A mutant Shiga-like toxin IIe bound to its receptor Gb(3): structure of a group II Shiga-like toxin with altered binding specificity

Background: Shiga-like toxins (SLTs) are produced by the pathogenic strains of Escherichia coli that cause hemorrhagic colitis and hemolytic uremic sy ndrome. These diseases in humans are generally associated with group II fam ily members (SLT-II and SLT-IIc), whereas SLT-IIe (pig edema toxin) is cent ral to edema disease of swine. The pentameric B-subunit component of the ma jority of family members binds to the cell-surface glycolipid globotriaosyl ceramide (Gb(3)), but globotetraosyi ceramide (Gb(4)) is the preferred rec eptor for SLT-IIe. A double-mutant of the SLT-IIe B subunit that reverses t wo sequence differences from SLT-II (GT3; Gln65-->Glu, Lys67-->Gln, SLT-I n umbering) has been shown to bind more strongly to Gb(3) than to Gb(4). Results: To understand the molecular basis of receptor binding and specific ity, we have determined the structure of the GT3 mutant B pentamer, both in complex with a Gb(3) analogue (2.0 Angstrom resolution; R = 0.155, R-free = 0.194) and in its native form (2.35 Angstrom resolution; R = 0.187, R-fre e = 0.232). Conclusions: These are the first structures of a member of the medically im portant group II Shiga-like toxins to be reported. The structures confirm t he previous observation of multiple binding sites on each SLT monomer, alth ough binding site 3 is not occupied in the GT3 structure. Analysis of the b inding properties of mutants suggests that site 3 is a secondary Gb(4)-bind ing site. The two mutated residues are located appropriately to interact wi th the extra beta GalNAc residue on Gb(4). Differences in the binding sites provide a molecular basis for understanding the tissue specificities and p athogenic mechanisms of members of the SLT family.