REFINEMENT OF NATIVE AND MUTANT VEROTOXIN B-SUBUNIT STRUCTURES. Allan M. Sharp, Penelope E. Stein, Amechand Boodhoo, and Randy J. Read. Departments of Biochemistry, and Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7

The ability of enterohemorrhagic E. coli strains to cause hemorrhagic colitis and hemolytic uremic syndrome is a result of the production of a shiga-like toxin or verotoxin. This is an AB₅ type toxin, with a catalytic A-subunit attached to a pentamer of B-subunits that bind the cell surface glycolipid globotriaosylceramide (Gb-3). The solution of the wild-type verotoxin B-subunit pentamer, at 2.2 Å resolution, revealed a predominantly [[beta]]-sheet structure, built around a central helix-lined pore [1]. Based on the distribution of conserved surface residues, the deep clefts at the interfaces between the subunits were proposed to be the sugar-binding sites. Mutation of a phenylalanine to an alanine residue in the cleft region (Mutant F30A) did eliminate the majority of glycoside binding [2]. Difference Fourier analysis of crystals of F30A at 2.0 Å resolution suggested that there were no major structural differences in the protein away from the mutation site. Further refinement of the native and mutant structures with XPLOR and TNT has reduced their R-factors to 0.195 and has allowed detailed analyses of the features of the structures, their distortion from ideal pentamers by crystal contacts, changes at their putative binding sites, and changes in occupancy at two crystallographic zinc binding sites.

A more recent 2.8 Å crystal structure of the verotoxin B-subunit complexed with Gb3 has shown that there are in fact three different classes of binding sites on the pentamer surface, one of them closely corresponding with the original prediction, and another contacting phenylalanine 30 [3]. The effects of the mutation may aid in elucidating the relative importance of the different sites.

Research supported by the AHMRF, MRC, HHMI.

[1] Stein, P. E. et al., Nature, 355, 748-750(1992).

[2] Clark, C. et al,. Molecular Microbiology, in press.

[3] Ling, H. et al , W127, p.179, ACA Annual Meeting, Montreal, 1995.