Myosin VI challenges the prevailing theory of how myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a 3-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two myosin VI monomers. This unprecedented mechanism generates an extension of the lever arm of myosin VI.

Due to a unique addition to the lever arm-positioning region (converter), class VI myosins move in the opposite direction (toward the minus-end of actin filaments) compared to other characterized myosin classes. However, the large size of the myosin VI lever arm swing (powerstroke) cannot be explained by our current view of the structural transitions that occur within the myosin motor. We have solved the crystal structure of a fragment of the myosin VI motor in the structural state that represents the starting point for movement on actin; the pre-powerstroke state. Unexpectedly, the converter itself rearranges to achieve a conformation that has not been seen for other myosins. This results in a much larger powerstroke than is achievable without the converter rearrangement. Moreover, it provides a new mechanism that could be exploited to increase the powerstroke of yet to be characterized plus-end-directed myosin classes.

It is unclear if the reverse-direction myosin, myosin VI, functions as a monomer or dimer in cells and how it generates large movements on actin. We deleted a stable, single α-helix (SAH) domain that has been proposed to function as part of a lever arm to amplify movements, without impact on in vitro movement or in vivo functions. A myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in vitro, but did not rescue in vivo functions. It was necessary for myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in vitro and rescue endocytosis and Golgi morphology in myosin VI-null fibroblasts. A model for myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in vivo functions.

SUMMARY Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C-terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30–36nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein we present new structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.

No abstract

Molecular motors such as myosins are allosteric enzymes that power essential motility functions in the cell and structural biology is an important tool to decipher how these motors work. Force is produced by myosins upon the actin-driven conformational changes that control the sequential release of the hydrolysis products of ATP (Pi followed by ADP). These conformational changes are amplified by a “lever arm” that includes the region of the motor known as the converter and the adjacent elongated light chain binding region. Analysis of four structural states of the motor provides a detailed understanding of the rearrangements and pathways of communication in the motor necessary for detachment from the actin track and repriming of the motor. However, the important part of the cycle in which force is produced remains enigmatic and awaits new high resolution structures. The value of a structural approach is particularly evident from the clues that have been provided from the structural states of the reverse myosin VI motor. Crystallographic structures have revealed that rearrangements within the converter subdomain occur which explains why this myosin can produce a large stroke in the opposite direction of all other myosins despite a very short lever arm. By providing detailed understanding of the motor rearrangements, structural biology will continue to reveal essential information to solve current enigma such as how actin promotes force production, how motors are tuned for specific cellular roles, or how motor/cargo interactions regulate myosin function in the cell.

In order to assess how lever arm length affects the three-dimensional motions of myosin V during processive motility, two constructs were studied using single molecule polarized total internal reflection fluorescence (polTIRF) microscopy. MyoV6IQ and MyoV4IQ contain 6 and 4 calmodulin (CaM) binding IQ motifs, and otherwise consist of the native myosin V excluding the tail domain. Bifunctional rhodamine labeled CaM replaced a native CaM, giving probe angles b P relative to the actin axis and a P , the azimuth around actin. As with other processive myosins, a P and b P exhibited tilting of the probe with each step. With MyoV6IQ, a P often returned to its initial value after two steps, as expected for nearly straight walking. This behavior enabled us to determine the orientation of the lever arm, a L and b L , as well as q L and f L , the probe angles relative to CaM. b L was 100 and 40 in the leading and trailing heads, respectively. In MyoV4IQ, b P was similar to 6IQ, but a P seldom returned to its earlier value after two steps. This indicates considerable net azimuthal rotation, as expected for smaller step sizes. Thus, lever arm length determines the azimuthal angular path, whereas the axial orientation is likely determined by structural constraints in the motor domain. Modified gliding filament assays were performed using polTIRF to detect twirling of actin about its axis during motility. MyoV6IQ twirled almost exclusively left-handed with a pitch of 1.4 mm. My-oV4IQ twirled with both right-and left-handed pitches of 1.0 and 1.2 mm, respectively. Bidirectional twirling of MyoV4IQ contrasts with every isoform of myosin previously tested (II, native V, VI and X) all of which twirled with a single handedness.

No abstract