Comparative Effects of the Proline-Alanine Rich Regions of Human and Murine Cardiac Myosin Binding Protein-C

Abstract

The N-terminus of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but it is unclear which sequences mediate the activating effects. Herron et al. (Circ Res, 98:1290-8, 2006) found that the Pro-Ala rich region (P-A) of human cMyBP-C could activate tension in the absence of Ca2+, whereas Razumova et al. (J Gen Physiol, 132:575-85, 2008) found that murine C1 and M domains activated tension. The different results might be explained by isoform differences, especially in P-A which is only 46% identical between mouse and human cMyBP-C. The goal of this study was to determine if species-specific differences in P-A account for the different activating effects of murine and human cMyBP-C. Recombinant chimeric proteins containing the C0, P-A, and C1 domains (C0C1) from either human or murine cMyBP-C were engineered and their activating effects assessed using in vitro motility and ATPase assays. Consistent with previous observations, human C0C1 activated actomyosin interactions in the absence of Ca2+, whereas murine C0C1 did not. However, substituting human P-A for murine P-A conferred activating properties to murine C0C1, whereas substituting murine P-A for human P-A depressed the activating effects of human C0C1. Activating effects of the chimera proteins were intermediate between those of murine and human C0C1, suggesting that C0 or C1 also contribute to activation properties. Further chimeric substitutions of C0 and C1 demonstrated that the human C1 domain also contributed to activation, whereas the C0 domain did not. These results suggest that the human P-A and C1 domains are sufficient to activate actomyosin interactions in the absence of Ca2+, and that species-specific differences are likely to contribute to functional differences of cMyBP-C. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.