Ezrin, a member of the ezrin, radixin, moesin (ERM) protein family, is a critical and ancient protein responsible for actin binding and organization. It anchors to the plasma membrane via its FERM domain and interfaces with the actin cortex through its C-terminal F-actin-binding domain (C-ERMAD), thereby regulating actin dynamics in processes such as phagocytosis, microvillus formation, and tumor cell metastasis. Although ezrin's interactions with plasma membrane receptors have been extensively characterized and are therefore well understood, the molecular details of the ezrin/F-actin interaction remain largely unexplored. To address this knowledge gap, we employed minimal actin cortices (MACs) based on planar supported lipid bilayers, to which ezrin T567D was tethered via its lipid receptor PtdIns4,5P2. The membrane-bound ezrin T567D mutant, being in an open, active conformation, is capable of binding F-actin through its C-ERMAD. Upon addition of prepolymerized F-actin, MACs were formed, which were subject to high-resolution fluorescence microscopy. This in vitro approach enabled us to quantitatively analyse the impact of single-point mutations in the C-terminal domain of ezrin on its F-actin binding capability and the resulting network architecture. Our findings reveal that the last helix of the C-ERMAD (αD-helix) is most important for the interaction with F-actin. The positively charged Lys-577, as well as the hydrophobic Ile-580 in the αD-helix, are pivotal for F-actin binding. Besides electrostatic interactions (Lys-577), Ile-580 appears to be crucial and might mediate the interaction with the hydrophobic cleft of G-actin.
Ackermann et al. (Mon,) studied this question.