Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions.
Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions.
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DOI：
10.1016/j.bpj.2022.05.013
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发表时间：
2022-06-21
影响因子：
3.4
通讯作者：
Sivaramakrishnan, Sivaraj
中科院分区：
文献类型：
--
作者：
Touma, Anja M.;Tang, Wanjian;Rasicci, David, V;Vang, Duha;Rai, Ashim;Previs, Samantha B.;Warshaw, David M.;Yengo, Christopher M.;Sivaramakrishnan, Sivaraj
心肌肌球蛋白结合蛋白C（cMyBP-C）通过与肌球蛋白S2尾和/或细丝的相互作用调节心肌收缩力。这些结合伴侣相互作用对cMyBP-C调节功能的相对贡献尚不清楚。因此，我们开发了一种“纳米冲浪者”测定作为模型系统来询问这些cMyBP-C结合伴侣相互作用。使用连接到DNA纳米管的重组人β-心肌肌球蛋白亚片段（HMM或S1）生成合成粗丝，间距为14或28 nm，对应于天然粗丝中的14.3 nm肌球蛋白间距。nanosurfer测定由添加到体外运动性测定的DNA纳米管组成，使得运动性表面上的肌球蛋白有效地将细丝递送到DNA纳米管，增强细丝在DNA纳米管上滑动的可能性。在肌球蛋白间距为14或28 nm的纳米管上，细丝的速度没有什么不同。然后，我们通过每14 nm在纳米管上交替HMM和cMyBP-C N-末端片段（C 0-C2或C1-C2）来表征cMyBP-C对细丝运动性的影响。相对于单独的HMM，C 0-C2和C1-C2都将细丝速度降低了四到六倍。使用肌球蛋白S1构建体也发生了类似的抑制，该构建体缺乏与cMyBP-C相互作用的肌球蛋白S2区域，这表明cMyBP-C N末端必须与其他肌球蛋白头部结构域和/或肌动蛋白相互作用以减慢细丝速度。细丝速度不受C 0-C1 f片段的影响，C 0-C1 f片段缺乏大部分M结构域，支持该结构域对抑制性相互作用的重要性。在M结构域中具有磷酸模拟替代的C 0-C2片段与其磷酸无效对应物相比，对细丝速度的抑制明显较少，突出了M结构域磷酸化对cMyBP-C功能的调节作用。因此，nanosurfer测定提供了一个平台，以精确地操纵空间依赖性cMyBP-C结合伴侣相互作用，揭示β-心肌肌球蛋白收缩性的分子调控。
Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a “nanosurfer” assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human β-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0–C2 or C1–C2) on nanotubes every 14 nm. Both C0–C2 and C1–C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0–C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0–C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of β-cardiac myosin contractility.
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影响因子：
20.1
作者：
Harris SP;Lyons RG;Bezold KL
通讯作者：
Bezold KL
影响因子：
38.3
作者：
Hariadi RF;Sommese RF;Adhikari AS;Taylor RE;Sutton S;Spudich JA;Sivaramakrishnan S
通讯作者：
Sivaramakrishnan S
影响因子：
5.6
作者：
Gruen, M;Gautel, M
通讯作者：
Gautel, M
影响因子：
20.1
作者：
Harris, SP;Rostkova, E;Moss, RL
通讯作者：
Moss, RL
影响因子：
2.9
作者：
Liu, Yingying;White, Howard D.;Forgacs, Eva
通讯作者：
Forgacs, Eva