The actin binding site of thymosin beta 4 mapped by mutational analysis

EMBO J. 1996 Jan 15;15(2):201-10.

Abstract

We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry*
  • Actins / isolation & purification
  • Actins / metabolism*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Binding, Competitive
  • Blood Proteins / chemistry
  • Calcium-Binding Proteins / chemistry
  • Carrier Proteins / chemistry
  • Chickens
  • Circular Dichroism
  • Conserved Sequence
  • Cross-Linking Reagents
  • Genetic Variation
  • Humans
  • Kinetics
  • Membrane Proteins / chemistry
  • Microfilament Proteins / chemistry
  • Molecular Sequence Data
  • Muscle, Skeletal / metabolism
  • Mutagenesis, Site-Directed
  • Peptide Fragments / chemistry
  • Phosphoproteins*
  • Protein Structure, Secondary*
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Thymosin / chemistry*
  • Thymosin / isolation & purification
  • Thymosin / metabolism*

Substances

  • Actins
  • Blood Proteins
  • Calcium-Binding Proteins
  • Carrier Proteins
  • Cross-Linking Reagents
  • DMTN protein, human
  • Membrane Proteins
  • Microfilament Proteins
  • Peptide Fragments
  • Phosphoproteins
  • Recombinant Proteins
  • villin
  • thymosin beta(4)
  • Thymosin