A Gain-of-Function Mutation in the M-domain of Cardiac Myosin-binding Protein-C Increases Binding to Actin |
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Authors: | Kristina L. Bezold Justin F. Shaffer Jaskiran K. Khosa Elaine R. Hoye Samantha P. Harris |
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Affiliation: | From the University of California, Davis, California 95618 |
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Abstract: | The M-domain is the major regulatory subunit of cardiac myosin-binding protein-C (cMyBP-C) that modulates actin and myosin interactions to influence muscle contraction. However, the precise mechanism(s) and the specific residues involved in mediating the functional effects of the M-domain are not fully understood. Positively charged residues adjacent to phosphorylation sites in the M-domain are thought to be critical for effects of cMyBP-C on cross-bridge interactions by mediating electrostatic binding with myosin S2 and/or actin. However, recent structural studies revealed that highly conserved sequences downstream of the phosphorylation sites form a compact tri-helix bundle. Here we used site-directed mutagenesis to probe the functional significance of charged residues adjacent to the phosphorylation sites and conserved residues within the tri-helix bundle. Results confirm that charged residues adjacent to phosphorylation sites and residues within the tri-helix bundle are important for mediating effects of the M-domain on contraction. In addition, four missense variants within the tri-helix bundle that are associated with human hypertrophic cardiomyopathy caused either loss-of-function or gain-of-function effects on force. Importantly, the effects of the gain-of-function variant, L348P, increased the affinity of the M-domain for actin. Together, results demonstrate that functional effects of the M-domain are not due solely to interactions with charged residues near phosphorylatable serines and provide the first demonstration that the tri-helix bundle contributes to the functional effects of the M-domain, most likely by binding to actin. |
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Keywords: | Cardiac Hypertrophy Cardiac Muscle Cardiomyopathy Contractile Protein Protein Phosphorylation Myosin-binding Protein C Tri-helix Bundle |
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