High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

Abstract

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin. The unprecedented number of high-resolution structures of a single myosin finally enabled us to assemble a nearly complete structural model of the myosin-V motor cycle and describe the molecular principles of force production.

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

“…Collectively, we observe numerous myosin-15–specific interactions with F-actin, consistent with specialization of this interface for myosin-15 function in stereocilia. At the actomyosin-15 interface, limited loop 2 interactions, unique loop 3 interactions, and primarily polar/electrostatic interactions through the CM loop likely predominate in determining myosin-15’s specific functional properties, as HLH-F-actin interactions are largely conserved in the actomyosin structures reported to date ( 21 – 23 , 34 , 41 , 50 ). However, as divergent myosins can have similar thermodynamic and kinetic tuning of their mechanochemical cycles, it remains challenging to delineate the roles of individual contacts at the actin-myosin interface, which are likely to modulate the intrinsic structural dynamics of the motor domain to control its function through complex mechanisms ( 51 ).…”

“…Similar properties are hallmarks of other well-characterized unconventional myosins whose F-actin engagement is modulated by force to regulate processive cargo trafficking (myosin-5a and myosin-6) and cytoskeleton-membrane tethering (myosin-1b) functions ( 17 , 18 ). Biophysical ( 19 , 20 ) and structural ( 21 – 23 ) studies of these myosins have revealed coupling between nucleotide binding pocket rearrangements enabling ADP release and minor lever-arm swings of differing magnitude and directionality, facilitating tuned mechanical gating of ADP release/ATP rebinding through their lever arms to mediate force-sensitive F-actin dissociation ( 24 ). By analogy to these motors, myosin-15 has also been speculated to be force sensitive ( 16 ), but this has not been experimentally examined and it remains unclear whether mechanical gating of ADP release through this mechanism is a universal feature of myosins.…”

Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin

“…Our cryo-EM structure of the TcART-F-actin complex revealed the extended interface between the toxin and its substrate (Fig S9). Interestingly, the position where TcART binds to F-actin partly overlaps with the position of other F-actin interacting proteins and peptides, such as myosin-V 41 , ExoY toxin from Pseudomonas aeruginosa 42 , and the Lifeact peptide 43 . In particular, all these proteins possess a hydrophobic residue that is homologous to Y183 of TcART (M515 in myosin-V, F374 in ExoY and F10 in Lifeact).…”

Structural basis of actin filament assembly and aging

“…Since then, numerous cryo-EM studies by our group and others have revealed the F-actin architecture in all nucleotide states 35,36 and in complex with a variety of ABPs such as cofilin 37,38 and myosin [39][40][41] . However, all previously published F-actin structures were solved at moderate resolutions of ~3 -4.5 Å, and therefore did not display sufficient details to allow for the modeling of solvent molecules and exact positions of amino-acid side chains.…”

“…In 2015, our group published the 3.7-Å single-particle cryo-EM structure of F-actin in complex with the ABP tropomyosin 34 , which represented the first complete atomic model of actin in the filamentous state. Since then, numerous cryo-EM studies by our group and others have revealed the F-actin architecture in all nucleotide states 35, 36 and in complex with a variety of ABPs such as cofilin 37, 38 and myosin 39–41 . However, all previously published F-actin structures were solved at moderate resolutions of ∼3 - 4.5 Å, and therefore did not display sufficient details to allow for the modeling of solvent molecules and exact positions of amino-acid side chains.…”