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

Abstract

Human recombinant ezrin, or truncated forms, were coated in microtiter plate and their capacity to bind actin determined. F-actin bound ezrin with a K(d) of 504 ± 230 nM and a molecular stoichiometry of 10.6 actin per ezrin. Ezrin bound both α- and β/γ-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.