The effects of two Leu-to-Pro substitutions, L57P and L43P, on structural and functional properties of cardiac...

Tropomyosin (Tpm) is an actin-binding protein that, together with troponin (Tn), mediates Ca²⁺-regulation of cardiac muscle contraction. Tpm coiled-coil dimers bind each other through overlap junctions between their N- and C-termini, forming a continuous strand along the actin filament. Among the many TPM1 mutations identified in cardiac Tpm (Tpm1.1), few substitute canonical residues with proline, the amino acid most disruptive to coiled-coil structure. We examined properties of recombinant cardiac Tpm with L43P or L57P substitutions in both chains and compared them with wild-type (WT) Tpm using differential scanning calorimetry, viscometry, molecular dynamics (MD) simulations, and an in vitro motility assay. Both mutations markedly destabilized the N-terminal part of the Tpm molecule. In MD simulations, Pro43 and Pro57 disrupted nearby backbone hydrogen bonds, and Pro43 promoted N-terminal unfolding, demonstrating a long-range effect within supercoiled Tpm molecules. Both substitutions strongly reduce Tpm's affinity for F-actin in the absence of Tn. In addition, viscometry showed that the L43P Tpm variant polymerizes less efficiently than WT and L57P Tpm, resulting in a reduced ability to assemble into a continuous strand along an actin filament. The L57P substitution increases maximum sliding velocity of thin filaments in in vitro motility assay and enhances Ca²⁺ sensitivity of actin-myosin interaction, a feature commonly associated with hypertrophic cardiomyopathy. In contrast, the L43P substitution hinders the formation of fully regulatory-competent thin filaments and severely impairs the Ca²⁺-regulatory function of reconstructed thin filaments in vitro. Our study reveals distinct mechanisms of pathogenic effects for these two largely similar amino acid substitutions.