Crystal structures reveal that Lewis-x and fucose bind to secondary cholera toxin binding site – in contrast to fucosyl-GM1

Abstract

13Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. 14 Infection occurs after ingestion of the bacteria, which colonize the human small intestine and 15 secrete their major virulence factorthe cholera toxin (CT). Recent studies suggest that the 16 GM1 receptor may not be the only target of the CT, and that fucosylated receptors such as 17 Lewis x (Le x ) and histo-blood group antigens may also be important for cellular uptake and 18 toxicity. However, where and how Le x binds to the CT remains unclear. Here we report the high-19resolution crystal structure (1.5 Å) of the receptor-binding B-subunit of the CT bound to the Le x 20 trisaccharide, and present matching SPR data for CT holotoxins. Le x , and also L-fucose alone (at 21 500-fold molar excess), bind to the secondary binding site of the toxin, distinct from the GM1 22 binding site. In contrast, fucosyl-GM1 binds to the primary binding sites due to high-affinity 23 interactions of its GM1 core. The two binding sites are likely connected by allosteric cross-talk. 24 rabbit small bowel segments lead to a concentration-dependent increase in the number of toxin 46 receptors and in the sensitivity to the diarrheogenic action of CT 10,13 . CT binds GM1 at the 47 bottom surface of the B-pentamer, which serves as a landing platform on the cell membrane 12 . 48More recently, CT was found to bind histo-blood group antigens (BGAs) at a secondary binding 49 site on the lateral side of the B-pentamer, which had originally been discovered for chimeras of 50 CTB and the homologous Escherichia coli heat-labile enterotoxin (LTB) 18 . Cholera toxin 51 naturally occurs in two variants -classical CT (cCT) and El Tor CT (ET CT), which are 52 produced by the corresponding V. cholerae biotypes 4 . However, since 2001 a new vibrio strain 53 has been observed with characteristics of the El Tor biotype that produces CT with the classical 54 amino acid sequence 19 . The two variants only differ in residues 18 and 47 in the secondary CT 55 binding site (cCT: H18, T47, ET CT: Y18, I47) 4,20 , which is why they have been in the focus of 56 cholera blood group binding studies [21][22][23][24] . Moreover, there is increasing evidence that GM1 is not 57 the only CT receptor and may not even be its main receptor 18,25-31 . 58 Current research shows that fucosylated structures have an important role in cholera 59 intoxication 29,30 . CT binds fucosylated human BGAs merely with millimolar affinity 21-24 , but 60 nevertheless causes blood-group-dependent cellular intoxication 32 . Patients with blood group O 61 are also more likely to get severe cholera symptoms [33][34][35][36] . BGAs are present in the intestine in 62 high concentrations, especially compared to GM1 37 . Most recently, Le x , a BGA precursor 63 expressed on human granulocytes and intestinal cells, has been shown to specifically bind to 64 CTB and was proposed to function as a cellular receptor 30 . Cholera intoxication in mice can be 65 independent of GM1 30 , and fucose-based ...

Specificity of <em>Escherichia coli</em> Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography

“…The crystal contained two B-pentamers in the asymmetric unit, giving us access to ten crystallographically distinct B-subunits. The B-pentamers are positioned top-to-top, with the primary binding sites on opposite ends, similarly to recent LTB and CTB structures [20,26] ( Figure 3a). The ligand LNnH is present in two of the ten primary binding sites, on opposite ends of the decamer, and characterized by high-quality electron density (Figure 3a and 3b).…”

“…In addition to Ile 58, which is conserved in all toxin variants, the largest effect was found upon substituting Asn94, followed by Ser95 and Glu7, and Tyr18. Intriguingly, these residues lie on two paths connecting the primary and secondary toxin binding sites, which have previously been implicated in allosteric cross-talk [10,26,27]. This cross-talk may not only be important for the communication between the sites, but also directly affect binding affinity and specificity, with implications for the core of the biological mechanism of the toxins.…”

Specificity of <em>Escherichia coli</em> Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis

“…The crystal contained two B-pentamers in the asymmetric unit, giving us access to ten crystallographically distinct B-subunits. The B-pentamers are positioned top-to-top, with the primary binding sites on opposite ends, similarly to the recent LTB and CTB structures [20,26] ( Figure 3a). The ligand LNnH is present in two of the ten primary binding sites, on opposite ends of the decamer, and characterized by high-quality electron density (Figure 3a and 3b).…”

“…The very same residues are present in a CT-like toxin from C. freundii, which exhibits the strongest multivalent binding to N-acetyllactosamine-terminated receptors known to date [15]. Intriguingly, these residues lie on two paths connecting the primary and secondary toxin binding sites, which have previously been implicated in allosteric cross-talk [10,26,27]. This cross-talk may not only be important for the communication between the sites, but also directly affect binding affinity and specificity, hence being at the core of the biological mechanism of the toxins.…”

“…The SPR experiments with analyte GM1 pentasaccharide (GM1a; Elicityl-oligotech, France, product code GLY096) were performed as described in [26]; briefly, the analyte was injected over the immobilized CM5 sensor chip with an increasing concentration up to 200 nM, and performed in duplicate. The dissociation constants for LNnT and LNnH were calculated by using a steady-state affinity model, while the GM1a data was fitted to a Langmuir 1:1 interaction model, using the Biacore T100 evaluation software.…”