Myosin VI dimerization triggers an unfolding of a three-helix bundle in order to extend its reach

Abstract

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 three-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 unconventional mechanism generates an extension of the lever arm of myosin VI.

Figure 1. Schematic of full-length myosin VI

A. The N-terminal motor domain is followed by a unique insert (Insert 2) that binds the structural calmodulin (CaM), followed by an IQ motif that is bound to an exchangeable CaM. This is adjacent to the proximal tail domain that may form an extension of the lever arm formed by the bound CaMs. The proximal tail is followed by the medial tail, a region that can weakly dimerize, as we have previously shown (Park et al., 2006). This same region has been predicted to contain a stable single alpha helix (SAH) by Spink et al. (2008). In this study we provide evidence that the medial tail likely contains a short dimerization domain (likely coiled coil) that may be followed by a SAH. The rest of the molecule is composed of a small distal tail region and a cargo-binding domain. B. Model of myosin VI dimer proposed by Spink et al. (2008), using stable single alpha helices (SAH) as lever arm extensions. The sites of truncations used for this study are noted by the C-terminal amino acid number.

Figure 2. The Myosin VI full-length lever arm

A. Structure of the myosin VI lever arm with the Insert 2 (purple, Ca²⁺-CaM pink) and the IQ motif (cyan, apo-CaM yellow) helix followed by the three helix bundle (blue) that forms the lever arm extension (LAE). Note the 30º kink at the junction between the IQ motif and the LAE at P835 (red). B. Details of the interactions between the apo-CaM bound to the IQ motif and the triple helix bundle. Two hydrogen bonds mediated by the helix 1 R836 also stabilize the bundle by interacting with two carbonyls of the loop between helices 2 and 3 (I885 and T888). C. Apolar interactions that stabilize the bundle in the proximal part. Note that helix 1 is shorter than the other two helices and the loop that follows is not visible in the electron density. Fewer interactions are found between the helices in the distal part of the bundle, which is not well stabilized. D. Sequence of the LAE with the residues found within the bundle marked by a colored dot. The loops between the helices are highlighted in blue.

Figure 3

A. Rotary shadowing EM of myosin VI constructs. Representative images for the MVI-940 construct following dimerization using actin clustering are shown in the first row and images of the zippered HMM (MVI-991-GCN4) are shown in the second row. These dimers show two closely spaced motor domains with an intervening link. The third row contains panel with representative images of MVI-940 monomers. B. Observed movements of myosin VI constructs. Representative stepping traces, based on FIONA values as a function of time, are shown for three truncated myosin VI constructs. Stepping traces for the labeled head (Cy3 on IQ-bound CaM) are shown for MVI-940 (red), MVI-919-GCN4 (blue), and for the full-length construct with the last two helices of the bundle deleted, MVI-FL 848–909 del (green). Positions of the dye were tracked and steps were calculated using a custom Student’s t-test algorithm coded for by IDL (ITT Visual Information Solutions, Boulder, CO). In all cases, the observed steps are actually two steps, since the probe is only on one head. C. Distribution of step sizes of Cy3-labeled truncated myosin VI constructs. For MVI-940, following dimerization by binding to actin, the average forward step (1/2 observed movement) was 27.2 ± 9.7 nm (n =87). The average backward step (1/2 observed movement) of MVI-940 was –12.3 ± 1.7 nm (n = 2). The distribution of the MVI-940 was similar to what we previously observed for both the full-length myosin VI dimer and the zippered HMM. The average forward step size of MVI-919-GCN4 was 23.7 ± 10.6 nm (n = 108) whereas the average backward step size was –12.5 ± 2.7 nm (n = 3). For the full-length construct with the second and third helices of the bundle deleted, the average forward step size was 16.1 ± 7.0 nm (n=61), and the average backward step size was −7.7 ± 4.3 nm (n=3),

Figure 4. Dimerization-mediated unfolding of the 3-helix bundle of myosin VI

Our working model for myosin VI in a cell is that the full-length protein exists as a monomer if not bound to cargo. Binding of myosin VI monomers to cargo alters the conformation of the molecule, possibly exposing a region capable of dimerization. The dimerization in turn triggers an unfolding of the 3-helix bundle that follows the CaM, generating a lever arm extension. A. The lead head (left) and the rear head (right) are bound to the actin filament (yellow spheres) 36 nm apart. If the triple helix is not unfolded, the bundle covers only 4 nm out of the 13 nm necessary. 18nm are thus unaccounted for. B. When the triple helix unfolds, three helices separated by segments of undefined structure account for the 13 nm necessary to extend the lever arm. C. Model of the dimerized molecule showing that the region immediately following the lever arm extension (LAE), the most proximal part of the medial tail, participates in dimerization, as do segments of the distal tail (orange) and the cargo-binding domain (magenta). Note that most of the medial tail (green) is shown as a SAH and acting as a spacer between motor and cargo. Thus the SAH is not contributing to the lever arm extension or to dimerization. The distance between the two cysteines introduced for fluorescence measurements at 845 and 880 are shown in the unfolded bundle with maximal extension of the helices. D. The positions of the cysteines that were introduced into the 3-helix bundle of myosin VI for fluorescence measurements (T845C and A880C) are shown in red. The distance between the two residues (Cα), to which two tetramethylrhodamine 5-iodoacetamide fluorophores were attached, is 18Å as depicted. E. Model of the dimerized full-length molecule in which the last two helices of the 3-helix bundle have been removed. If dimerization occurs immediately following this remaining helix, then the step size will be greatly decreased, as illustrated.