Edda M. Twiddy

Crystal structure of a new heat-labile enterotoxin, LT-IIb.

BACKGROUND Cholera toxin from Vibrio cholerae and the type I heat-labile enterotoxins (LT-Is) from Escherichia coli are oligomeric proteins with AB5 structures. The type II heat-labile enterotoxins (LT-IIs) from E. coli are structurally similar to, but antigenically distinct from, the type I enterotoxins. The A subunits of type I and type II enterotoxins are homologous and activate adenylate cyclase by ADP-ribosylation of a G protein subunit, G8 alpha. However, the B subunits of type I and type II enterotoxins differ dramatically in amino acid sequence and ganglioside-binding specificity. The structure of LT-IIb was determined both as a prototype for other LT-IIs and to provide additional insights into structure/function relationships among members of the heat-labile enterotoxin family and the superfamily of ADP-ribosylating protein toxins. RESULTS The 2.25 A crystal structure of the LT-IIb holotoxin has been determined. The structure reveals striking similarities with LT-I in both the catalytic A subunit and the ganglioside-binding B subunits. The latter form a pentamer which has a central pore with a diameter of 10-18 A. Despite their similarities, the relative orientation between the A polypeptide and the B pentamer differs by 24 degrees in LT-I and LT-IIb. A common hydrophobic ring was observed at the A-B5 interface which may be important in the cholera toxin family for assembly of the AB5 heterohexamer. A cluster of arginine residues at the surface of the A subunit of LT-I and cholera toxin, possibly involved in assembly, is also present in LT-IIb. The ganglioside receptor binding sites are localized, as suggested by mutagenesis, and are in a position roughly similar to the sites where LT-I binds its receptor. CONCLUSIONS The structure of LT-IIb provides insight into the sequence diversity and structural similarity of the AB5 toxin family. New knowledge has been gained regarding the assembly of AB5 toxins and their active-site architecture.

Antigenic Heterogeneity among Heat-Labile Enterotoxins from Escherichia Coli

We have previously described a solid phase radioimmunoassay for quantitative measurements of cholera enterotoxin (CT) and other antigenically related enterotoxins (1). The heat-labile enterotoxin (LT) of Escherichia coli cross-reacts with CT and can be measured in this assay system. The solid phase radioimmunoassay is performed in polyvinyl microtiter plates and involves four sequential steps: immobilization of purified equine anticholeragenoid antibody; binding of enterotoxin antigen; binding of rabbit antienterotoxin; and binding of radioiodinated goat anti-rabbit IgG (γ) (hereafter designated [125I]GARG).

The Escherichia Coli/Vibrio Cholerae Family Of Enterotoxins

The heat-labile enterotoxins (LT-I and LT-II) of Escherichia coli and cholera enterotoxin (CT) from Vibrio cholerae belong to a family of related protein toxins. Each toxin consists of an A subunit that activates adenylate cyclase in target cells by ADP ribosylation of the regulatory protein Gs and an oligomeric B subunit that binds to plasma membrane receptors on susceptible target cells. The E. coli/V. cholerae enterotoxin family is divided into serogroups based on neutralization tests. LT-I and CT belong to serogroup I, whereas LT-II belongs to serogroup II. Antigenic variants of CT, LT-I and LT-II are produced by V. cholerae and E. coli strains from natural sources. Genes encoding the A polypeptides for all of these toxins are homologous, and both ADP-ribosyl transferase activity and stimulation of the activity by ADP-ribosylation factor (ARF) are conserved functions of the A subunits. Genes for the B polypeptides of the toxins in serogroup I are also homologous, but they have no significant homology with the B subunit genes of the toxins in serogroup II. Gangliosides to which toxins bind with highest affinity are GM1 for LT-I and CT, GDlb for LT-lIa and GDla for LT- Ilb. This paper presents an overview of the E. coli/V. cholerae enterotoxin family and summarizes recent work from our laboratory on these toxins.