的晶体结构。该结构揭示了 Ca 结合到 CaM 的四个 EF 手以及第五个 Ca 结合到包含 Asp81 和 Glu85 的 CaM 结构域连接区。CaM 突变体 E85A 消除了第五个 Ca 的结合并削弱了 CaMBD3 与 Ca 结合的 CaM 的结合。因此，第五个 Ca 的结合对于稳定溶液中的复合物很重要，而不是晶体假象。复合物中的 CaMBD3 肽采用 α-螺旋（位于 Phe4246 和 Val4271 之间），与 CaM 的两个叶相互作用。CaMBD3 螺旋中的疏水性残基（Leu4255 和 Leu4259）与 CaM 的 N-叶形成分子间接触，而 CaMBD3 突变（L4255A 和 L4259A）都削弱了 CaM 与 RyR2 的结合。CaMBD3 螺旋相反侧的芳基残基（Phe4246 和 Tyr4250）与 CaM 的 C-叶相互作用，但突变体（F4246A 和 Y4250A）在溶液中对 CaM 结合没有可检测的影响。我们认为，CaM 与 CaMBD3 的结合以及第五个 Ca 与 CaM 的结合可能有助于调节 RyR2 通道功能。
