Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2R3, Canada.
Department of Medicine, University of Alberta, Edmonton, AB T6G 2R3, Canada.
J Mol Biol. 2021 Jun 25;433(13):167010. doi: 10.1016/j.jmb.2021.167010. Epub 2021 Apr 24.
Cardiac troponin (cTn) is made up of three subunits, cTnC, cTnI, and cTnT. The regulatory N-terminal domain of cTnC (cNTnC) controls cardiac muscle contraction in a calcium-dependent manner. We show that calcium-saturated cNTnC can adopt two different orientations, with the "active" orientation consistent with the 2020 cryo-EM structure of the activated cardiac thin filament by Yamada et al. Using solution NMR N R relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R 10 s), being connected by a flexible linker. However, upon addition of cTnI, the complex tumbles as a rigid unit (R 30 s). cTnI phosphomimetic mutants S22D/S23D, S41D/S43D and dilated cardiomyopathy- (DCM-)associated mutations cTnI K35Q, cTnC D75Y, and cTnC G159D destabilize the active orientation of cNTnC, with intermediate N R rates (R 17-23 s). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI and cTnI. Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI, cTnC-cTnT, or cTnC-cTnI-cTnT), the cNTnC domain is still immobilized, revealing a new interaction between cNTnC and the IT-arm that stabilizes a "dormant" orientation. We propose that the calcium sensitivity of the cardiac troponin complex is regulated by an equilibrium between active and dormant orientations, which can be shifted through post-translational modifications or DCM-associated mutations.
肌钙蛋白（cTn）由三个亚基组成，即 cTnC、cTnI 和 cTnT。cTnC 的调节 N 端结构域（cNTnC）以钙离子依赖的方式控制心肌收缩。我们表明，饱和钙离子的 cNTnC 可以采取两种不同的取向，其中“活性”取向与 Yamada 等人 2020 年冷冻电镜结构的激活心肌细肌丝一致。通过溶液 NMR N R 弛豫分析，我们证明 cTnC 的两个结构域独立旋转（平均 R 10 s），由一个柔性连接连接。然而，当加入 cTnI 时，复合物作为刚性单元旋转（R 30 s）。cTnI 磷酸模拟突变体 S22D/S23D、S41D/S43D 和扩张型心肌病（DCM）相关突变 cTnI K35Q、cTnC D75Y 和 cTnC G159D 破坏 cNTnC 的活性取向，具有中间 N R 速率（R 17-23 s）。cNTnC 的活性取向由 cTnI、cTnI 和 cTnI 的柔性尾部稳定。令人惊讶的是，当 cTnC 被掺入缺乏这些尾部的复合物中（cTnC-cTnI、cTnC-cTnT 或 cTnC-cTnI-cTnT）时，cNTnC 结构域仍然被固定，揭示了 cNTnC 与 IT 臂之间的新相互作用，稳定了一种“休眠”取向。我们提出，心肌钙蛋白复合物的钙离子敏感性受活性和休眠取向之间的平衡调节，这种平衡可以通过翻译后修饰或 DCM 相关突变来改变。
