The N-terminus of Cardiac Myosin Binding Protein-C Contains Multiple Binding Sites for F-actin

Abstract

Cardiac myosin binding protein-C (cMyBP-C), long known to interact with thick filaments, also interacts with thin filaments (actin) through its N-terminus. However, a single actin binding site has not been identified and it is unclear whether one or more N-terminal domains of cMyBP-C interact with actin. In this study we aimed to characterize the interaction of the N-terminus of cMyBP-C with actin using recombinant proteins consisting of various cMyBP-C N-terminal domains. Results from high speed cosedimentation binding assays showed that recombinant proteins containing the C1 domain and the MyBP-C motif bound to F-actin at a 1:1 molar ratio with a dissociation constant (Kd) ~ 10 uM. In contrast, proteins containing either C1 or the motif showed reduced binding at a 1:2 molar ratio. Proteins containing both C1 and the motif also bundled actin filaments, suggesting multiple actin interaction sites. Binding of recombinant proteins to Ca2+ regulated thin filaments was similar to binding to F-actin alone. Strongly bound myosin cross-bridges (myosin S1, no ATP) abolished cMyBP-C binding to actin, while weakly bound crossbridges (myosin S1 plus ATP) diminished, but did not abolish, binding. Recombinant myosin ΔS2, which binds to the MyBP-C motif in vitro (~6 uM), did not affect cMyBP-C binding to actin. However, phosphorylation of the motif or alkaline pH both reduced binding. Together, these results suggest that the N-terminus of cMyBP-C contains at least two binding sites for actin and that binding is modulated through electrostatic interactions. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.