Klebl David P, McMillan Sean N, Risi Cristina, Forgacs Eva, Virok Betty, Atherton Jennifer L, Harris Sarah A, Stofella Michele, Winkelmann Donald A, Sobott Frank, Galkin Vitold E, Knight Peter J, Muench Stephen P, Scarff Charlotte A, White Howard D
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
Nature. 2025 Apr 9. doi: 10.1038/s41586-025-08876-5.
Myosins produce force and movement in cells through interactions with F-actin. Generation of movement is thought to arise through actin-catalysed conversion of myosin from an ATP-generated primed (pre-powerstroke) state to a post-powerstroke state, accompanied by myosin lever swing. However, the initial, primed actomyosin state has never been observed, and the mechanism by which actin catalyses myosin ATPase activity is unclear. Here, to address these issues, we performed time-resolved cryogenic electron microscopy (cryo-EM) of a myosin-5 mutant having slow hydrolysis product release. Primed actomyosin was predominantly captured 10 ms after mixing primed myosin with F-actin, whereas post-powerstroke actomyosin predominated at 120 ms, with no abundant intermediate states detected. For detailed interpretation, cryo-EM maps were fitted with pseudo-atomic models. Small but critical changes accompany the primed motor binding to actin through its lower 50-kDa subdomain, with the actin-binding cleft open and phosphate release prohibited. Amino-terminal actin interactions with myosin promote rotation of the upper 50-kDa subdomain, closing the actin-binding cleft, and enabling phosphate release. The formation of interactions between the upper 50-kDa subdomain and actin creates the strong-binding interface needed for effective force production. The myosin-5 lever swings through 93°, predominantly along the actin axis, with little twisting. The magnitude of lever swing matches the typical step length of myosin-5 along actin. These time-resolved structures demonstrate the swinging lever mechanism, elucidate structural transitions of the power stroke, and resolve decades of conjecture on how myosins generate movement.
肌球蛋白通过与F-肌动蛋白相互作用在细胞中产生力和运动。运动的产生被认为是通过肌动蛋白催化肌球蛋白从ATP产生的启动（预动力冲程）状态转变为动力冲程后状态，同时伴有肌球蛋白杠杆摆动。然而，最初的启动肌动球蛋白状态从未被观察到，并且肌动蛋白催化肌球蛋白ATP酶活性的机制尚不清楚。在这里，为了解决这些问题，我们对具有缓慢水解产物释放的肌球蛋白-5突变体进行了时间分辨低温电子显微镜（cryo-EM）研究。启动肌动球蛋白主要在启动肌球蛋白与F-肌动蛋白混合后10毫秒被捕获，而动力冲程后肌动球蛋白在120毫秒时占主导地位，未检测到丰富的中间状态。为了进行详细解释，将cryo-EM图谱与伪原子模型进行拟合。启动的马达通过其较低的50 kDa亚结构域与肌动蛋白结合时会发生微小但关键的变化，肌动蛋白结合裂隙打开且磷酸盐释放被阻止。肌动蛋白与肌球蛋白的氨基末端相互作用促进上部50 kDa亚结构域的旋转，关闭肌动蛋白结合裂隙，并使磷酸盐释放。上部50 kDa亚结构域与肌动蛋白之间相互作用的形成创造了有效产生力所需的强结合界面。肌球蛋白-5杠杆主要沿肌动蛋白轴摆动93°，几乎没有扭转。杠杆摆动的幅度与肌球蛋白-5沿肌动蛋白的典型步长相匹配。这些时间分辨结构展示了杠杆摆动机制，阐明了动力冲程的结构转变，并解决了数十年来关于肌球蛋白如何产生运动的猜想。
