{"doi":"10.1101/2025.11.07.687285","title":"On the Mechanism of Ezrin Activation","abstract":"ABSTRACT 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":559472,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9608,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1245499,"name":"Jeriann Beiter","orcid":null,"position":1,"is_corresponding":false},{"id":1412810,"name":"Sahithya S. Iyer","orcid":"0009-0000-5591-5177","position":2,"is_corresponding":false},{"id":577140,"name":"Andrew T. Lombardo","orcid":"0000-0002-1814-5748","position":3,"is_corresponding":false},{"id":566371,"name":"Michelle C. Mendoza","orcid":"0000-0002-6490-1794","position":4,"is_corresponding":false},{"id":246697,"name":"Gregory A. Voth","orcid":"0000-0002-3267-6748","position":5,"is_corresponding":false},{"id":911998,"name":"Dovydas Vasiliauskas","orcid":null,"position":0,"is_corresponding":true}],"reference_count":54,"raw_metadata":null,"created_at":"2026-07-19T02:55:34.849815Z","pmid":"41279471","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}