Structural Destabilization of Tropomyosin Induced by a Cardiomyopathy-Linked Mutation

Abstract

In striated muscle, thin filaments, composed of F-actin, and thick filaments constitute the basic contractile units called sarcomeres. Tropomyosin (Tpm), a two-stranded coiled-coil protein, binds along the actin filaments through head-to-tail polymerization, protects and stabilizes the thin filaments. The N-terminus of Tpm orients toward the pointed end of thin filament, where it interacts with pointed end-binding proteins such as tropomodulin (Tmod) and leiomodin (Lmod) to maintain the uniform filament length critical for proper sarcomeric functions. Recently, a hypertrophic cardiomyopathy-associated mutation, R21H, has been identified in striated muscle Tpm (Tpm1.1) with molecular mechanism of perturbing muscular function unknown. We designed, expressed, and purified the Tpm chimeric peptide αTM1a1-28Zip. The peptide consists of 28 N-terminal residues of Tpm1.1 followed by 18 C-terminal residues of the GCN4 leucine zipper domain. The peptide was crystallized and its structure was solved. To study how this mutation affects Tpm1.1, we introduced the mutation R21H in the peptide. An effect of the mutation was studied in silico using molecular dynamics simulation (MDS) and in vitro by circular dichroism (CD). Temperature measurements using CD were conducted to characterize the effect of the mutation R21H on thermal stability of the αTM1a1-28Zip peptide alone and its complexes with Tmod and Lmod fragments containing Tpm-binding sites. CD data showed that the mutation R21H caused a significant decrease in the helical content and structural stability of αTM1a1-28Zip. Complexes formed between the αTM1a1-28Zip[R21H] peptide and Tmod or Lmod fragments were less stable than those formed with wild-type αTM1a1-28Zip. All CD data were in agreement with MDS results which showed that the mutation R21H significantly altered the coiled-coil structure of αTM1a1-28Zip. We suggest that the mutation R21H destabilizes Tpm structure by disrupting local salt bridges formed between residues Arg21 and Glu26 on opposite strands.