On the Mechanism of Ezrin Activation
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Ezrin is a peripheral membrane protein that contributes to the organization and stability of cellular membrane structures by reversibly linking the plasma membrane to actin filaments. The formation of this membrane-actin linkage has been experimentally shown to require ezrin N-terminal (FERM) domain binding to PI(4,5)P 2 phospholipid-enriched membrane sites and the phosphorylation of the ezrin C-terminal domain (CTD) at residue T567. Collectively, membrane association and T567 phosphorylation are believed to promote separation of the FERM and CTD domains; however, the underlying molecular mechanism remains less clear. In this study, we investigate the mechanistic steps of ezrin activation and the thermodynamic free energy landscape of FERM-CTD dissociation using enhanced sampling molecular dynamics (MD). We find that upon ezrin attachment to a lipid membrane, PI(4,5)P 2 molecules outcompete other phospholipids at the surface of the FERM F1 and F3 subdomains. This interaction triggers a major conformational rearrangement within the FERM domain that destabilizes the FERM F2-CTD interface and initiates dissociation between the FERM and CTD. By employing well-tempered metadynamics (WTMetaD) with a contact-map collective variable, we determine that the principal barrier to FERM-CTD dissociation comes from F3-CTD interactions and that this dissociation can happen spontaneously with a moderate free energy barrier. We also show that the FERM-CTD reassociation after ezrin T567 phosphorylation is impeded due to reduced dissociation energy barrier. The free energy profile of dissociation between FERM and the CTD-replacing EBP50 protein is similar to that of the FERM-CTD system with nonphosphorylated T567, which agrees well with an in vivo experimental observation that EBP50 competes with the CTD for F2-F3 binding after CTD is dissociated. Together, our results help establish a revised view on the ezrin activation mechanism where FERM binding to PI(4,5)P 2 enables spontaneous dissociation of the nonphosphorylated CTD.
SIGNIFICANCE
Ezrin and related ERM proteins control how cells link their plasma membrane to the actin cytoskeleton, a process fundamental to cell shape, signaling and motility. Despite decades of study, the molecular basis of ezrin activation – how it transitions from a self-inhibited to an active membrane-bound state – has remained unresolved. Using atomistic and enhanced sampling molecular dynamics together with biochemical validation, we show that binding of the FERM domain to PI(4,5)P 2 -enriched membranes alone is sufficient to trigger spontaneous dissociation of the nonphosphorylated C-terminal domain. Phosphorylation of T567 subsequently stabilizes the open conformation and prevents domain reassociation, enabling actin engagement and binding of FERM partners such as EBP50. Collectively, these findings advance a more integrated view of ezrin activation, highlighting how membrane interactions, conformational flexibility and phosphorylation act in concert to regulate membrane-cytoskeleton coupling.
