Topographical Studies of Smooth and Nonmuscle Myosins

平滑肌和非肌肉肌球蛋白的地形学研究

• 批准号: 7662357
• 负责人: Christine R Cremo
• 金额: $ 29.64万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7662357
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Acrylamides; Active Sites; Address; Adopted; Affinity; Binding; Catalytic Domain; Cysteine; Electron Spin Resonance Spectroscopy; Equilibrium; Filament; Fluorescence; Goals; H-Meromyosin; Head; Hydrogen Bonding; Kinetics; Knowledge; Length; Light; Link; Location; Maps; Mass Spectrum Analysis; Measurement; Mediating; Molecular Conformation; Motor; Myosin ATPase; Myosin Type II; N-terminal; Nucleic Acid Regulatory Sequences; Nucleotides; Phosphorylation; Physiologic pulse; Publishing; Regulation; Resolution; Scanning; Series; Serine; Smooth Muscle Myosins; Structural Models; Structure; Techniques; Testing; Work; base; crosslink; intermolecular interaction; research study; retinal rods

DESCRIPTION (provided by applicant): Phosphorylation of smooth muscle myosin II (SMM) and nonmuscle myosin II (NMM) is required to activate cellular contractile functions. Phosphorylation of a single serine in the regulatory light chain (RLC) of each of the two "head" domains of myosin is sufficient to activate the ATPase by more than 1000 fold (1). The current knowledge suggests that this remarkable regulatory switch is mediated through large conformational changes in the myosin structure. Our long-term goal is to determine the structural basis of the phosphorylation-dependent regulatory switch. We have demonstrated that both head domains (2) and both catalytic domains (3) of SMM and NMM are required to retain an intact regulatory switch. However, atomic resolution structures of double-headed myosin constructs have not been forthcoming, even after more than 10 years since the initial myosin head structure was solved (4). And, all atomic resolution structures of single-head constructs have limited or no structural information about the RLC and no information about the RLC domain in which the phosphorylated serine is found. There are no structures available for the RLC of SMM or NMM. To address this problem, we have applied transient kinetic (1,5,6), fluorescence (7), electron paramagnetic resonance (EPR) (8) and photo-crosslinking (5,9,10) approaches toward an understanding of the structural basis of the regulatory switch. We have shown that 1) there is an interaction between the two RLC in the switched-off state that appears to be weakened or altered in the phosphorylated switched-on state (9,10), 2) the N-terminal region of the regulatory domain (RD) containing serine-19 appears to undergo a large conformational change from an extended to folded structure (10) and 3) this conformational change requires the presence of nucleotide at the active site approximately 15 nm away (7). Hypotheses We have published an atomic resolution structural model of the RD of SMM in the switched-off state (10), which serves as one of our initial structural hypotheses for this proposal. Here we extend this initial work to propose the Hydrogen Bonding Network Hypothesis to explain how the regulatory domain switches between the on- and off-states. We also propose a structural hypothesis to explain how RLC phosphorylation destabilizes the full-length SMM 10S conformation. Aims We have devised a series of experiments that directly test the above-mentioned structural hypotheses in addition to structural proposals by others (11-13). We propose to use a truncated myosin construct (HMM) and full-length myosin, both of which are capable of adopting a fully switched-off and fully switched-on conformation. We also address key unanswered questions about the effect of phosphorylation upon the kinetic mechanism of ATP hydrolysis.

描述（由申请人提供）：平滑肌肌球蛋白II（SMM）和非肌肉肌球蛋白II（NMM）的磷酸化需要激活细胞收缩功能。肌球蛋白两个“头”结构域的调节轻链（RLC）中单个丝氨酸的磷酸化足以激活ATPase超过1000倍（1）。当前的知识表明，这种非凡的调节开关是通过肌球蛋白结构的巨大构象变化来介导的。我们的长期目标是确定磷酸化依赖性调节开关的结构基础。 我们已经证明，SMM和NMM的催化域（3）均需要保持完整的调节开关。然而，即使在最初的肌球蛋白头结构被解决了十多年之后，双头肌球蛋白构建体的原子分辨率也没有到来（4）。而且，单头构建体的所有原子分辨率结构都有有关RLC的有限或没有结构信息，并且没有找到磷酸化丝氨酸的RLC结构域的信息。 SMM或NMM的RLC没有可用的结构。 为了解决这个问题，我们已经应用了瞬态动力学（1,5,6），荧光（7），电子顺磁共振（EPR）（8）（8）和光相连接（5,9,10）方法来理解调节开关的结构基础。我们已经表明，1）在关闭状态下两个RLC之间存在相互作用，在磷酸化的开关状态下似乎被削弱或改变了（9,10），2）含有丝氨酸-19的调节域（RD）的N末端区域，其中包含丝网的N-末端区域似乎是从扩展到折叠结构（10）和3）的构造（10）和3），并且是3）。大约15 nm（7）。 假设 我们已经在关闭状态下发布了SMM RD的原子分辨率结构模型（10），该模型是我们对此提案的最初结构假设之一。在这里，我们扩展了这项最初的工作，以提出氢键网络假设，以解释调节域如何在境内和非国家之间切换。我们还提出了一个结构假设，以解释RLC磷酸化如何破坏全长SMM 10S构象。 目标 我们已经设计了一系列实验，这些实验除了其他人的结构建议外，除了结构性建议外，直接测试了上述结构假设（11-13）。我们建议使用截短的肌球蛋白构建体（HMM）和全长肌球蛋白，它们都能够采用完全关闭的构象和完全关闭的构象。我们还解决了有关磷酸化对ATP水解动力学机制的影响的关键未解决问题。

项目成果

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation.

肌球蛋白 II 磷酸化调节的广泛紊乱和变构机制。

• DOI: 10.1073/pnas.1014137108
• 发表时间: 2011
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Vileno,Bertrand;Chamoun,Jean;Liang,Hua;Brewer,Paul;Haldeman,BrianD;Facemyer,KevinC;Salzameda,Bridget;Song,Likai;Li,Hui-Chun;Cremo,ChristineR;Fajer,PiotrG
• 通讯作者: Fajer,PiotrG