Volume 17, Issue 4, Pages (February 2005)

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Ron Rock University of Chicago The Role of the Proximal Tail in the Large Steps of Myosin VI.
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Volume 17, Issue 4, Pages 603-609 (February 2005) A Flexible Domain Is Essential for the Large Step Size and Processivity of Myosin VI  Ronald S. Rock, Bhagavathi Ramamurthy, Alexander R. Dunn, Sara Beccafico, Bhadresh R. Rami, Carl Morris, Benjamin J. Spink, Clara Franzini-Armstrong, James A. Spudich, H.Lee Sweeney  Molecular Cell  Volume 17, Issue 4, Pages 603-609 (February 2005) DOI: 10.1016/j.molcel.2005.01.015

Figure 1 Domain Organization of Myosin VI (A) Rotary shadowing EM of M6HMM. All observed M6HMM molecules were dimeric. Representative images showing the full range of head positions are shown. Scale bar, 50 nm. The illustration shows the domain organization of this construct as a dimer of heavy chains, associated calmodulin (CaM) light chains, and engineered C-terminal GCN4 and YFP domains. M6HMM is presumed to dimerize via a portion of the tail domain. The histogram shows the distribution of interhead distances, measured as the distance between the approximate centroids of each head. This distance ranges from 15–45 nm, with a mean of 27 ± 6 nm (SD, n = 37). Note that the farthest ends of the heads are separated by a larger distance than the measured center-to-center distance. (B) PAIRCOIL scores for myosin VI tail sequences. The scores of myosin VI sequences from S. scrofa and D. melanogaster are shown in black and red. The dashed black line shows the PAIRCOIL score for M6HMM, where it differs from S. scrofa. Color bars above the sequence indicate the locations of the domains of M6HMM. The tail domain has been divided into proximal and distal subdomains. The proximal tail has a low coiled-coil probability, whereas the distal tail likely contains a coiled coil. The boundary between the tail subdomains is arbitrarily defined by the onset of near-unit probability of coiled-coil formation. This boundary is particularly sharp in the D. melanogaster sequence. Note that the S. scrofa score shown is for the wild-type sequence not the engineered M6HMM sequence. For M6HMM, the score climbs back to unit probability in the GCN4 domain. Also shown in blue is the average PAIRCOIL score for members of the myosin VI family. Accession numbers: H. sapiens, Q9UM54; S. scrofa, Q29122; G. gallus, Q9I8D1; D. melanogaster, Q01989; M. musculus, Q64331; S. purpuratus, Q9NGM0; M. saxatilis, Q9PWF6, Q9PWF5. (C) Two models for the structure of the proximal tail. At left, a rigid domain of unknown structure that serves as a lever arm extension is shown. At right, the proximal tail is shown as an unstructured, flexible element (curved lines). Molecular Cell 2005 17, 603-609DOI: (10.1016/j.molcel.2005.01.015)

Figure 2 Stepping of Single-Headed Myosin VI (A) Step size distribution of M6S1. The step size was 12.1 ± 1.0 nm (SEM, n = 291). The domain structure of M6S1 is shown at right, along with representative rotary-shadowed EM images showing that this construct is single headed. The length of M6S1, measured as the distance between the approximate centers of the head domain and of the YFP domain, is 15 ± 2 nm (SD, n = 32). Scale bar, 50 nm. (B) Same for M6longS1. The step size is 11.9 ± 1.2 nm (SEM, n = 195) not significantly different from M6S1. As for M6S1, EM images reveal that this construct is single headed, showing that the proximal tail is not sufficient for dimerization. The length of M6longS1 is 17 ± 1 nm (SD, n = 34). Scale bar, 50 nm. Molecular Cell 2005 17, 603-609DOI: (10.1016/j.molcel.2005.01.015)

Figure 3 Stepping of a Myosin VI with a Constrained Proximal Tail (A) Rotary shadowing EM of M6-2hepzip. Example images show that the two heads are closer in this construct than in M6HMM, and the range of head-to-head distances is smaller. The two heads are separated by 19 ± 2 nm (SD, n = 36). The domain structure illustration shows that the two heads are held together by the GCN4 substitution in the proximal tail. (B) Optical trapping time trace of M6-2hepzip stepping. Bead position is shown in black, and a trace median-filtered over a 100 ms window is shown in red. Isolated single events such as those at 3 s and 28 s are the most common, but processive runs (here at 6 s, 35 s, and 48 s) are also observed. The bead variance trace (blue, 10 ms window) and bead-to-bead correlation trace (green, 10 ms window) show no apparent decrease in system stiffness between processive steps. (C) Measurement of the processive step size. The small step size of M6-2hepzip makes manual tabulation of mechanical transitions difficult. Therefore, the pairwise distance distribution was analyzed as follows. The pairwise distance distribution was calculated by isolating the processive runs, then reducing the bead position data by averaging over a 30 ms window. All pairwise distances between data points were then calculated within the reduced data set. This pairwise distance distribution (black curve) was then fit to the sum of eight Gaussians (blue curves). The free parameters for the fit were the amplitudes of each Gaussian, the variance (the same for all Gaussians), an offset (<1 nm), and a uniform spacing between Gaussians. The spacing between Gaussians is 12.0 ± 2 nm. Molecular Cell 2005 17, 603-609DOI: (10.1016/j.molcel.2005.01.015)

Figure 4 Structural Model of Myosin VI Stepping (A) Model for M6HMM and M6-2hepzip, each stepping from right to left. For M6HMM (left), the unstructured proximal tails allow a broad diffusive search. The lever arm swing biases the diffusive search 12 nm toward the pointed end. The long-pitch helix of the actin filament provides an additional bias toward sites that are at a nearby azimuth. For M6-2hepzip (right), the coiled-coil insert imposes a significant constraint upon the diffusive search. The two heads span four actin monomers, with regular steps. Such a stepping pattern requires tight spiraling around the actin filament. (B) Mechanical model of M6HMM, with the trailing head (T) attached to actin and the leading head (L) free. The 12 nm working stroke of the trailing head is indicated. The proximal tails are shown as springs. Dark circles indicate junctions that are expected to act as free swivels. (C) Mechanical model of M6HMM after the lead head binds. To span 36 nm, the proximal tails must together extend 24 nm. The head separations before and after a step do not have to be equal, as is assumed in this illustration. Note that the observed step size is half the distance traversed by the trailing head when it becomes the new leading head. (D) Calculated first-passage time (black) and dwell time (red) for a 36 nm step as a function of load. The equilibrium position of the free head is given by −F/2k, where k is the combined stiffness of both segments of proximal tail (0.12 pN/nm). In this model, the proximal-tail stiffness remains constant, but as the external load increases, the distance traversed by the free head also increases. The free head must therefore diffuse over an energy barrier given by U0 = k(24 + F/2k)2/2. This energy barrier is used to find a first-passage time as γ/k(π/4)1/2(kbT/U0)1/2exp(U0/kbT), where γ is the viscous drag coefficient, k is the stiffness, and kbT is thermal energy. The dwell time curve adds a zero-load, rate-limiting transition at ∼3 s−1 under these conditions (Altman et al., 2004). Molecular Cell 2005 17, 603-609DOI: (10.1016/j.molcel.2005.01.015)