Quantitative analysis of the binding of ezrin to large unilamellar vesicles containing phosphatidylinositol 4,5 bisphosphate.
The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP(2)) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP(2). For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be approximately 5 microM for PIP(2)-LUVs and 20- to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP(2)-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of approximately 30 nm(2)) binding to a single PIP(2) can block access to neighboring PIP(2) molecules and thus contributes to lower the accessible PIP(2) concentration. In addition, no evidence exists for a clustering of PIP(2) induced by ezrin addition.
Blin
Guillaume
G
DIMNP, Universités Montpellier II et I, CNRS, Montpellier cedex 05, France.
Margeat
Emmanuel
E
Carvalho
Kévin
K
Royer
Catherine A
CA
Roy
Christian
C
Picart
Catherine
C
D001665:Binding Sites; D003198:Computer Simulation; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008560:Membrane Fluidity; D008956:Models, Chemical; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D011485:Protein Binding; D053835:Unilamellar Liposomes / Q000737:chemistry*; D000099149:Ezrin