Proteolytic processing induces a conformational switch required for antibacterial toxin delivery

• Published in: Nature communications 2022-08, Vol.13 (1), p.5078-17, Article 5078
• Main Authors: Bartelli, Nicholas L., Passanisi, Victor J., Michalska, Karolina, Song, Kiho, Nhan, Dinh Q., Zhou, Hongjun, Cuthbert, Bonnie J., Stols, Lucy M., Eschenfeldt, William H., Wilson, Nicholas G., Basra, Jesse S., Cortes, Ricardo, Noorsher, Zainab, Gabraiel, Youssef, Poonen-Honig, Isaac, Seacord, Elizabeth C., Goulding, Celia W., Low, David A., Joachimiak, Andrzej, Dahlquist, Frederick W., Hayes, Christopher S.
• Format: Article
• Language: English
• Subjects: 101/58; 101/6; 631/326/41/1319; 631/326/41/2536; 631/45/470; 631/45/612/1242; 82/29; 82/80; 82/83; Anti-Bacterial Agents - metabolism; Anti-Bacterial Agents - pharmacology; Antiinfectives and antibacterials; Bacteria; Bacterial structural biology; Bacterial toxins; BASIC BIOLOGICAL SCIENCES; Biochemistry; Cleavage; Conformation; Conserved sequence; Cytoplasm; Cytosol; Domains; E coli; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Gram-negative bacteria; Humanities and Social Sciences; Laboratories; Membrane Proteins - metabolism; Membranes; Molecular biology; multidisciplinary; Nucleases; Peptides; Periplasm; Protein folding; Proteins; Proteolysis; Science; Science (multidisciplinary); Thermodynamic properties; Toxins; Translocation
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-32795-y

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. Contact-dependent growth inhibition (CDI) is an important mechanism of bacterial competition. Here, Bartelli et al. show that proteolytic processing of a CDI toxin induces a conformational switch required for translocation into target bacteria.