A new functional site W115 in CdtA is critical for Aggregatibacter actinomycetemcomitans cytolethal distending toxin

Aggregatibacter actinomycetemcomitans, a specific pathogen of localized aggressive periodontitis, produces a cytolethal distending toxin (CDT) that arrests eukaryotic cells irreversibly in G0/G1 or G2/M phase of the cell cycle. Although structural studies show that the aromatic patch region of CdtA plays an important role in its biological activity, the functional sites of CdtA have not been firmly established. In this study, site-specific mutagenesis strategy was employed for cdtA point mutations construction so as to examine the contributions of individual amino acids to receptor binding and the biological activity of holotoxin. The binding ability was reduced in CdtA(Y181A)BC holotoxin and the biological function of CDT was not weaken in CdtA(Y105A)BC, CdtA(Y125A)BC, CdtA(F109A)BC and CdtA(S106N)BC holotoxin suggesting that these sites were not critical to CDT. But the binding activity and cell cycle arrest ability of holotoxin complexes were inhibited in CdtA(W115G)BC. And this site did not affect the holotoxin assembly by size exclusion chromatography. Therefore, W115 might be a critical site of CdtA binding ability. These findings suggest that the functional sites of CdtA are not only in the aromatic patch region. W115, the new functional site is critical for receptor binding and cell cycle arrest, which provides potential targets for pharmacological disruption of CDT activity.