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• W2029986004 abstract "Article10 May 2007free access Atomic resolution insight into host cell recognition by Toxoplasma gondii Tharin MA Blumenschein Tharin MA Blumenschein Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Nikolas Friedrich Nikolas Friedrich Department of Microbiology and Genetics, Faculty of Medicine, University of Geneva CMU, Geneva, Switzerland Search for more papers by this author Robert A Childs Robert A Childs Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK Search for more papers by this author Savvas Saouros Savvas Saouros Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Elisabeth P Carpenter Elisabeth P Carpenter Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Maria A Campanero-Rhodes Maria A Campanero-Rhodes Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK Search for more papers by this author Peter Simpson Peter Simpson Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Wengang Chai Wengang Chai Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK Search for more papers by this author Theodoros Koutroukides Theodoros Koutroukides Medical Science Division, Fox Chase Cancer Centre, Philadelphia, PA, USA Search for more papers by this author Michael J Blackman Michael J Blackman Division of Parasitology, National Institute for Medical Research, London, UK Search for more papers by this author Ten Feizi Ten Feizi Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK Search for more papers by this author Dominique Soldati-Favre Dominique Soldati-Favre Department of Microbiology and Genetics, Faculty of Medicine, University of Geneva CMU, Geneva, Switzerland Search for more papers by this author Stephen Matthews Corresponding Author Stephen Matthews Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Tharin MA Blumenschein Tharin MA Blumenschein Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Nikolas Friedrich Nikolas Friedrich Department of Microbiology and Genetics, Faculty of Medicine, University of Geneva CMU, Geneva, Switzerland Search for more papers by this author Robert A Childs Robert A Childs Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK Search for more papers by this author Savvas Saouros Savvas Saouros Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Elisabeth P Carpenter Elisabeth P Carpenter Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Maria A Campanero-Rhodes Maria A Campanero-Rhodes Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK Search for more papers by this author Peter Simpson Peter Simpson Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Wengang Chai Wengang Chai Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK Search for more papers by this author Theodoros Koutroukides Theodoros Koutroukides Medical Science Division, Fox Chase Cancer Centre, Philadelphia, PA, USA Search for more papers by this author Michael J Blackman Michael J Blackman Division of Parasitology, National Institute for Medical Research, London, UK Search for more papers by this author Ten Feizi Ten Feizi Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK Search for more papers by this author Dominique Soldati-Favre Dominique Soldati-Favre Department of Microbiology and Genetics, Faculty of Medicine, University of Geneva CMU, Geneva, Switzerland Search for more papers by this author Stephen Matthews Corresponding Author Stephen Matthews Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author Author Information Tharin MA Blumenschein1,‡, Nikolas Friedrich2,‡, Robert A Childs3, Savvas Saouros1, Elisabeth P Carpenter1, Maria A Campanero-Rhodes3, Peter Simpson1, Wengang Chai3, Theodoros Koutroukides4, Michael J Blackman5, Ten Feizi3, Dominique Soldati-Favre2 and Stephen Matthews 1 1Division of Molecular Biosciences, Imperial College London, London, UK 2Department of Microbiology and Genetics, Faculty of Medicine, University of Geneva CMU, Geneva, Switzerland 3Glycosciences Laboratory, Division of Medicine, Imperial College London, Middlesex, UK 4Medical Science Division, Fox Chase Cancer Centre, Philadelphia, PA, USA 5Division of Parasitology, National Institute for Medical Research, London, UK ‡These authors contributed equally to this work *Corresponding author. Division of Molecular Biosciences, Imperial College of Science, Technology and Medicine, South Kensington Campus, London SW7 2AZ, UK. Tel.: +44 207 594 5315; Fax: +44 207 594 5207; E-mail: [email protected] The EMBO Journal (2007)26:2808-2820https://doi.org/10.1038/sj.emboj.7601704 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Micronemal proteins (MICs) are released onto the parasite surface just before invasion of host cells and play important roles in host cell recognition, attachment and penetration. Here, we report the atomic structure for a key MIC, TgMIC1, and reveal a novel cell-binding motif called the microneme adhesive repeat (MAR). Using glycoarray analyses, we identified a novel interaction with sialylated oligosaccharides that resolves several prevailing misconceptions concerning TgMIC1. Structural studies of various complexes between TgMIC1 and sialylated oligosaccharides provide high-resolution insights into the recognition of sialylated oligosaccharides by a parasite surface protein. We observe that MAR domains exist in tandem repeats, which provide a highly specialized structure for glycan discrimination. Our work uncovers new features of parasite–receptor interactions at the early stages of host cell invasion, which will assist the design of new therapeutic strategies. Introduction Toxoplasma gondii is a protozoan parasite that is uniquely adapted to infect a wide range of hosts, including virtually all warm-blooded animals and up to 50% of the world's human population. The primary transmission route in humans is via contact with feces from infected domestic cats or ingestion of undercooked contaminated meat, particularly lamb. Toxoplasmosis causes a variety of disease states in humans, including severe disseminated disease in immunosuppressed individuals due to reactivation of encysted parasites and birth defects in infants, where mothers are exposed during pregnancy (Hill and Dubey, 2002). Infection is rapidly established in the host by the fast-replicating form of the parasite, the tachyzoite, which can invade an extremely broad range of cell types. Unlike other pathogens that hijack existing host cell uptake pathways, Toxoplasma and other apicomplexan parasites, including Plasmodium, actively force entry into host cells. The process is initiated by contact with the host cell plasma membrane, followed by reorientation and then the generation of a motive force, which drives penetration into a novel, parasite-induced structure called the parasitophorous vacuole (Carruthers and Boothroyd, 2006). The rapid and smooth transition through these stages requires a highly regulated release of proteins from several parasite organelles, namely micronemes, rhoptries and dense granules (Carruthers and Sibley, 1997). Microneme discharge occurs first and their contents participate in the attachment of parasites to the host cell surface (Carruthers et al, 1999), and the formation of a connection with the parasite actinomyosin system (Jewett and Sibley, 2003), thereby providing the platform from which to drive motility and invasion (Soldati and Meissner, 2004). One of the first micronemal proteins (MICs) to be discovered in T. gondii was MIC1 (TgMIC1), which functions in cell adhesion (Fourmaux et al, 1996). TgMIC1 is a remarkable, multifunctional protein that in addition to binding host cell receptors, interacts with two other microneme proteins (TgMIC4, TgMIC6) (Brecht et al, 2001; Reiss et al, 2001) and is essential for transport of the entire complex through the early secretory pathways (Reiss et al, 2001; Huynh et al, 2004; Saouros et al, 2005). Deletion of the mic1 gene in T. gondii has also confirmed the specific and critical role played by TgMIC1 in host cell attachment and invasion in vitro and has provided evidence for its role in virulence in vivo (Cerede et al, 2005). Recent studies have shown that a purified TgMIC1 subcomplex is a potent antigen and acts as an effective vaccine in the mouse model (Lourenco et al, 2006). Unlike the battery of other MICs, the sequence of TgMIC1 does not exhibit an obvious likeness to vertebrate adhesive motifs. However, a recent nuclear magnetic resonance (NMR) structure revealed a previously unidentified and novel galectin-like domain within the C-terminus of TgMIC1 that promotes proper folding of TgMIC6 and contributes to complex formation (Saouros et al, 2005). Although studies have clearly highlighted the importance of MICs in apicomplexan invasion, the finer structural details of host cell recognition remain largely unknown. So far, two main studies have addressed this issue in Plasmodium falciparum: erythrocyte-binding antigen (EBA-175) binds sialic acid (Tolia et al, 2005) and TRAP binds heparin (Tossavainen et al, 2006), although high-resolution information regarding carbohydrate recognition was not forthcoming. Combining atomic resolution studies with data from carbohydrate microarrays we reveal a novel interaction between T. gondii and a variety of sialylated oligosaccharides. The binding mode is attributed to a new family of domains named the micronemal adhesive repeat (MAR) that exists in tandem repeats and provides a highly specialized structure for glycan recognition. Our work presented here addresses many long-standing issues and uncovers new features regarding parasite–receptor interactions in the early stages of host cell invasion. Furthermore, an understanding of the atomic resolution details of how T. gondii invades host cells opens the way to the design of therapeutic strategies. Results and discussion The overall structure of cell-binding region of TgMIC1 To resolve the atomic structure of the host cell-binding region from TgMIC1 we expressed the N-terminal 246 amino acids (residues 17–262, hereafter termed TgMIC1-NT) in Escherichia coli fused with thioredoxin to aid disulfide bond formation and solubility. Binding assays revealed that our bacterially and Pichia-produced TgMIC1-NT bound independently to host cells (Figure 1A). The high binding efficiency observed for E. coli-derived material is likely due to the higher purity of this reagent (Figure 1A). Our earlier experiments on the C-terminal domain from TgMIC1 (TgMIC1-CT) had excluded a role in cell binding for this region, (Saouros et al, 2005); therefore, we can conclude that TgMIC1-NT possesses the cell-binding properties of the full-length TgMIC1. Figure 1.Host cell binding by TgMIC1-NT. (A) Cell binding assays on HFFs were performed using supernatant of P. pastoris cultures expressing TgMIC1, TgMIC1-NT, or TgMIC1MAR2, bacterially produced TgMIC1-NT and PfProfillin, the latter being used as a negative control. Anti-His antibodies are used as the probe for Western blots and the asterisk indicates background from host cells. Samples of input (I), supernatant (S), wash (W) and cell-binding fraction (CB) were run on each gel (see Materials and methods). Molecular weight markers in kDa. The Pichia-produced material is less efficient as it is a crude cell supernatant, whereas that from E. coli is a purified protein. These data show that bacterially produced TgMIC1-NT retains the cell-binding activity of native TgMIC1. Increasing the concentration of the input (I) up to 50 times results in enhanced binding of bacterially produced TgMIC1NT but not of PfProfilin to HFFs. Loading has been adjusted for detection in the linear range. Note: in all cell binding assays, the total volume of the input, supernatant and the wash is 500 μl, whereas the total volume of the cell bound fraction is 50 μl. (B) Cell binding competition experiments were performed using bacterially produced HisTgMIC1-NT and supernatants of P. pastoris cultures expressing TgMIC1myc. Anti-myc and Anti-His antibodies were used as probes for Western blots. Samples are named as follows: input (I), supernatant (S), wash (W) and cell binding fraction (CB). For the different conditions of competition only the cell-bound fraction is shown. These data confirm that no inhibition of TgMIC1 binding to HFFs is observed in the presence of lactose, galactose or heparin. Note: in all cell binding assays, the total volume of the input, supernatant and the wash is 500 μl, whereas the total volume of the cell-bound fraction is 50 μl. Download figure Download PowerPoint After subsequent removal of the purification tag, we crystallized the recombinant protein and a selenomethionine (SeMet)-substituted form of TgMIC1-NT, and resolved the structure using the MAD (multiple-wavelength anomalous dispersion) method. The atomic structure for residues 29–259 was solved at a resolution of 1.9 Å (Figure 2A; Table I and Supplementary Figure 1). Residues 17–28 and 262 likely exhibit a degree of flexibility and therefore could not be observed in the crystal structure. The new structure reveals a repeated domain consisting of a distorted barrel arrangement of five β-strands, which is flanked on one side by an antiparallel helical bundle comprising one helix from each terminus. Two disulfide bonds, C1–C4 and C5–C7 (namely C45–C85 and C103–C113 in repeat 1 and C154–C179 and C193–C203 in repeat 2; Figure 3A), are absolutely conserved between the repeats and stabilize the core structure, one connecting helix α1 to the β-barrel and the other between strands β3 and β4. Although the two repeats (amino-acid residues 16–144 and 145–237) have a sequence identity of 27% and superimpose with a backbone r.m.s.d. of 2.2 Å over 89 residues (Figure 2B), some notable differences are apparent. The first helix and the subsequent loop to strand β1 are significantly longer in the first repeat and are stabilized by an additional disulfide bond, C53–C61 (Figure 3A). Most strikingly, the second repeat is elaborated at its C-terminus by a short ‘β-finger’ (amino-acid residues 238–256), which is pinned to the main body of the domain by a new arrangement of two disulfide bonds (C6–C9 and C8–C10; Figures 2C and 3A) replacing the single connection observed in repeat 1 (C6–C8; Figures 2C and 3A). A further intriguing aspect of this region is the presence of a cis-proline within the 248NPPL251 motif (Supplementary Figure 1), which enables unusual positioning of both backbone and side-chain elements that may correspond to an interaction site, possibly for its partner in the complex MIC4 (Saouros et al, 2005). Figure 2.Host cell binding and structure of TgMIC1-NT. (A) Ribbon representation of a representative structure for TgMIC1-NT. Repeat 1 (MAR1) is shown in green and repeat 2 (MAR2) in blue. The orientation shown on the right represents a 180° rotation. (B) Ribbon representation showing the superimposition of Repeat 1 (MAR1, green) and Repeat 2 (MAR2, blue). The orientation shown is the same as the representation on the left in (A). The calculated r.m.s.d. is 2.2 Å over 89 amino-acid residues. (C) Zoomed region illustrating the altered disulfide bond patterns at the C-terminus of repeat 1 (MAR1, green) and repeat 2 (MAR2, blue). The additional ‘β-finger’ (amino-acid residues 238–256) is pinned to the main body of MAR2 by a new arrangement of two disulfide bonds C6–C9 and C8–C10 (namely C197–C242 and C236–C252; see Figure 3A) replacing the single connection observed in repeat 1, C6–C8 (namely C107–C143; Figure 3A). Download figure Download PowerPoint Figure 3.MICs from apicomplexan parasites contain the TgMIC1 repeat. (A) Structure-based sequence alignment of MAR1 and MAR2 domains from other MICs. Including TgMIC1 (MAR1 amino-acid residues 32–144, MAR2 amino-acid residues 145–263), two of the three MIC1-like proteins from T. gondii (MAR1 amino-acid residues 114–222, MAR2 amino-acid residues 223–336 in TgMIC1a and MAR1 amino-acid residues 114–222, MAR2 amino-acid residues 223–335 in TgMIC1b), NcMIC1 (MAR1 amino-acid residues 30–142, MAR2 amino-acid residues 143–261) and EtMIC3 (MAR1 amino-acid residues 42–149, MAR1a amino-acid residues 150–274, MAR1b amino-acid residues 291–425, MAR1c amino-acid residues 36–158 and MAR1d amino-acid residues 180–280). For clarity, the third MIC1-like protein, TgMIC1c, from T. gondii, has been omitted. Conserved residues are shaded in blue. Cysteines and disulfide bond connectivities are highlighted in orange. Cis-proline within the 232NPPL235 motif is shown in red. Secondary structure elements are indicated above the sequences. (B) A schematic representation of a model for the seven sequential MAR1 domains from EtMIC3 is shown in two orientations (left, perpendicular to the helical axis and right, along the helical axis). Download figure Download PowerPoint Table 1. Data collection, phasing and refinement statistics (also see Supplementary Figure 1) Native SeMet Peak SeMet Inflection SeMet Remote 2,6-sialyl-N-acetyllactosamine 2,3-sialyl-N-acetyllactosamine Data collection and phasing Space group P43212 P43212 P43212 P43212 P43212 P43212 Cell dimensions a, c (Å) 66.2, 172.3 66.3, 172.6 66.3, 172.6 66.3, 172.6 65.9, 173.0 66.1, 172.7 Beamline 10.1 SRS 10.1 SRS 10.1 SRS 10.1 SRS 14.1 SRS 14.1 SRS Wavelength 0.980 Å 0.980 Å 0.980 Å 0.972 Å 1.488 Å 1.488 Å Resolution (Å)a 18.0–1.9 (1.97–1.90) 18.0–2.8 (2.90–2.80) 18.0–2.8 (2.90–2.80) 18.0–2.8 (2.90–2.80) 20.0–2.0 (2.07–2.00) 18.0–2.3 (2.38–2.30) Total observations 154 515 91 673 90 740 93 048 542 909 351 534 Unique reflections 301 53 10 069 10 030 10 070 49 275 17 796 Redundancya 5.1 (3.9) 9.1 (9.0) 9.0 (7.5) 9.2 (9.0) 7.6 (7.6) 7.6 (7.8) Rsym (%)a,b, a,b 7.5 (31.1) 9.1 (23.3) 10.2 (29.1) 9.3 (25.1) 10.5 (53.6) 8.6 (41.3) I/σIa 19.2 (3.4) 20.5 (9.5) 18.6 (6.4) 20.6 (9.0) 43.54 (6.7) 38.0 (6.7) Completeness (%)a 96.6 (76.2) 99.9 (100) 99.6 (96.6) 99.9 (100) 99.9 (99.7) 100 (99.9) Refinement Native 2,6-sialyl-N-acetyllactosamine soak 2,3-sialyl-N-acetyllactosamine soak Resolution (Å)a 17.5–1.9 (1.95–1.90) 18.29–2.07 (2.12–2.07) 17.5–2.3 (2.36–2.30) Number of reflections 28 518 22 744 16 806 Rfactorc/Rfreed(%)a 17.8/20.4 (29.4/31.3) 18.3/21.2 (28.4/35.6) 17.3/21.3 (20.2/25.5) Number of atoms Protein 1840 1745 1744 Ligand/ion 74 98 83 Water 181 181 153 B-factors Protein 26.3 27.1 35.0 Ligand/ion 42.5 52.9 51.8 Water 41.4 39.1 45.8 R.m.s. deviations Bond lengths (Å) 0.018 0.021 0.023 Bond angles (deg) 1.6 1.8 1.9 a Values in parentheses correspond to the highest-resolution shell. b Rsym=∑h∑i∣I i (h)−〈I(h)〉∣/∑h ∑i (h), where Ii(h) is the ith measurement. c R-factor=∑∣∣F(h)obs∣−∣F(h)calc ∣∣/∑∣F(h)obs. d Rfree is calculated in the same way as the R-factor, using only 5% of reflections randomly selected to be excluded from the refinement. The MAR domain—a new fold unrelated to thrombospondin type1 repeats In the initial characterization of TgMIC1 it was postulated that a tandem arrangement of degenerate thrombospondin type I repeats (TSR1s) was present at the N-terminus (Fourmaux et al, 1996). TRS1s adopt an antiparallel, three-stranded fold comprising alternating stacked layers of tryptophan and arginine residues. Our structure of TgMIC1-NT now enables us to reassess this domain classification and reveals a fold that bears no resemblance to the TSR1s. Moreover, it is unrelated to the classical β-sandwich structure of the prototypic surface antigen, SAG1, from Toxoplasma (He et al, 2002), and to the dimeric arrangement of EBA-175 (Tolia et al, 2005). A search of the DALI database reporting no structural hits confirms this and the fact that the structure of TgMIC1-NT presents a previously unknown protein fold (Holm and Sander, 1995). Based on these observations, we have named this repeat domain the ‘MAR domain’, after micronemal adhesive repeat. A search against other apicomplexan genomes identified tandem MAR domains in MIC1 from Neospora caninum (Keller et al, 2002) and in three other MIC1-like proteins in T. gondii (Figure 3A), which may help to endow this parasite with the ability to invade a wide range of cell types as well as evade host immune responses. MIC3 proteins from Eimeria tenella, the cause of coccidiosis in poultry, contain between four and seven consecutive MAR1 domains constructed from five distinct MAR1 sequences (Labbe et al, 2005) (Figure 3A). A model of an uninterrupted stretch of MAR1 domains, as in EtMIC3, generated using our structure of TgMIC1-NT, gives rise to a stalk structure comprising a left-handed helical axis with 70° rotations and 11 Å translations relating adjacent MARs (Figure 3B). This arrangement would project from the parasite surface, presenting an array of MAR domains that could provide increased cell-binding avidity (Supplementary Figure 2). Future studies will be aimed at unveiling other parasite surface proteins that possess members of the MAR family. The MAR domain—a novel carbohydrate-binding domain A long-standing question is how T. gondii can infect and replicate within all nucleated cells. The broad host range suggests that complementary receptors exist on a wide variety of host cell types. Carbohydrate recognition and discrimination provide excellent means to facilitate such interactions and often play an important role in early recognition events by invasive pathogens. Although carbohydrate-binding properties have been described for microneme proteins from Toxoplasma (Cerede et al, 2002; Harper et al, 2004), studies on the nature of these interactions have not been forthcoming. Monteiro et al (1998) showed that sialic acid plays a role in host cell invasion, but the identity of the parasite ligand has been the subject of much speculation. Indirect evidence exists for a lactose-binding activity for TgMIC1 and the TgMIC1–TgMIC4 subcomplex (Lourenco et al, 2001, 2006). However, cell binding inhibition (Figure 1B) and NMR titration experiments performed in the present study failed to detect lactose recognition by TgMIC1. To reassess the carbohydrate-binding properties of TgMIC1, microarray analyses (Campanero-Rhodes et al, 2006; Palma et al, 2006) were carried out using the fusion proteins TgMIC1-NT and TgMIC1-CT, and lipid-linked oligosaccharide probes (Feizi and Chai, 2004), as described. The microarrays encompassed a panel of >200 oligosaccharide probes representing diverse mammalian glycan sequences and their analogues, as well as some derived from fungal and bacterial polysaccharides (Palma et al, 2006) (Supplementary Table 1). The C-terminal galectin-like domain, TgMIC1-CT, neither showed binding to galactose-terminating glycans in the array, consistent with our early NMR evidence (Saouros et al, 2005), nor was there binding to any of the other probes in the microarray (results not shown). In contrast, significant binding signals with fluorescence intensities between 150 and approximately 8000 were elicited by TgMIC1-NT and were observed only among terminally sialylated structures. (Figure 4 and Supplementary Table 1). All the non-sialylated structures tested had numerical scores below 150 (Supplementary Table 1). Figure 4.Carbohydrate microarray data on sialyl glycan binding by TgMIC1-NT. Numerical scores are shown of the binding signals, means of duplicate spots at 7 fmol/spot (with error bars) for the 58 sialyl oligosaccharide probes examined. Sixty-nine positions are shown, as 11 of the probes were printed more than once (Supplementary Table 1). Selected sialo-oligosaccharide sequences are annotated, with designations of Neu5Acα-2,3-gal linkage as pink; Neu5Acα-2,6Gal, blue; NeuGcα-2-3Gal, green and Neu5Acα-2, 8 linkage yellow. The scores for the non-sialylated probes in the microarray are not shown. These are provided in Supplementary Table 1 (positions 70–218). Download figure Download PowerPoint More than 40 out of the 69 sialylated probes arrayed gave numerical binding scores greater than 150 with TgMIC1-NT; among them were several N- and O-glycans, and gangliosides, in groups D, G and F, respectively, and others representing sialylated capping structures on backbones of mammalian glycoconjugates in groups A, B, C and E (Figure 4). Where close comparisons could be made (Table II), a mild preference is apparent for the Neu5Acα2–3Gal linkage over the α-2,6 and α-2,8 linkages (Neu5Ac denotes N-acetylneuraminic acid). The N-acetyl group of the sialic acid was found to be required for binding (cf for example probes 19 and 27 in Table II). No effects on binding were observed when downstream residues were either sulfated or fucosylated (Supplementary Table 1). Table 2. Comparisons of TgMIC1 binding signals elicited by selected sialyl probes in the carbohydrate microarray Name (position in Figure 4) Sequence Fluorescence intensity at 7 fmol per spot SA (3′) LacNAc (3) NeuAcα-3Galβ-4GlcNAc 886 SA (6′) LacNAc (6) NeuAcα-6Galβ-4GlcNAc 124 Sialyl Lex (21) NeuAcα-3Galβ-4GlcNAcβ-3Galβ-4Glc 2520 ∣ Fucα-3 LSTc (22) NeuAcα-6Galβ-4GlcNAcβ-3Galβ-4Glc 100 6′ SU-Sialyl Lex (19) HSO3-6 1692 ∣ NeuAcα-3Galβ-4GlcNAcβ-3Galβ-4Glcβ-Cer ∣ Fucα-3 de-N-acatyl 6′ SU-Sialyl Lex (27) HSO3-6 <1 ∣ Neuα-3Galβ-4GlcNAcβ-3Galβ-4Glcβ-Cer ∣ Fucα-3 A2F(A2-3) (36) NeuAcα-3Galβ-4GlcNAcβ-2Manα-6Fucα-3 4170 ∣∣ Manβ-4GlcNAcβ-4GlcNAc ∣ NeuAcα-3Galβ-4GlcNAcβ-2Manα-3 A2 (37) NeuAcα-6Galβ-4GlcNAcβ-2Manα-6 1197 ∣ Manβ-4GlcNAcβ-4GlcNAc ∣ NeuAcα-6Galβ-4GlcNAcβ-2Manα-3 NeuAcα-6Galβ-4GlcNAcβ-2Manα-6 ∣ Manβ-4GlcNAcβ-4GlcNAc ∣ A3 (38) NeuAcα-6Galβ-4GlcNAcβ-2Manα-3 6777 ∣ NeuAcα-6Galβ-4GlcNAcβ-2 GT1b (53) NeuAcα-3Galβ-4GalNAcβ-4Galβ-4Glcβ-Cer 8254 ∣ NeuAcα-8 NeuAcα-3 GD1a (54) NeuAcα-3Galβ-4GalNAcβ-4Galβ-4Glcβ-Cer 4492 ∣ NeuAcα-3 GT1a (55) NeuAcα-8NeuAcα-3Galβ-4GalNAcβ-4Galβ-4Glcβ-Cer 204 ∣ NeuAcα-3 To locate the receptor-binding surfaces on TgMIC1 and investigate the sialic acid binding mode in more detail, crystals were soaked with either sialic acid, α-2,3-sialyl-N-acetyllactosamine or α-2,6-sialyl-N-acetyllactosamine. The crystallographic structures of the complexes showed that the protein structure was largely unchanged and in all cases a single glycan was shown to be bound to the MAR2 domain of TgMIC1-NT (Figure 5 and Supplementary Figure 1). A shallow binding pocket is formed by a contiguous stretch of residues between Lys216 and Glu221, most of which makes specific direct contacts with the sialyl moiety that represents a novel binding mode. The interactions between the protein and sugar molecules do not appear to involve any tightly bound water molecules. Most notably, the two oxygen atoms from the carboxyl group are recognized by hydrogen bonds to the amide and side-chain hydroxyl protons of Thr220, rather than the side chain of an arginine or lysine, which is often the case in other sialic acid-binding proteins (May et al, 1998; Alphey et al, 2003). Recognition of the acetyl group in sialic acid is enabled by a hydrogen bond between the NH group and the backbone carbonyl of His218. The side chain of Tyr219 contacts the glycerol moiety, while His218 stacks with the underside of the ring. Electron density was observed for the galactose unit in both α-2,3-sialyl-N-acetyllactosamine and α-2,6-sialyl-N-acetyllactosamine complexes, but no specific protein contacts could account for its preferred orientation (Figure 5A). The glucose ring was not clearly observed in these electron density maps. Figure 5.Structure of TgMIC-NT in complex with sialyl oligosaccharides. (A) Simulated annealing (Fo–Fc) OMIT map contoured at 3σ (left) and 2σ levels (right) for the TgMIC-NT–glycan complex showing the unambiguous orientation of the sialic acid moiety and the position of the galactose unit of α-2,3-sialyl-N-acetyllactosamine. The omit map was calculated with the glycan omitted; stick models of key side chains (green) and sialic acid (Magenta) are overlaid on the map. (B) Structure of α-2,3-sialyl-N-acetyllactosamine in complex with the MAR2 domain from TgMIC1-NT. Ribbon representation of MAR2 is shown in green. Key interacting side chains and the oligosaccharide are shown as stick representations. Oxygen and nitrogen atoms participating in hydrogen bonds are colored in red and blue, respectively. Note: the structure of the α-2,6-sialyl-N-acetyllactosamine complex is shown in Supplementary Figure 1. (C) Structure of the MAR1 domain from TgMIC1-NT shows the position of the acetate molecul" @default.
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• W2029986004 date "2007-05-10" @default.
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• W2029986004 title "Atomic resolution insight into host cell recognition by Toxoplasma gondii" @default.
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