title: A gain-of-function mutation in the M-domain of cardiac myosin-binding protein-C increases binding to actin.
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.
authors.affiliation: University of California, Davis, California 95618, USA.
authors.forename: Kristina L
authors.initials: KL
authors.lastname: Bezold
authors.forename: Justin F
authors.initials: JF
authors.lastname: Shaffer
authors.forename: Jaskiran K
authors.initials: JK
authors.lastname: Khosa
authors.forename: Elaine R
authors.initials: ER
authors.lastname: Hoye
authors.forename: Samantha P
authors.initials: SP
authors.lastname: Harris
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D000099163:Myosin Binding Protein C
chemical_list: D000199:Actins; D002118:Calcium; D002352:Carrier Proteins; D011994:Recombinant Proteins; D000099163:Myosin Binding Protein C
keywords: Cardiac Hypertrophy; Cardiac Muscle; Cardiomyopathy; Contractile Protein; Myosin-binding Protein C; Protein Phosphorylation; Tri-helix Bundle