Pennsylvania Muscle Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104.
Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104.
Proc Natl Acad Sci U S A. 2018 Feb 6;115(6):1292-1297. doi: 10.1073/pnas.1718316115. Epub 2018 Jan 22.
Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.
肌球蛋白以依赖于同种型的方式响应机械负荷来调节其功率输出，从而使其能够动态适应一系列运动挑战。在这里，我们通过冷冻电子显微镜确定了肌球蛋白-IB（myo1b）与肌动蛋白结合的一个僵硬状态和两个 ADP 结合状态的近原子分辨率结构，揭示了基于力感应的结构基础。这两个 ADP 结合状态通过杠杆的 25°旋转分开。第一个 ADP 状态的杠杆向肌动蛋白丝的尖端旋转，并与核苷酸结合口袋的上半部分的 N 端亚结构域形成一个以前未识别的界面。该杠杆的尖端取向通过阻止 N 端亚结构域进行打开核苷酸结合位点所需的枢转而阻止 ADP 释放，从而揭示了机械负荷如何通过限制杠杆旋转来抑制 myo1b。第二个 ADP 状态的杠杆采用类似于僵硬的构象，由 myo1b 的特异性元素稳定。我们确定这种构象作为 ADP 释放途径中的中间态的作用。此外，我们将这些结构与其他肌球蛋白的结构进行比较，揭示了肌球蛋白超家族的肌动球蛋白结合位点的结构多样性，并且我们揭示了与纤维状肌动蛋白结合的鬼笔环肽的高分辨率结构，鬼笔环肽是一种有效的肌动蛋白稳定因子。这些结果为理解肌球蛋白超家族的力感应能力范围提供了一个框架。
