Journal of Molecular Biology
Volume 406, Issue 3, 25 February 2011, Pages 387-402
Journal home page for Journal of Molecular Biology

Crystal Structures Exploring the Origins of the Broader Specificity of Escherichia coli Heat-Labile Enterotoxin Compared to Cholera Toxin

https://doi.org/10.1016/j.jmb.2010.11.060Get rights and content

Abstract

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally and functionally related and share the same primary receptor, the GM1 ganglioside. Despite their extensive similarities, these two toxins exhibit distinct ligand specificities, with LT being more promiscuous than CT. Here, we have attempted to rationalize the broader binding specificity of LT and the subtle differences between the binding characteristics of LTs from human and porcine origins (mediated by their B subunit pentamers, hLTB and pLTB, respectively). The analysis is based on two crystal structures of pLTB in complexes with the pentasaccharide of its primary ligand, GM1, and with neolactotetraose, the carbohydrate determinant of a typical secondary ligand of LTs, respectively. Important molecular determinants underlying the different binding specificities of LTB and CTB are found to be contributed by Ser95, Tyr18 and Thr4 (or Ser4 of hLTB), which together prestabilize the binding site by positioning Lys91, Glu51 and the adjacent loop region (50–61) containing Ile58 for ligand binding. Glu7 and Ala1 may also play an important role. Many of these residues are closely connected with a recently identified second binding site, and there appears to be cross-talk between the two sites. Binding to N-acetyllactosamine-terminated receptors is further augmented by Arg13 (present in pLT and some hLT variants), as previously predicted.

Introduction

Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are bacterial pathogens responsible for the severe diarrheal diseases cholera and travelers' diarrhea, respectively. They cause hundreds of thousands of deaths annually, mostly in developing countries. While cholera infections affect only humans, ETEC infections can also affect pigs and other farm animals. The major virulence factors of these bacteria are their highly homologous enterotoxins, cholera toxin (CT) and heat-labile enterotoxin (LT), respectively. These toxins are examples of bacterial AB5 toxins, which consist of one enzymatically active A subunit anchored to the center of a doughnut-shaped B-pentamer.1 While CT and LT share the same protein fold,2, 3 the mechanisms by which the toxins are delivered differ. For example, LT remains associated to lipopolysaccharide (LPS)-coated vesicles upon internalization,4 whereas CT is secreted as a soluble protein.

The B-pentamers of CT and LT (i.e., CTB and LTB, respectively) harbor five primary receptor binding sites located at the interface between adjacent B subunits. The primary receptor for the two toxins is the GM1 ganglioside [Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer, where GalNAc is 2′-N-acetyl galactosamine, NeuAc is N-acetyl neuraminic acid/sialic acid and Cer is ceramide].5, 6 GM1 has been found not only to be critical for anchoring the toxin to the host tissues, but also for internalization of the toxin and its subsequent transport within the target cells.7, 8, 9, 10 The primary receptor binding sites are almost identical in sequence and three-dimensional structure between the two toxins,3, 11, 12 and their interaction with GM1 is highly specific and extremely strong.13, 14, 15, 16, 17 Holotoxin formation further enhances GM1 binding, increasing avidity approximately 100-fold compared to B-pentamers alone.17

Both CTB and LTB bind GM1 with similar affinity;14 however, LTB is more promiscuous than CTB.6, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 For example, LTB also binds glycoconjugates carrying terminal N-acetyllactosamine (Galβ4GlcNAcβ, where GlcNAc is 2′-N-acetyl glucosamine) epitopes, albeit with significantly weaker affinity,20, 21, 22, 28 whereas CTB does not bind to these structures. Furthermore, there are subtle but distinct variations in the ligand-binding properties of the highly homologous enterotoxins from human (hLT) and porcine (pLT) isolates of LT.28, 29 In a recent study, we also demonstrated that hLTB, but not pLTB, possesses a second, previously unidentified binding site capable of binding carbohydrate epitopes of the blood group ABO system.30, 31 The molecular characteristics of this second binding site clarify why CTB does not interact as strongly with the human blood group antigens as hLTB,30, 32 but there is still no explanation as to why LTs possess a broader specificity than CT with respect to N-acetyllactosamine-terminated ligands, such as neolactotetraosylceramide (Galβ4GlcNAcβ3Galβ4GlcβCer), and why pLT generally shows stronger binding to these structures than hLT.

Several attempts have been made to identify the residues responsible for these differences.11, 21, 28, 29, 33, 34 A sequence comparison of pLTB and hLTB reveals only one nonconserved amino acid residue in the direct vicinity of the GM1 binding site, at position 13. This residue has been proposed to play a prominent role in the broader specificity of pLTB11, 21, 28, 29 by providing additional interactions to the oligosaccharide moiety of the ligand. In pLTB, an arginine is present at position 13, as well as in several strains of hLT,35, 36 while in CT and other hLT strains, the shorter histidine residue is found at this position.37, 38, 39, 40 In addition, LT-specific residues in the sequence range from 1 to 25, as well as the residues at positions 94 and 95, which are in the vicinity of the binding site, have also been shown to be important for binding to N-acetyllactosamine-terminated glycoconjugates,33, 34 although no explanation for this has been proposed so far.

The interaction between GM1 and CT has been thoroughly investigated, both functionally and structurally. In comparison, LT has received less attention. In the present study, we present the first crystal structure of LTB in complex with the GM1 pentasaccharide, and we explore the molecular basis for the increased ligand promiscuity at the primary receptor site in LTB. To test the role of the residue at position 13, we introduced an H13R mutation into the sequence of hLTB such that it resembles pLTB, and we evaluated the binding to N-acetyllactosamine-terminated oligosaccharides, using solid-phase glycosphingolipid binding assays. In addition, we have also solved the structure of pLTB in complex with the terminal tetrasaccharide of a secondary, N-acetyllactosamine-terminated receptor, neolactotetraosylceramide (NEO). The structures presented here were compared with the other toxin structures in the Protein Data Bank (PDB)41 to rationalize the ligand-binding differences between CT, hLT and pLT.

Section snippets

Crystal structures

The crystal structures of pLTB in complex with the GM1 pentasaccharide and neolactotetraose (NEO) were determined at 2.4 and 1.8 Å resolution, respectively. They exhibit the well-known fold of AB5 toxin B subunits,1, 2 consisting of a five-stranded antiparallel β-sheet and two α-helices, one of them lining the central pore of the assembled homopentamer. The two ligand complexes were crystallized under different conditions and in two different crystal forms (Table 1). Both structures are

Concluding Remarks

LTs are more promiscuous than CT; however, no structural studies have yet elucidated the underlying molecular mechanisms. Here, we present the crystal structure of pLT in complex with NEO, representing a typical secondary ligand of heat-labile enterotoxins, and compare it to the structure of its complex with the carbohydrate determinant of its primary receptor, GM1. Our results suggest that entropic effects play a prominent role in LT binding to the relatively flexible linear N

Production and purification of pLTB and hLTB/H13R

Recombinant B subunits were produced and purified as described.33 In brief, the plasmids encoding pLTB and hLTB/H13R were expressed in V. cholerae strain JS156962 carrying the expression plasmid pMLpLTBtac and pML-hLTBtac, respectively. Cell cultures were grown for 20–24 h at 37 °C in modified syncase medium. The medium for hLTB/H13R was supplemented with ampicillin (100 μg/ml). The cells were removed by centrifugation (20 min at 7000 rpm). The B subunits were recovered by precipitation with

Acknowledgements

We wish to warmly thank Gergely Katona for data collection on the pLTB–GM1 complex at the ESRF in Grenoble. This study was supported by grants from the Swedish Research Council [621-2003-4057 (U.K.), 12628 (S.T.)], the Norwegian Research Council [171631/V40 and 183613/S10 (FUGE-GlycoNor) (A.M. and U.K.)], the Swedish Cancer Foundation (S.T.), the Swedish Medical Society/The Foundations of the National Board of Health and Welfare (S.T.), Volvo Assar Gabrielssons Foundation (S.T.) and by the

References (89)

  • ZhangR.-G. et al.

    The three-dimensional crystal structure of cholera toxin

    J. Mol. Biol.

    (1995)
  • HolmnerÅ. et al.

    Novel binding site identified in a hybrid between cholera toxin and heat-labile enterotoxin: 1.9 Å crystal structure reveals the details

    Structure

    (2004)
  • HorstmanA.L. et al.

    Lipopolysaccharide 3-deoxy-d-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins

    J. Biol. Chem.

    (2004)
  • HorstmanA.L. et al.

    Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles

    J. Biol. Chem.

    (2000)
  • HorstmanA.L. et al.

    Bacterial surface association of heat-labile enterotoxin through lipopolysaccharide after secretion via the general secretory pathway

    J. Biol. Chem.

    (2002)
  • KarlssonK.-A.

    Preparation of total nonacid glycolipids for overlay analysis of receptors for bacteria and viruses and for other studies

    Methods Enzymol.

    (1987)
  • MatthewsB.W.

    Solvent content of protein crystals

    J. Mol. Biol.

    (1968)
  • MerrittE.A. et al.

    The 1.25 Å resolution refinement of the cholera toxin B-pentamer: evidence of peptide backbone strain at the receptor-binding site

    J. Mol. Biol.

    (1998)
  • Matković-CalogovićD. et al.

    Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity

    J. Biol. Chem.

    (1999)
  • SixmaT.K. et al.

    Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli

    Nature

    (1991)
  • MerrittE.A. et al.

    Galactose-binding site in Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT)

    Mol. Microbiol.

    (1994)
  • KestyN.C. et al.

    Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells

    EMBO J.

    (2004)
  • HolmgrenJ. et al.

    Interaction of cholera toxin and membrane GM1 ganglioside of small intestine

    Proc. Natl Acad. Sci. USA

    (1975)
  • HolmgrenJ. et al.

    Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin

    Infect. Immun.

    (1982)
  • ChinnapenD.J.-F. et al.

    Rafting with cholera toxin: endocytosis and trafficking from plasma membrane to ER

    FEMS Microbiol. Lett.

    (2007)
  • WolfA.A. et al.

    Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia

    J. Cell Biol.

    (1998)
  • PangH. et al.

    Ganglioside GM1 levels are a determinant of the extent of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus

    J. Cell Sci.

    (2004)
  • De HaanL. et al.

    Cholera toxin: a paradigm for multi-functional engagement of cellular mechanisms (Review)

    Mol. Membr. Biol.

    (2004)
  • MerrittE.A. et al.

    Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide

    Protein Sci.

    (1994)
  • MinkeW.E. et al.

    Structure of m-carboxyphenyl-α-d-galactopyranoside complexed to heat-labile enterotoxin at 1.3 Å resolution: surprising variations in ligand-binding modes

    Acta Crystallogr., Sect. D: Biol. Crystallogr.

    (2000)
  • KuziemkoG.M. et al.

    Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance

    Biochemistry

    (1996)
  • SinghA.K. et al.

    Gangliosides as receptors for biological toxins: development of sensitive fluoroimmunoassays using ganglioside-bearing liposomes

    Anal. Chem.

    (2000)
  • LauerS. et al.

    Analysis of cholera toxin-ganglioside interactions by flow cytometry

    Biochemistry

    (2002)
  • HolmgrenJ.

    Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid

    Infect. Immun.

    (1973)
  • ÅngströmJ. et al.

    Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes

    Proc. Natl Acad. Sci. USA

    (1994)
  • TenebergS. et al.

    Comparison of the glycolipid-binding specificities of cholera toxin and porcine Escherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

    Glycoconj. J.

    (1994)
  • OrlandiP.A. et al.

    The heat-labile enterotoxin of Escherichia coli binds to polylactosaminoglycan-containing receptors in CaCo-2 human intestinal epithelial cells

    Biochemistry

    (1994)
  • BalanzinoL.E. et al.

    Differential interaction of Escherichia coli heat-labile toxin and cholera toxin with pig intestinal brush border glycoproteins depending on their ABH and related blood group antigenic determinants

    Infect. Immun.

    (1994)
  • BalanzinoL.E. et al.

    Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: relationship with ABH blood group determinants

    Mol. Cell. Biochem.

    (1999)
  • GalvánE.M. et al.

    Participation of ABH glycoconjugates in the secretory response to Escherichia coli heat-labile toxin in rabbit intestine

    J. Infect. Dis.

    (1999)
  • GalvánE.M. et al.

    Ability of blood group A-active glycosphingolipids to act as Escherichia coli heat-labile enterotoxin receptors in HT-29 cells

    J. Infect. Dis.

    (2004)
  • GalvánE.M. et al.

    Functional interaction of Escherichia coli heat-labile enterotoxin with blood group A-active glycoconjugates from differentiated HT29 cells

    FEBS J.

    (2006)
  • TenebergS. et al.

    Common architecture of the primary galactose binding sites of Erythrina corallodendron lectin and heat-labile enterotoxin from Escherichia coli in relation to the binding of branched neolactohexaosylceramide

    J. Biochem.

    (2000)
  • Holmner-Rocklöv, Å. (2005). Molecular recognition of carbohydrates-structural and functional characterisation of...
  • Cited by (22)

    • Ganglioside binding domains in proteins: Physiological and pathological mechanisms

      2022, Advances in Protein Chemistry and Structural Biology
      Citation Excerpt :

      Overall, given the critical role of the NH of the amide group of GalNAc to “internalize” the negative charge of gangliosides inside their oligosaccharide part, this mechanism could be coined the “NH trick.” Interestingly, the crystal structures of the sugar moiety of various gangliosides bound to proteins seem to display a similar NH group position (Berntsson, Peng, Dong, & Stenmark, 2013; Buch et al., 2015; Davies, Masuyer, & Stenmark, 2020; Fotinou et al., 2001; Hamark et al., 2017; Holmner et al., 2011; Merritt et al., 1998; Neu, Woellner, Gauglitz, & Stehle, 2008; Ng et al., 2013; Reiss et al., 2012). Taken together, these data suggest that a key function of nitrogen atoms in gangliosides is to prevent the electrostatic repulsion between two vicinal gangliosides in lipid raft domains.

    • Escherichia coli

      2021, Foodborne Infections and Intoxications
    • Vibrio cholerae and Escherichia coli heat-labile enterotoxins and beyond

      2015, The Comprehensive Sourcebook of Bacterial Protein Toxins
    • EcxAB is a founding member of a new family of metalloprotease AB <inf>5</inf> toxins with a hybrid cholera-like B subunit

      2013, Structure
      Citation Excerpt :

      The similarities of the glycan binding sites in EcxAB and CtxAB suggest that they target similar cellular subtypes. Comparisons made between cholera and heat-labile toxins have shown that minor differences within the glycan binding sites nevertheless leads to substantial effects on specificity (Holmner et al., 2011); the affinity of EcxAB for GM1 was thus determined by surface plasmon resonance (SPR) (Figure 4C). A kinetic analysis of EcxAB binding as measured by SPR could not be modeled with an appropriate fit due to the multivalent nature of the EcxB pentamer.

    • Escherichia coli

      2013, Foodborne Infections and Intoxications
    • The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis

      2012, Cellular Immunology
      Citation Excerpt :

      The LTB standard (soluble LTB expressed in E. coli) [14] exhibited markedly greater binding avidity for CHO-K1GM1+ cells (Fig. 3B). Since broader oligosaccharide specificity was observed for LTB [27] from the results of GD1a-ELISA this ganglioside was discarded as a putative non-GM1 receptor for LTB. Internalization of LTBABC, LTB and CTB into CHO-K1GM1+ cells was investigated by confocal fluorescent microscopy.

    View all citing articles on Scopus
    1

    Present addresses: Å. Holmner, Department of Biomedical Engineering and Informatics, Västerbotten County Council, SE-901 85 Umeå, Sweden; M. Ökvist, ESRF, 6 Rue Jules Horowitz, BP 220, 38043 Grenoble Cedex 9, France.

    View full text