The actin binding site of thymosin beta 4 mapped by mutational analysis.
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.
Van Troys
M
M
Department of Biochemistry, University of Ghent, Belgium.
Dewitte
D
D
Goethals
M
M
Carlier
M F
MF
Vandekerckhove
J
J
Ampe
C
C
D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001798:Blood Proteins / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D002645:Chickens; D002942:Circular Dichroism; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D008840:Microfilament Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013947:Thymosin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000099201:Thymosin beta(4)
D000199:Actins; D001798:Blood Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D013947:Thymosin; D000099201:Thymosin beta(4); C044805:DMTN protein, human; C026615:villin