Calcium-Calmodulin Competes with Actin for Binding to the M-Domain of Cardiac Myosin Binding Protein-C

Abstract

The regulatory M-domain of cardiac myosin binding protein-C (cMyBP-C) binds to myosin, actin, and to calmodulin when calcium is present (i.e., calcium-calmodulin), but it is unclear whether binding of all three ligands is independent or if binding interactions are competitive. Here we investigated whether calcium-calmodulin (Ca-Cam) binding to the M-domain affected the ability of the M-domain to bind to actin using cosedimentation binding and calmodulin-sepaharose pull-down assays. Results of actin cosedimentation binding assays showed that Ca-Cam significantly reduced specific binding of a recombinant protein containing three N-terminal domains of cMyBP-C (i.e., C1-M-C2, referred to as C1C2) when 10 μM calmodulin was present in the presence of calcium (pCa 3.0). In the absence of calcium (at pCa 10.0) calmodulin had no effect on C1C2 binding to actin. Increasing Ca-Cam concentrations to achieve higher molar ratios with respect to C1C2 further reduced the amount of C1C2 that bound to actin. Conversely, in calmodulin-sepharose pull-down experiments, binding of C1C2 to calmodulin was only modestly reduced in the presence of increasing concentrations of F-actin. Taken together, these results indicate that binding of Ca-Cam can compete with actin for binding to the M-domain. These results suggest a potential mechanism whereby the functional effects of cMyBP-C binding to actin can be regulated by calcium. This work supported by NIH HL080367.