Molecular Cardiac Physiology Group, Simon Fraser University, Burnaby, British Columbia, Canada.
Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, USA.
J Biol Chem. 2021 Jan-Jun;296:100350. doi: 10.1016/j.jbc.2021.100350. Epub 2021 Feb 3.
Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca and Mg and a regulatory site (II) that has been thought to bind only Ca. Binding of Ca at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca/Mg through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca/Mg interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg caused a 1.4-fold lower affinity for Ca. These experiments strongly suggest that cytosolic-free Mg occupies a significant population of the available site II. Interaction of Mg with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg, such as secondary hyperparathyroidism or ischemia, respectively.
心肌细肌丝由肌动蛋白、原肌球蛋白和肌钙蛋白组成，肌钙蛋白在 Ca 结合时改变构象，引发肌肉收缩。人心脏肌钙蛋白 C（cTnC）是细肌丝的 Ca 感应成分。它包含结构位点（III/IV），既能结合 Ca 又能结合 Mg，还有一个调节位点（II），之前认为该位点只结合 Ca。该位点的 Ca 结合引发了一系列构象变化，最终导致力的产生。然而，支持 II 位点结合调节的机制仍不清楚。在这里，我们通过等温滴定量热法和热力学积分模拟对 II 位点与 Ca/Mg 的相互作用进行了量化。用 WT N 端 cTnC 和全长 cTnC 进行直接和竞争结合滴定表明，生理相关浓度的 Ca/Mg 与相同的部位相互作用。此外，已发现两个配位残基在 II 位点被去除的 D67A/D73A N 端 cTnC 构建体对两种阳离子的亲和力显著降低。此外，1 mM Mg 导致 Ca 的亲和力降低 1.4 倍。这些实验强烈表明，细胞溶质游离的 Mg 占据了 II 位点的很大一部分。Mg 与 cTnC 的 II 位点的相互作用可能对心脏具有重要的功能意义，无论是在基线状态还是在减少或增加 Mg 可用性的疾病状态下，例如继发性甲状旁腺功能亢进或缺血。
