A dual involvement of the amino-terminal domain of ezrin in F- and G-actin binding.

Human recombinant ezrin, or truncated forms, were coated in microtiter plate and their capacity to bind actin determined. F-actin bound ezrin with a Kd of 504 +/- 230 nM and a molecular stoichiometry of 10.6 actin per ezrin. Ezrin bound both alpha- and beta/gamma-actin essentially as F-form. F-actin binding was totally prevented or drastically reduced when residues 534-586 or 13-30 were deleted, respectively. An actin binding activity was detected in amino-terminal constructs (ezrin 1-310 and 1-333) provided the glutathione S-transferase moiety of the fusion protein was removed. Series of carboxyl-terminal truncations confirmed the presence of this actin-binding site which bound both F- and G-actin. The F- and G-actin-binding sites were differently sensitive to various chemical effectors and distinct specific ezrin antibodies. The internal actin-binding site was mapped between residues 281 and 333. The association of ezrin amino-terminal fragment to full-length ezrin blocked F-actin binding to ezrin. It is proposed that, in full-length ezrin, the F-actin-binding site required the juxtaposition of the distal-most amino- and carboxyl-terminal residues of the ezrin molecule.