We copurified the merlin-1 head and tail domains and crystallized the head:tail complex. SDS-PAGE (SDY, unpublished data) and mass spectrometry analyses confirmed the presence of the tail domain in these crystals (Supporting Information Table). However, electron density was only observed for the head domain. The final model is comprised of residues 20–82, 91–152, 178–312 (chain “A”); 20–82, 91–152, 178–312 (“B”); 20–82, 91–158, 178–312 (“C”); and 20–82, 91–150, and 199–312 (“D”). As seen in other isolated FERM domain structures, and in the moesin head:tail structure,15 the structure of the merlin head domains harbors three subdomains (F1, F2, and F3) [Fig. 1(A)] having fold similarities to known single-domain proteins.32 The F1 subdomain resembles ubiquitin, whereas F2 shares structural similarities with the acyl-CoA binding protein, and F3 has structural homology to phosphotyrosine binding (PTB), pleckstrin homology (PH), and Enabled/VASP Homology 1 (EVH1) signaling domains. In particular, in all reported structures, the F2 FERM subdomain is comprised of four α-helices that form a compact bowl-like structure. To our surprise, the F2 subdomain of the merlin FERM domain is unfurled and the F2 α3b α-helix is rotated away from the remainder of this subdomain. The unfurled F2 subdomain is seen in all four subunits in the symmetric unit and all four subunits are very similar. The F2 α3b α-helix (residues 151–201) does not interact with the remainder of this subdomain as seen in the native structure of the merlin head domain alone17 but with the α-helix α1c of the F3 subdomain of a two-fold related molecule [Fig. 1(B); Supporting Information Fig. S1]. Further, the loop that follows the F2 α-helix α2b (residues 151–158) engages in hydrophobic interactions with the side chains of Lys44, Asp45, Asp48, and Arg52 of the α-helix α1a of the F1 subdomain, and there are also electrostatic interactions between Asp152 and Arg52. In addition, the extended F2 α-helix α3b and its preceding region (residues 178–192) engage in hydrophobic contacts with Asn263, Ile264, Ser265, Leu297, Cys300, Ile301, Gly302, Asp305, and Leu306, which are located on the β-strand β5c (262–267) and α-helix α1c (290–311) of the two-fold related F3 subdomain [Fig. 1(C)]. Hydrogen-bond interactions of Met179 with Tyr266, Ile188 with Asp305, and Tyr192 with Arg309 are also manifest. Finally, the new extended loop connecting F2 α-helices α3b and α4b (residues 194–202) engages in hydrophobic interactions not seen in other FERM structures with the side chains of Cys51, Arg52, Arg57, Thr59, and Trp60, which are located on the two-fold related F1 subdomain α-helix α1a and its following loop. Hydrogen bond interactions of His195 with Arg309 and Arg198 with Leu56, Arg57, and Thr59 are also found in this contact area.
Figure 1. The merlin FERM domain structure is unfurled. (A) Cartoon drawing of the human merlin head FERM domain. The F1 subdomain (residues 20–82 and 91–100) is shown in yellow, the F2 subdomain (residues 101–158 and 178–215) is shown in green, and the F3 motif (residues 216–313) is shown in magenta. Some termini (21, 82, 158, and 178) and secondary structure elements (“a” belonging to the F1, “b” to F2, and “c” to the F3 subdomains) are labeled in several panels. (B) The unfurled F2 subdomain engages in additional contacts with another monomer, which is shown as a surface representation. The FERM subdomains are colored as in panel (A) (F1, yellow or black; F2, green; and F3, magenta). (C) Detailed view of the intermolecular interactions of the extended F2 α3b α-helix (F2, green) with a two-fold related molecule (F1, yellow; and F3, magenta). A surface representation is also shown for the F2 subdomain. (D) Superposition of our unfurled merlin head domain (molecule “C”; F1, yellow; F2, green; and F3, magenta) onto the closed, unbound FERM domain structure of merlin (PDB entry 1h4r; white and red) is shown. The two molecules in the closed FERM structure superimpose with r.m.s.d. of 1.3 and 1.4 Å for 1965 atoms of our unfurled merlin structure. The large movement of the α-helix α3b of the F2 subdomain (red) is indicated by the arrow. (E) Superposition of the unfurled merlin structure (molecule “C,” orange) onto the moesin head:tail complex structure (PDB entry 1ef1; F1 and F2, white; F2 α-helix α1b, moesin residues 95–112, red; F2 α2b α-helix, moesin residues 118–135, yellow; F2 α3b α-helix, moesin residues 164–179, green; F2 α-helix α4b, moesin residues 183–196, blue; tail, black) with r.m.s.d. of 1.9 Å for 1780 atoms of the two moesin FERM domains in the asymmetric unit. The large movement of α-helix α3b is indicated by a double arrow. The movement of the β6c-β7c loop that seems necessary to allow tail binding is indicated by an arrow. (F) Close-up view of the movement of the α-helix α3b upon tail binding. Trp191 residing on the F2 α-helix α3b of the superimposed closed, unbound merlin FERM conformation clashes with the tail domain, in particular with His529.
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Superposition of our unfurled merlin head domain structure onto the 1.8 Å structure of the merlin head domain alone17 shows that the F1 and F3 subdomains, and the α-helices α1b, α2b, and α4b regions of F2, are almost identical with r.m.s.d. of less than 0.6 Å for 1,704 atoms of residues 20–147 and 202–312 [Fig. 1(D)]. Similar results are obtained in a superposition with the mouse merlin FERM domain crystal structure.18 However, in our structure the last turn of the α2b α-helix of the F2 subdomain unfurls, thereby extending the following loop region and moving α-helix α3b to a completely new position, which also results in movement of the N-terminus of the F2 α-helix α4b.
Superposition with the 3 Å full-length moesin crystal structure33 (Supporting Information Fig. S2A) shows that the C-terminal region of the additional α-helix A of the central domain in moesin and the A–B loop prevents unfurling of its FERM domain. However, the central α-helical region, harboring α-helices A and B, is divergent between merlin and moesin with only 30% sequence identity.
Superposition with the moesin head:tail complex crystal structure [Fig. 1(E)] shows additional novel features of the F3 β6c-β7c loop (merlin residues 275–283), where this loop in our unfurled merlin FERM domain is located further away from the tail domain-binding site present in moesin, presumably to allow binding of the merlin-1 tail. Further, superposition of the closed, merlin structure, the moesin head–tail structure, and our unfurled head domain established that the β6c-β7c loop displays the conformation seen in the moesin head:tail complex structure allowing tail binding (Supporting Information Fig. S2B). Importantly, the F2 α-helix α3b, in particular Trp191 residing on α3b, prevents tail domain binding in the unbound merlin structure [Fig. 1(F)]. Indeed, the F2 α-helix α3b is shifted in the moesin head:tail structure to allow binding of the tail domain. Moreover, crystal contacts are not compatible with the tail binding as seen for moesin. We conclude that binding of the tail domain induces movements in the FERM domain, which could be initiating events for further unfurling of this region in merlin. Interestingly, there is only 43% identity in regions of divergent conformation (merlin residues 150–201), yet there is 53 and 74% identity in the 51 residues before (merlin residues 98–149) or after (merlin residues 202–253) this unfurled region (Supporting Information Fig. S3).