Proteolytic processing induces a conformational switch required for antibacterial toxin delivery
Nicholas L. Bartelli, Victor J. Passanisi, K. Michalska, Ki‐Ho Song, Dinh Q. Nhan, Hongjun Zhou, Bonnie J. Cuthbert, Lucy Stols, William H. Eschenfeldt, Nicholas G. Wilson, Jesse S. Basra, Ricardo Cortés, Zainab Noorsher, Youssef Gabraiel, Isaac Poonen-Honig, Elizabeth C. Seacord, Celia W. Goulding, David A. Low, A. Joachimiak, Frederick W. Dahlquist, Christopher S. Hayes
Abstract
Many Gram-negative bacteria use CdiA effector proteins to inhibit the growth of neighboring competitors. CdiA transfers its toxic CdiA-CT region into the periplasm of target cells, where it is released through proteolytic cleavage. The N-terminal cytoplasm-entry domain of the CdiA-CT then mediates translocation across the inner membrane to deliver the C-terminal toxin domain into the cytosol. Here, we show that proteolysis not only liberates the CdiA-CT for delivery, but is also required to activate the entry domain for membrane translocation. Translocation function depends on precise cleavage after a conserved VENN peptide sequence, and the processed ∆VENN entry domain exhibits distinct biophysical and thermodynamic properties. By contrast, imprecisely processed CdiA-CT fragments do not undergo this transition and fail to translocate to the cytoplasm. These findings suggest that CdiA-CT processing induces a critical structural switch that converts the entry domain into a membrane-translocation competent conformation.