Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Structural studies of the membrane-bound protein Ezrin using cryo-electron microscopy (#225)

Hasti Iranmanesh 1 , Katharine A. Michie 2 , Paul M. Curmi 1
  1. School of Physics and School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, 2052, NSW, Australia
  2. Structural Biology Facility, Mark Wainwright Analytical Centre, The University of New South Wales, Sydney, 2052, NSW, Australia

Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family. It plays a key role in variety of cell functions, such as signal transduction, cell migration, and coupling of the actin cytoskeleton to the cell membrane1. ERM proteins exist as monomers, dimers, and presumably oligomers within cells. The structure of ezrin consists of three main domains: a N-terminal FERM domain which directly/indirectly binds to the membrane, a central a helical domain, and a C-terminal domain (CTD) with an actin filament binding site. Ezrin regulates the shape and dynamics of a cell by switching between an auto-inhibited “closed form” and active “open form”2. In its dormant state ezrin’s FERM domain and CTD form a complex, masking the membrane and actin binding sites. Activation of ezrin has been associated with sequential binding of the FERM domain to the membrane, release of CTD, and the phosphorylation of threonine Thr567 in CTD3. The transition between active and inactive states requires ezrin to undergo significant conformational changes which are poorly understood.

 

We are using cryo-electron microscopy (cryo-EM) and tomography to determine the atomic structures of free ezrin and membrane-bound ezrin. Structural comparison between these two atomic models should give us information on ezrin’s varied conformations and insight into ezrin function. Our initial tomograms of a phosphomimetic ezrin mutant (T567D) in the presence of membrane vesicles show the formation of ordered ezrin assemblies between the surfaces of adjacent vesicles where the length of ezrin corresponds to that of dimer models. We have used single particle analysis to determine the structure of the wild type ezrin.  Our maps show the globular FERM:CTD complex and parts of the central helical domain (ca. 51 kDa). Our preliminary results show that cryo-EM is a promising technique to study the structural transitions within ezrin associated with membrane binding.

  1. Neisch, A. L.; Fehon, R. G., Ezrin, Radixin and Moesin: key regulators of membrane–cortex interactions and signaling. Current Opinion in Cell Biology 2011, 23 (4), 377-382.
  2. Michie, K. A.; Bermeister, A.; Robertson, N. O.; Goodchild, S. C.; Curmi, P. M. G., Two Sides of the Coin: Ezrin/Radixin/Moesin and Merlin Control Membrane Structure and Contact Inhibition. Int J Mol Sci 2019, 20 (8), 1996.
  3. Fievet , B. T.; Gautreau , A.; Roy , C.; Del Maestro , L.; Mangeat , P.; Louvard , D.; Arpin , M., Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin. Journal of Cell Biology 2004, 164 (5), 653-659.