Myosin VI: an innovative motor that challenged the swinging lever arm hypothesis

Abstract

The swinging crossbridge hypothesis states that energy from ATP hydrolysis is transduced to mechanical movement of the myosin head while bound to actin. The light chain-binding region of myosin is thought to act as a lever arm that amplifies movements near the catalytic site. This model has been challenged by findings that myosin VI takes larger steps along actin filaments than early interpretations of its structure seem to allow. We now know that myosin VI does indeed operate by an unusual approximately 180 degrees lever arm swing and achieves its large step size using special structural features in its tail domain.

Figure 1. The swinging lever arm hypothesis

Schematic (a) and crystal structures (b,c) of the myosin II head domain in pre-stroke and post-stroke states. The crystal structure of myosin is based on that of Rayment et al (protein data bank identifier 2MYS), with its lever arm extended on the basis of the structure obtained by Dominguez et al (protein data bank identifier 1BR2). All myosins are composed of a globular catalytic head domain, a converter and a lever arm. Conformational changes in the catalytic head are amplified by the lever arm during the ATPase cycle through an ~ 70° rotation of the converter. The lever arm of myosin II consists of an α-helix wrapped with two light chains (not shown here). The end of the lever arm is connected by the tail domains to a myosin thick filament in the case of myosin II, or to vesicular cargo in the case of the unconventional myosin V and myosin VI.

Figure 2. Schematic of myosin VI structure and some of its cellular functions

a | Myosin VI consists of (sequentially from the amino terminus to the carboxy terminus) a catalytic head with a converter domain, two light chain-binding domains, a proximal tail, a medial tail and a cargo-binding domain (the distal tail (see REF. 32) has been incorporated into the cargo-binding domain here). b | Myosin VI has been implicated in several cellular processes, some of which are depicted here. For example, myosin VI is involved in the transfer of endocytic vesicles from clathrin-coated pits to endosomes along the cortical actin filament network. It is also found at the base of stereociliary bundles of hair cells (specialized filipodia) of the inner ear, where it is essential for their structural integrity, and has been found to have a role in maintaining normal Golgi morphology.

Figure 3. Schematic of the myosin VI lever arm and tail domains

The amino acid numbers of the head, unique insert (UI), calmodulin-binding IQ motif, proximal tail, medial tail and cargo-binding domain of human myosin VI. The amino acid sequence of the ER/K motif-containing α-helix in the medial tail is shown in the expansion. The colours in this motif are used to depict the charge of the different amino acids based on standard CPK representation: positively charged amino acids (containing the primary amine and, therefore, nitrogen) are blue, negatively charged amino acids (containing an acid, for example oxygen) are orange and hydrophobic residues are green.

Figure 4. Reversing the direction of myosin VI

The unique insert in the myosin VI lever arm changes the orientation of the lever arm as it emerges from the converter. a | Myosin VI in the post-stroke conformation (protein data bank identifier 2BK1). b | Myosin VI in the pre-stroke conformation (protein data bank identifier 2V26). The converter of myosin VI rotates ~ 180°, compared with ~ 70° in other myosins, resulting in an ~ 180° swing in the myosin VI lever arm from pre-stroke to post-stroke states. The blown up structures of the converters at the bottom of panels a and b show the change in the converter conformation between post-stroke and pre-stroke states. The individual α-helices and β-sheets are shown in different colours. c | The stroke size of myosin VI with one versus two light chain-binding domains, and swinging through 180°and 70°.

Figure 5. Schematic of lever arms in different myosins examined as single-headed species

Myosin II has a short lever arm with two light chains bound that swings ~ 70°, resulting in an ~ 10 nm stroke (left). Myosin V has a longer lever arm with six light chains bound that also swings ~ 70°, resulting in an ~ 20 nm stroke (centre). Myosin VI has a lever arm with two light chains bound, a globular proximal tail and a single, rigid α-helix, all of which swing ~ 180°, resulting in a large stroke of ~ 30 nm (right).

Figure 6. The ER/K motif-containing α-helix in the myosin VI lever arm

a | Molecular dynamic simulations of the ER/K motif-containing α-helix in the medial tail of myosin VI show dynamic charge–charge interactions between the glutamic acid (E) and lysine (K) or arginine (R) residues. The colours used to depict the charge of the different amino acids are based on standard CPK representation. Positively charged amino acids (containing the primary amine and, therefore, nitrogen) are dark blue, and negatively charged amino acids (containing an acid, for example oxygen) are red. Carbon atoms in the side chains are green and hydrogen is white. b | Schematic of myosin VI showing the ~ 30 nm stroke that results from the rigid ER/K α-helix extension. The faded structures indicate the pre-stroke state as well as the movement of the lever arm until it reaches the post-stroke state. c | Our model of the ER/K motif-containing α-helix as a tensegrity structure,. The attractive forces between side chains can be considered to be under tension, whereas the CO–NH hydrogen bonds in the backbone can be considered to be under compression. The balance between these forces stabilizes the ER/K α-helix and enables it to withstand the bending forces that arise when the myosin VI lever arm strokes against an external load. P T, proximal tail.

Figure 7. Myosin VI as a monomer and a dimmer

Myosin VI is a monomer when isolated from cells. In the cytoplasm, the cargo-binding domain is probably folded back, and interacts with the head, with a potential regulatory function that has yet to be clarified. Myosin VI is depicted as a monomer in the cytoplasm (centre), as monomers that are part of clusters on a vesicle (left) or as a dimer on a vesicle (right). Although this schematic shows that the medial tail domains of myosin VI dimers do not interact, it has been suggested that the medial tail does in fact dimerize. Clustering or dimerization on vesicles is probably mediated by adaptor proteins, which are bound to integral membrane proteins (together represented as a binding intermediate).