The Structural Basis of Myosin V Processive Movement as Revealed by Electron Cryomicroscopy

The Program of Cell Adhesion, The Burnham Institute, La Jolla, California 92037, USA.
Molecular Cell (Impact Factor: 14.02). 10/2005; 19(5):595-605. DOI: 10.1016/j.molcel.2005.07.015
Source: PubMed


The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.

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    • "They found a tight one-to-one coupling of nucleotide utilization for each step in the single molecule fluorescence assay and found that the leading head had little tendency to release nucleotide as long as the trail head was attached. This is consistent with load dependence effectively gating the kinetics of the two heads to aid in the processive movement, as has been suggested in the earlier single molecule mechanical experiments (Veigel et al., 2001). "
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    ABSTRACT: Striated muscle is an elegant system for study at many levels. Much has been learned about the mechanism of contraction from studying the mechanical properties of intact and permeabilized (or skinned) muscle fibers. Structural studies using electron microscopy, X-ray diffraction or spectroscopic probes attached to various contractile proteins were possible because of the highly ordered sarcomeric arrangement of actin and myosin. However, to understand the mechanism of force generation at a molecular level, it is necessary to take the system apart and study the interaction of myosin with actin using in vitro assays. This reductionist approach has lead to many fundamental insights into how myosin powers muscle contraction. In addition, nature has provided scientists with an array of muscles with different mechanical properties and with a superfamily of myosin molecules. Taking advantage of this diversity in myosin structure and function has lead to additional insights into common properties of force generation. This review will highlight the development of the major assays and methods that have allowed this combined reductionist and comparative approach to be so fruitful. This review highlights the history of biochemical and biophysical studies of myosin and demonstrates how a broad comparative approach combined with reductionist studies have led to a detailed understanding of how myosin interacts with actin and uses chemical energy to generate force and movement in muscle contraction and motility in general.
    Frontiers in Physiology 03/2014; 5:90. DOI:10.3389/fphys.2014.00090 · 3.53 Impact Factor
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    • "Evidence suggests that the answer may be that strong binding to actin stiffens the head. 3-D reconstructions of actin filaments saturated with myosin heads from various sources [32] [46] [47] [48] consistently show strong features in the levers, and a lack of contact between them, out to high radius from the actin, implying that the lever maintains a rather fixed geometric relationship to the actin helix, i.e. it is not flexing at the MD-lever junction. NMR studies of myosin heads found over 20% of mobile structure and all this became immobile upon binding actin [49]. "
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    ABSTRACT: We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~23pN·nm/rad(2) is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor-lever junction.
    Journal of Molecular Biology 12/2013; 426(4). DOI:10.1016/j.jmb.2013.11.028 · 4.33 Impact Factor
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    • "Image analysis of actin filaments bound by MVt. A hybrid procedure (Volkmann et al., 2005) that combines single-particle reconstruction approaches with helical symmetry (Egelman, 2000) was used to obtain the reconstructions. 14,396 small overlapping segments of actin filaments with bound chicken MVt were selected from the micrographs. "
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    ABSTRACT: Vinculin and its splice variant, metavinculin (MV), are key elements of multiple protein assemblies linking the extracellular matrix to the actin cytoskeleton. Vinculin is expressed ubiquitously, whereas MV is mainly expressed in smooth and cardiac muscle tissue. The only difference in amino acid sequence between the isoforms is a 68-residue insert in the C-terminal tail domain of MV (MVt). Although the functional role of this insert remains elusive, its importance is exemplified by point mutations that are associated with dilated and hypertrophic cardiomyopathy. In vinculin, the actin binding site resides in the tail domain. In this paper, we show that MVt binds actin filaments similarly to the vinculin tail domain. Unlike its splice variant, MVt did not bundle actin filaments. Instead, MVt promoted severing of actin filaments, most efficiently at substoichiometric concentrations. This surprising and seemingly contradictory alteration of vinculin function by the 68-residue insert may be essential for modulating compliance of vinculin-induced actin bundles when exposed to rapidly increasing external forces.
    The Journal of Cell Biology 05/2012; 197(5):585-93. DOI:10.1083/jcb.201111046 · 9.83 Impact Factor
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