The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency.

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function.

A gain-of-function mutation in the M-domain of cardiac myosin-binding protein-C increases binding to actin.

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.

Baculovirus-mediated overexpression of the phosphorylase b kinase holoenzyme and alpha gamma delta and gamma delta subcomplexes.

Recombinant baculoviruses were created and used to coexpress rat phosphorylase kinase (Phk) alpha, gamma, and delta subunits and rabbit beta subunit in insect cells. Coexpression allowed creation of the (alphabetagammadelta)4 hexadecamer, the alphagammadelta heterotrimer, and the gammadelta heterodimeric subcomplexes. Neither the individual alpha, beta, or gamma subunit nor any complex containing the beta subunit other than the hexadecameric holoenzyme was obtained in soluble form. The expressed complexes exhibited pH- and [Ca2+]-dependent specific activities that were similar to those of the Phk holoenzyme purified from rabbit skeletal muscle (SkM Phk). SkM Phk, expressed Phk, and the alphagammadelta subcomplex were activated by exogenous calmodulin and underwent Ca(2+)-dependent autophosphorylation. In some of these features there were subtle differences that could likely be attributed to differences in the covalent modification state of the baculovirus-driven expressed protein. Our results provide an important avenue to probe the detailed characterization of the structure of Phk and the function of the individual domains of the subunits using baculovirus-mediated expression of Phk and Phk subcomplexes.