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

M Van Troys, D Dewitte, M Goethals, MF Carlier… - The EMBO …, 1996 - embopress.org
M Van Troys, D Dewitte, M Goethals, MF Carlier, J Vandekerckhove, C Ampe
The EMBO journal, 1996embopress.org
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 …
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.
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