Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography

Abstract

Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can 'walk' hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5-7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head-S2 junction places the cargo-binding domain near the motor domain's ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.

The globular tail domain puts on the brake to stop the ATPase cycle of myosin Va

“…Because the assigned GTD-binding site has no direct interaction with the ATP-binding site, they proposed that the GTD regulates motor activity allosterically (10). However, Taylor and colleagues assigned the GTD-binding site to loop 1, which is near the entrance of the ATP-binding pocket in the motor domain (11). They proposed that binding of the GTD to loop 1 decreases the rates of nucleotide exchange, thus inhibiting the ATPase activity of the motor domain (11).…”

“…However, Taylor and colleagues assigned the GTD-binding site to loop 1, which is near the entrance of the ATP-binding pocket in the motor domain (11). They proposed that binding of the GTD to loop 1 decreases the rates of nucleotide exchange, thus inhibiting the ATPase activity of the motor domain (11). However, the recently solved crystal structure of the motor domain of chicken myosin Va (12,13) and the GTD of Myo2p, a yeast myosin V (14), show that it is unlikely that the GTD binds to lobe P117-P137 and loop-1 simultaneously.…”

Switching of myosin-V motion between the lever-arm swing and Brownian search-and-catch

“…The minimal unit for myosin-V force generation is a motor or head domain, which includes the actin and nucleotide-binding site, and a lever-arm, which has six calmodulin-binding motifs 10,11 . A crystal structure and electron micrographs have suggested the lever-arm tilts in conjunction with ATP hydrolysis cycle 12,13 , leading to the model assumes that the 'lever-arm swing' is the dominant process for force generation 14,15 .…”

Antiparallel coiled-coil–mediated dimerization of myosin X

“…In this prevailing model, the parallel dimerization allows the proper transduction of the mechanic forces generated by ATP hydrolysis and is thus necessary for the "walking" of myosins along actin filaments (1-6). However, the parallel coiled-coil dimerization model has been directly demonstrated only in myosin II and V (7,8). Instead of coiled-coil-mediated dimerization, certain unconventional myosins have been shown to undergo cargo bindingmediated dimerization (9,10).…”