Title: Structural basis for recognition of the translocated intimin receptor (Tir) by intimin from enteropathogenic Escherichia coli
Abstract: Article1 June 2000free access Structural basis for recognition of the translocated intimin receptor (Tir) by intimin from enteropathogenic Escherichia coli Miranda Batchelor Miranda Batchelor Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Sunil Prasannan Sunil Prasannan Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Sarah Daniell Sarah Daniell Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Stephen Reece Stephen Reece Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Ian Connerton Ian Connerton University of Nottingham, School of Biological Sciences, Division of Food Sciences, Sutton Bonington Campus, Loughborough, LE12 5RD UK Search for more papers by this author Graham Bloomberg Graham Bloomberg Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD UK Search for more papers by this author Gordon Dougan Gordon Dougan Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Gad Frankel Gad Frankel Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Stephen Matthews Corresponding Author Stephen Matthews Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Centre for Structural Biology, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Miranda Batchelor Miranda Batchelor Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Sunil Prasannan Sunil Prasannan Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Sarah Daniell Sarah Daniell Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Stephen Reece Stephen Reece Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Ian Connerton Ian Connerton University of Nottingham, School of Biological Sciences, Division of Food Sciences, Sutton Bonington Campus, Loughborough, LE12 5RD UK Search for more papers by this author Graham Bloomberg Graham Bloomberg Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD UK Search for more papers by this author Gordon Dougan Gordon Dougan Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Gad Frankel Gad Frankel Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Stephen Matthews Corresponding Author Stephen Matthews Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Centre for Structural Biology, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Author Information Miranda Batchelor1, Sunil Prasannan1, Sarah Daniell1, Stephen Reece1, Ian Connerton3, Graham Bloomberg4, Gordon Dougan1, Gad Frankel1 and Stephen Matthews 1,2 1Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK 2Centre for Structural Biology, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK 3University of Nottingham, School of Biological Sciences, Division of Food Sciences, Sutton Bonington Campus, Loughborough, LE12 5RD UK 4Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD UK ‡M. Batchelor and S.Prasannan contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2452-2464https://doi.org/10.1093/emboj/19.11.2452 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Intimin is a bacterial adhesion molecule involved in intimate attachment of enteropathogenic and enterohaemorrhagic Escherichia coli to mammalian host cells. Intimin targets the translocated intimin receptor (Tir), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study we localized the Tir-binding region of intimin to the C-terminal 190 amino acids (Int190). We have also determined the region's high-resolution solution structure, which comprises an immunoglobulin domain that is intimately coupled to a novel C-type lectin domain. This fragment, which is necessary and sufficient for Tir interaction, defines a new super domain in intimin that exhibits striking structural similarity to the integrin-binding domain of the Yersinia invasin and C-type lectin families. The extracellular portion of intimin comprises an articulated rod of immunoglobulin domains extending from the bacterium surface, conveying a highly accessible ’adhesive tip' to the target cell. The interpretation of NMR-titration and mutagenesis data has enabled us to identify, for the first time, the binding site for Tir, which is located at the extremity of the Int190 moiety. Introduction Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli constitute a significant risk to human health worldwide. EPEC is the cause of severe infantile diarrhoeal disease in many parts of the developing world, while EHEC are the etiological agents of a food-borne disease that can cause acute gastro-enteritis, bloody diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome (HUS) (reviewed by Nataro and Kaper, 1998). EPEC colonize the small intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the ‘attaching and effacing’ (A/E) lesion (Moon et al., 1983). The A/E lesion is characterized by localized destruction (effacement) of brush border microvilli, intimate attachment of the bacillus to the host cell membrane and the formation of an underlying pedestal-like structure in the host cell consisting of polymerized actin, α-actinin, ezrin, talin and myosin (reviewed by Frankel et al., 1998a). A/E lesion formation is essential for pathogenicity and similar lesions have been associated with several other bacterial mucosal pathogens, most notably in EHEC (Donnenberg et al., 1993a,b). The first gene to be associated with A/E activity was eae encoding the intimate EPEC and EHEC adhesin, intimin (Jerse et al., 1990). Intimin exists as at least five antigenically distinct subtypes that have been named intimin α, β, γ, δ and ϵ (Adu-Bobie et al., 1998; Oswald et al., 2000). EPEC/EHEC intimins exhibit homology at their N-termini to the invasin polypeptides of Yersinia (Isberg et al., 1987) and, like Yersinia invasin (Leong et al., 1990), intimin harbours receptor-binding activity at the C-terminus of the polypeptide (Frankel et al., 1994, 1995). A 76-amino acid motif enclosed by a disulfide bridge between two cysteines lies within the C-terminal domain of intimin. This is absolutely required for intimin binding to the host cell, A/E lesion formation and colonization of mucosal surfaces (Frankel et al., 1995, 1996, 1998b; Hicks et al., 1998; Higgins et al., 1999a,b). The C-terminal domain of invasin also harbours two Cys residues, in similar locations to those found in intimin (Leong et al., 1993). Recently, we have determined the global fold of the C-terminal 280 amino acids of intimin α (Int280α) by a combination of perdeuteration, site-specific protonation and multidimensional nuclear magnetic resonance (NMR) (Kelly et al., 1998, 1999). The structure shows that Int280α is ∼90 Å in length and is built from three globular domains. The first two domains (residues 1–91 and 93–181) each comprise β-sheet sandwiches that resemble the immunoglobulin super family (IgSF). Despite no significant sequence homology, the topology of the C-terminal domain (residues 183–280) is reminiscent of the C-type lectins, a family of proteins responsible for cell-surface carbohydrate recognition. A domain between residues 558 and 650 within the extracellular portion of intimin aligns with 35% identity (44% conservation) with the first domain of Int280, identifying a further Ig-like domain (Kelly et al., 1999). This produces at least four domains that protrude from the bacterial membrane for interaction with the host cell. For the purposes of future discussion, the domains in Int280 are therefore renamed as D2, D3 and D4 respectively. The presence of a disulfide bridge in the C-type lectin-like module of Int280α is essential for correct folding of this domain and for carbohydrate binding by other C-type lectins (Weis and Drickamer, 1996). Modelling other intimin types (including the EHEC intimin γ) (Yu and Kaper, 1992; Adu-Bobie et al., 1998) would suggest they have similar structures, and define a new family of bacterial adhesion molecule. The intimin-encoding eae gene is part of the large pathogenicity islands found in EPEC and EHEC, termed the LEE (locus of enterocyte effacement) (McDaniel et al., 1995; Perna et al., 1998). In addition to intimin, the LEE also encodes a type III secretion system (Jarvis et al., 1995; Jarvis and Kaper, 1996), an intimin receptor [translocated intimin recepor (Tir)/EspE] (Kenny et al., 1997; Deibel et al., 1998) and three secreted proteins, EspA, EspB and EspD, which are required for signal transduction in host cells and A/E lesion formation (Donnenberg et al., 1993c; Kenny et al., 1996; Lai et al., 1997). EspA is a structural protein and a major component of a large filamentous organelle that is transiently expressed on the bacterial surface and interacts with the host cell during the early stage of A/E lesion formation (Ebel et al., 1998; Knutton et al., 1998). EspA filaments may contribute to bacterial adhesion but, of greater significance, they appear to be a component of a translocation apparatus and as such are essential for the translocation of EspB (Knutton et al., 1998; Wolff et al., 1998) and Tir (Kenny et al., 1997) into host cells. Int280 can bind directly to uninfected host cells (Frankel et al., 1994, 1996). Moreover, a recent study has demonstrated that intimin can induce a T helper cell-type 1 immune response in the colonic mucosa and colonic hyperplasia in mice (Higgins et al., 1999a). In addition, the bacterial protein Tir (EspE) can also act as an EPEC/EHEC intimin receptor (Kenny et al., 1997; Deibel et al., 1998). Recently, the intimin-binding region of Tir has been localized to a stretch of amino acids residues that resides between the two putative membrane-spanning domains of the polypeptide (termed Tir-M) (de Grado et al., 1999; Hartland et al., 1999; Kenny, 1999). Immunofluorescence staining of infected cells using polyclonal antisera raised against the N- and C-terminal peptides of Tir (Tir-N and Tir-C, respectively) demonstrate that these two regions are located within the host cell where they can induce the polymerization of actin and other cytoskeletal proteins to produce the characteristic pedestal-like structure (Kenny et al., 1997; Hartland et al., 1999). We report that an active Tir-binding fragment of intimin spans the IgSF-like domain, D3, and the lectin-like domain, D4. We have also determined the high-resolution structure of the portion, comprising the C-terminus 190 amino acids, which form a super domain capable of interaction with Tir. Using chemical shift titration experiments we have also further localized, for the first time, the binding site of Tir to a concerted patch of residues at the tip of the structure (D4), and propose a plausible model for the intimin–Tir interaction. Results and discussion Localization of the minimal Tir-binding region of intimin Based on low resolution structural information reported previously (Kelly et al., 1998, 1999), a number of truncated Int280α derivatives, expressed as maltose-binding protein (MBP) fusion proteins, were constructed and used together with purified Tir-M (Hartland et al., 1999) in a gel overlay protein-binding assay. The MBP–Int280α derivatives are shown schematically in Figure 1 and include Int280, Int190, Int150 and Int120. We have shown before that the D3 domain forms numerous hydrophobic contacts with D4 (Kelly et al., 1999). Accordingly, we included the first 42 amino acids of D4 in the derivatives designed to assess the potential Tir-binding activity of the IgSF-like domains (IntD2 and D3; Figure 1). MBP–Int146, which harbours D3 and 62 amino acids of D4, encompasses both conserved WLQYGQ and WGAANKY motifs (Adu-Bobie et al., 1998) (Figure 1). The gel overlay protein-binding assays revealed that Tir-M bound to the immobilized Int280α and Int190α fusion proteins (Figure 2A). However, no binding was observed with any other MBP–Int fusion proteins or MBP alone (Figure 2A; data not shown). Similar results were obtained when the binding assay was performed with immobilized Tir-M and soluble MBP–Int fusion proteins (data not shown). These results are consistent with those reported recently for intimin γ from EHEC (Liu et al., 1999). Figure 1.Schematic representation of the overlapping Int280-derived polypeptides. The two IgSF-like domains (D2 and D3), the C-type lectin-like domain (D4) and the conserved motifs in Int280 are shown at the top. The position of W150 within Int190 is indicated. Numbers on both sides of the fragments mark the first and last amino acids of each fragment within the Int280 domain. Download figure Download PowerPoint Figure 2.(A) Detection of Int–Tir interactions using gel overlays. Western blots of MBP–Int derivatives were reacted with a rabbit MBP antiserum (top) or overlayed with Tir-M (bottom). Similar levels of MBP–Int280 (lane 1), MBP–Int190 (lane 2), MBP–Int150 (lane 3), MBP–Int146 (lane 4), MBP–IntD3&D4 (lane 5) and MBP–IntD3 (lane 6); fusion proteins were detected with polyclonal antiserum (top), while Tir-M only bound to MBP–Int280 (bottom, lane 1) and MBP–Int190 (bottom, lane 2). (B) Detection of Int–Tir interactions using the yeast two-hybrid system. β-galactosidase assays showing a 14-fold increase in enzymatic activity in strains co-expressing the whole Tir polypeptide and Int280 and a 7.5-fold increase when Int190 was co-expressed with Tir compared with the parent, cured and the other single and double transformants. Download figure Download PowerPoint The interaction of the different Int280α derivatives with Tir was also investigated using the yeast two-hybrid system, designed to identify protein–protein interactions through the functional restoration of the yeast GAL4 transcriptional activator in vivo (James et al., 1996). For this, selected DNA fragments encoding truncated Int280α polypeptides were sub-cloned into pGAD424 to generate plasmids pICC39, pICC40, pICC41 and pICC42 (Table I). We previously reported that in the yeast two-hybrid system, the interaction of Int280 (pICC19) with the whole Tir polypeptide is more efficient than the interaction of Int280 with Tir-M (Hartland et al., 1999). Accordingly, the DNA fragment encoding the whole Tir polypeptide, expressed from the second yeast two-hybrid system vector, pGBT9 (pICC10; Hartland et al., 1999), was used in this part of the study. Table 1. List of plasmids Plasmid Description Source/reference pGBT9 A yeast GAL4 DNA-BD cloning vector Clontech pGAD424 A yeast GAL4 DNA-AD cloning vector Clontech pMal-C2 Vector for expression of MBP-tagged proteins NEB pET3d Vector for expression of proteins for purification without a tag Novagen pICC10 pGBT9 expressing Tir Hartland et al. (1999) pICC19 pGAD424 expressing Int280 Hartland et al. (1999) pICC39 pGAD424 expressing Int190 this study pICC40 pGAD424 expressing Int150 this study pICC41 pGAD424 expressing Int146 this study pICC42 pGAD424 expressing Int120 this study pICC44 pGAD424 expressing Int190W150A this study pICC45 pMal expressing MBP–Int280 Frankel et al. (1994) pICC46 pMal expressing MBP–Int190 this study pICC47 pMal expressing MBP–Int150 Frankel et al. (1994) pICC48 pMal expressing MBP–Int146 this study pICC49 pMal expressing MBP–Int120 Frankel et al. (1994) pICC50 pMal expressing MBP–IntD2+D3 this study pICC51 pMal expressing MBP–IntD3 this study pICC52 pMal expressing Int190W150A this study pCVD438 pACYC184 containing the eae gene of E2348/69 Donnenberg and Kaper (1991) pICC53 pCVD438W899A this study pICC54 pET3a expressing Int190 this study pICC59 pET3a expressing Int188 this study pICC62 pGAD424 expressing Int280A240 this study pICC63 pMal expressing Int280A240 this study Plasmid pICC10 was co-transformed with each of the different pGAD424-based plasmids into a derivative of the yeast stain PJ69-4A selected previously as a reporter for intimin–Tir interaction (Hartland et al., 1999). Replica plating these colonies onto selective media yielded vigorously growing colonies, and hence a positive two-hybrid phenotype, in yeast strains expressing both Tir and Int280 (pICC10 and pICC19) and Tir and Int190 (pICC10 and pICC39). No yeast colonies were observed using Tir with any of the other Int280 truncations or single plasmid transformants (data not shown). The function of the non-selective reporter, lacZ, was also assessed in these strains by measuring β-galactosidase activity (Figure 2B). The host or single plasmid-bearing strains exhibited low levels of β-galactosidase activity, whereas the strains expressing Int280 or Int190 and Tir showed a 14- or 7.5-fold induction of β-galactosidase units, respectively. Based on the β-galactosidase levels of the strains bearing the plasmids pICC10 and pICC19, and pICC10 and pICC39, Int280 appears to interact with Tir with greater affinity than Int190. This implies that regions outside Int190 may be involved in interaction with the whole Tir polypeptide. Nevertheless, taken together, these results show that the region of intimin that spans the C-terminal 190 amino acids is capable of interacting with Tir. In the low-resolution structure of Int280 it can be seen that the D3–D4 junction comprises a small surface area. Therefore, it is unlikely that a truncation at this point will severely affect stability, whereas truncations elsewhere will probably compromise its stability. Localizing the Tir-binding region of intimin to the C-terminal 190 amino acids using gel overlay and yeast two-hybrid system assays defines the minimal functional fragment. Solution structure of Int190 Using a combined perdeuteration/site-specific protonation and multi-dimensional NMR approach we were able to define the fold of the original 280-amino acid fragment, which is 30.1 kDa (Kelly et al., 1999). The shorter Int190 fragment is ∼20 kDa and its size falls within the applicability of standard multi-resonance NMR methods for determining highly defined structures. The DNA fragment encoding Int190 was sub-cloned into pET3a and overexpressed in BL21. However, Int190 was expressed at levels significantly lower than Int280. We hypothesized that this might be due to the presence of two hydrophobic amino acids (Phe1 and Phe2) at the N-terminus of the Int190 polypeptide. In order to try to improve expression and solubility we removed these two amino acids and cloned into pET3a a DNA fragment encoding Int188. The level of expression was vastly improved but Int188 concentrated into insoluble inclusion bodies. Accordingly, following centrifugation, the inclusion body material was precipitated, solubilized and refolded as described in Materials and methods. Comparison of the 1H-15N heteronuclear single quantum coherence (HSQC) NMR spectra for Int188 and Int280 indicates that Int188 is fully structured. Peaks corresponding to amides within the N-terminal 92 amino acids of Int280 are absent in the Int188 spectrum but the rest remain largely unchanged. 1H, 15N, 13C sequence-specific backbone and side-chain assignments were completed using the standard methodology (Bax et al., 1990; Grzesiek and Bax, 1992a,b; Kay et al., 1994; Muhandiram and Kay, 1994). Structures were calculated on the basis of 1226 nuclear Overhauser effect (NOE) distance, 115 H-bond distance and 307 dihedral angle restraints. The calculations used a hybrid torsion angle and Cartesian co-ordinate dynamics protocol, executed within the program XPLOR (Nilges et al., 1988; Brünger, 1993). A final family of 15 structures (shown in Figure 3) were produced which contained no distance violations >0.2 Å. Int188 is ∼60 Å in length and comprises two intimate globular domains: D3 (residues 3–90) and D4 (residues 91–190, numbered according to Int190). The overall root-mean square deviations (r.m.s.d.) between the family and mean co-ordinate position are 0.7 ± 0.1 and 0.9 ± 0.1 Å for the backbone atoms of secondary structure element in D3 and D4, respectively. For all heavy atoms of the same region the r.m.s.d. values rise to 1.2 ± 0.1 and 1.4 ± 0.1 Å, respectively. Numerous contacts between D3 and D4 are observed that define the relative orientation of the two domains. The r.m.s.d. for all the backbone atoms of D3–D4 (residues 1–188) from the average structure is 1.5 ± 0.2 Å, indicating the relative orientation of these domains is well defined. The complete set of structural statistics is shown in Table II. Figure 3.(A) Cα traces representing the superimposition of the 15 refined Int188 structures. (B) Cα traces representing the superimposition of the 15 refined Int188 structures. The orientations of (A) and (B) are related by a 90° rotation. (C) Schematic representation of Int188 for the orientation displayed in (A). (D) Schematic representation of Int188 domains for the orientation displayed in (B). (E) A ‘flattened’ illustration highlighting the topology of Int188. Helices are represented as open tubes and β-strands as arrows. Download figure Download PowerPoint Table 2. Results from structure calculations for Int188 Statistic <SA>a Restraints medium and short range (1 < |i − j| < 5) 771 0.030 ± 0.003 long range (|i − j| > 4) 455 0.025 ± 0.002 hydrogen bonds 151 0.027 ± 0.003 dihedral angles 307 0.94 ± 0.03 Idealized geometry bonds (Å) 0.0024 ± 0.0003 angles (°) 0.58 ± 0.009 improper angles (°) 0.47 ± 0.02 Coordinate positionsb backbone atoms for secondary structure residues in D3: (5–8, 8–14, 20–23, 32–38, 43–48, 54–57, 61–65, 70–77, 82–87) 0.7 ± 0.1c heavy atoms for secondary structure residues in D3: (5–8, 8–14, 20–23, 32–38, 43–48, 54–57, 61–65, 70–77, 82–87) 1.2 ± 0.1 backbone atoms for secondary structure residues in D4: (92–96, 104–113, 122–132, 146–151, 162–167, 173–175, 186–190) 0.9 ± 0.1 heavy atoms for secondary structure residues in D4: (92–96, 104–113, 122–132, 146–151, 162–167, 173–175, 186–190) 1.4 ± 0.1 backbone atoms for all residues 3–190 1.5 ± 0.2 all heavy atoms for all residues 3–190 1.9 ± 0.2 a The average r.m.s.ds for the final 15 structures. b Numbered according to Int190, see Figure 4C. c The average r.m.s.ds from the average structure. The topology of Int188 is shown in Figure 3. D3 displays little sequence homology with known IgSF members, but belongs to the Type C set of the IgSF. Interestingly, D4 contains a unique feature that is not seen in mammalian IgSF domains. A prominent β-insertion (A′, A″) between strands A and A″′ extends a platform on top of D3 that contacts D4 and helps to define the relative orientation of the two domains. This feature was poorly defined in our earlier determination of the fold of Int280, but in the current study is now characterized by numerous representative β-sheet NOEs and is therefore well defined. D4 comprises four helices that surround two anti-parallel β-sheets. The C-terminal strand is disulfide-bonded to helix I, and together with the N-terminus of D4 forms the two principal strands of the first sheet. Helix III protrudes from the main structure into the solvent and therefore was not observed in our low-resolution structure of Int280 (Kelly et al., 1999). Interestingly, this helix contains an unusual kink at residue 139, which is replaced by proline in other intimin types. Despite no significant sequence homology (i.e. <10%), the α/β topology of D4 is reminiscent of the C-type lectins domains (CTLDs). Seventy-seven structurally equivalent Cα atoms from D4 of intimin superimpose with a r.m.s.d. of 3.0 Å on the lectin domain from E-selectin (Graves et al., 1994). The CTLDs form a family of calcium-binding proteins responsible for cell-surface carbohydrate recognition (CRD) that includes animal cell-receptors and bacterial toxins. However, several variants on the CTLD theme have recently been characterized. One example of a CTLD that lacks calcium co-ordination but recognizes carbohydrate is the TSG-6 link module that binds haluronan (Kohda et al., 1996). Conversely, the type II antifreeze protein from sea raven, which does co-ordinate calcium, inhibits the formation of ice crystals, hypothesized to bind ice surfaces via the second β-sheet and calcium loop (Gronwald et al., 1998). Intriguingly, the CTLDs are also structurally related to proteins that are involved in protein recognition. These include CD94/NKG2 heterodimer, in which two CTL-like domains form an extensive flat surface involving the second β-sheet that is postulated to interact with the type 1b human leukocyte antigen (Boyington et al., 1999). In CTLDs, the carbohydrate recognition site lies on the exposed face of the second β-sheet and an extensive loop between strands C and D (30 residues) in which co-ordinated calcium is directly involved in binding. No evidence for calcium binding is available for intimin and, moreover, intimin D4 lacks the extensive calcium binding loop, which is replaced by a six-residue helix (helix IV). Further differences between the CTL-like domain from intimin and archetypal CTLDs also exist. D4 from intimin contains a single disulfide bond whereas CTLDs retain several that are conserved. Intimin also contains a larger proportion of helical structure than known CTLDs and the relative orientations of these regions are subtly different. However, the similarity with CTLDs does raise the question of an intimin function involving carbohydrate recognition, but evidence has yet to be provided. Therefore, this remains highly speculative. Structural comparison with invasin from Yersinia pseudotuberculosis Intimin from EPEC and invasin from enteropatho genic Yersinia pseudotuberculosis are probably the best-characterized bacterial cell-adhesion paradigms. Research has now culminated in the high-resolution three- dimensional structure information on both proteins. Intimin and invasin belong to a family of outer membrane proteins that mediate bacterial adherence. Members of this family of proteins are ∼900 amino acids in length and possess highly similar N-termini: >36% identity exists within the first 500 amino acids, part of which is hypothesized to be sequestered within the bacterial outer membrane. Despite being highly related, these proteins perform remarkably divergent tasks, and this is illustrated by the marked difference in pathogenesis between the organisms. Invasin causes invasion and translocation of the bacterium across the intestinal epithelium deeper into host tissue. Intimin also promotes adherence to epithelial cells, but not internalization; instead, it induces the A/E lesion. Invasin recognizes members of the integrin super-family from mammalian cells in order to facilitate bacterial adherence to and invasion of host cells. Moreover, two aspartic acid residues, reminiscent of the integrin-binding and synergy regions of fibronectin, are required for recognition (Hamburger et al., 1999). It has also been established that homo-multimerization of invasin from Y.pseudotuberculosis facilitates receptor clustering, tight adherence and the subsequent invasion signal (Dersch and Isberg, 1999). In contrast, intimin binds the Tir, which is introduced into the host cell membrane by the bacterium via a type III protein secretion/translocation system. For both intimin and invasin a C-terminal fragment of ∼190 amino acids is sufficient for function in which no significant sequence homology is present (<10%). Remarkable likenesses can be revealed between the structures of intimin and invasin (Figure 4). Hamburger et al. determined the crystal structure of an extracellular portion of invasin (Inv497) (Hamburger et al., 1999). Inv497 is ∼180 Å in length and composed of five distinct domains (D1, D2, D3, D4 and D5), the first four of which resemble eukaryotic members of the IgSF. D3–D5 in invasin are analogous to the domains of Int280 (280 amino acid C-terminal fragment of intimin), which was revealed by earlier NMR studies (Kelly et al., 1998, 1999). It has also been reported that sequence alignment indicates the existence of a further IgSF domain within intimin. Also, secondary structure