Escherichia coli cytolethal distending toxin subunit CdtB: Structure, function and catalytic mechanism

Cytolethal distending toxin (CDT) is a DNA-damaging toxin which is secreted by several gram-negative bacterial pathogens. The CDT holotoxin is a heterotrimer composed of CdtA, CdtB and CdtC subunits. CDT is an AB2 type toxin with the catalytically active A subunit of the AB2 toxin corresponding to the CdtB subunit, and the binding B subunit mediated by the CdtA and CdtC subunits. The toxic effects associated with CDT include cellular distension and cell cycle arrest. This dissertation reports the first X-ray crystal structure of an isolated CdtB subunit from Escherichia coli. Comparison of the EcCdtB crystal structure to previously reported holotoxin crystal structures combined with NMR dynamics data of free EcCdtB revealed three significant insights. First, in conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtB suggest that CDT intoxication is contingent upon holotoxin disassembly. Second, solution NMR structural and 15N-relaxation studies of free EcCdtB reveal disorder at a region (residues G233-D242) that occupies the holotoxin interface. Third, EcCdtB residues L186-T209, containing tandem arginine residues essential for nuclear translocation and intoxication, are also disordered in solution. In contrast to the observed solution disorder, nearly identical well-defined alpha-helix and beta-strand structures are observed in this region of the free and holotoxin-bound CdtB crystallographic models. These observations suggest distinct changes in structure involved in subunit assembly-disassembly and nuclear localization. Finally, this work has sought to dissect the catalytic mechanism of CdtB. Comparison of the EcCdtB and DNase I active site in conjunction with experimental data reveal that the primary catalytic residues are conserved and nearly identical in position. CdtB was found to contain three ionizing groups required for catalysis similar to DNase I. In addition, residues D174 and E44 are not critical for catalysis individually, but together are absolutely necessary for enzymatic activity. Together these data suggest CdtB shares a similar catalytic mechanism with DNase I. The lower catalytic activity seen in CdtB may be due to the combined effect of a lack of a homolog for DNase I residue E78, less distinct roles played by D174 and E44, and possible differences in substrate binding.