Institute of Immunology and Physiology of the Russian Academy of Sciences, 620049 Yekaterinburg, Russia.
Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.
Int J Mol Sci. 2024 Oct 18;25(20):11195. doi: 10.3390/ijms252011195.
About half of the mutations that lead to hypertrophic cardiomyopathy (HCM) occur in the gene. However, the molecular mechanisms of pathogenicity of point mutations in cardiac myosin-binding protein C (cMyBP-C) remain poorly understood. In this study, we examined the effects of the D75N and P161S substitutions in the C0 and C1 domains of cMyBP-C on the structural and functional properties of the C0-C1-m-C2 fragment (C0-C2). Differential scanning calorimetry revealed that these mutations disorder the tertiary structure of the C0-C2 molecule. Functionally, the D75N mutation reduced the maximum sliding velocity of regulated thin filaments in an in vitro motility assay, while the P161S mutation increased it. Both mutations significantly reduced the calcium sensitivity of the actin-myosin interaction and impaired thin filament activation by cross-bridges. D75N and P161S C0-C2 fragments substantially decreased the sliding velocity of the F-actin-tropomyosin filament. ADP dose-dependently reduced filament sliding velocity in the presence of WT and P161S fragments, but the velocity remained unchanged with the D75N fragment. We suppose that the D75N mutation alters nucleotide exchange kinetics by decreasing ADP affinity to the ATPase pocket and slowing the myosin cycle. Our molecular dynamics simulations mean that the D75N mutation affects myosin S1 function. Both mutations impair cardiac contractility by disrupting thin filament activation. The results offer new insights into the HCM pathogenesis caused by missense mutations in N-terminal domains of cMyBP-C, highlighting the distinct effects of D75N and P161S mutations on cardiac contractile function.
大约一半导致肥厚型心肌病（HCM）的突变发生在基因中。然而，肌球蛋白结合蛋白 C（cMyBP-C）点突变的致病分子机制仍知之甚少。在这项研究中，我们研究了 cMyBP-C 的 C0 和 C1 结构域中的 D75N 和 P161S 取代对 C0-C1-m-C2 片段（C0-C2）的结构和功能特性的影响。差示扫描量热法显示这些突变使 C0-C2 分子的三级结构无序。从功能上讲，D75N 突变降低了体外运动分析中调节的薄丝的最大滑动速度，而 P161S 突变增加了它。这两种突变都显著降低了肌动球蛋白相互作用的钙敏感性，并损害了横桥对薄丝的激活。D75N 和 P161S C0-C2 片段大大降低了 F-肌动蛋白-原肌球蛋白丝的滑动速度。ADP 剂量依赖性地降低了 WT 和 P161S 片段存在下的丝滑动速度，但 D75N 片段的速度保持不变。我们假设 D75N 突变通过降低 ADP 对 ATP 酶口袋的亲和力并减慢肌球蛋白循环来改变核苷酸交换动力学。我们的分子动力学模拟意味着 D75N 突变影响肌球蛋白 S1 的功能。这两种突变通过破坏薄丝的激活来损害心脏收缩力。研究结果为 cMyBP-C N 端结构域中错义突变导致 HCM 的发病机制提供了新的见解，突出了 D75N 和 P161S 突变对心脏收缩功能的不同影响。
