BPS2026 – Mechanistic PI(4,5)P2 binding and T567 phosphorylation effects on ezrin activation

Ezrin is a peripheral membrane protein that participates in the maintenance of cellular membrane structures and prevents cancer progression by reversibly linking the membrane to actin filaments. The event of membrane-actin linkage has been experimentally found to be enabled by the attachment of the ezrin N-terminal (FERM) domain to PI(4,5)P 2 -enriched membrane sites and the phosphorylation of ezrin C-terminal (CTD) T567 residue. Taken together, membrane binding and phosphorylation are proposed to induce dissociation between the FERM and CTD domains; however, the mechanistic effects of the process remain obscure. In this study, we examine the mechanistic steps of ezrin activation and the thermodynamic free-energy profiles of FERM-CTD dissociation via molecular dynamics. We find that upon ezrin association with a model lipid membrane, PI(4,5)P 2 displaces other phospholipids at the FERM surface and induces a substantial conformational change in the FERM domain that destabilizes the FERM F2-CTD interface and initiates dissociation between FERM and CTD. Further, using well-tempered metadynamics with a contact-map collective variable we find that the barrier to FERM-CTD dissociation comes primarily from F3-CTD interactions, and that FERM-CTD reassociation is hindered after T567 phosphorylation due to a significantly reduced dissociation barrier. The free-energy profile of dissociation between FERM and the CTD-replacing EBP50 protein closely matches that of the FERM-CTD system with non-phosphorylated T567, which agrees well with in vivo experimental observations that EBP50 competes with CTD for F2-F3 binding after CTD dissociation. Together, our results help establish a new ezrin activation mechanism in which FERM binding to PI(4,5)P 2 enables spontaneous dissociation of the non-phosphorylated CTD. The FERM-CTD separation then provides space for kinase phosphorylation of the CTD which, once phosphorylated, does not reassociate with FERM and ultimately enables membrane-actin linkage as well as other downstream FERM interaction effects.