- To assess how the phosphorylation influences ezrin conformation and its binding to PIP2 membranes, we purified recombinant wild type ezrin (ezrinWT) and a phosphomimetic mutant where the threonine at position 567 was replaced by an aspartate (ezrinTD), mimicking the open configuration of ezrin.
- We showed that ezrinTD has a higher binding affinity to membranes containing PIP2 (a lipid to which ezrin interacts specifically in cells) compared to that of ezrinWT. The dissociation constant K_d is equal to 1.2 M and 4.2 M for ezrinTD and ezrinWT, respectively.
- By using large unilamellar vesicles (LUVs) combined with cryo-electron microscopy (cryo-EM), to our surprise, we observed that both ezrinWT and ezrinTD assemble in an anti-parallel manner to tether adjacent membranes. Furthermore, the distances measured between the centers of the globular domains of the two opposing ezrin molecules sandwiched between the lipid layers were different: 24.1 ± 1.3 nm for ezrinTD and 28.7 ± 1.2 nm for ezrinWT (error bar represents standard deviation).
- Inspired by the membrane tethering of ezrinWT and ezrinTD observed by cryo-EM, we measured their membrane tethering strengths by dual-micropipette experiments in which two opposing giant unilamellar vesicles (GUVs) were held by micropipettes and tethered by ezrinWT or ezrinTD. We found that the tethering energy of ezrinWT was on average higher than that of ezrinTD.
- We confirmed that the open configuration of ezrinTD can bridge actin filaments to membranes.
- We assessed ezrin binding to positively curved membranes by injecting ezrin adjacent to membrane nano-tubes (10nm-100nm in radius) pulled outward from PIP2-containing GUVs by optical tweezers. We observed that only the phosphomimetic mutant of ezrin, ezrinTD, senses positive membrane curvature, likely due to its different conformation compared to ezrinWT.
- To assess ezrin binding to negatively curved membranes, we performed that same GUV-nano-tubes experiments while encapsulating ezrin inside the GUVs. We showed that neither ezrinWT nor ezrinTD senses negative membrane curvature.
- By cell biology experiments, we identified IRSp53 is a binding partner of ezrin in cells. IRSp53 is involved in the initiation of filopodia and is enriched inside model membrane nanotubes, having negative membrane curvature. We demonstrated that both ezrinWT and ezrinTD interact directly with the I-BAR domain of IRSp53. And this interaction further facilitate the enrichment of ezrinWT or ezrinTD in I-BAR domain decorated membrane tubules.
- Overall, our work reveals new mechanisms, specific conformation or binding to a curvature sensor partner, for targeting curvature insensitive proteins to curved membranes. The results of this work is currently under reviewed in Proceedings of the National Academy of Sciences for publication.
