The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module
2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês
10.1038/emboj.2009.323
ISSN1460-2075
AutoresShin‐ichi Terawaki, Ken Kitano, Tomoyuki Mori, Yan Zhai, Yoshiki Higuchi, Norimichi Itoh, Takashi Watanabe, Kozo Kaibuchi, Toshio Hakoshima,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoArticle5 November 2009Open Access The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module Shin-ichi Terawaki Shin-ichi Terawaki Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Department of Life Science, University of Hyogo, Kamigori-cho, Ako-gun, Hyogo, Japan Search for more papers by this author Ken Kitano Ken Kitano Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Tomoyuki Mori Tomoyuki Mori Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Yan Zhai Yan Zhai Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Yoshiki Higuchi Yoshiki Higuchi Department of Life Science, University of Hyogo, Kamigori-cho, Ako-gun, Hyogo, Japan Search for more papers by this author Norimichi Itoh Norimichi Itoh Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan Search for more papers by this author Takashi Watanabe Takashi Watanabe Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan Search for more papers by this author Kozo Kaibuchi Kozo Kaibuchi Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan Search for more papers by this author Toshio Hakoshima Corresponding Author Toshio Hakoshima Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Shin-ichi Terawaki Shin-ichi Terawaki Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Department of Life Science, University of Hyogo, Kamigori-cho, Ako-gun, Hyogo, Japan Search for more papers by this author Ken Kitano Ken Kitano Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Tomoyuki Mori Tomoyuki Mori Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Yan Zhai Yan Zhai Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Yoshiki Higuchi Yoshiki Higuchi Department of Life Science, University of Hyogo, Kamigori-cho, Ako-gun, Hyogo, Japan Search for more papers by this author Norimichi Itoh Norimichi Itoh Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan Search for more papers by this author Takashi Watanabe Takashi Watanabe Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan Search for more papers by this author Kozo Kaibuchi Kozo Kaibuchi Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan Search for more papers by this author Toshio Hakoshima Corresponding Author Toshio Hakoshima Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Author Information Shin-ichi Terawaki1,2, Ken Kitano1, Tomoyuki Mori1, Yan Zhai1, Yoshiki Higuchi2, Norimichi Itoh3, Takashi Watanabe3, Kozo Kaibuchi3 and Toshio Hakoshima 1 1Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan 2Department of Life Science, University of Hyogo, Kamigori-cho, Ako-gun, Hyogo, Japan 3Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan *Corresponding author. Structural Biology Laboratory, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Tel.: +81 743 72 5570; Fax: +81 743 72 5579; E-mail: [email protected] The EMBO Journal (2010)29:236-250https://doi.org/10.1038/emboj.2009.323 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 ephrinB's 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. Introduction T-lymphoma invasion and metastasis 1 (Tiam1) was originally isolated as an invasion-inducing gene product from T-lymphoma (Habets et al, 1994). Tiam1 belongs to the Dbl-family of guanine nucleotide-exchange factors (GEFs) that contain the Dbl-homology (DH) and Pleckstrin-homology (PH) tandem (DH–PH) domain, which directly catalyses the GDP/GTP exchange reaction in the GTPase cycle and thus facilitating the switch between inactive GDP-bound and active GTP-bound states (Mertens et al, 2003). The structures and functions of DH–PH domains have been extensively studied (Hakoshima et al, 2003; Rossman et al, 2005). Tiam1 acts as a GEF that specifically activates Rac GTPases to induce membrane ruffling and cell motility through reorganization of actin cytoskeletons and the establishment of cell polarity, which are believed to be processes closely related with cancer cell invasion and metastasis (Michiels et al, 1995). Crystallographic investigations have elucidated at atomic resolution the mechanism by which the Tiam1 DH–PH domain catalyses the GTP/GDP exchange of Rac1 (Worthylake et al, 2000). Tiam1 has a closely related homolog Tiam2, also referred to as STEF (SIF and Tiam1 like-exchange factor), which was isolated by RT–PCR using mRNA extracted from mouse adult brain (Hoshino et al, 1999). SIF (still life) is a Drosophila Tiam1 homolog that has a function in the changes in morphology of axon terminals that occur with differentiation into mature synapses (Sone et al, 1997). All of these GEFs possess two PH domains, which are often referred to as PHn and PHc domains. The PHc domain is part of the well-characterized DH–PH tandem domain, whereas PHn is featured as part of a novel N-terminal PH–CC–Ex region that functions as a protein interaction module. This region is composed of three parts: a PH subdomain, a putative coiled-coil (CC) region and a conserved extra (Ex) region whose structural characteristics are undefined (Figure 1A). Stimulation of cells by treatment with serum resulted in membrane localization of these GEFs and subsequent induction of membrane ruffling (Michiels et al, 1997; Stam et al, 1997). This translocation from the cytoplasm to plasma membranes is an essential step for Rac activation. Interestingly, this PH–CC–Ex region, but not the PHn domain alone or the PHc domain of the DH–PH tandem domain, is essential for this process and has a crucial function in the redistribution of Tima1/2 in cells by interacting with plasma membranes (Michiels et al, 1997; Stam et al, 1997). Notwithstanding the extensive studies that have focused on this medically and biologically important GEF, the precise mechanism by which the PH–CC–Ex region contributes to membrane localization remains unknown. Figure 1.Domains in Tiam 1 and Tiam2. (A) Tiam1 and Tiam2 molecules possess five common domains/regions: the PH–CC–Ex region (orange), a Ras-binding domain (RBD), a PSD-95/DlgA/ZO-1 (PDZ) domain, a DH domain and the C-terminal PH (PHc) domain are indicated. The PH–CC–Ex region contains the N-terminal PH (PHn) domain. Two PEST sequences (ellipses) and the myristoylation site in Tiam1 are indicated. (B) Three subdomains of the Tiam2 PH–CC–Ex region used in this study. Download figure Download PowerPoint The PH subdomain of the Tiam1/2 PH–CC–Ex region preferentially binds the products of phosphatidylinositol (PI)-3-kinase, PI-3,4,5-trisphosphate (PI(3, 4, 5)P3) and PI-3,4-bisphosphate (PI (3, 4)P2), as well as PI-4,5-bisphosphate (PI(4, 5)P2) (Ceccarelli et al, 2007). More importantly, it has been suggested that the PH–CC–Ex region may function as a protein–protein interaction module. The region has been shown to directly bind to integral membrane proteins that contain the important cell adhesion molecule CD44, the hyaluronic acid (HA) receptor critical for cell motility and cell proliferation (Bourguignon et al, 2000), the axon guidance cue protein ephrin B1 that mediates neurite outgrowth (Tanaka et al, 2004), and the ephrin receptor Eph B2 (Tolias et al, 2007). Interacting with ephrins and Ephs, Tiam1 participates in the forward signalling of the Eph B receptor and the backward signal of ephrin B1. In addition to these membrane proteins, the PH–CC–Ex region has been reported to directly bind scaffolding proteins that bind Rac effectors such as JNK-interacting protein-1 and -2 (JIP-1 and JIP-2) (Buchsbaum et al, 2002), spinophillin/neurabin II (Buchsbaum et al, 2003) and Par3 of the Par3–Par6–aPKC complex (Chen and Macara, 2005; Nishimura et al, 2005). Moreover, Tiam1 has been found to form a complex with the Rac effector IRSp53 (insulin receptor substrate p53) by itself (Connolly et al, 2005). These findings suggest that some Rac GEFs can influence Rac GTPase signalling specificity in addition to promoting their activation (Buchsbaum et al, 2002). Although biochemical studies have indicated a potential cooperative function of the PH subdomain and the extended CC and Ex segments of the Tiam1/2 PH–CC–Ex region for protein–protein interactions, the structural and functional roles of the extended regions remained elusive. Moreover, it was unclear whether the Tiam1/2-binding proteins possessed common structural and/or sequence characteristics. Here, we report the crystal structures of the PH–CC–Ex region of Tiam1 and Tiam2. The structures revealed that the three regions are combined into a unique globular structure, the PHCCEx domain, which creates a positively charged groove for binding to proteins along with the PI-binding site in the PH subdomain. Furthermore, we identified a negatively charged peptide region as the PHCCEx-binding site of CD44, which preserves the Motif-I sequence motif that is conserved in the PHCCEx-binding site of ephrin Bs. In Par3 and JIP2, we also identified another sequence motif, the acidic cluster Motif-II, for PHCCEx binding. Our results provide valuable clues towards understanding the molecular mechanism by which the PHCCEx domain mediates the Rac-specific GEFs to translocate towards plasma membranes through the dual recognition mode of PIs and membrane proteins, and also to recruit Rac effector proteins through binding to scaffolding proteins. Results Structural determination The PH–CC–Ex regions of Tiam1 and Tiam2 display high sequence similarity (65% identity). The N-terminal boundary of the PH–CC–Ex regions was determined by comparison with other canonical PH domains, whereas the C-terminal domain boundary was not determined uniquely using sequences. Therefore, we prepared protein molecules that possessed different C-terminal ends for the crystallization. We first succeeded in crystallizing the Tiam1 PH–CC–Ex region, which comprised 274 residues (residues 429–702), although the crystals diffracted poorly (∼4.5 Å resolution) (Terawaki et al, 2008). Crystals suitable for structure determination were obtained from a construct of the Tiam2 PH–CC–Ex region that included residues 500–757 (Figure 1B). Protein samples derived from the construct were crystallized into two different crystal forms, both of which contained four molecules per asymmetric unit. The structure of the initially obtained P43212 crystals was determined using the multiwavelength anomalous dispersion (MAD) method combined with single isomorphous replacement using the native and selenomethione-substituted protein crystals, and subsequently refined using the P21 crystal to 2.1-Å resolution. The low-resolution structure of the Tiam1 PH–CC–Ex region was then determined by molecular replacement using the Tiam2 PH–CC–Ex structure as a search model and refined to 4.5-Å resolution. The PH–CC–Ex region is folded into a single globular domain, PHCCEx domain In our crystal structures, the PH–CC–Ex region is folded into a single globular domain (Figure 2A and B) that contains three-folded subdomains: an N-terminal PH subdomain (residues 504–625), a CC subdomain (residues 630–700) forming an antiparallel coiled coil with two long α-helices (α2 and α3) and a C-terminal Ex subdomain (residues 707–741) forming a small globular domain comprising three α-helices (α4–α6) (Figures 2C and 3). The PH and CC subdomains are positioned so as to form a V-shape, and the Ex subdomain is folded back to wedge into the gape of the V-shape. The resultant globular domain has overall dimensions of 35 × 55 × 70 (Å), and a thickness of 30 Å. Thus, this PH–CC–Ex region is hereafter referred to as the PHCCEx domain. The compact globular domain structure is consistent with our sedimentation equilibrium data, which suggest a monomeric globular domain for the PH–CC–Ex region in solution with estimated molecular masses of 32.9 kDa (Tiam1) and 30.4 kDa (Tiam2) (Supplementary Figure S1). Figure 2.Structure of the Tiam2 PHCCEx domain. (A) A stereo view of the Tiam2 PHCCEx domain comprising three subdomains: the N-terminal PH subdomain (pink), a CC region (blue) and an Ex region (green) with three variable loops (red). Residues 553–560 (a dotted line) of loop β3–β4 are not defined in the current map. (B) As in (A) but with a rotation (90°) to show the putative PI-binding site. (C) Topology diagram of the Tiam2 PHCCEx domain. The missense mutation site (Ala441 of Tiam1) found in Tiam1 is marked by M. (D) The Tiam2 PH subdomain (pink and red) was superposed on the β-spectrin PH domain (grey and cyan) bound to Ins(1, 4, 5)P3. Side chains important for direct interactions with Ins(1, 4, 5)P3 are shown with the stick models with hydrogen bonds (dotted lines) between β-spectrin and Ins(1, 4, 5)P3. Download figure Download PowerPoint Figure 3.Sequence alignment of PHCCEx domains with secondary structure elements of the Tiam2 PHCCEx domain at the top. Conserved and semi-invariant (E=D, R=K=H, T=S, F=Y, V=L=I=M=C) residues are highlighted in yellow and blue-green, respectively. Acidic and basic residues are in red and blue, respectively. Residues predicted to be involved in interactions with phosphoinositides are indicated by red circles. Residues that were shown to be important for CD44 binding are indicated by blue circles. The Tiam1 missense mutation is indicated by a box. Download figure Download PowerPoint In the asymmetric unit of the present crystal, the four PHCCEx domains essentially exhibit the same structure. Pairwise superposition on each subdomain indicates a small average root-mean-square (r.m.s.) deviation (0.3 Å) for both PH and Ex subdomains. The CC subdomain, however, displays a relatively large deviation (0.9 Å), which is associated with bending of a long CC structure and resulted in large average r.m.s. deviations (0.52–1.47 Å) for the overall PHCCEx domain (Supplementary Figure S2). The current structure contains no models for the loop between strands β3 and β4 (β3–β4 loop), which was poorly defined in the current electron density map. N-terminal PH subdomain The PH subdomain of the Tiam2 PHCCEx domain comprises 122 residues and displays a typical PH domain fold, a β-sandwich capped at one end by a C-terminal α-helix (Figure 2C). At the β1–β2 loop, the PH subdomain forms an additional β1′ strand, which is located at the edge of the canonical seven-stranded β-sandwich and is associated with β1 strand. A DALI database search indicated the highest score for mouse β-spectrin PH domain (Hyvönen et al, 1995), which exhibits 30% sequence identity to the Tiam2 PH subdomain, with a small r.m.s. deviation (1.4 Å). On the other hand, low scores are obtained for phosphotyrosine-binding (PTB) domains with r.m.s. deviations larger than ∼3.2 Å. Similarly, poor structural similarity was observed with the EVH1/WH1 domain of Mena (Prehoda et al, 1999) and PHear domain of NECAP1 (Ritter et al, 2007). All of these domains possess peptide-binding sites for target proteins on the β-sandwich folds. It is well known that PTB domains possess a wide groove between β5 strand and α1 helix for peptide binding. In our PHCCEx domain structure, CC and Ex subdomains completely cover the corresponding groove of the PH subdomain. Like canonical PH domains, our PH subdomain possesses the narrow groove that seems not to accommodate a peptide chain. In fact, the isolated PH subdomain exhibited no CD44-binding activity (data not shown). Thus, it is unlikely that the peptide-binding ability is specially conferred by the PH subdomain of our PHCCEx domain. Conventional PH domains, which were first identified as phosphoinositide-binding domains, have important functions in the spatial and temporal regulation of protein localization (Lemmon, 2008). The PI-binding affinities of PH domains were found to be dependent on three variable loops (β1–β2, β3–β4 and β6–β7 loops) that flank the open end of the β-sandwich (DiNitto and Lambright, 2006). The Tiam2 PH subdomain possesses a long β1–β2 loop similar to that of the β-spectrin PH domain and its closely related PH domains (Figure 2B). Indeed, structural comparison with the β-spectrin PH domain reveals that the Tiam2 PH subdomain exhibits similar conformations in terms of the β1–β2 and β5–β6 loops (Supplementary Figure S3A). For PI binding, β-spectrin and its closely related ArhGAP9 PH domains preserve two short sequences, K-Xn-R-S-W in β1–β2 loop and Y-X-K in β5–β6 loop (Hyvönen et al, 1995; Ceccarelli et al, 2007). These sequences are fairly well conserved in the Tiam1/2 PH subdomains. Overlay of Ins(1, 4, 5)P3 bound to β-spectrin on the current structure suggests that similar PI binding could occur in the PH subdomain of the Tiam2 PHCCEx domain (Figure 2D). Structural comparison with the Akt PH domain reveals conformational deviations in the β1–β2 and β5–β6 loops (Supplementary Figure S3B). In particular, loop β1–β2 of the Tiam1/2 PH subdomains lacks the K-Xn-K/R-X-R motif, which is characteristic in Akt, Btk and Grp1 PH domains that exhibit canonical PI binding with a high affinity using loops β1–β2 and β3–β4. Moreover, the β3–β4 loop of the Tiam2 PH subdomain displays profound conformational flexibility. These structural considerations support the earlier notion that the Tiam1/2 PH subdomain would use a non-canonical PI-binding mechanism like the case with β-spectrin (Rameh et al, 1997; Ceccarelli et al, 2007). In fact, like the ArhGAP9 PH domain, both the Tiam1 PH subdomain alone and the PHCCEx domain have been shown to exhibit strong preferences for PI(4, 5)P2 as well as PI(3, 4, 5)P3 and PI(3, 4)P2 (Ceccarelli et al, 2007). This is in sharp contrast with the Akt PH domain, which exhibits a well-established preference for PI(3, 4, 5)P3 and PI(3, 4)P2 over PI(4, 5)P2. CC subdomain The CC subdomain of the Tiam2 PHCCEx domain follows the C-terminal α1 helix of the PH subdomain through a four-residue linker (residues 626–629). Two long α-helices, α2 and α3, of the CC domain form extended antiparallel CC helices that are stabilized by being N-terminally capped by conserved Asp residues and C-terminally capped by main-chain amide groups of the following loops. In addition, α2 helix is C-terminally capped by Arg668 from α3 helix at the tip of the coiled coil. The antiparallel CC association is stabilized primarily by non-polar contacts involving the leucine zipper-like motif (Supplementary Figure S4). At the C-terminal half of α2 helix, polar residues sparsely occupy the positions for leucine or non-polar residues of the heptad repeat and contribute towards stabilization of the coiled coil by polar contacts. These interactions include three hydrogen bonds (one between Asp648 and Asn681 at the centre of the coiled coil and two from Arg668 to carbonyl groups of Ser660 and Val662 at the tip of the coiled coil) and two salt bridges (one between Lys644 and Asp688 and the other between Lys652 and Glu679). These irregular interactions may confer a more flexible conformation of the CC subdomain compared with regular coiled coils such as the leucine zipper, which is stabilized by regular non-polar interactions between two intertwined helices. Ex subdomain is folded into a novel small-helical domain The CC subdomain is tethered to the Ex subdomain by a linker comprising six residues (residues 701–706). The compact fold of the Ex subdomain is achieved by close non-polar interactions of α6 helix with the groove formed by α4 and α5 helices (Supplementary Figure S5A). In addition to non-polar contacts, α6 helix is anchored to this groove by formation of a hydrogen bond between Arg740 of α6 helix and Ser718 of α5 helix. Three helices are N-terminally capped by Asn706 (α4 helix), Ser715 (α5) and Ser729 (α6). Moreover, α6 helix is C-terminally capped by Arg523 from β1’ strand of the PH subdomain. The topology of the three helices is classified as a novel globular fold, an irregular array fold of short three-helices in the SCOP protein structure database (Murzin et al, 1995). This observation is consistent with its unique amino acid sequence, which exhibits no homology to any known helical domains such as the homeodomain, which consists of three short helices featuring the helix-turn-helix motif (Supplementary Figure S5B). Inter-subdomain interactions in the PHCCEx domain The globular assembly of the PHCCEx domain is constructed by direct interactions between each pair of the three subdomains. The interactions at each interface comprise a mixture of non-polar and polar contacts, including hydrogen bonds and salt bridges. The Ex subdomain is involved in close interactions with both the PH and CC subdomains, with a total buried accessible surface area of both interfaces of 2127 Å2 (Figure 4A). At the interface with the PH subdomain, the Ex subdomain packs α5–α6 loop and α6 helix into the concave region of the PH subdomain formed by one β-sheet (β1′–β1–β7–β6–β5) of the β-sandwich. Three strands of the β-sheet (β1′, β1 and β7) predominantly make non-polar contacts with α5 and α6 helices of the Ex subdomain through conserved aliphatic residues (Figure 4B). In particular, β1′ strand is packed in the groove formed by α5 and α6 helices of the Ex subdomain and Leu525 is located deep inside the groove. In addition to non-polar contacts, Glu526 from β1′ strand forms a salt bridge with Arg724 from α5 helix (Ex). Thus, PH subdomain-specific β1′ strand has an important function in stabilization of the PH–Ex interactions. β5 and β6 strands make polar contacts with His734 and Ser731 from α6 helix through direct or water-mediated hydrogen bonds. Figure 4.Inter-subdomain interactions. (A) Inter-subdomain interfaces are in blue (PH–Ex interfaces), yellow (PH–CC interfaces) and red (Ex–CC interfaces). The shared region for the two subdomains is in green. (B) A close-up view of the interface between the PH (red) and Ex (green) subdomains. Hydrogen bonds are indicated by broken lines. Residues whose side chains participate in polar interactions are labelled with boxes. (C) A close-up view of the interface between the CC (blue) and Ex (green) subdomains. (D) A close-up view of the interface between the PH (red) and CC (blue) subdomains. Download figure Download PowerPoint The Ex subdomain forms another groove comprising α4 and α6 helices, which accommodates the CC subdomain (Figure 4A). The middle region (Glu679-Phe690) of α3 helix of the CC subdomain is docked into this groove through the insertion of two projected non-polar residues, Met687 and Phe690 (Figure 4C). The interface between the Ex and CC subdomains, however, extends direct and water-mediated polar contacts including a salt bridge between Arg740 (α6) and Glu683 (α3). The interface between the PH and CC subdomains is created primarily with α1 helix, β4–β5 loop and β5 strand of the PH subdomain, and makes contact with the tip of the CC subdomain (Figure 4A). The total buried accessible surface area of the PH–CC interface is 1237 Å2, and contains hydrophobic contacts between non-polar side chains from α1 helix of the PH subdomain and both helices of the CC subdomain (Figure 4D). The interface contains two hydrogen bonds involving side chains and one salt bridge between Asp570 and Arg691. Interaction between the PHCCEx domain and membrane proteins We have outlined the PHCCEx domain structure above. Prior to efforts attempting to map the binding site on the PHCCEx structure, we tried to define the essential regions on membrane proteins for binding to the PHCCEx domain. CD44 was the first membrane protein identified that specifically binds to the Tiam1 PHCCEx domain (Bourguignon et al, 2000). The cytoplasmic tail of CD44 comprises 72 amino-acid residues (mouse residues 292–363) and has recently been characterized by spectroscopic and hydrodynamic methods (Mori et al, 2008). The study revealed that the cytoplasmic tail peptide adopts a random-coil structure in solution, which suggests that PHCCEx-CD44 binding may be mediated by protein–peptide rather than protein–protein interactions. Indeed, the N-terminal juxtamembrane region of the cytoplasmic tail contains a short peptide region (KKKLVIN at 300–306) responsible for binding to ezrin/radixin/moesin (ERM) proteins that acts as linker between plasma membranes and actin cytoskeletons (Mori et al, 2008). Besides the juxtamembrane region, the functional roles of the C-terminal long stretch are largely unknown. In an effort to define the peptide region of CD44 responsible for binding to the PHCCEx domain, we synthesized a series of N-terminal biotinylated CD44 tail peptides for surface plasmon resonance (SPR) measurements (Figure 5A and B). The Tiam2 PHCCEx domain did not bind the N-terminal CD44 peptide (residues 294–323) that contains the binding site for ERM proteins, but did bind the C-terminal peptides, which contain the acidic peptide comprising 14 residues (337–350). Next, we compared sequences of the PHCCEx-binding site of CD44s from different sources with the ephrin Bs cytoplasmic peptides. Interestingly, the sequence alignment reveals that CD44s conserve the acidic sequence motif, hereafter referred to as Motif-I, but also ephrin B's exhibit detectable homology to Motif-I (Figure 5C). Figure 5.Interaction between the Tiam2 PHCCEx domain and CD44 cytoplasmic peptides. (A) Deletion mapping suggests 14 residues (337–350) of the CD44 cytoplasmic tail as the minimal-binding region as determined from the binding assay using SPR measurements. The yellow rectangle indicates the suggested minimum region. The bold rectangle indicates the ERM-binding region (residues 294–323) at the juxtamembrane region. (B) Interactions between the Tiam2 PHCCEx domain and the CD44 cytoplasmic peptides as determined from equilibrium SPR measurements. (C) Sequence alignment of PHCCEx-binding regions of CD44 and ephrin Bs using the program CLUSTALW. The suggested minimal region is indicated at the top. Invariant residues (E=D, T=S, C=L=F=Y) are highlighted in light green. Acidic and basic residues are in red and blue, respectively. Sequences are from the ExPASy database (human CD44: P16070, rat: P26051-2, mouse: P15379-2, hamadryas: P14745, horse: Q05078, bovine: Q29423, Chinese hamster: P20944, golden hamster: Q60522-2, dog: Q28284, human ephrin B1: P98172, mouse: P52795, rat: P52796, human ephrin B2: P52799, mouse: P52800, and human ephrin B3: Q15768, mouse: O35393.) Download figure Download PowerPoint In our SPR measurements, the acidic peptide of CD44 comprising 24 residues (327–350) exhibited strongest binding to the Tiam2 PHCCEx domain with a dissociation constant KD of 0.92 μM (Figure 6A). Similar results were obtained for the Tiam1 PHCCEx domain although the binding affinity is somewhat weaker than those of Tiam2. Moreover, our measurements showed that the acidic peptides of ephrin B1 and 2 indeed bind the Tiam2 PHCCEx domain with similar affinity (in the micromolar range) to that of CD44, whereas ephrin B3 exhibited weaker binding. At present, we presume that this may be caused by the insertion of residues GGG at the C-terminal half of the motif. Figure 6.Interaction between the Tiam2 PHCCEx domain and peptides. (A) The obtained KD values obtained from SPR measurements. The peptide sequences are aligned with basic (blue) and acidic (red) residues. Residues conserved among CD44s and ephrin Bs are in bold. Our mutation studies suggested CD44 residues (highlighted in yellow) critical for binding. (B) Binding assay of the Tiam1 PHCCEx domain to the mutant CD44 peptides. Reduction of binding affinity by mut
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