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Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site - PubMed

  • ️Tue Jan 01 2019

Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site

Joel B Heim et al. Sci Rep. 2019.

Abstract

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor - the cholera toxin (CT). The GM1 ganglioside is considered the primary receptor of the CT, but recent studies suggest that also fucosylated receptors such as histo-blood group antigens are important for cellular uptake and toxicity. Recently, a special focus has been on the histo-blood group antigen Lewisx (Lex), however, where and how the CT binds to Lex remains unclear. Here we report the high-resolution crystal structure (1.5 Å) of the receptor-binding B-subunits of the CT bound to the Lex trisaccharide, and complementary quantitative binding data for CT holotoxins. Lex, and also L-fucose alone, bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. Lex is the first histo-blood group antigen of non-secretor phenotype structurally investigated in complex with CT. Together with the quantitative binding data, this allows unique insight into why individuals with non-secretor phenotype are more prone to severe cholera than so-called 'secretors'.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Schematic drawing of CT and its glycan receptors. (a) Toxin binding to host cell. The AB5 toxin CT consists of one catalytically active A subunit (CTA; dark blue) and a pentamer of the receptor-binding B subunits (CTB; light blue). The toxin’s primary and secondary binding sites (for GM1 and HBGAs, respectively) are indicated on one of the five B subunits with a green circle and an orange rectangle, respectively. (b) Structures of the oligosaccharides Lewisx triaose and tetraose, Lewisy tetraose and fucosyl-GM1 hexaose. Fucose residues are highlighted in red. Carbohydrate symbols follow the nomenclature of the Consortium for Functional Glycomics (Nomenclature Committee, Consortium for Functional Glycomics;

d

-galactose (Gal)–yellow circle, N-acetylgalactosamine (GalNAc)–yellow square,

d

-glucose (Glc)–blue circle, N-acetylglucosamine (GlcNAc)–blue square,

l

-fucose (Fuc)–red triangle, N-acetylneuraminic acid (Neu5Ac)–purple filled diamond.

Figure 2
Figure 2

Lewisx binds to the secondary binding site of the cholera toxin. (a) X-ray structure of CTB in complex with Lex (PDB ID: 6HJD, this work); side and top views (rotated by 90°). The toxin B-pentamer is shown in cartoon and transparent surface representation, and the ligands depicted in stick representation. Lex only binds to secondary binding sites on the lateral side of the toxin. (b) Close-up view of the secondary binding site (chain B), with σA-weighted Fo-Fc electron density map for Lex (grey mesh, contoured at 3.0 σ, generated before placing the ligand) and selected residues depicted in green stick representation. (c) Superimposition of Ley tetraose (blue sticks; PDB ID: 5ELB) on CTB complex with Lex triaose (green sticks; PDB ID: 6HJD, this work). Carbohydrate residues are labelled in italics, and fucose carbons are numbered. (d) Stereo image of the carbohydrate-toxin interactions. Hydrogen bonds are shown as yellow dashed lines, amino acid residues are labelled with 3-letter code. A hash (#) identifies residues contributed by a neighbouring subunit in the toxin B-pentamer. The figure was prepared with MacPyMol (Schrödinger LLC (

www.schrodinger.com/pymol

); version 1.8.0.3).

Figure 3
Figure 3

CTB complexes with

l

-fucose and fucosyl-GM1os.

l

-fucose binds to the secondary CT binding site, while fucosyl-GM1os binds to the primary binding site, facing the cell membrane. The cholera toxin B-pentamer is shown in cartoon and transparent surface representations, and the ligands depicted in stick representation. (a) X-ray structure of CTB in complex with

l

-fucose (orange sticks; PDB ID: 6HMW, this work); side view. (b) Close-up view of the secondary binding site, with σA-weighted Fo-Fc electron density map for

l

-fucose (grey mesh, contoured at 3.0 σ, generated before placing the ligand), and selected residues shown in stick representation, amino acid residues are labelled with 3-letter code. (c) X-ray structure of CTB in complex with fucosyl-GM1os (green sticks, with the terminal fucose highlighted in orange; PDB ID: 6HMY, this work). (d) Close-up view of the primary binding site, with σA-weighted Fo-Fc electron density map for fucosyl-GM1os (grey mesh, contoured at 3.0 σ, generated before placing the ligand) and Trp88 shown in stick representation. Carbohydrate residues are labelled in italics. The terminal fucose and glucose residues show weaker electron density compared to the four core residues of fucosyl-GM1os.

Figure 4
Figure 4

SPR sensorgrams and affinity plots for cholera toxin variants. SPR experiments were performed with CT holotoxins coupled to the sensor chip and using Lex tetraose (sensorgrams and corresponding plots of steady state response against concentration) or GM1os (sensorgrams with model fit) as analytes, as indicated in the panel legends. Kd values were calculated from the steady state plots (Lex) or using a Langmuir 1:1 model (GM1os). The Kd value for H18A + Lex could not be calculated since saturation was not reached upon addition of 40 mM ligand. H18AH94A only showed weak interaction for the highest Lex concentrations used; therefore, the affinity plot does not show a saturation-binding curve.

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