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. 2003 Feb 3;22(3):502-14.
doi: 10.1093/emboj/cdg039.

Structural basis of adhesion-molecule recognition by ERM proteins revealed by the crystal structure of the radixin-ICAM-2 complex

Keisuke Hamada  1 , Toshiyuki Shimizu, Shigenobu Yonemura, Shoichiro Tsukita, Sachiko Tsukita, Toshio Hakoshima
Affiliations

Structural basis of adhesion-molecule recognition by ERM proteins revealed by the crystal structure of the radixin-ICAM-2 complex

Keisuke Hamada et al. EMBO J. .

Abstract

ERM (ezrin/radixin/moesin) proteins recognize the cytoplasmic domains of adhesion molecules in the formation of the membrane-associated cytoskeleton. Here we report the crystal structure of the radixin FERM (4.1 and ERM) domain complexed with the ICAM-2 cytoplasmic peptide. The non-polar region of the ICAM-2 peptide contains the RxxTYxVxxA sequence motif to form a beta-strand followed by a short 3(10)-helix. It binds the groove of the phosphotyrosine-binding (PTB)-like subdomain C mediated by a beta-beta association and several side-chain interactions. The binding mode of the ICAM-2 peptide to the FERM domain is distinct from that of the NPxY motif-containing peptide binding to the canonical PTB domain. Mutation analyses based on the crystal structure reveal the determinant elements of recognition and provide the first insights into the physical link between adhesion molecules and ERM proteins.

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Figures

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Fig. 1. Overall structure of the radixin FERM domain bound to the ICAM-2 tail peptide. (A) Views of the radixin FERM domain bound to the ICAM-2 peptide by ribbon representations. The ICAM-2 peptide is shown in blue. The radixin FERM domain consists of subdomains A (light blue), B (red) and C (brown). The linkers A–B (residues 83–95) and B–C (residues 196–203) are colored in gray and the C-terminal linker in green. (B) The ICAM-2 peptide model in a 2Fo–Fc electron density map countered at the 1σ level. The amino acid residues are indicated with labels of one-letter codes. Labels in parentheses indicate the terminal residues whose side chains were not defined in the map. (C) The 28-residue peptide synthesized based on the sequence of the mouse ICAM-2 cytoplasmic tail was used for the structural work. Basic residues are in blue. This peptide has two basic regions and a non-polar region between them. The 16 residues of the peptide defined on the current map are boxed. Key residues in binding to the radixin FERM domain are underlined (see text). The short β-strand (residues 7–10) and one 310 helix (resides 12–15) are indicated with an arrow and a cylinder.
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Fig. 2. The ICAM-2 tail peptide recognition by the radixin FERM domain. (A) Surface electrostatic potentials of the radixin FERM domain viewed from the same direction as in Figure 11A. Positive (blue, +14 kT/e) and negative (red, –14 kT/e) potentials are mapped on the van der Waals surfaces. The ICAM-2 peptide found in the complex crystal is shown in a stick model. The disordered C-terminal basic region is indicated by an arrow of dotted lines. (B) The ICAM-2 binding groove on subdomain C is formed primarily by hydrophobic residues from helix α1C and strand β5C. The bound ICAM-2 peptide is shown in a transparency ribbon model. (C) Schematic representation of the interactions between the ICAM-2 peptide (blue) and subdomain C (brown). Hydrogen bonds are shown by broken lines. (D) The ICAM-2 peptide found in the FERM–ICAM-2 complex is shown in a stick model (light blue) with their interacting residues from subdomain C (brown). Hydrogen bonds are shown by dotted lines. (E) A close-up view of the hydrophobic and hydrogen bonding interactions between the ICAM-2 peptide and the FERM domain mediated by His288 from subdomain C.
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Fig. 2. The ICAM-2 tail peptide recognition by the radixin FERM domain. (A) Surface electrostatic potentials of the radixin FERM domain viewed from the same direction as in Figure 11A. Positive (blue, +14 kT/e) and negative (red, –14 kT/e) potentials are mapped on the van der Waals surfaces. The ICAM-2 peptide found in the complex crystal is shown in a stick model. The disordered C-terminal basic region is indicated by an arrow of dotted lines. (B) The ICAM-2 binding groove on subdomain C is formed primarily by hydrophobic residues from helix α1C and strand β5C. The bound ICAM-2 peptide is shown in a transparency ribbon model. (C) Schematic representation of the interactions between the ICAM-2 peptide (blue) and subdomain C (brown). Hydrogen bonds are shown by broken lines. (D) The ICAM-2 peptide found in the FERM–ICAM-2 complex is shown in a stick model (light blue) with their interacting residues from subdomain C (brown). Hydrogen bonds are shown by dotted lines. (E) A close-up view of the hydrophobic and hydrogen bonding interactions between the ICAM-2 peptide and the FERM domain mediated by His288 from subdomain C.
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Fig. 3. Sequence alignments of subdomain C and the FERM-binding peptides. (A) Secondary structure elements and sequence alignments of subdomains C of FERM domains with the related PTB domains. The FERM subdomain C of mouse radixin (mRAD) and the related proteins are aligned with the secondary structure elements of the radixin FERM subdomain C at the top: α-helix (blue rectangles) and β-strands (red arrows). Identical residues are highlighted in yellow. The aligned FERM subdomains C are mouse radixin (mRAD), moesin (mMOE), ezrin (mEZR), merlin (mMRL), human band 4.1 (hB41), talin (hTAL), protein tyrosine phosphatase D1 (hPTPD1) and Fas-associated protein tyrosine phosphatase 1 (hFAP1). The aligned PTB domains are the IRS-1 PTB domain (hIRS1), the X11 PTB domain (hX11). The secondary structure elements of these PTB domains are boxed with blue lines (α-helices) and with red lines (β-strands). The mouse FERM domains exhibit 100% identities with those of human and >99.7% identities with other mammalian homologues. Blue circles mark the radixin subdomain C residues interacting with the ICAM-2 peptide. (B) Sequence alignment of the juxtamembrane cytoplasmic regions of adhesion molecules that bind ERM proteins and of the related adhesion molecules. Basic and acidic residues are shown in blue and red, respectively. The basic clusters located at the N-terminal region are underlined. Glutamines frequently appearing in this N-terminal basic region are shown in green. Key residues of the ICAM-2 peptide for binding to the radixin FERM domain are boxed with hydrophobic residues in brown. The sources are mouse (m), rat (r), human (h), bovine (b) and nematode (c). The numbers on the right-hand side of the column indicate the C-terminal residue numbers in the sequences. The N-terminal regions of the C-terminal tail domains of ERM proteins are also aligned at the bottom of the figure with an arrow indicating strand 1.
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Fig. 4. Membrane-targeting and unmasking of ERM proteins. (A) Model of ERM proteins bound to both PIP2 in a membrane and the ICAM-2 cytoplasmic tail. The N-terminal FERM, the central helical and the C-terminal tail domains are shown with a triangle in blue, a rectangle in yellow and a block in red, respectively. ERM proteins partition between the cell cortex and cytosol. ICAM-2 in plasma membranes colocalized with PIP2. PIP2 molecules recruit masked ERM proteins from the cytosol to plasma membranes and unmask the masked ERM proteins for subsequent binding to the cytoplasmic tails of adhesion molecules such as ICAM-2 and actin filaments. (B) Comparison of the FERM–ICAM-2 complex with the masked form of moesin. Superposition of the C-terminal tail domain (pink) in the moesin FERM domain–C-tail complex on the radixin FERM domain (gray) bound to the ICAM-2 peptide (blue). (C) Comparison of the β-strand of ICAM-2 (blue green) and strand 1 of the moesin C-terminal tail domain (pink).
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Fig. 5. Comparison of the FERM–ICAM-2 complex with the related PTB–peptide complexes. (A) Superposition of the radixin FERM subdomain C (gray) bound to the ICAM-2 tail (blue) on the IRS-1 PTB domain (light gray) bound to the IR peptide (pink). (B) Peptide alignment based on the superimposed complex structures. The β-strands formed in the complex formations are underlined. The 310 helix in ICAM-2 and the NPxY motif sequences in the PTB domain-binding (IR, IL-4R and APP) peptides are boxed. The phosphotyrosine residues are in bold in the IR and IL-4R peptides. In the APP peptide, two Phe residues anchoring the peptide to the X11 PTB domain are also in bold. (C) Comparison of the 310 helix in the ICAM-2 peptide bound to the radixin FERM domain with the β-turn of the NPxY motif in the IR peptide bound to the IRS-1 PTB domain. The intra-peptide hydrogen bonds are shown as dotted lines.
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