Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D., et al. (1999) Nature (London) 400, 590 -593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin. Myosin V was identified in chicken brain cytoplasmic extracts as a calmodulin binding protein with actin-activated MgATPase activity and the ability to translocate actin filaments (1-3). By electron microscopy of rotary shadowed images, native chicken brain myosin V has two heads and two heavy chains that associate through a long coiled-coil domain in its tail region. Unlike myosin II, myosin V does not form filaments and is believed to act as a single molecule (4).The unusual structure, cellular functions (5, 6), and steady-state biochemical properties (7), as well as single molecule mechanics (4) of myosin V support it being a processive motor. That is, for each diffusional encounter, a single (two-headed) myosin V molecule may be capable of going through multiple ATPase cycles and traveling long distances, equivalent to many individual steps of the motor, along its actin track before dissociating.The myosin molecule, whether in the sarcomere of a muscle (myosin II) or moving vesicles on actin tracks (myosin V), goes through a characteristic cyclic interaction with actin (Scheme 1; predominant pathway is shown in bold). The key steps include the rapid binding of ATP to actin-bound myosin, the hydrolysis of ATP, the sequential release of phosphate (P i ) and ADP, and the rebinding of ATP. During the cycle, the myosin populates either the weak-binding states or strong-binding states (Scheme 1). Weakbinding myosin states dynamically detach and rebind to actin with a low affinity (K d Ͼ 1 M), whereas the strong-binding myosin states remain bound to actin with a high affinity (K d Ͻ Ͻ 1 M). Mechanical force generation, work, and directed movement on actin are possible only during periods when the myosin is strongly bound to actin. The fraction of the ATPase cycle that the myosin spends in ...

The ability to sense molecular tension is crucial for a wide array of cellular processes, including the detection of auditory stimuli, control of cell shape, and internalization and transport of membranes. We show that myosin-I, a motor protein which has been implicated in powering key steps in these processes, dramatically alters its motile properties in response to tension. We measured the displacement generated by single myosin-I molecules, and we determined the actin-attachment kinetics with varying tensions using an optical trap. The rate of myosin-I detachment from actin decreases > 75-fold within 2 pN of tension, resulting in myosin-I transitioning from a low (< 0.2) to a high duty-ratio motor (> 0.9). This impressive tension sensitivity supports a role for myosin-I as a molecular force sensor.Myosin-Is are the widely expressed, single-headed, and membrane-associated members of the myosin superfamily that participate in regulating membrane dynamics and structure in nearly all eukaryotic cells. Eight myosin-I isoforms are expressed in humans, making it the largest "unconventional" myosin family (1). One specific and well characterized molecular function of a myosin-I isoform (myo1c) is to dynamically provide tension to sensitize mechano-sensitive ion channels responsible for hearing (2-4). Myosin-Is also power the transport and deformation of membranes in the cell cortex and in apical cell projections (5-8). To perform these roles, myosin-Is have been proposed to act as tension-sensing proteins that alter their ATPase and mechanical properties in response to changes in loads imparted by their cellular cargos (3,9). Biochemical, structural, and single molecule experiments suggest that some myosin-I isoforms (myo1a, myo1b, myo1c) are adapted to sense tension. Specifically, it has been shown that myosin-I produces its working stroke displacement in two substeps (10). An initial displacement of the lever arm is followed by an additional ∼ 32° rotation that accompanies ADP release (11). Since ADP release kinetically limits the rate of the detachment of myosin from actin (12,13), the extra lever arm rotation has been proposed to be a force-sensing substep, with the presence of resisting loads preventing this lever arm rotation, and thus inhibiting ADP release and actin detachment. A similar model has been proposed for gating of myosin-V motor activity during processive motility (14).We characterized the motor activity of myosin-I by measuring single-molecule forcegenerating events using the three-bead configuration, in which a single actin filament, suspended between two beads held separate by optical traps, is brought close to the surface of a pedestal-bead that is sparsely coated with myosin (15). A recombinant myo1b splice isoform containing five IQ motifs and a C-terminal biotinylation tag (16) was attached to streptavidincoated pedestal beads (17). Single-molecule actomyosin interactions at low loads were acquired using low trap stiffness (∼ 0.022 pN/nm; Fig. 1A) in the presence of 1 -50 μM ATP. NI...

Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of P i release, which lowers the K ATPase but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins.Myosin VI is unique among members of the myosin superfamily of molecular motors in that it moves toward the pointed ends of actin filaments as opposed to the barbed ends (1). Although the cellular roles of myosin VI are not defined, it has been implicated in membrane trafficking and organelle transport (2-4) as well as maintaining the structural integrity of inner ear hair cells (5).Native myosin VI has two "heads," or catalytic domains, that are thought to be regulated by p21-activated kinase phosphorylation in vivo (3). Although the phosphorylation site was not identified directly, it was mapped between amino acids 308 and 631 and believed to be Thr 406 of the actin-binding interface, because flanking sequences have p21-activated kinase recognition sites, and phosphorylation of Thr 406 would be consistent with the TEDS rule (6). All myosins have an acidic residue (Asp or Glu) at this position and are constitutively active or, in the case of Acanthamoeba myosin I, have a serine or threonine that when phosphorylated increases the ATPase rate more than 20-fold (7). Myosin I heavy chain kinases are p21-activated kinase homologues (8, 9). Mutagenesis of Acanthamoeba myosin I demonstrates that the phosphorylated and unphosphorylated states are mimicked by replacement of the phosphorylatable threonine with a glutamate or alanine, respectively (10).The molecular mechanism by which myosin VI achieves its reverse directionality is not known, but it might be linked to unique aspects of the converter domain, the structural element between the motor and light chain binding domains (1). In this study, we define the kinetic mechanism of the actomyosin VI ATPase cycle to ascertain if pointed end-directed motility requires unique biochemical adaptations. In addition, to determine whether myosin VI is regulated by h...

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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.