Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain

• Published in: Journal of molecular and cellular cardiology 2012-01, Vol.52 (1), p.219-227
• Main Authors: Weith, Abbey, Sadayappan, Sakthivel, Gulick, James, Previs, Michael J, VanBuren, Peter, Robbins, Jeffrey, Warshaw, David M
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Actomyosin - metabolism; Amino Acid Motifs; Animals; Cardiovascular; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Chickens; Contractile proteins; Contractility; Heart; Laser trap; Mice; Phosphorylation; PKA phosphorylation; Protein Binding - drug effects; Protein Structure, Tertiary; Single molecule biophysics
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2011.09.019

Abstract Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~ 20 ms) and long (~ 300 ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.