addgene kanamycin ezrmcherry addgene  (Addgene inc)

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a) Ezrin protein sequence: membrane binding FERM domain consists of three lobes – F1 (residues P2-P86), F2 (residues E87-G202), and F3 (residues I203-K296); linker region consists of residues P297-P496; and the actin-binding C-terminal domain (CTD) consists of residues T497-L586. b) AlphaFold 3.0 predicted closed-form structure of the full-length ezrin protein . In the predicted closed-form ezrin structure, C-terminal CTD domain interacts with FERM over the F2-F3 subdomain surface. c) System setup for the FERM-CTD structure (PDB ID: 4RM9) at a DOPC:DOPS:PIP 2 (80:16:4ratio) membrane that was used in the molecular dynamics simulations in this study. d) Number of PIP 2 head groups phosphorus atoms within 10 Å of any atoms in the residues in FERM. Asterisks (*) indicate significant difference (α = 0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns, e) Aggregation of PIP 2 (orange), DOPS (blue) and DOPC (gray) phospholipids in a PIP 2 /DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark. f) Number of DOPS within 10 Å of the residues in FERM. Asterisks (*) indicate significant difference (independent sample t-test, α=0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns. Double asterisk (**) indicates significant difference (ANOVA, α = 0.05) between the means in systems with DOPC/DOPS/PIP 2 membranes and DOPC/DOPS membranes in the 990-1000 ns interval. g) Aggregation of DOPS phospholipids (blue) in a DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark.

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Sequencing, Membrane, Binding Assay

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a) Hydrogen bond counts between ezrin F2-CTD lobes (left) and ezrin F3-CTD lobes (right). Significantly more hydrogen bonds are broken in the system where ezrin has a phosphorylated T567 residue. b) Significant opening between CTD residues AA538-AA546 and FERM F2 lobe is only observed in the FERM-CTD system with phosphorylated T567 at a PIP 2 /DOPS/DOPC membrane. Left – crystal structure of ezrin FERM and C-ERMAD (PDB ID:4RM9). Right – distance between alpha carbon atoms of residue pairs K162-R542, Q160-K546, R156-T548 in the beginning of the equilibrated unbiased MD simulations (t=0 ns) and the end of the 1 µs simulation (t=1000 ns) for versions of the system that contain either a nonphosphorylated T567 (npT567, top) or phosphorylated T567 (pT567, bottom). b)-d) Distances of CTD residue Cα atoms from the Cα atoms of their nearest FERM F2-F3 residues. b) distances of CTD residues AA538-AA542 increase only for a system phosphorylated T567 at a PIP 2 /DOPS/DOPC membrane (red), c) large changes in distances are observed between Cα atoms of CTD helix H3 (residues AA549-AA559) and FERM F3 residues K230-T235 an CTD helix H3, d) no significant change in distance is observed between CTD helix H5 (residues T576-L586) and nearest FERM atoms. e) Coulombic potentials between CTD helices H1-H5 and nearest secondary structures in FERM F2-F3 lobes. Helices H1-H5 correspond to amino acid residues as follows: H1 – residues E525-Q540, H2 – residues A541-R547, H3 – residues H549-R559, H4 – residues K564-R572, H5 – residues T576-L586. Notation: pT567 – system with a phosphorylated T567, wt – system with a nonphosphorylated T567, soln – simulation carried out in a KCl solution. Asterisks (*) indicate a statistically significant coulombic attraction strength difference for a certain CTD helix between the FERM-CTD pT567 system at a PIP 2 /DOPC/DOPS membrane against any other systems. The inset provides a visualization of H1-H5 CTD helices in the crystal structure of closed state ezrin (PDB ID: 4RM9).

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Residue, Membrane

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a) Well-tempered metadynamics contact map collective variable can be successfully used to make the ezrin system undergo a full dissociation between FERM and CTD domains in about 20 ns. Image on the right is the schematic of how the contact map CV includes contacts across the entire surface of CTD and FERM F2-F3 lobes. b) The free energy surfaces of well-tempered contact map WTMetaD show that ezrin system with nonphosphorylated T567 has a mean barrier of 11.2 ± 0.3 kcal/mol for the transition from closed to dissociated FERM-CTD (blue) and a 3.2 ± 0.5 kcal/mol transition barrier between the closed and open states when the system has a phosphorylated T567 residue. The barrier of EBP50-FERM dissociation (11.7 ± 1.1 kcal/mol) is similar to that of FERM-wtCTD. Results are reported as mean (± SE) of sample free energy values corresponding to certain contact map collective variable values. The free energy profiles converged in 50-100 ns of the WTMetaD runs (Supplementary Figure S8). c) Ezrin FERM-CTD crystal structure (PDB ID: 4RM9). d) Crystal structure of ezrin FERM domain and EBP50 C-terminal residues (PDB ID: 1SGH). The C-terminal end of ezrin CTD domain has very similar alignment to F3 lobe of FERM as does the end helix of EBP50. e)-f) Distance between the C α atoms of different contact map pairs at contact map values 0.1-0.9 obtained from WTMetaD runs between F3 and CTD (e), and between F2 and CTD (f). Results in e)-f) are presented as mean distance and standard error for each of the contact map coordinate values collected from all WTMetaD simulation frames that have a contact map value in a certain range. g) RMSF plots for lobe F2 (left) and F3 (right) during the WTMetaD simulations for the system with phosphorylated T567 (red) and the system with nonphosphorylated T567 (blue).

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Residue

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a) Coomassie staining of purified proteins isolated from either HEK-293T cells (LOK-GFP-Flag) or Bacterial expression (EBP50 constructs and Ezrin). b) Western blotting of biochemical in vitro kinase assays blotting for total Ezrin vs. Ezrin phosphorylated at T567 (pT567). Purified full length untagged Ezrin and LOK-GFP-Flag kinase and were added at a constant concentration 1.5mg/ml and 0.05mg/ml respectively. Prior to the addition of ATP, the proteins were incubated with 0.5mg/ml of EBP50 or EBP50 variants and PI(4,5)P 2 was either added or withheld from duplicates of each EBP50 condition. Mutations in the two PDZ domains activate EBP50 leading to constitutively active binding between EBP50 and Ezrin’s FERM domain while EBP50’s tail domain is an unregulated Ezrin FERM binding motif. Phosphorylation of Ezrin at pT567 was PI(4,5)P 2 dependent but independent from EBP50 binding. c) Western blotting of lysate from human WT Jeg-3 epithelial cells and Jeg-3 CRISPR knockouts of EBP50 and LOK/SLK. Cells were treated with either the phosphatase inhibitor Calyculin A (Cal. A), the kinase inhibitor Staurosporine (Staursp) or no treatment prior to lysing and blotted for total Ezrin vs. Ezrin phosphorylated at T567 (pT567). Cells lacking EBP50 have less pT567 than their WT counterparts. The effect of treatment with Cal. A or Staursp. is not altered in cells lacking EBP50 vs. WT cells indicating that EBP50 influences the turnover but not the capacity of phosphorylation/dephosphorylation of ezrin at T567. Cells lacking the endogenous ERM kinases LOK/SLK have no phosphorylation at T567.

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Staining, Purification, Isolation, Expressing, Construct, Western Blot, In Vitro, Concentration Assay, Incubation, Binding Assay, Phospho-proteomics, CRISPR, De-Phosphorylation Assay

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a)-b) Closed state ezrin is attracted to apical cell membranes due to highly negatively charged PIP 2 phospholipids. c) Upon attachment to membrane, FERM F1 and F3 subdomains recruit more PIP 2 . d) Ezrin wtCTD spontaneously dissociates from FERM over long timescales. e) Dissociation of CTD leaves space between FERM and CTD for LOK kinase to phosphorylate the T567 residue. f) Upon T567 phosphorylation, ezrin primarily remains in the open state due to the lower propensity to inhabit the closed state. g) CTD is enabled to float away and interact with actin filaments, thus leaving space for downstream FERM F2-F3 interactions with EBP50 or other cellular targets.

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Membrane, Residue, Phospho-proteomics