Comparison of dormant human and Sfmoesin structures. (a) The human FERM-C-terminal domain complex (PDB code 1EF1). The three lobes of the ERM domain (F1, F2 and F3) are colored cyan and the C-terminal domain red. The β1 strand of the C-terminal domain is contributed by a crystal-packing interaction. (b) The 2.1 Å Sfmoesin structure. The α-helical domain (yellow) folds into three extended helices (αA, αB and αC), each of which containing elements that pack against the FERM domain. The αB and αC helices form an anti-parallel coiled-coil. (c) In the 3.0 Å structure, 67 more residues of the ∼70Å αB/αC coiled-coil are revealed.

Extent and sequence conservation of the surfaces buried by the α-helical domain and linker region. (a) The Sfmoesin FERM domain. The view is rotated by ∼180° around a vertical axis from that in Fig. 2. (b) Molecular surface of the FERM domain. Yellow regions are those in contact with the α-helical domain and linker region (∼1800 Ų of buried accessible surface area). (c) Conservation of the FERM domain. Magenta regions correspond to residues that are either identical or conservatively substituted (e.g. Asp/Glu, Arg/Lys, Ser/Thr) in all ERM-merlin proteins. Green regions correspond to residues conserved only in the ERM family.

Comparison of the Sfmoesin α-helical domain with active ERM domain structures and other coiled-coil domains. (a) The structures of “activated” merlin, radixin, and moesin (PDB entries 1ISN, 1J19, 1E5W, respectively) superimposed on the 2.1 Å structure of Sfmoesin, showing that the αA helix does not greatly alter its orientation upon activation. (b) The Rad50 coiled-coil (PDB entry 1L8D) superimposed on that of the 3.0 Å structure of Sfmoesin in the same orientation as panel (a). In this structure, Rad50 is dimerized through a hook-like structure at the turn of the coiled-coil 48. (c) Superposition of the methyl-accepting chemotaxis protein coiled-coil (PDB entry 2CH7) 49. This coiled-coil is half of an antiparallel four-helix bundle. Note that the N-terminal segment of Sfmoesin αB, which does not participate in coiled-coil with αA, maintains proper coiled-coil geometry. Superpositions are the optimal fits reported by a search of the PDB using the DALI server 50.

The known intermolecular binding sites of ERM proteins are masked in the dormant structure. (a) The ICAM-2 (orange cartoon), IP₃ (ball and sticks), and EBP50 (green coil) ligands mapped onto the structure of Sfmoesin (from PDB entries 1J19, 1GC6 and 1SGH, respectively). (b) Solvent accessible surface of the FERM domain of Sfmoesin with the α-helical domain and C-terminal domain superimposed. The surface is oriented as in panel (a) and colored by its electrostatic potential, contoured from −6.0 (red) to 6.0 kT/e^- (blue). The intensely basic surface likely helps moesin to bind negatively charged lipid bilayers, such as those that contain PIP₂ (see panel a). (c) The electrostatic surface of dormant Sfmoesin. The association of the α-helical domain and the linker region ablates the positive charge of the surface and masks the PIP₂ site. The ICAM-2 peptide binding site is masked by the β1-strand of the C-terminal domain. The electrostatic calculation does not take into account the negatively charged, disordered loop (residues 473-485) that connects the linker to the C-terminal domain.

Domain structure of Sfmoesin and primary sequence of the ERM-merlin α-helical domain. (a) Domain structure of Sfmoesin. Residue numbers at the domain boundaries are indicated. (b) Alignment of ERM-Merlin α-helical domains. The sequence for αC is folded back (runs right to left) to indicate its register with the αB helix. Helical regions are indicated by a yellow coil, and the β1 strand of the C-terminal tail by a red arrow. The a and d positions of the coiled-coil heptad repeat 16 are shown with orange and cyan backgrounds. These positions interact with the other helix of the coil, as shown. Residues that are disordered in both Sfmoesin structures are shown with lower case italics. Sequence numbering corresponds to that of Sfmoesin, and the asterisks indicate invariant or highly conserved residues. Sites sensitive to trypsin digestion in the radixin α-helical domain (black arrows), and positions in human merlin associated with cancer (purple arrows) are indicated. Sequences used are as follows: human merlin (HsMerlin), SwissProt accession no. P35240; human radixin (HsRadixin), P35241; human ezrin (HsEzrin), P15311; human moesin (HsMoesin), P26038; and D. melanogaster moesin (DmMoesin), GenBank accession no. NP_996392.

Crystal packing interactions and mobility of the α-helical domain. (a) Stereo view along the crystallographic 2-fold axis of a D₃ center in the crystals of the 3.0 Å structure. Each asymmetric unit in the cluster is colored uniquely. In this structure, differences in lattice packing tilt the αB/αC coiled-coil region by 7°, allowing it to form crystal contacts with a 2-fold related coiled-coil via the side chains of Gln426 and Leu430 (ball and stick models). Only residues 400-409 are missing in its helical turn. This structure eliminates the possibility of a dimer mediated by the coiled-coil in the crystalline lattice. (b) Temperature factors as a function of residue position in the 2.1 and 3.0 Å structures of Sfmoesin. The regions of the α-helical domain that contact the FERM domains are as stable as the FERM domain, while those at greater distances from the ERM domain have gradually increasing mobility. The change in crystal contacts between the two structures orders more of the αB/αC coiled-coil in the low resolution structure, but also disorders part of the αA-αB loop (residues 312-325).

Stereo views of interdomain contacts of the Sfmoesin α-helical domain and linker region. (a) The αA helix (yellow) interacts with the F1 and F2 lobes (cyan) in a bipartite fashion. Carbon atoms are shown with the same color as the backbone, oxygens red, nitrogens blue and sulfurs green. Specific hydrogen bonds are shown as black dashed lines. Residues colored with white carbons indicate side chains that are disordered or exist in multiple conformations in the 2.1 Å crystal structure. (b) The launching pad involves the hydrophobic face of αB and a highly conserved surface of the F1 lobe. Met347 is analogous to the site of tyrosine phosphorylation in ezrin 35;36, which leads to activation of ezrin in response to growth factor stimulation. Mutation of the residue analogous to Leu344 to proline in merlin is associated with NF2 39. Both modifications in the context of the Sfmoesin structure likely disrupt this interface. (c) The landing pad is formed by the end of the αC helix and the linker region. Contacts between the linker region and the F1 lobe are dominated by backbone-side chain interactions, possibly explaining the lack of strong sequence conservation in this region of the α-helical domain. Mutation of the residue analogous to Trp43 and residues in the F1 αA helix in merlin are also associated with NF2 39; 51. These changes likely disrupt the observed interdomain contacts.