Evolutionarily conserved surface residues constitute actin binding sites of tropomyosin

Bipasha Barua, Melissa C. Pamula, Sarah E. Hitchcock-DeGregori

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

Tropomyosin (Tm) 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 in eukaryotic muscle and nonmuscle cells. Mutations in Tm cause skeletal and cardiac myopathies. We applied a neoteric molecular evolution approach to gain insight into the fundamental unresolved question of what makes the Tm coiled coil an actin binding protein. We carried out a phylogenetic analysis of 70 coding sequences of Tm genes from 26 animal species, from cnidarians to chordates, and evaluated the substitution rates (ω) at individual codons to identify conserved sites. The most conserved residues at surface b, c, f heptad repeat positions were mutated in rat striated muscle αTm and expressed in Escherichia coli. Each mutant had 3-4 sites mutated to Ala within the first half or the second half of periods 2-6. Actin affinity and thermodynamic stability were determined in vitro. Mutations in the first half of periods 2, 4, and 5 resulted in the largest reduction in actin affinity (>4-fold), indicating these mutations include residues in actin-binding sites. Mutations in the second half of the periods had a ≤2-fold effect on affinity indicating these residues may be involved in other conserved regulatory functions. The structural relevance of these results was assessed by constructing molecular models for the actin-Tm filament. Molecular evolution analysis is a general approach that may be used to identify potential binding sites of a protein for a conserved protein.

Original languageEnglish (US)
Pages (from-to)10150-10155
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number25
DOIs
StatePublished - Jun 21 2011

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Actin filament structure
  • Cytoskeleton
  • Muscle regulation
  • Protein evolution

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