Cryo-EM reveals multiple mechanisms of ribosome inhibition by doxycycline

Abstract Antimicrobial resistance is driving the search for new antibiotics and a greater understanding of their mechanism of action. Doxycycline is amongst the most-prescribed antimicrobials. It demonstrates a particularly low minimum inhibitory concentration against the zoonotic pathogen Coxiella burnetii. Doxycycline canonically targets the bacterial ribosome by blocking tRNA binding at the decoding centre (A-site) of the small subunit. Using cryo electron-microscopy, we analysed doxycycline binding to C. burnetii and Escherichia coli ribosomes. Both structures reveal unexpected binding at the exit tunnel in the large subunit. In C. burnetii three doxycycline molecules stack to block the tunnel. In E. coli one doxycycline molecule triggers a major uncharacterised conformation of the ribosome. This fundamentally reorganises the peptidyl transferase centre and blocks tRNA binding, challenging the concept that this region is largely static. We identify a new ribosomal protein in the C. burnetii large subunit and characterise an additional member of the prokaryotic ribosome hibernation promoting factor family. These insights into ribosome function and antibiotic action may aid the development of new ribosome inhibitor antibiotics. Competing Interest Statement The authors have declared no competing interest. Data availability The structures associated with this manuscript have been submitted to the Protein Data Bank with the codes: 9SLG, 9ST6, 9SX2, 9T0Z, 9T1N, 9TY2, and 9T61. The cryo-EM data have been submitted to the Electron Microscopy Data Bank (EMDB) with the codes EMD-55001, EMD-55212, EMD-55330, EMD-55416, EMD-55439, EMD-55479, and EMD-55601.