Structural basis for CD44 recognition by ERM proteins

Abstract

CD44 is an important adhesion molecule that functions as the major hyaluronan receptor which mediates cell adhesion and migration in a variety of physiological and pathological processes. Although full activity of CD44 requires binding to ERM (ezrin/radixin/moesin) proteins, the CD44 cytoplasmic region, consisting of 72 amino acid residues, lacks the Motif-1 consensus sequence for ERM binding found in intercellular adhesion molecule (ICAM)-2 and other adhesion molecules of the immunoglobulin superfamily. Ultracentrifugation sedimentation studies and circular dichroism measurements revealed an extended monomeric form of the cytoplasmic peptide in solution. The crystal structure of the radixin FERM domain complexed with a CD44 cytoplasmic peptide reveals that the KKKLVIN sequence of the peptide forms a beta strand followed by a short loop structure that binds subdomain C of the FERM domain. Like Motif-1 binding, the CD44 beta strand binds the shallow groove between strand beta5C and helix alpha1C and augments the beta sheet beta5C-beta7C from subdomain C. Two hydrophobic CD44 residues, Leu and Ile, are docked into a hydrophobic pocket with the formation of hydrogen bonds between Asn of the CD44 short loop and loop beta4C-beta5C from subdomain C. This binding mode resembles that of NEP (neutral endopeptidase 24.11) rather than ICAM-2. Our results reveal a characteristic versatility of peptide recognition by the FERM domains from ERM proteins, suggest a possible mechanism by which the CD44 tail is released from the cytoskeleton for nuclear translocation by regulated intramembrane proteolysis, and provide a structural basis for Smad1 interactions with activated CD44 bound to ERM protein.

FIGURE 1.

Results of analytical ultracentrifugation of the CD44 cytoplasmic peptide. A, sedimentation equilibrium data with a fitted curve for the 0.25 mg ml^-1 CD44 cytoplasmic peptide solution at 38,000 rpm. The upper panel shows residuals representing the difference between the calculated fit and the experimental data using XLA/XL-I data analysis software. The molecular mass of the CD44 cytoplasmic tail was estimated to be 8.8 kDa. This result shows that the CD44 cytoplasmic tail adopts a monomeric form in the solution state. B, the distribution of sedimentation coefficients obtained from sedimentation velocity analysis of the CD44 cytoplasmic tail. The single peak is found at 0.73 S. C, the distribution of apparent molecular mass obtained from sedimentation velocity analysis of the CD44 cytoplasmic tail. The single peak is found at 10.4 kDa. D, CD spectra of the 72-residue CD44 cytoplasmic tail. Spectra of the CD44 peptide at 50 mm NaCl (blue) and 150 mm (yellow) are shown with a difference spectrum between the FERM domain and the 1:1 complex of the radixin FERM domain and CD44 (cyan). E, sensorgrams for each concentration of the merlin (left) or radixin (right) FERM domain. The signals from the control surface were subtracted. F, distribution of apparent molecular mass obtained from sedimentation velocity analysis of the FERM domain in the free and the CD44-bound forms. Estimated molecular masses are 38.7 kDa (free form) and 44.1 kDa (CD44-bound form), suggesting monomers.

FIGURE 2.

Crystal structure of the FERM-CD44 complex. A, ribbon representations of the radixin FERM domain complexed with the CD44 cytoplasmic peptide (blue). The radixin FERM domain comprises three subdomains: A (residues 3-82 in green), B (residues 96-195 in red), and C (residues 204-297 in yellow). Linkers A-B (residues 83-95) and B-C (residues 196-203) are colored gray. B, surface electrostatic potentials of the FERM domain and a close-up view of the CD44 cytoplasmic peptide docked into the groove formed between helix α1C and strand β5C of subdomain C. The peptide is shown as a stick model (labeled with one-letter codes), and the four side chain-binding sites (S1--S4) for the bound β strand of CD44 and the deep hydrophobic pocket (P1) are labeled and indicated with yellow dashed circles. Site S4 adjoins pocket P1. C, a stick model of the CD44 cytoplasmic peptide is shown in the omit electron density map for the CD44 cytoplasmic peptide at the contour level of 1σ.

FIGURE 3.

CD44 binding to subdomain C of the FERM domain and sequence alignment of subdomain C of ERM and related proteins. A, the CD44 cytoplasmic peptide (blue stick model) binds the groove between helix α1C (yellow cylinder) and strand β5C (yellow stick) of subdomain C of the FERM domain. Hydrogen bonds are shown as red dashed lines. B, schematic representation of the interactions between the CD44 cytoplasmic peptide (blue) and subdomain C (yellow). The nonpolar contacts involving side chains are shown with contacted residues. Two hydrogen bonds formed by the main chain carbonyl group of FERM Ile²⁴⁵ with the main and side chains of CD44 Asn¹⁵ are relatively long (3.45 Å). C, sequence alignment of subdomain C of mouse (m) radixin, ezrin, moesin, human band 4.1 protein (h P 4.1), and talin (h Talin). The secondary structure of the radixin FERM subdomain C is shown at the top. Green rectangle, α-helix; red arrows, β-strands. Residues that participate in nonpolar and polar interactions with the CD44 peptide are highlighted in yellow and cyan, respectively.

FIGURE 4.

Comparison of ICAM-2, NEP, and CD44 peptides bound to the FERM domain. Shown is superposition of ICAM-2 (magenta) from the FERM-ICAM-2 complex (28) and NEP (green) from the FERM-NEP complex (31) on the FERM-CD44 complex. The N-terminal extensions of ICAM-2 and CD44 or the C terminus of NEP that would be linked to the transmembrane helix in the plasmamembrane are indicated with dotted lines.

FIGURE 5.

Comparison of FERM-binding peptide sequences. A, alignment of the juxtamembrane regions of CD44 cytoplasmic tails is compared with that of ICAM-2. The secondary structures found in our FERM-CD44 complexed structure are shown at the top with those found in the FERM-ICAM-2 complex (28). The solid boldface lines indicate peptide residues defined in the maps, and the dotted lines indicate residues that were not defined in the maps. Basic and acidic residues are shown in blue and red, respectively, and key residues for binding are marked with boxes and highlighted in yellow. Two conserved basic clusters of CD44 are highlighted in blue. B, alignment of the juxtamembrane regions of NEP cytoplasmic tails is shown together with the secondary structures found in the FERM-NEP complex (31).

FIGURE 6.

Comparison of CD44 and ICAM-2 peptides bound to the radixin FERM domain. A, comparison of the FERM-bound peptide conformations of the CD44 (cyan) and ICAM-2 (magenta) peptides. Two peptide structures are overlaid in the middle. The side chains and main chains are shown as stick and line-tracing models, respectively. B, close-up view of the loop region of the CD44 cytoplasmic peptide (cyan) bound to the FERM domain (gray). The CD44 loop comprising Val¹³-Ile¹⁴-Asn¹⁵-Gly¹⁶ is docked into pocket P1 on subdomain C. Hydrogen bonds are shown as red dashed lines. The side chain of Val¹³ is not shown for clarity. CD44 Asn¹⁵ forms three hydrogen bonds to the main chain carbonyl groups of Trp²⁴², Ser²⁴³, and Ile²⁴⁵ of the FERM domain. C, close-up view of the 3₁₀ helix of the ICAM-2 peptide (magenta) bound to the FERM domain (gray) in the FERM-ICAM-2 complex (28). The 3₁₀ helix comprising Val¹²-Leu¹³-Ala¹⁴-Ala¹⁵ is docked into pocket P1 on subdomain C. The side chain of Ala¹⁵ is not shown for clarity. The water molecule mediating the hydrogen bond between ICAM-2 Trp¹⁶ and FERM His²⁸⁸ is shown as a green sphere.

FIGURE 7.

Comparison of CD44 and NEP peptides bound to the FERM domain. A, comparison of the FERM-bound peptide conformations of the CD44 (cyan) and NEP (green) peptides. The side chains and main chains are shown as stick and line-tracing models, respectively. B, two peptide structures are overlaid. C, close-up view of the hairpin of the NEP peptide (green) bound to the FERM domain (gray). The hairpin comprising Asp¹¹-Ile¹²-Asn¹³-Ala¹⁴ is docked into pocket P1 on subdomain C. NEP Asn¹³ forms a hydrogen bond to the main chain carbonyl group of FERM Trp²⁴².

FIGURE 8.

Pull-down assays of the FERM domain with wild-type and phosphorylated CD44. A, eluted samples analyzed by SDS-PAGE. Ser² is replaced with Asp (S2D) and phosphoserine (S2p). B, summary of pull-down assays of wild-type and phosphorylated CD44 to the radixin FERM domain. The bar graph documents the relative binding affinity.