AbstractCytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY. CNFY consists of five domains (D1-D5), and by integrating structural and functional data we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) or fused β-lactamase reporter proteins. We further found that domain D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY, leading to higher deamidation activity. Finally, sequence comparisons identified the CNF translocation module in many uncharacterized bacterial proteins, suggesting its usability as a universal drug delivery tool.
Smaller Building Blocks Form Larger Assemblies: Aggregation Behavior of Biaryl-Based Dendritic Facial Amphiphiles