Cardiac muscle contraction depends on interactions between thick (myosin) and thin (actin) filaments (TFs). TFs are regulated by intracellular Ca²⁺ levels. Under activating conditions Ca²⁺ binds to the troponin complex and displaces tropomyosin from myosin binding sites on the TF surface to allow actomyosin interactions. Recent studies have shown that in addition to Ca²⁺, the first four N-terminal domains (NTDs) of cardiac myosin binding protein C (cMyBP-C) (e.g. C0, C1, M and C2), are potent modulators of the TF activity, but the mechanism of their collective action is poorly understood. Previously, we showed that C1 activates the TF at low Ca²⁺ and C0 stabilizes binding of C1 to the TF, but the ability of C2 to bind and/or affect the TF remains unknown. Here we obtained 7.5 Å resolution cryo-EM reconstruction of C2-decorated actin filaments to demonstrate that C2 binds to actin in a single structural mode that does not activate the TF unlike the polymorphic binding of C0 and C1 to actin. Comparison of amino acid sequences of C2 with either C0 or C1 shows low levels of identity between the residues involved in interactions with the TF but high levels of conservation for residues involved in Ig fold stabilization. This provides a structural basis for strikingly different interactions of structurally homologous C0, C1 and C2 with the TF. Our detailed analysis of the interaction of C2 with the actin filament provides crucial information required to model the collective action of cMyBP-C NTDs on the cardiac TF.

心肌收缩取决于粗（肌球蛋白）和细（肌动蛋白）细丝 (TF) 之间的相互作用。TF受细胞内Ca ²⁺水平的调节。在激活条件下，Ca ²⁺与肌钙蛋白复合物结合，并从 TF 表面的肌球蛋白结合位点置换原肌球蛋白，以允许肌钙蛋白相互作用。最近的研究表明，除了 Ca ²⁺之外，心脏肌球蛋白结合蛋白 C (cMyBP-C) 的前四个 N 端结构域 (NTD)（例如 C0、C1、M 和 C2）是 TF 的有效调节剂。活动，但对它们的集体作用机制知之甚少。以前，我们发现 C1 在低 Ca ^{2+下激活 TF}C0 稳定 C1 与 TF 的结合，但 C2 结合和/或影响 TF 的能力仍然未知。在这里，我们获得了 C2 修饰的肌动蛋白丝的 7.5 Å 分辨率冷冻电镜重建，以证明 C2 以单一结构模式与肌动蛋白结合，与 C0 和 C1 与肌动蛋白的多态性结合不同，该模式不会激活 TF。C2 的氨基酸序列与 C0 或 C1 的比较表明，参与与 TF 相互作用的残基之间的同一性水平较低，但参与 Ig 倍数稳定化的残基具有高水平的保守性。这为结构同源的 C0、C1 和 C2 与 TF 的显着不同相互作用提供了结构基础。