High-resolution landscape of an antibiotic binding site

Kevin B. Yang, Maria Cameranesi, Manjunath Gowder, Criseyda Martinez, Yosef Shamovsky, Vitaliy Epshtein, Zhitai Hao, Thao Nguyen, Eric Nirenstein, Ilya Shamovsky, Aviram Rasouly () and Evgeny Nudler ()
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Kevin B. Yang: New York University Grossman School of Medicine
Maria Cameranesi: New York University Grossman School of Medicine
Manjunath Gowder: New York University Grossman School of Medicine
Criseyda Martinez: New York University Grossman School of Medicine
Yosef Shamovsky: New York University Grossman School of Medicine
Vitaliy Epshtein: New York University Grossman School of Medicine
Zhitai Hao: New York University Grossman School of Medicine
Thao Nguyen: New York University Grossman School of Medicine
Eric Nirenstein: New York University Grossman School of Medicine
Ilya Shamovsky: New York University Grossman School of Medicine
Aviram Rasouly: New York University Grossman School of Medicine
Evgeny Nudler: New York University Grossman School of Medicine

Nature, 2023, vol. 622, issue 7981, 180-187

Abstract: Abstract Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug–enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription–replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.

Date: 2023
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DOI: 10.1038/s41586-023-06495-6

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