Title: Identification of Amino Acid Residues in the Cardiac Myosin Binding Protein-C Motif Important for Actin Binding
Abstract: N-terminal domains of cardiac myosin binding protein-C (cMyBP-C) can activate actomyosin interactions in the absence of Ca2+ and bind to actin in a phosphorylation dependent manner. We have previously shown that two N-terminal domains, C1 and the MyBP-C motif ("M") domain, bind specifically to actin and to thin filaments; however, the sequences or residues that mediate actin binding have not been identified. The goal of this study was to identify residues in the M-domain that contribute to actin binding and to investigate whether interactions between the M-domain and actin mediate the activating properties of cMyBP-C. We therefore used alanine-scanning mutagenesis to target candidate actin binding sites in the M-domain that bear homology to the actin binding motifs in other known actin binding proteins and to assess the effects of mutations on the ability of recombinant proteins to bind actin and activate actomyosin interactions in motility assays. Results demonstrate that mutation of select positively-charged amino acids in the M-domain that are homologous to binding motifs in known actin binding proteins reduced binding of cMyBP-C to actin. The mutations also reduced or eliminated the activating properties of recombinant cMyBP-C in in vitro motility assays. However, mutation of other positively-charged amino acids did not affect actin binding or protein functional properties. These results indicate that specific residues within the M-domain confer actin binding and that interactions with actin contribute to the functional effects of recombinant cMyBP-C N-terminal proteins. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.