Institute of Biology, Cellular Biophysics, Humboldt Universität zu Berlin, Berlin, Germany.
Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.
Protein Sci. 2023 Nov;32(11):e4798. doi: 10.1002/pro.4798.
Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild-type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium-dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV-induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto-inhibitory role of the regulatory segment, inter-subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKII in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.
利用非天然氨基酸诱变技术，我们构建了一个突变型 CaMKII，该突变体能在紫外光照射下与钙调蛋白形成共价键。与野生型 CaMKII 一样，L308BzF 突变体以钙依赖性方式与钙调蛋白形成化学计量比的结合。利用该构建体，我们证明了钙调蛋白与 CaMKII 的结合，即使在这些化学计量条件下，也不会干扰 CaMKII 的寡聚状态。此外，我们能够通过紫外光诱导的钙调蛋白结合来实现 CaMKII L308BzF 的激活，一旦建立，其对钙耗竭就进一步不敏感。除了调节片段的典型自动抑制作用外，在没有 CaM 的情况下的亚基间交联表明，激酶结构域和调节片段在基础条件下具有很大的移动性。CaMKII 的体外特性及其在哺乳动物细胞中的表达表明，它可能是用光控制哺乳动物细胞中 CaMKII 活性的有前途的候选物。
