Role of Aromatic Amino Acids in Receptor Binding Activity and Subunit Assembly of the Cytolethal Distending Toxin of Aggregatibacter actinomycetemcomitans

Departments of Microbiology, and Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6030

^* To whom correspondence should be addressed. Email: dirienzo{at}pobox.upenn.edu.

	Abstract

The periodontal pathogen Aggregatibacter actinomycetemcomitans produces a cytolethal distending toxin (Cdt) that inhibits the proliferation of oral epithelial cells. Structural models suggest that the CdtA and CdtC subunits of the heterotrimer form two putative lectin domains with a central groove. A region of CdtA rich in heterocyclic amino acids (aromatic patch) appears to play an important role in receptor recognition. In this study site-specific mutagenesis was used to assess the contributions of aromatic amino acids (tyrosine and phenylalanine) to receptor binding and CdtA-CdtC assembly. Predominant surface-exposed aromatic residues that are adjacent to the aromatic patch region in CdtA or are near the groove located at the junction of CdtA and CdtC were studied. Separately replacing residues Y105, Y140, Y188 and Y189 with alanine in CdtA resulted in differential effects on binding related to residue position within the aromatic region. These data indicate that an extensive receptor binding domain extends from the groove across the entire face of CdtA that is orientated 180° from the CdtB subunit. Substitution for residues Y105 in CdtA and Y61 and F141 in CdtC, that are located in or at the periphery of the groove, inhibited toxin assembly. Taken together, these data along with the lack of an aromatic amino acid rich region in CdtC, similar to that in CdtA, suggests that binding of the heterotoxin to its cell surface receptor is mediated predominantly by the CdtA subunit. These findings are important for developing strategies designed to block the activity of this prominent virulence factor.