Dataset Information

Reverse conformational changes of the light chain-binding domain of myosin V and VI processive motor heads during and after hydrolysis of ATP by small-angle X-ray solution scattering.

ABSTRACT: 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.

SUBMITTER: Sugimoto Y

PROVIDER: S-EPMC2852323 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Reverse conformational changes of the light chain-binding domain of myosin V and VI processive motor heads during and after hydrolysis of ATP by small-angle X-ray solution scattering.

Sugimoto Yasunobu Y Sato Osamu O Watanabe Shinya S Ikebe Reiko R Ikebe Mitsuo M Wakabayashi Katsuzo K

Journal of molecular biology 20090714 2

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 4 ...[more]

PMID: 19607837

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Project description:Conventional myosin is representative of biomolecular motors in which the hydrolysis of adenosine triphosphate (ATP) is coupled to large-scale structural transitions both in and remote from the active site. The mechanism that underlies such "mechanochemical coupling," especially the causal relationship between hydrolysis and allosteric structural changes, has remained elusive despite extensive experimental and computational analyses. In this study, using combined quantum mechanical and molecular mechanical simulations and different conformations of the myosin motor domain, we provide evidence to support that regulation of ATP hydrolysis activity is not limited to residues in the immediate environment of the phosphate. Specifically, we illustrate that efficient hydrolysis of ATP depends not only on the proper orientation of the lytic water but also on the structural stability of several nearby residues, especially the Arg238-Glu459 salt bridge (the numbering of residues follows myosin II in Dictyostelium discoideum) and the water molecule that spans this salt bridge and the lytic water. More importantly, by comparing the hydrolysis activities in two motor conformations with very similar active-site (i.e., Switches I and II) configurations, which distinguished this work from our previous study, the results clearly indicate that the ability of these residues to perform crucial electrostatic stabilization relies on the configuration of residues in the nearby N-terminus of the relay helix and the "wedge loop." Without the structural support from those motifs, residues in a closed active site in the post-rigor motor domain undergo subtle structural variations that lead to consistently higher calculated ATP hydrolysis barriers than in the pre-powerstroke state. In other words, starting from the post-rigor state, turning on the ATPase activity requires not only displacement of Switch II to close the active site but also structural transitions in the N-terminus of the relay helix and the "wedge loop," which have been proposed previously to be ultimately coupled to the rotation of the converter subdomain 40 A away.

Dataset Information

Reverse conformational changes of the light chain-binding domain of myosin V and VI processive motor heads during and after hydrolysis of ATP by small-angle X-ray solution scattering.

Publications

Reverse conformational changes of the light chain-binding domain of myosin V and VI processive motor heads during and after hydrolysis of ATP by small-angle X-ray solution scattering.

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