The structure of the myosin VI motor reveals the mechanism of directionality reversal

Menetrey, J.; Bahloul, Amel; Wells, Amber L.; Yengo, Christopher M.; Morris, Carl; Sweeney, H. Lee; Houdusse, Anne

doi:10.1038/nature03592

Cited by 205 publications

(249 citation statements)

References 41 publications

(51 reference statements)

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“…This observation raises the question as to whether the extended light chain conformation plays a role in determining processivity and step size. The crystal structure of myosin VI, including two CaM molecules, a Ca 2ϩ -free CaM bound to the IQ motif and a Ca 2ϩ -loaded CaM bound to a unique reverse gear insert present in the converter domain of this myosin, has just been reported (44). However, the CaM molecule bound to the IQ motif is mostly disordered and its conformation could not be unambiguously determined.…”

Section: Light Chain-light Chain Interactions As a Function Of The Spmentioning

confidence: 99%

Structure of the light chain-binding domain of myosin V

Terrak

Rębowski

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

118

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Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of Ϸ36 nm along the actin helical track, depend on its unusually long light chainbinding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2-IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM-IQ complexes, comparative sequence analysis, and the previously determined structures calmodulin ͉ IQ motif ͉ x-ray crystallography ͉ FRET M yosin V is a molecular motor involved in a range of organelle-transporting functions, including the transport of melanosomes and synaptic vesicles in mammals and vacuoles and mRNA in yeast (1-4). Myosin V is composed of two identical heavy chains and 12 light chains. Each heavy chain consists of an N-terminal motor domain, containing the actinbinding and ATP catalytic sites, followed by the light chainbinding domain (LCBD), formed by six IQ motifs in tandem, and the tail domain, composed of regions of coiled-coil and a globular domain involved in cargo binding. The coiled-coil regions mediate the association of the heavy chains into dimers. The IQ motifs are Ϸ25-aa segments, centered around the consensus sequence IQxxxRGxxxR, and constitute the binding sites for the light chains, which can be either calmodulin (CaM) or CaM-related molecules (5, 6).A number of features distinguish myosin V from other myosin families. Myosin V has a high duty cycle, defined as the property to remain attached to actin for a large fraction of the mechanochemical cycle (7-9). The high duty cycle of myosin V is explained by a slow rate of ADP release, which becomes the rate-limiting step in the ATPase cycle (7). This kinetic adaptation allows myosin V to take multiple steps without dissociating from the actin filament, that is, myosin V is a processive motor (10-12). Linked with processivity is the ability of myosin V to take large steps of Ϸ36 nm (10), a distance equal to the helical repeat of the actin filament. Such a step size allows myosin V to walk in a straight line on the actin filament, in a hand-over-hand fashion (13-16). These characteristics seem to adapt myosin V for its cellular function, the transport of large cargoes atop the actin filament while avoiding collisions with cellular structures (3). Yet, central to this motor's uniqueness is its unusually long LCBD (4). A number of laboratories have recently established a direct connection between the length and structural integrity of the LCBD and the step size and processivity of myosin V (4,(17)(18)(19)(20). These studies focus on the role of the LCBD as a passive structural device whose function is to amplify small nucleotidedependent motions originating in the motor doma...

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Section: Light Chain-light Chain Interactions As a Function Of The Spmentioning

confidence: 99%

Structure of the light chain-binding domain of myosin V

Terrak

Rębowski

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

118

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“…Employing the reasonable assumption that the insert and IQ domain remain rigidly attached to the converter domain, Ménétrey et al (12) modeled the unknown prestroke conformation of myosin VI by rotating the converter to match the prestroke conformation of (ϩ)-end-directed myosins. This model predicted a power stroke that was directed toward the (Ϫ) end but far smaller (Ϸ2.5 nm) than the measured myosin VI stroke size.…”

Section: Myosin VI Must Have An Unusual Power Strokementioning

confidence: 99%

The power stroke of myosin VI and the basis of reverse directionality

Bryant

Altman

Spudich

2007

Proc. Natl. Acad. Sci. U.S.A.

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Myosin VI supports movement toward the (؊) end of actin filaments, despite sharing extensive sequence and structural homology with (؉)-end-directed myosins. A class-specific stretch of amino acids inserted between the converter domain and the lever arm was proposed to provide the structural basis of directionality reversal. Indeed, the unique insert mediates a 120°redirection of the lever arm in a crystal structure of the presumed poststroke conformation of myosin VI [Mé né trey J, Bahloul A, Wells AL, Yengo CM, Morris CA, Sweeney HL, Houdusse A (2005) Nature 435:779 -785]. However, this redirection alone is insufficient to account for the large (؊)-end-directed stroke of a monomeric myosin VI construct. The underlying motion of the myosin VI converter domain must therefore differ substantially from the power stroke of (؉)-end-directed myosins. To experimentally map out the motion of the converter domain and lever arm, we have generated a series of truncated myosin VI constructs and characterized the size and direction of the power stroke for each construct using dual-labeled gliding filament assays and optical trapping. Motors truncated near the end of the converter domain generate (؉)-end-directed motion, whereas longer constructs move toward the (؊) end. Our results directly demonstrate that the unique insert is required for directionality reversal, ruling out a large class of models in which the converter domain moves toward the (؊) end. We suggest that the lever arm rotates Ϸ180°between pre-and poststroke conformations.actin ͉ molecular motor ͉ pointed end ͉ swinging cross-bridge T he basic actomyosin motor has been embellished, altered, and reused many times through the evolution of diverse members of the myosin superfamily (1). Class VI myosins are highly specialized (Ϫ)-end-directed motors involved in a growing list of functions in animal cells, including endocytosis, cell migration, and maintenance of stereociliar membrane tension (2). Initial biophysical characterization (3-5) of myosin VI raised two important questions concerning the structural basis of its unique motor properties. First, how does the motor achieve reverse directionality? And second, how does dimeric myosin VI take long steps along actin, matching the stride of myosin V without the apparent benefit of a long lever arm?A Backward-Moving Myosin Myosin VI attracted attention as a candidate for a (Ϫ)-enddirected motor after the identification of a class-specific insert following the converter domain. In the swinging cross-bridge model of actomyosin function, the converter domain transmits motion from the myosin head to the C-terminal lever arm, generating a directed power stroke. Wells et al. (3) hypothesized that a structure inserted between the converter domain and the lever arm could redirect the stroke and lead to backward movement. The predicted reverse directionality of myosin VI was experimentally confirmed (3), but the precise mechanism of this remarkable adaptation remained unclear, including whether the unique insert was necess...

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“…Thus, reversal of direction in Kinesin-14 motors is accomplished by the evolution of a unique mechanical element that can take advantage of existing conformational changes in the catalytic core, as is also true for direction reversal by the myosin VI motor 26 . Unlike conventional kinesin, which is built for long-distance processive movement, Ncd is a nonprocessive motor27 , 28 designed for microtubule crossbridging and tension development in meiotic or mitotic spindles29.…”

mentioning

confidence: 99%

A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd

Endres

Yoshioka

Milligan

et al. 2005

Nature

156

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Kinesins are microtubule-based motor proteins that power intracellular transport 1,2 . Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described [3][4][5] . By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors (such as Drosophila Ncd6 , 7) are less well understood. Using cryo-electron microscopy, here we demonstrate that a coiled-coil mechanical element of microtubule-bound Ncd rotates ~70° towards the minus end upon ATP binding. Extending or shortening this coiled coil increases or decreases velocity, respectively, without affecting ATPase activity. An unusual Ncd mutant that lacks directional preference 8 shows unstable nucleotide-dependent conformations of its coiled coil, underscoring the role of this mechanical element in motility. These results show that the force-producing conformational change in Ncd occurs on ATP binding, as in other kinesins, but involves the swing of a lever-arm mechanical element similar to that described for myosins.Crystal structures of the Ncd dimer show a coiled coil extending directly from the pair of catalytic cores (termed the 'neck')9 -11, and mutagenesis experiments indicate that the proximal portion of the neck functions as the mechanical element that powers minus-enddirected motility9 , 12. Previous cryo-electron microscopy (cryo-EM) studies of dimeric Ncd suggested that the neck extends towards the microtubule plus end in the nucleotide-free, microtubule-bound complex. This assignment was based on the position of an amino-terminal Src homology domain 3 (SH3) domain used as a marker for the neck, because the neck itself was not visible in this study13. Neither the neck nor the SH3 marker was observed in the presence of AMPPNP, however, which led to the proposal that ATP binding causes the Ncd neck to transition into a detached and mobile state and that the shift in the average position of this mobile neck towards the microtubule minus end is the driving force for Ncd motility13. A conflicting model of the nature and timing of the power stroke has been proposed on the basis of the crystal structure of a motility-deficient Ncd point mutant (NcdN600K)10, which shows the neck in a different orientation that is predicted to point towards the microtubule minus end. It has been proposed that this structure represents the nucleotide-free state of the motor and that a minus-end-directed power stroke occurs on release of ADP. Thus, the exact nature and timing of the motility-producing conformational change in Ncd remains uncertain.Here we have used cryo-EM to investigate the position of the neck in dimeric Ncd (residues 281-700; see Methods) bound to a microtubule in the absence of nucleotide and in the presence of two mimics of an ATP-like state, AMPPNP (a non-hydrolysable ATP analogue) and (a transition-state analogue). Helical image analysis of 15 protofilament microtubules was used to ca...

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The structure of the myosin VI motor reveals the mechanism of directionality reversal

Cited by 205 publications

References 41 publications

Structure of the light chain-binding domain of myosin V

Structure of the light chain-binding domain of myosin V

The power stroke of myosin VI and the basis of reverse directionality

A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd

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