High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing

Abstract

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.

Converter domain mutations in myosin alter structural kinetics and motor function

“…slowing the detachment rate at higher loads to increase the time myosin is attached to actin). The structural mechanism of myosin mechanosensitivity has been investigated in recent studies using X-ray crystallography and cryo-EM, which demonstrated different conformations of the lever arm in nucleotide states that are thought to play a crucial role in mediating force production (35)(36)(37)(38). The two converter mutants we investigated in the current study demonstrated little difference in unloaded sliding velocity but displayed increased sensitivity to external loads examined with load-dependent mixed motor motility assays (Fig.…”

“…There is a second power stroke that occurs during the transition between actomyosin. ADP states, which was identified in structural studies of smooth muscle myosin, myosin V, and myosin IB (35,38,53). Kinetic simulations demonstrate that the power stroke and phosphate release transients are best fit by a fast power stroke (k ϩPWF ) followed by rapid phosphate release (k ϩPi ) ( Figs.…”

Actin's N‐terminal acetyltransferase uncovered

“…Of particular interest is myosin, which binds in a cleft between two actin subunits along the long‐pitch helix of the actin filament, within contact distance to the N‐termini of both actin subunits. The figure shows the recently determined cryo‐EM structure of ADP‐myosin‐IB bound to the actin filament (Mentes et al, ) [Color figure can be viewed at wileyonlinelibrary.com]…”

Class I myosins: Highly versatile proteins with specific functions in the immune system

“…Analysis by cryo‐electron microscopy of Myo1b shows that this protein has two binding states to ADP, separated by a 25° lever arm; the first ADP state is important to open the nucleotide‐binding site, the second state allows the stabilization of myosin with F‐actin. In accordance with this information, conformational changes in myosin superfamily are regulated by rotation of the lever arm that affects their association with filamentous actin …”