Ezrin is a critical component in the formation of membrane structures in epithelial cells, where it can simultaneously link the membrane and various membrane proteins with the actin cytoskeleton. Ezrin is highly post-translationally modified, allowing for dynamic regulation of membrane-actin structures. Ezrin contains three structural domains: an N-terminal FERM domain, a central α-helical domain containing a coiled-coil, and a C-terminal domain (CTD). The protein is thought to exist in two states: an inactive form in which the FERM and CTD interact with one another, and an active state in which the CTD is released from the FERM and able to bind actin. The current model for the activation of Ezrin involves the disruption of the FERM:CTD interaction initiation the FERM domain binding to PI(4,5)P2 and the phosphorylation of T567 in the CTD, both of which cause conformational changes in the protein to release the CTD. There is evidence, however, to suggest that this model is not entirely correct, as previous mutagenesis and structural data suggests the CTD partially masks the proposed lipid-binding surface on the FERM domain, and little is understood about any conformational changes that occur in full length Ezrin.
We have undertaken a series of in vitro lipid-binding experiments using Ezrin, as well as the isolated CTD and FERM domain, in order to interrogate the structural changes that occur upon contacting membranes. Using cosedimentation experiments we have demonstrated that the FERM domain can simultaneously interact with membranes and the CTD. Limited proteolysis experiments have indicated that Ezrin undergoes a conformational change in the presence of lipids, with similar rates of proteolysis between the wildtype protein and a T567D phosphomimetic mutant construct. Together, these results challenge the general model of Ezrin activation, and provide the basis for further mechanistic studies by cryo-electron microscopy, single molecule fluorescence and mass spectrometry.