Electron Microscopy and 3D Reconstruction of F-Actin Decorated with Cardiac Myosin-Binding Protein C (cMyBP-C)
Electron Microscopy and 3D Reconstruction of F-Actin Decorated with Cardiac Myosin-Binding Protein C (cMyBP-C)
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DOI：
10.1016/j.jmb.2011.05.010
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发表时间：
2011-07-08
影响因子：
5.6
通讯作者：
Craig, Roger
中科院分区：
文献类型：
--
作者：
Mun, Ji Young;Gulick, James;Craig, Roger
肌球蛋白结合蛋白 C (MyBP-C) 是一种类似于脊椎动物横纹肌粗丝（含有肌球蛋白）的 130 kDa 杆状蛋白。它由来自免疫球蛋白和纤连蛋白 III 型家族的 10 或 11 个球状 10 kDa 结构域以及一个额外的 MyBP-C 特异性基序组成。心脏异构体 cMyBP-C 在 p-肾上腺素能刺激时发生的磷酸化依赖性心脏功能增强中发挥着关键作用，MyBP-C 的突变会导致骨骼肌和心脏疾病。除了与肌球蛋白结合外，MyBP-C 还可以通过其 N 末端与肌动蛋白结合，从而可能通过这种粗细丝桥以一种新颖的方式调节收缩。为了了解肌动蛋白结合的结构基础，我们使用负染色电子显微镜和三维重建来研究用细菌表达的 N 末端 cMyBP-C 片段装饰的 F-肌动蛋白的结构。在 25 至 180 mM KCl 浓度的各种盐条件下获得了清晰的装饰。在 180 mM KCl 水平下进行三维螺旋重建以尽量减少非特异性结合，显示 MyBP-C 密度分布在肌动蛋白子域 1 外围的大部分区域，并从其表面沿肌动蛋白尖端方向切向延伸。与 MyBP-C Ig 结构域原子结构的分子拟合表明，肌动蛋白上的大多数 N 末端结构域可能是有序的。结合位置可以调节原肌球蛋白位置并干扰肌球蛋白头与肌动蛋白的结合。 (C) 2011 Elsevier Ltd. 保留所有权利。
Myosin-binding protein C (MyBP-C) is an similar to 130-kDa rod-shaped protein of the thick (myosin containing) filaments of vertebrate striated muscle. It is composed of 10 or 11 globular 10-kDa domains from the immunoglobulin and fibronectin type III families and an additional MyBP-C-specific motif. The cardiac isoform cMyBP-C plays a key role in the phosphorylation-dependent enhancement of cardiac function that occurs upon p-adrenergic stimulation, and mutations in MyBP-C cause skeletal muscle and heart diseases. In addition to binding to myosin, MyBP-C can also bind to actin via its N-terminal end, potentially modulating contraction in a novel way via this thick-thin filament bridge. To understand the structural basis of actin binding, we have used negative stain electron microscopy and three-dimensional reconstruction to study the structure of F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions varying from 25 to 180 mM KCl concentration. Three-dimensional helical reconstructions, carried out at the 180-mM KCl level to minimize nonspecific binding, showed MyBP-C density over a broad portion of the periphery of subdomain 1 of actin and extending tangentially from its surface in the direction of actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it could modulate tropomyosin position and would interfere with myosin head binding to actin. (C) 2011 Elsevier Ltd. All rights reserved.