The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner

• DOI: 10.1074/jbc.m808850200
• Publication Date: 2009-03-07T01:14:26Z
• Authors: Justin F. Shaffer; Robert W. Kensler; Samantha P. Harris
• Keywords: Sarcomeres; 0303 health sciences; Binding Sites; Myocardium; Amino Acid Motifs; Actins; Protein Structure, Tertiary; Mice; 03 medical and health sciences; Animals; Cattle; Rabbits; Phosphorylation; Carrier Proteins; Protein Binding

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 (K(d)) of approximately 10 microm and a molar binding ratio (B(max)) 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 B(max)) 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.