W0029

The Crystal Structure of a Novel 79 kDa, Six Domain B.t. Toxin. T. Rydel, E. Sturman, J. Williams^#, F. Moshiri, E. Krieger, and B. Isaac. Monsanto, Biotechnology Sector, 700 Chesterfield Pkwy W, Chesterfield, MO 63017-1732; ^#Pharmacia, 700 Chesterfield Pkwy W, Chesterfield, MO 63017-1732.

Bacillus thuringiensis (B.t.) bacteria produce crystalline inclusions that contain ‘Cry’ proteins or insecticidal crystal proteins (ICPs) that are toxic to insect pests of agriculture, forestry, and public health. Monsanto is interested in B.t. ICPs for use in genetically-modified (GM), insect-resistant crop plants. We have solved the crystal structure of a novel 79 kDa, six-domain B.t. toxin to 3.0 Å resolution by the multiple isomorphous replacement (MIR) method. This B.t. toxin is a member of the Cry22 class.

This structure is noteworthy for several reasons. It is distinct from other crystallography-characterized B.t. ICPs, such as the Cry1’s, Cry2’s Cry3’s, the ET33/ET34 binary toxin, and CytB. It contains six clear folding domains: a 242-residu N-terminal domain, which is similar to p58/ERGIC-53, a protein involved in glycoprotein export from the endoplasmic reticulum; four 80-residue cadherin-like repeat domains; and a 142-residue C-terminal domain, similar to domain III of the Cry1’s, Cry2’s, and Cry3’s. The N-terminal domain and repeat domains have never been observed in a B.t. ICP structure. Finally, this toxin possesses an unusually slender and elongated shape, extending ~235 Å from end-to-end.

The 20-3.0 Å structure (R-factor = 26.7%, R-free = 33.9%) contains no solvent molecules yet. The structure will eventually be extended to 2.3 Å resolution. Switching the crystal storage solution from ammonium to lithium sulfate was key to getting derivatives suitable for phasing. This presentation will focus on the structure solution challenges and the unique toxin features.