CRYSTAL STRUCTURE OF A NEW HEAT-LABILE ENTEROTOXIN, LT-IIb, THAT CAN ADP-RIBOSYLATE Gs-ALPHA. F. van den Akker^1,2, S. Sarfaty^1,3, E. Twiddy⁴, R.K. Holmes⁴, W.G.J. Hol1³, ¹Departments of Biological Structure and Biochemistry, ²Biomolecular Structure Center, ³Howard Hughes Medical Research Institute, University of Washington, Seattle, WA 98195, and ⁴Dep. of Microbiology, University of Colorado Health Science Center, Denver, CO 80262, USA

The LT-IIb crystal structure is the latest addition to structures determined in the cholera toxin (CT) family which encompasses a common theme of conserved structural similarities despite little sequence identity. The LT-IIb subunit organization is identical to CT and heat-labile enterotoxin (LT-I): a catalytic A subunit, capable of ADP-ribosylating Gsalpha, and a B pentamer which serves to bind to the ganglioside receptor located on the outside of the target cells. The sequence similarity of LT-IIb and LT-I is substantial when comparing their A subunits but was undetectable when comparing their B subunits. The crystal structure was determined by single isomorphous replacement (SIR) using a K₂PtCl₄ derivative. An initial 15-5 Å[[Omega]] SIR map, without anomalous data, showed features of a 5-fold arrangement of long rods. These rods could be superimposed onto the 5 long a-helices found in the LT-I B pentamer and provided the first evidence of structural similarity between the B subunits of the two toxins. Solvent flattening combined with phase extension and 5-fold averaging improved the electron density dramatically. The LT-IIb structure is currently refined to 2.25 Å resolution with an R-factor of 19.1% with good geometry. The B-pentamer of LT-IIb shows the same 'OB-fold' as the pentamers of LT-I, CT, and other AB5 toxin structures reported to date, such as pertussis toxin and shiga toxin. This constitutes a remarkable level of structural homology even in the absence of detectable sequence identity. The nature of the B pentamer pore, which is involved in binding the A subunit, among members of the cholera toxin family is very different except for a conserved ring of solvent accessible hydrophobic surface present in all members of the family. We speculate that this hydrophobic ring is critical for AB5 assembly in the periplasm of the pathogens producing these toxins.