Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics
Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics
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DOI：
10.1073/pnas.1521281113
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发表时间：
2016-03-22
影响因子：
11.1
通讯作者：
Thomas, David D.
中科院分区：
文献类型：
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作者：
Colson, Brett A.;Thompson, Andrew R.;Thomas, David D.
我们已经使用了时间分辨荧光共振能量转移（TR-FRET）和双电子-电子共振（DEER）的定点光谱，结合互补分子动力学（MD）模拟，以解决心肌肌球蛋白结合蛋白C（cMyBP-C）的结构和动力学，专注于N-末端区域。这些结果对这种蛋白质在心肌收缩中的作用有影响，特别是与β-肾上腺素能信号，心力衰竭和肥厚性心肌病有关。N-末端cMyBP-C结构域C 0-C2（C 0 C2）含有与粗丝和细丝潜在相互作用的结合区域。MyBP-C基序中PKA的磷酸化调节这些结合相互作用。我们的光谱分析检测cMyBP-C上的定点探针对之间的距离。我们设计了cMyBP-C内的分子内标记位点对，使用TR-FRET和DEER以高分辨率测量蛋白质柔性区域的距离和无序。磷酸化降低了分子紊乱的水平，C 0 C2分子内距离的分布变得更加紧凑，探针侧接C1和C2之间的基序或C 0和C1之间的富含Pro/Ala的接头（PAL）。从微秒MD模拟中获得了进一步的见解，其揭示了无序基序区域中的大的结构变化，其中磷酸化暴露了基序内具有高潜力的蛋白质-蛋白质相互作用位点的稳定α-螺旋上的一系列残基的表面。这些实验和计算结果阐明了cMyBP-C的柔性和动态部分的结构转变，为这种蛋白质在心肌收缩性中的调节作用提供了以前未识别的分子见解。
We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiacmyosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to beta-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable a-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility.