HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technicka 5 166 28 Prague 6 Czech Republic

Department of Clinical Microbiology Rigshospitalet 2200 Copenhagen Denmark

Department of Experimental Medical Science Lund University 221 00 Lund Sweden

Department of Molecular Biology and Umeå Centre for Microbial Research Umeå University 90187 Umeå Sweden

Institute for Biochemistry and Molecular Biology University of Hamburg Martin Luther King Platz 6 20146 Hamburg Germany

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i Flemingovo nam 2 166 10 Prague 6 Czech Republic

University of Tartu Institute of Technology 50411 Tartu Estonia

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Structural conservation of antibiotic interaction with ribosomes