Swinging lever mechanism of myosin directly demonstrated by time-resolved cryoEM
Abstract Myosins are essential for producing force and movement in cells through their interactions with F-actin. Generation of movement is proposed to occur through structural changes within the myosin motor domain, fuelled by ATP hydrolysis, that are amplified by a lever swing1, transitioning myosin from a primed (pre-powerstroke) state to a post-powerstroke state. However, the initial, primed actomyosin state, proposed to form prior to lever swing, has never been observed. Nor has the mechanism by which actin catalyses myosin ATPase activity been resolved. To address this, we performed time-resolved cryoEM of a myosin-5 mutant having slow hydrolysis product release. Primed actomyosin was captured 10 ms after mixing primed myosin with F-actin, whereas post-powerstroke actomyosin predominated at 120 ms, with no abundant intermediate structures. The structures were solved to 4.4Å and 4.2Å global resolution respectively. The primed motor binds to actin through its lower 50 kDa subdomain, with the actin-binding cleft open and Pi release prohibited. N-terminal actin interactions with myosin promote rotation of the upper 50 kDa subdomain, which closes the actin-binding cleft, and enables Pi release. Formation of upper 50 kDa subdomain interactions with actin creates the strong-binding interface required for effective force production. The myosin-5 lever swings through an angle of 93°, predominantly along the actin axis, with little twisting, to produce the post-powerstroke state. The magnitude of the lever swing matches the typical step length of myosin-5 walking along actin. These time-resolved structures directly demonstrate the swinging lever mechanism, ending decades of conjecture on how myosin produces force and movement..
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Preprint| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
bioRxiv.org - (2024) vom: 10. Jan. Zur Gesamtaufnahme - year:2024 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Klebl, David P. [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2024.01.05.574365 |
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| PPN (Katalog-ID): |
XBI042099692 |
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| 100 | 1 | |a Klebl, David P. |e verfasserin |0 (orcid)0000-0002-3581-8334 |4 aut | |
| 245 | 1 | 0 | |a Swinging lever mechanism of myosin directly demonstrated by time-resolved cryoEM |
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| 520 | |a Abstract Myosins are essential for producing force and movement in cells through their interactions with F-actin. Generation of movement is proposed to occur through structural changes within the myosin motor domain, fuelled by ATP hydrolysis, that are amplified by a lever swing1, transitioning myosin from a primed (pre-powerstroke) state to a post-powerstroke state. However, the initial, primed actomyosin state, proposed to form prior to lever swing, has never been observed. Nor has the mechanism by which actin catalyses myosin ATPase activity been resolved. To address this, we performed time-resolved cryoEM of a myosin-5 mutant having slow hydrolysis product release. Primed actomyosin was captured 10 ms after mixing primed myosin with F-actin, whereas post-powerstroke actomyosin predominated at 120 ms, with no abundant intermediate structures. The structures were solved to 4.4Å and 4.2Å global resolution respectively. The primed motor binds to actin through its lower 50 kDa subdomain, with the actin-binding cleft open and Pi release prohibited. N-terminal actin interactions with myosin promote rotation of the upper 50 kDa subdomain, which closes the actin-binding cleft, and enables Pi release. Formation of upper 50 kDa subdomain interactions with actin creates the strong-binding interface required for effective force production. The myosin-5 lever swings through an angle of 93°, predominantly along the actin axis, with little twisting, to produce the post-powerstroke state. The magnitude of the lever swing matches the typical step length of myosin-5 walking along actin. These time-resolved structures directly demonstrate the swinging lever mechanism, ending decades of conjecture on how myosin produces force and movement. | ||
| 650 | 4 | |a Biology |7 (dpeaa)DE-84 | |
| 650 | 4 | |a 570 |7 (dpeaa)DE-84 | |
| 700 | 1 | |a McMillan, Sean N. |0 (orcid)0000-0002-6833-5523 |4 aut | |
| 700 | 1 | |a Risi, Cristina |4 aut | |
| 700 | 1 | |a Forgacs, Eva |4 aut | |
| 700 | 1 | |a Virok, Betty |4 aut | |
| 700 | 1 | |a Atherton, Jennifer L. |4 aut | |
| 700 | 1 | |a Stofella, Michele |4 aut | |
| 700 | 1 | |a Winkelmann, Donald A. |4 aut | |
| 700 | 1 | |a Sobott, Frank |4 aut | |
| 700 | 1 | |a Galkin, Vitold E. |4 aut | |
| 700 | 1 | |a Knight, Peter J. |0 (orcid)0000-0002-8850-9500 |4 aut | |
| 700 | 1 | |a Muench, Stephen P. |0 (orcid)0000-0001-6869-4414 |4 aut | |
| 700 | 1 | |a Scarff, Charlotte A. |0 (orcid)0000-0001-6168-0060 |4 aut | |
| 700 | 1 | |a White, Howard D. |4 aut | |
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