Shin-ichi Terawaki

Structural basis for CD44 recognition by ERM proteins.

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 β strand followed by a short loop structure that binds subdomain C of the FERM domain. Like Motif-1 binding, the CD44 β strand binds the shallow groove between strand β5C and helix α1C and augments the β sheet β5C-β7C 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 β4C-β5C 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.

Structural Basis for Type II Membrane Protein Binding by ERM Proteins Revealed by the Radixin-neutral Endopeptidase 24.11 (NEP) Complex

ERM (Ezrin/Radixin/Moesin) proteins mediate formation of membrane-associated cytoskeletons by simultaneously binding actin filaments and the C-terminal cytoplasmic tails of adhesion molecules (type I membrane proteins). ERM proteins also bind neutral endopeptidase 24.11 (NEP), a type II membrane protein, even though the N-terminal cytoplasmic tail of NEP possesses the opposite peptide polarity to that of type I membrane proteins. Here, we determined the crystal structure of the radixin FERM (Four point one and ERM) domain complexed with the N-terminal NEP cytoplasmic peptide. In the FERM-NEP complex, the amphipathic region of the peptide forms a β strand followed by a hairpin that bind to a shallow groove of FERM subdomain C. NEP binding is stabilized by β-β interactions and docking of the NEP hairpin into the hydrophobic pocket of subdomain C. Whereas the binding site of NEP on the FERM domain overlaps with the binding site of intercellular adhesion molecule (ICAM)-2, NEP lacks the Motif-1 sequence conserved in ICAM-2 and related adhesion molecules. The NEP hairpin, although lacking the typical inter-chain hydrogen bond but is stabilized by hydrogen bonds with the main chain and side chains of subdomain C, directs the C-terminal basic region of the NEP peptide away from the groove and toward the membrane. The overlap of the binding sites on subdomain C for NEP and Motif-1 adhesion molecules such as CD44 provides the structural basis for the suppression of cell adhesion through interaction between NEP and ERM proteins.

Structural basis of PSGL-1 binding to ERM proteins

P‐selectin glycoprotein ligand‐1 (PSGL‐1), an adhesion molecule with O‐glycosylated extracellular sialomucins, is involved in leukocyte inflammatory responses. On activation, ezrin–radixin–moesin (ERM) proteins mediate the redistribution of PSGL‐1 on polarized cell surfaces to facilitate binding to target molecules. ERM proteins recognize a short binding motif, Motif‐1, conserved in cytoplasmic tails of adhesion molecules, whereas PSGL‐1 lacks Motif‐1 residues important for binding to ERM proteins. The crystal structure of the complex between the radixin FERM domain and a PSGL‐1 juxtamembrane peptide reveals that the peptide binds the groove of FERM subdomain C by forming a β‐strand associated with strand β5C, followed by a loop flipped out towards the solvent. The Motif‐1 310 helix present in the FERM–ICAM‐2 complex is absent in PSGL‐1 given the absence of a critical Motif‐1 alanine residue, and PSGL‐1 reduces its contact area with subdomain C. Non‐conserved positions are occupied by large residues Met9 and His8, which stabilize peptide conformation and enhance groove binding. Non‐conserved residues play an important role in compensating for loss of binding energy resulting from the absence of conserved residues important for binding.

The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide‐exchange factors that possess the PH–CC–Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH–CC–Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three‐helical globular Ex subdomain. The PH subdomain resembles the β‐spectrin PH domain, suggesting non‐canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2‐interacting proteins for binding to PHCCEx domain, Motif‐I in CD44 and ephrinBs and the NMDA receptor, and Motif‐II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.

Structural basis of the cytoplasmic tail of adhesion molecule CD43 and its binding to ERM proteins

CD43/leukosialin/sialophorin is the major adhesion molecule in most hematopoietic cells and belongs to the sialomucin superfamily. In leukocyte emigration and activation, the exclusion of CD43 from the immunological synapse is an essential step. While the exclusion requires binding of the cytoplasmic region to ERM (ezrin/radixin/moesin) proteins, the detailed specific nature of the interaction between CD43 and ERM proteins is obscure. We have characterized the conformational properties of the CD43 cytoplasmic region, consisting of 124 amino acid residues, by hydrodynamic and spectroscopic measurements. Sedimentation equilibrium and velocity studies of ultracentrifugation revealed that the CD43 cytoplasmic peptide exists in a monomeric and extended form in solution. The crystal structure of the complex between the radixin FERM (4.1 and ERM) domain and the CD43 juxtamembrane region peptide reveals that the nonpolar region of the peptide binds subdomain C of the FERM domain. CD43 lacks the Motif-1 sequence for FERM binding found in the FERM-intercellular adhesion molecule-2 complex but possesses two conserved leucine residues that dock into the hydrophobic pocket of subdomain C without forming a 3(10)-helix. The FERM-binding site on CD43 is overlapped with the functional nuclear localization signal sequence. Our structure suggests that regulation of ERM binding may be coupled with regulated intramembrane proteolysis of CD43 followed by the nuclear transfer of the cytoplasmic peptide.

Crystallographic characterization of the radixin FERM domain bound to the C-terminal region of the human Na+/H+-exchanger regulatory factor (NHERF)

Radixin is a member of the ERM proteins, which cross-link plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins interact with Na(+)/H(+)-exchanger regulatory factors (NHERFs), which are PDZ-containing adaptor proteins, to modulate the ion-channel activity. Here, crystals of complexes between the radixin FERM domain and the C-terminal regions of NHERF and NHERF2 have been obtained. The crystals of the FERM-NHERF complex were found to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 69.38 (2), b = 146.27 (4), c = 177.76 (7) A. The crystal contains four complexes in the asymmetric unit. An intensity data set was collected to a resolution of 2.50 A.

Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry

Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicD CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP)

ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6(1)22, with unit-cell parameters a = b = 122.7, c = 128.3 A, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 A.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1.

Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM domain bound to CD43 belong to space group P4(3)22, with unit-cell parameters a = b = 68.72, c = 201.39 A, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM domain bound to PSGL-1 belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 80.74, b = 85.73, c = 117.75 A, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9 A for the FERM-CD43 complex and 2.8 A for the FERM-PSGL-1 complex.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44

CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell-cell and cell-matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane-cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 A, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 A.