Ezrin interacts with S100A4 via both its N- and C-terminal domains.

Beáta Biri-Kovács,László Nyitray,György Török,Gyula Pálfy,Andrea Bodor,Gergő Gógl,László Homolya,Henrietta Vadászi,Bence Kiss,Eugene A Permyakov

doi:10.1371/journal.pone.0177489

Abstract

Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process.

Highlights

Ezrin is a member of the ERM protein family and is responsible for linking the plasma membrane and the cytoskeleton; it has important roles in cell adhesion, migration and cell growth [1]
Recent results demonstrated that S100P, like S100A4, binds to non-muscle myosin isoforms [28] and this finding triggered the study of other ezrin-S100 protein interactions involving S100 paralogs A2, A4, A6 and B in the binding assays
S100A4 and S100P interact with the N-terminal ERM domain (N-ERMAD) with micromolar dissociation constants (2.2 ± 0.2 μM and 1.7 ± 0.3 μM, respectively), while S100A2, S100A6 and S100B bind with low affinity (Kd % 10–20 μM)

Summary

Introduction

Ezrin is a member of the ERM protein family and is responsible for linking the plasma membrane and the cytoskeleton; it has important roles in cell adhesion, migration and cell growth [1]. It participates in pathological processes such as cancer cell invasion and metastasis [2]. Ezrin consists of three domains: the FERM domain that is located at the N-terminal region (called N-ERMAD, ~300 amino acids), an α-helical linker region (~160 residues). Ezrin-S100A4 interaction supported through the New National Excellence Program of the Ministry of Human Capacities

Methods

Results

Conclusion