Strategies for the structural determination of the catalytic domain of Escherichia coli cytotoxic necrotizing factor 1

Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa toxin produced by certain pathogenic strains of Escherichia coli, constitutively activates members of the Rho GTPase family, leading to cytopathic effects. The toxin inhibits GTP turnover by Rho proteins through site-specific deamidation of a Rho glutamine required for GTP hydrolysis. To understand the basis for catalytic activity and target specificity of CNF1, the structure of a catalytically active fragment of CNF1 was sought. Here, strategies that led to successful expression of a soluble 33 kDa active fragment, growth and improvement in the quality of the crystals and determination of phases using a quadruple methionine-substitution mutant of the fragment are presented.