Topographical Studies of Smooth and Nonmuscle Myosins

说明(申请人提供)：激活细胞收缩功能需要肌球蛋白II(SMM)和非肌球蛋白II(NMM)的磷酸化。肌球蛋白的两个“头”结构域的调节轻链(RLC)中的单个丝氨酸的磷酸化足以激活ATPase 1000倍以上(1)。目前的知识表明，这种显着的调控开关是通过肌球蛋白结构的大量构象变化来调节的。我们的长期目标是确定依赖磷酸化的调控开关的结构基础。 我们已经证明，SMM和NMM的头部结构域(2)和催化结构域(3)都需要保持完整的调控开关。然而，双头肌球蛋白结构的原子分辨结构还没有出现，即使在最初的肌球蛋白头部结构被解决10多年后也是如此(4)。而且，所有单头结构的原子分辨率结构都具有有限的或没有关于RLC的结构信息，并且没有关于其中发现磷酸化丝氨酸的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，我们应用了瞬时动力学(1，5，6)、荧光(7)、电子顺磁共振(EPR)(8)和光交联法(5，9，10)来了解调控开关的结构基础。我们已经证明：1)两个RLC之间在关闭状态下存在相互作用，在磷酸化打开状态下(9，10)似乎减弱或改变，2)含有丝氨酸-19的调节域(RD)的N-末端区域似乎经历了从延伸到折叠结构(10)的较大构象变化，以及3)这种构象变化需要在大约15 nm外的活性部位存在核苷酸(7)。 假设 我们已经发表了SMM的RD在关断状态下的原子分辨结构模型(10)，这是我们对这一提议的初始结构假设之一。在这里，我们扩展了这一初步工作，提出了氢键网络假说，以解释调控结构域如何在开启和关闭状态之间切换。我们还提出了一个结构假说来解释RLC磷酸化如何破坏全长SMM 10S构象的稳定性。 目标 除了其他人提出的结构假设外，我们还设计了一系列实验来直接检验上述结构假设(11-13)。我们建议使用截断肌球蛋白结构(HMM)和全长肌球蛋白，两者都能够采用完全关闭和完全开启的构象。我们还解决了关于磷酸化对ATP水解动力学机制的影响的关键未回答的问题。

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.