Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain.
Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain.
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心肌肌球蛋白结合蛋白-C 与肌动蛋白的独特单分子结合以及肌动球蛋白运动的磷酸化依赖性抑制需要基序结构域的 17 个氨基酸。
DOI：
10.1016/j.yjmcc.2011.09.019
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发表时间：
2012
影响因子：
5
通讯作者：
Warshaw,DavidM
中科院分区：
文献类型：
--
作者：
Weith,Abbey;Sadayappan,Sakthivel;Gulick,James;Previs,MichaelJ;Vanburen,Peter;Robbins,Jeffrey;Warshaw,DavidM
心肌肌球蛋白结合蛋白C（cMyBP-C）具有11个免疫球蛋白或纤连蛋白样结构域，C 0至C10，其结合肌节蛋白，包括肌联蛋白、肌球蛋白和肌动蛋白。使用细菌表达的小鼠N-末端片段（C 0至C3）在肌球蛋白产生的肌动蛋白运动的体外运动性测定和评估单分子肌动蛋白结合能力的激光陷阱测定中，我们确定cMyBP-C基序的前N-末端17个氨基酸（C1和C2之间的接头）含有强的立体特异性肌动蛋白结合位点，该位点依赖于由于一簇丝氨酸而产生的正电荷。基序内4个丝氨酸的磷酸化降低了片段的肌动蛋白结合能力和肌动球蛋白抑制作用。使用激光陷阱分析，我们观察到单个cMyBP-C片段与单个肌动蛋白丝瞬时结合，具有短（~ 20 ms）和长（~ 300 ms）的附着寿命，类似于已知的肌动蛋白结合蛋白α-辅肌动蛋白。这些实验表明，含有cMyBP-C基序的cMyBP-C N-末端结构域束缚肌动蛋白丝，并提供了一种cMyBP-C调节肌动球蛋白运动产生的机制，即通过在肌节内施加有效的粘性负荷。
Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~20ms) and long (~300ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.