1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

Hatlem, Daniel; Heggelund, J.E.; Burschowsky, D.; Krengel, Ute; Kristiansen, Per Eugen

doi:10.1007/s12104-017-9728-9

Cited by 8 publications

(11 citation statements)

References 30 publications

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“…The partial competition of GM1 and Le x observed by Cervin et al . 30 is most likely due to allosteric cross-talk, in line with results from a recent NMR study of the homologous LTB 46 . Similarly, we found that the CT secondary binding site variant H18A exhibits increased GM1os binding.…”

Section: Discussionsupporting

confidence: 84%

“…GM1 binding to CTB is known to be positively cooperative 56–58 , and the small structural changes associated with cooperative binding to the primary site could easily be transmitted to the secondary binding site, for example via the helix connecting the two sites or by stabilization of the loop region comprising residues 55–60 59 . Recent NMR data for the homologous LTB from E. coli (ETEC) are consistent with such a cross-talk between binding sites 46 . Likewise, CT variant H18A shows increased GM1os affinity compared to wild-type CT, even though the mutation is in the secondary binding site.…”

Section: Discussionmentioning

confidence: 82%

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

Hodnik

et al. 2018

Preprint

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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 ...

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 82%

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

Hodnik

et al. 2018

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“…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.…”

Section: Discussionmentioning

confidence: 88%

“…Intriguingly, binding to N-acetyllactosamine-terminated structures was found to be synergistically enhanced when LTB-specific residues Asn94 and Ser95 were introduced together with hLTB residues 1-25 in an LTB/CTB chimera called LCTBH [14,17]. This sequence contains important determinants of the secondary toxin binding site, which have already been in the focus of previous studies [10,17,18,20,27]. In particular, residue 4 was attributed major significance since back-substitution of LTB-specific Ser4 to CTB-specific Asn4 resulted in a complete loss of the chimera's favourable binding properties [17].…”

Section: Secondary Binding Site Residues 4 7 and 18mentioning

confidence: 85%

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

Heggelund¹,

Heim²,

Bajc³

et al. 2018

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Diarrhoea caused by enterotoxigenic Escherichia coli is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhoea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed eleven single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched Lacto-N-neohexaose (Galb4GlcNAcb6[Galb4GlcNAcb3]Galb4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding, enhancing avidity.

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“…Intriguingly, binding to N-acetyllactosamine-terminated structures was found to be synergistically enhanced when LTB-specific residues Asn94 and Ser95 were introduced together with hLTB residues 1-25 in an LTB/CTB chimera called LCTBH [14,17]. This sequence contains important determinants of the secondary toxin binding site, which have already been in the focus of previous studies [10,17,18,20,27]. In particular, residue 4 was attributed major significance since back-substitution of LTB-specific Ser4 to CTB-specific Asn4 (creating toxin variant LCTBK) resulted in a complete loss of the chimera's favourable binding properties [17].…”

Section: Secondary Binding Site Residues 4 7 and 18mentioning

confidence: 85%

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

Heggelund

Heim

Bajc

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Diarrhoea caused by enterotoxigenic Escherichia coli is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhoea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed eleven single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched Lacto-N-neohexaose (Galbeta4GlcNAcbeta6[Galbeta4GlcNAcbeta3]Galbeta4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.

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1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

Cited by 8 publications

References 30 publications

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

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

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

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

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