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.

We studied the effect of mutations in an alpha-helical region of actophorin (residues 91-108) on F-actin and PIP(2) binding. As in cofilin, residues in the NH(2)-terminal half of this region are involved in F-actin binding. We show here also that basic residues in the COOH-terminal half of the region participate in this interaction whereby we extend the previously defined actin binding interface [Lappalainen, P., et al. (1997) EMBO J. 16, 5520-5530]. In addition, we demonstrate that some of the lysines in this alpha-helical region in actophorin are implicated in PIP(2) binding. This indicates that the binding sites of F-actin and PIP(2) on actophorin overlap, explaining the mutually exclusive binding of these ligands. The Ca(2+)-dependent F-actin binding of a number of actophorin mutants (carrying a lysine to glutamic acid substitution at the COOH-terminal positions of the actin binding helical region) may mimic the behavior of members of the gelsolin family. In addition, we show that PIP(2) binding, but not actin binding, of actophorin is strongly enhanced by a point mutation that leads to a reinforcement of the positive electrostatic potential of the studied alpha-helical region.

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