Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle.
Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle.
复制标题
肌动球蛋白跨桥循环中预力产生状态的表征。
DOI：
10.1073/pnas.0710793105
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发表时间：
2008
影响因子：
11.1
通讯作者：
Yengo,ChristopherM
中科院分区：
文献类型：
--
作者：
Sun,Mingxuan;Rose,MichaelB;Ananthanarayanan,ShobanaK;Jacobs,DonaldJ;Yengo,ChristopherM
肌球蛋白是一种基于肌动蛋白的马达蛋白，在其ATPase周期中，通过在肌动蛋白附着(强结合：ADP或僵硬)和肌动蛋白分离(弱结合：ATP或ADP·PI)状态之间循环来产生力。然而，目前还不清楚肌动蛋白结合部位发生了哪些具体的构象变化，以及这些结构变化是如何导致产物释放和作用力和运动的。我们用荧光共振能量转移(FRET)技术研究了肌球蛋白V的肌动蛋白结合区在弱和强结合状态下肌动蛋白结合裂隙上50 kDa区域的构象变化。监测FRET信号的稳态和寿命数据表明，在肌动蛋白结合较弱的状态下，裂隙处于更开放的构象。瞬时动力学实验表明细胞发生了快速的构象变化，这与肌动蛋白激活的磷酸盐释放之前的裂隙闭合是一致的。我们的结果已经确定了力产生前的肌动球蛋白ADP·Pistate，并表明力产生可能发生在一种结晶学尚未看到的状态，在这种状态下，肌动蛋白结合裂隙和核苷酸结合口袋是关闭的。计算模型揭示了在不同的核苷酸状态下，上50 kDa结构域的刚性发生了戏剧性的变化，这表明该结构域的内在灵活性允许肌球蛋白马达同时完成紧密核苷酸结合(闭合核苷酸结合口袋)和高亲和力肌动蛋白结合(闭合肌动蛋白结合裂隙)。
Myosin is an actin-based motor protein that generates force by cycling between actin-attached (strong binding: ADP or rigor) and actin-detached (weak binding: ATP or ADP·Pi) states during its ATPase cycle. However, it remains unclear what specific conformational changes in the actin binding site take place on binding to actin, and how these structural changes lead to product release and the production of force and motion. We studied the dynamics of the actin binding region of myosin V by using fluorescence resonance energy transfer (FRET) to monitor conformational changes in the upper-50-kDa domain of the actin binding cleft in the weak and strong actin binding states. Steady-state and lifetime data monitoring the FRET signal suggest that the cleft is in a more open conformation in the weak actin binding states. Transient kinetic experiments suggest that a rapid conformational change occurs, which is consistent with cleft closure before actin-activated phosphate release. Our results have identified a pre-force-generation actomyosin ADP·Pistate, and suggest force generation may occur from a state not yet seen by crystallography in which the actin binding cleft and the nucleotide binding pocket are closed. Computational modeling uncovers dramatic changes in the rigidity of the upper-50-kDa domain in different nucleotide states, which suggests that the intrinsic flexibility of this domain allows myosin motors to accomplish simultaneous tight nucleotide binding (closed nucleotide binding pocket) and high-affinity actin binding (closed actin binding cleft).