Abstract
In cardiac muscle, regulation of actin polymerization at the thin filament pointed end is controlled by two structurally similar but functionally antagonistic proteins, leiomodin-2 and tropomodulin-1. Both proteins contain tropomyosin-binding site 1, which is essential for their recruitment to the pointed end. Using circular dichroism, we determined changes in melting temperatures (ΔTm) for complexes of tropomyosin and leiomodin-2 fragments containing several hypomorphic mutations, which moderately affect binding to tropomyosin. We ran molecular dynamics simulations for the complexes and calculated standard Gibbs free energies of binding, which we found to strongly correlate with the ΔTm. We found that the E34Q mutation in leiomodin-2 resulted in a decrease in the melting temperature of the complex of tropomyosin and leiomodin-2 fragments, indicating a decrease in the affinity of leiomodin-2 for tropomyosin. Although modest, this change in in vitro affinity made leiomodin-2 a weaker competitor for tropomyosin than tropomodulin-1 in cardiomyocytes. This mutation significantly reduced the ability of leiomodin-2 to displace tropomodulin-1 at thin filament pointed ends and affected the ability of leiomodin-2 to elongate thin filaments. Our results highlight the essential role of the tropomyosin-binding site in the dynamic equilibrium between tropomodulin-1 and leiomodin-2 at the pointed end of thin filaments. Our data also suggest the potential use of the correlation between ΔTm and the modeled standard Gibbs free energies of binding to predict changes in the stability of complexes between tropomyosin and leiomodin or tropomodulin isoforms.