Movement is one of the hallmarks of life. Myosins are molecular motors that use cellular ATP to power interactions with actin filaments and create force and directed movement. Myosin II is essential for muscle contraction and cytokinesis but other members of the myosin superfamily play roles in phagocytosis, cortical cell tension, signal transduction, endocytosis, exocytosis, and intracellular vesicle transport. Conformational changes in the myosin motor allow it to cycle through defined structural states which differ in their nucleotide- and actin- affinities (Figure 23) [1].


Fig. 23: Structural states of myosin during the contractile cycle. 1-Without bound nucleotide, myosin is strongly bound to actin (rigor state). 2-ATP binding dissociates the complex actin-myosin. 3-ATP is then hydrolysed in ADP+Pi. There is a swing of the lever arm (green). 4-Myosin can rebind to actin, release its hydrolysis products and produce its force. 5- Myosin is again strongly bound to actin without nucleotide bound.

Myosin hydrolyses ATP in states that have a weak affinity for actin, and strain is produced when myosin rebinds to the actin filament, which accelerates the release of the ATPase products from the motor. The proposed mechanism for force generation, the swinging lever arm hypothesis (Figure 23), is largely based on high-resolution structures of the weakly bound states. It postulates that the swing of the lever arm is responsible for the force production. However, since force production occurs when myosin is strongly bound to actin, details of the strongly-bound states of myosin are essential to understand how force is produced.

The affinity of myosin for actin depends directly on the bound nucleotide in the myosin active site. So far, high-resolution structures of myosin have provided atomic details of three structural states of the motor that correspond to weak actin-binding states. While these studies provided a detailed description of how ATP hydrolysis occurs, they were insufficient to describe the details of the structural changes that lead to the strong actin-affinity states. Based on kinetic evidence that an unconventional myosin, class V myosin, populates a unique state in the absence of nucleotide and actin, we hypothesised that its high resolution structure could for the first time reveal features of a strong actin-binding, force generating state.

We expressed and crystallised a class V myosin containing only a motor domain and the first calmodulin-binding site (first part of the lever arm). The 2.0 Å refined structure (from a dataset collected on ID29) revealed a novel conformation for the myosin head (without bound nucleotide) in which all of the key features that were predicted to occur in the myosin state with the strongest affinity for F-actin (i.e. rigor state) are realised. The nucleotide-binding site has adopted new conformations of the nucleotide-binding elements that reduce the affinity for the nucleotide. The major cleft in the molecule has closed resulting in a drastically different actin-binding interface (Figure 24), and the lever arm has assumed a position consistent with that in an actomyosin rigor complex. However, the way in which this was achieved was totally unexpected. The distortion of the central seven-stranded beta-sheet of the motor domain is essential to allow large relative movements of the subdomains. These changes in the motor reveal elements of the structural communication between the actin-binding interface and the nucleotide binding site of myosin that underlie the mechanism of chemo-mechanical transduction.

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Fig. 24: Comparison of the rigor-like state of myosin V and the ATP-bound state of myosin II. The myosin V structure reveals at atomic resolution how the major cleft in the molecule closes and leads to a new actin-binding interface.

n conclusion, the high-resolution structure of the motor domain of class V myosin provides critical information for our understanding of how myosin produces force. In revealing unexpected rearrangements in the molecule, this new structure reveals the structural basis of the coupling between the binding and release of actin and nucleotide, and the strong inverse linkage that has remained enigmatic. While closure of the 50 kDa cleft generates a strong actin-binding interface, ATP binding in the myosin active site allows myosin to re-open this large cleft and to separate the actin-binding interface into two separate entities which allow myosin dissociation from actin.

[1] A. Houdusse, and H.L. Sweeney, Curr. Opin. Struct. Biol. 11,182-194 (2001).
[2] A.D.E. Mehta et al., Nature 400, 590-593 (1999).

Principal Publication and Authors
P.D. Coureux (a), A.L. Wells (b), J. Menetrey (a), C.M. Yengo (b), C.A. Morris (b), H.L. Sweeney (b), A. Houdusse (a). Nature 425, 419-23 (2003).
(a) Institut Curie CNRS, UMR144, Paris (France)
(b) University of Pennsylvania School of Medicine (USA)