A MUTANT SHIGA-LIKE TOXIN IIv BOUND TO ITS RECEPTOR. Hong Ling*, Amechand Boodhoo$, Glen D. Armstrong$, Clifford G. Clark#, James L.Brunton# and Randy J. Read*$ *Department of Biochemistry & $Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, T6G 2H7, Canada, #Department of Microbiology and Medicine, University of Toronto, Toronto Ontario M5S 1A1, Canada
Shiga-like toxin II variant (SLT-IIv) is a member of the Shiga toxin family. SLT-IIv is produced by certain strains of E.coli that cause edema in pigs. Like other family members, it is a bipartite molecule composed of an enzymatic (A) subunit, and five copies of a binding (B) subunit. The B pentamers of Shiga-like toxins mediate receptor binding, cytotoxic specificity and extracellular localization of the holotoxin. The functional receptor of the B subunits for most family members is the glycolipid Gb3 (globotriaosyl ceramide), but SLT-IIv has a preference for the glycolipid Gb4 (globotetraosyl ceramide). Interestingly, a double mutant of SLT-IIv (designated as GT3: Gln65/Glu, Lys67/Gln in the B subunit) loses its preference for Gb4 and instead binds most strongly to Gb3.
In order to understand the molecular basis for the receptor specificity, we have determined the structure of the GT3 mutant B pentamer complexed with Gb3 at 2Å. The structure was solved by molecular replacement using the Shiga-like toxin I B subunit as a search model (64% identity with the SLT-IIv B subunit). Refinement consisted of XPLOR runs combined with 5-fold averaging in DEMON, and manual rebuilding in O. The refined structure has excellent stereochemistry and an R-factor of 17.5% (Rfree=22.8%).
The B subunit structure has a typical oligomer binding (OB) motif which consists of a five-stranded antiparallel [[beta]]-barrel capped by an alpha helix. The five identical B subunits form a symmetric pentamer. The structure reveals two Gb3 binding sites per monomer on the bottom surface (opposite to the interface with the A subunit) of the B pentamer.
Research supported by AHFMR, MRC and HHMI