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Nucleotide-dependent shape changes in the reverse direction motor, myosin VI.

ABSTRACT: We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca(2+)). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-?S resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ?140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO(4), which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ?1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended ?-helical structure in the tail, but molecules viewed by metal shadowing had a tail ?3× longer, 29 vs. 9 nm, part of which is likely to be a single ?-helix.

SUBMITTER: Song CF

PROVIDER: S-EPMC2980756 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Nucleotide-dependent shape changes in the reverse direction motor, myosin VI.

Song Chun Feng CF Sader Kasim K White Howard H Kendrick-Jones John J Trinick John J

Biophysical journal 20101101 10

We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca(2+)). The shape of truncated apo m ...[more]

PMID: 21081082

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Project description:We used small-angle X-ray solution scattering (SAXS) technique to investigate the nucleotide-mediated conformational changes of the head domains [subfragment 1 (S1)] of myosin V and VI processive motors that govern their directional preference for motility on actin. Recombinant myosin V-S1 with two IQ motifs (MV-S1IQ2) and myosin VI-S1 (MVI-S1) were engineered from Sf9 cells using a baculovirus expression system. The radii of gyration (R(g)) of nucleotide-free MV-S1IQ2 and MVI-S1 were 48.6 and 48.8 A, respectively. In the presence of ATP, the R(g) value of MV-S1IQ2 decreased to 46.7 A, while that of MVI-S1 increased to 51.7 A, and the maximum chord length of the molecule decreased by ca 9% for MV-S1IQ2 and increased by ca 6% for MVI-S1. These opposite directional changes were consistent with those occurring in S1s with ADP and Vi or AlF(4)(-2) bound (i.e., in states mimicking the ADP/Pi-bound state of ATP hydrolysis). Binding of AMPPNP induced R(g) changes of both constructs similar to those in the presence of ATP, suggesting that the timing of the structural changes for their motion on actin is upon binding of ATP (the pre-hydrolysis state) during the ATPase cycle. Binding of ADP to MV-S1IQ2 and MVI-S1 caused their R(g) values to drop below those in the nucleotide-free state. Thus, upon the release of Pi, the reverse conformational change could occur, coupling to drive the directional motion on actin. The amount of R(g) change upon the release of Pi was ca 6.4 times greater in MVI-S1 than in MV-S1IQ2, relating to the production of the large stroke of the MVI motor during its translocation on actin. Atomic structural models for these S1s based upon the ab initio shape reconstruction from X-ray scattering data were constructed, showing that MVI-S1 has the light-chain-binding domain positioned in the opposite direction to MV-S1IQ2 in both the pre- and post-powerstroke transition. The angular change between the light chain-binding domains of MV-S1IQ2 in the pre- to post-powerstroke transition was approximately 50 degrees, comparable to that of MII-S1. On the other hand, that of MVI-S1 was approximately 100 degrees or approximately 130 degrees much less than the currently postulated changes to allow the maximal stroke size of myosin VI-S1 but still significantly larger than those of other myosins reported so far. The results suggest that some additional alterations or elements are required for MVI-S1 to take maximal working strokes along the actin filament.

Dataset Information

Nucleotide-dependent shape changes in the reverse direction motor, myosin VI.

Publications

Nucleotide-dependent shape changes in the reverse direction motor, myosin VI.

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