Swinging lever mechanism of myosin directly shown by time-resolved cryo-EM

• 成果类型: Article
• 署名作者: 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.
• 署名单位: University of Leeds; University of Leeds; University of Leeds; University of Leeds; Old Dominion University; University of Sheffield; Rutgers University System; Rutgers University New Brunswick; Rutgers University Biomedical & Health Sciences; Max Planck Society
• 刊物名称: Nature
• ISSN/ISSBN: 0028-2166
• DOI: 10.1038/s41586-025-08876-5
• 发表日期: 2025-06-12
• 关键词: grid preparation; actin; muscle; MODEL; complex; refinement; BEHAVIOR; binding; charmm; amber

摘要

Myosins produce force and movement in cells through interactions with F-actin1. 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 swing2,3. 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)4 of a myosin-5 mutant having slow hydrolysis product release5,6. 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 degrees, predominantly along the actin axis, with little twisting. The magnitude of lever swing matches the typical step length of myosin-5 along actin7. 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.