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.
Roy
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Laboratoire de Dynamique Moléculaire des Interactions Membranaires, CNRS UMR 5539, Université Montpellier II, Bât. 24, CC107, place Eugène Bataillon, 34095 Montpellier Cedex 5, France. roy@univ-montp2.fr
Martin
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Mangeat
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D000199:Actins / Q000378:metabolism*; D001665:Binding Sites; D003598:Cytoskeletal Proteins; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D000099149:Ezrin
D000199:Actins; D003598:Cytoskeletal Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000099149:Ezrin