An Evolutionary Structural Bioinformatics Analysis of the Actin Binding Protein, Tropomyosin

Abstract

Tropomyosin is a two-chained, α-helical coiled-coil protein that associates end-to-end to form a continuous strand along actin filaments and regulates the functions and stability of actin. Mutations in tropomyosin cause skeletal and cardiomyopathies. We have carried out a phylogenetic analysis of tropomyosin to identify its conserved residues and to elucidate their importance for binding actin. Actin is a highly conserved protein and our hypothesis is that the actin binding sites of tropomyosin have been conserved through evolution to retain its actin-binding function. Phylogenetic trees of 60 coding sequences were constructed from tropomyosin genes from 19 species within the phyla Chordata, Hemichordata and Echinoderm. The rates of substitution (ω) at amino acid sites (codons) in the protein sequence were calculated to identify the most conserved sites (lowest ω) using CODEML in PAML 4.1. A total of 103 out of 284 residues were identified as highly conserved (ω ≤ 0.015), of which 24 are in a and d positions of the heptad repeat involved in the hydrophobic coiled coil interface, 38 are in potential interhelical e-g position salt bridges important for folding of the coiled coil, and 41 are in b, c, or f surface positions available for binding to other proteins, such as actin. The conserved residues at the b, c, and f surface positions were selected for initial mutagenesis studies with mutations to alanine. Preliminary actin binding co-sedimentation assays carried out for three tropomyosin mutants showed a 2- to 3-fold decrease in actin-binding affinity compared to the wild-type protein. Further analysis of mutations at other conserved surface positions and their effect on actin-binding affinity and stability of the mutant proteins are in progress. Supported by Muscle Dystrophy Association.