The EBP50-moesin interaction involves a binding site regulated by direct masking on the FERM domain

Abstract

Members of the ezrin-radixin-moesin (ERM) protein family serve as regulated microfilament-membrane crosslinking proteins that, upon activation, bind the scaffolding protein ERM-phosphoprotein of 50 kDa (EBP50). Here we report a 3.5 Å resolution diffraction analysis of a complex between the active moesin N-terminal FERM domain and a 38 residue peptide from the C terminus of EBP50. This crystallographic result, combined with sequence and structural comparisons, suggests that the C-terminal 11 residues of EBP50 binds as an α-helix at the same site occupied in the dormant monomer by the last 11 residues of the inhibitory moesin C-terminal tail. Biochemical support for this interpretation derives from in vitro studies showing that appropriate mutations in both the EBP50 tail peptide and the FERM domain reduce binding, and that a peptide representing just the C-terminal 14 residues of EBP50 also binds to moesin. Combined with the recent identification of the I-CAM-2 binding site on the ERM FERM domain (Hamada, K., Shimizu, T., Yonemura, S., Tsukita, S., and Hakoshima, T. (2003) EMBO J. 22, 502-514), this study reveals that the FERM domain contains two distinct binding sites for membrane-associated proteins. The contribution of each ligand to ERM function can now be dissected by making structure-based mutations that specifically affect the binding of each ligand.

Sip1, theDrosophilaorthologue of EBP50/NHERF1, functions with the sterile 20 family kinase Slik to regulate Moesin activity

“…Sip1 binds Moesin in both two-hybrid and co-immunoprecipitation analyses, as has been observed for EBP50/NHERF1 in mammalian cells (Finnerty et al, 2004;Morales et al, 2004;Morales et al, 2007;Nguyen et al, 2001;Reczek and Bretscher, 1998). Consistent with this result, loss of Moesin and/or ezrin results in mislocalization of Sip1/EBP50 in both the fly and in the mouse (Fig.…”

“…One potential problem with this model is that previous work has suggested that EBP50/NHERF1 is unable to bind the folded, inactive form of ezrin (Finnerty et al, 2004;Reczek and Bretscher, 1998;Morales et al, 2007), raising the question of how Sip1 can facilitate activation of Moesin if it cannot bind the inactive, folded form. A possible answer to this question comes from recent observations that ERMs are regulated by several factors that might operate in a step-wise fashion.…”

“…The Cterminal region or 'EB domain' of EBP50/NHERF1, which has previously been shown to bind ezrin in mammals ( Fig. 1) (Finnerty et al, 2004;Reczek and Bretscher, 1998) is also conserved in Sip1. There was 23% identity (61% similarity) between the EB domain of Sip1 and human EBP50/NHERF1 ( Fig.…”

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

“…which is masked by C-ERMAD in the closed form of full-length ezrin (33,65,66). Using the monomer fraction from gel filtration, we find that an insignificant 0.4% fraction of the wild-type ezrin is capable of binding to NHERF1, whereas 16.2% ezrin(T567D), 18.3% ezrin(S249D), and about 27% ezrin(S249D/ T567D) are capable of binding NHERF1 (supplemental Table S2).…”

Distribution dynamics and functional importance of NHERF1 in regulation of Mrp-2 trafficking in hepatocytes

“…On the basis of ERM protein (moesin and ezrin) and NHERF1 structural studies, the COOHterminal 11 residues of NHERF1 appear to bind to the NH 2 -terminal portion of moesin (11). Further sequence analyses and biochemical studies with different NH 2 -terminal NHERF1 mutations suggest that the phenylalanine F355 mutant (F355R) abolished interaction with the ezrin NH 2 -terminal domain (5,11). We have created a NHERF1 F355R mutation that alters key residues necessary for interaction with radixin on the basis of known moesin structures and sequence comparison.…”