Abstract
We have used site-directed spin and fluorescence labeling to test molecular models of the actin-myosin interface. Force is generated when the actin-myosin complex undergoes a transition from a disordered weak-binding state to an ordered strong-binding state. Actomyosin interface models, in which residues are classified as contributing to either weak or strong binding, have been derived by fitting individual crystallographic structures of actin and myosin into actomyosin cryo-EM maps. Our goal is to test these models using site-directed spectroscopic probes on actin and myosin. Starting with Cys-lite constructs of both yeast actin (ActC) and the Dictyostelium myosin II motor domain (S1dC), site-directed labeling (SDL) mutants were generated by mutating residues to Cys in the proposed weak and strong-binding interfaces. This report focuses on the effects of forming the strong-binding complex on four SDL mutants, two located in the proposed weak-binding interface (ActC5 and S1dC619) and two located in the proposed strong-binding interface (ActC345 and S1dC401). Neither the mutations nor labeling prevented strong actomyosin binding or actin-activation of myosin ATPase. Formation of the strong-binding complex resulted in decreased spin and fluorescence probe mobility at all sites, but both myosin-bound probes showed remarkably high mobility even after complex formation. Complex formation decreased solvent accessibility for both actin-bound probes, but increased it for the myosin-bound probes. These results are not consistent with a simple model in which there are discrete weak and strong interfaces, with only the strong interface forming under strong-binding conditions, nor are they consistent with a model in which surface residues become rigid and inaccessible upon complex formation. We conclude that all four of these residues are involved in the strong actin-myosin interface, but this interface is remarkably dynamic, especially on the surface of myosin.
Original language | English (US) |
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Pages (from-to) | 1107-1117 |
Number of pages | 11 |
Journal | Journal of Molecular Biology |
Volume | 356 |
Issue number | 5 |
DOIs | |
State | Published - Mar 10 2006 |
Bibliographical note
Funding Information:This work was supported by grants to D.D.T. from NIH (AR32961) and the Muscular Dystrophy Association, and to V.L.K. from NIH (AR47755) and the American Heart Association (to V.L.K). We thank Dr Peter Rubenstein and Dr James Spudich for the gift of the Cys-lite yeast actin construct and Cys-lite Dicty. myosin motor domain construct, respectively. We thank Yuri Nesmelov for assistance with EPR spectroscopy, and Igor V. Negrashov for his work on the fluorescence instrumentation and analysis software. We thank Zach Ryan, Shawn Galdeen, and Luke Olsen for their advice and assistance in Dicty. myosin mutagenesis, expression, and purification.
Keywords
- Actin
- Interface
- Myosin
- Site-directed labeling