The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner*
- ‡Department of Neurobiology, Physiology, and Behavior, University of California, Davis, California 95616, the §Department of Bioengineering, University of Washington, Seattle, Washington 98195, and the ¶University of Puerto Rico Medical School, San Juan, Puerto Rico 00936
- 1 To whom correspondence should be addressed: 196 Briggs Hall, One Shields Ave., Davis, CA 95616-8519. Tel.: 530-752-0642; Fax: 530-752-5582; E-mail: samharris{at}ucdavis.edu.
Abstract
Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (Kd) of ∼10 μm and a molar binding ratio (Bmax) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced Bmax) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase cross-bridge cycling.
Footnotes
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↵2 The abbreviations used are: cMyBP-C, cardiac myosin-binding protein C; NTF, native thin filaments; PKA, protein kinase A; DTT, dithiothreitol.
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↵* This work was supported, in whole or in part, by National Institutes of Health Grants SC1HL096017 (to R. W. K.) and HL080367 (to S. P. H.). This work was also supported by a National Science Foundation Graduate Research Fellowship (to J. F. S.).
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- Received November 21, 2008.
- Revision received February 18, 2009.
- The American Society for Biochemistry and Molecular Biology, Inc.
