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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.01038

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

¹ Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY14853, USA
² Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA

View larger version (55K): [in a new window]   Fig. 1. The overall structure of dormant moesin. The N-terminal FERM domain (cyan) is associated with the C-terminal tail (red) which binds as an extended peptide inhibitor to block a large part of the surface of the FERM domain (from Pearson et al., 2000). The three FERM domain lobes are labeled as F1, F2 and F3 (F for FERM domain), and the major secondary structural elements of the C-terminal tail are labeled with the nomenclature used in this paper: ß1 for the single ß-strand, and A, B, C and D for the four -helices. Fig. 1 was created with the programs Swiss-PdbViewer (Guex and Peitsch, 1997) and Pov-Ray (http://www.povray.org).

View larger version (35K): [in a new window]   Fig. 2. Visualization of the binding of P38 to the FERM domain. (A) Final Fo-Fc electron density map, contoured at 3, of the P38: FERM complex (blue) shown with the C traces of two refined FERM domains (cyan and magenta) as they pack in the crystal. Boxes highlight the two major features of the difference map discussed in the text. The twofold crystallographic symmetry can be clearly seen in the density highlighted by the larger rectangle. A and B were prepared with the program PyMOL (DeLano, 2002). (B) View onto the F2-F3 face of the FERM domain (cyan surface) showing the same Fo-Fc electron density map contoured at 2 s (blue). The C-terminal tail (rendered as a C backbone in red) is shown as it is seen bound in the dormant moesin structure (Pearson et al., 2000). (C) Alignment of the 38 C-terminal residues of EBP50 and E3KARP with the last 38 residues of the ERM proteins. Based on the dormant moesin complex, the secondary structures are drawn as a cartoon, and the residues buried at the interface with the FERM domain are marked with an asterisk. The eleven C-terminal residues that fit the difference density associated with the F3 lobe are indicated by a black bar, and the residues discussed in the text are highlighted in yellow.

View larger version (65K): [in a new window]   Fig. 3. Biochemical support that the C-terminal residues of EBP50 bind as proposed. (A) Residues F355 and L358 in the EBP50 tail are important for the interaction with the FERM domain. Bacteria containing plasmids for the expression of MBP fused to the C-terminal 39 residues of EBP50 (WT), or containing the F355R mutation (F355R) or the last residue deleted (L358), were grown to log phase and either subjected to induced protein expression with IPTG (+) or not induced (-). Lysates were prepared and applied to beads on which the ezrin FERM domain had been immobilized. After washing, bound proteins were eluted and analyzed. The three eluates show proteins recovered from the induced lysates; essentially no material was recovered from parallel uninduced lysates (not shown). (B) The C-terminal 14 residue peptide of EBP50 binds to the FERM domain. Purified wild-type ezrin FERM domain (Wild type) or purified ezrin mutant N210F/T214A FERM domain (N210F/T214A) was mixed with beads containing covalently linked peptide (Peptide) or beads lacking the peptide (Control). The load (L), unbound (U), and bound and eluted (B) fractions were analyzed by SDS-PAGE.

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