Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA

• Type of Material: Article
• Authors: Neeraja Marathe, Pennsylvania State University; Ha An Nguyen, Emory University; John N. Alumasa, Pennsylvania State University; Alexandra B. Kuzmishin Nagy, Emory University; Michael Vazquez, Pennsylvania State University; Christine Dunham, Emory University; Kenneth C. Keiler, Pennsylvania State University
• Language: English
• Date: 2023-09-08
• Publisher: American Society for Microbiology
• Publication Version: Final Published Version
• Copyright Statement: © 2023 Marathe et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): https://doi.org/10.1128/mbio.01461-23
• Title of Journal or Parent Work: mBio
• Volume: 14
• Issue: 5
• Start Page: e01461
• End Page: 23
• Grant/Funding Information: Support for this work was provided by NIH IRACDA 2K12GM000680 (A.B.K.N.) and NIH R01GM121650 (K.C.K., C.M.D.). This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by the Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
• Keywords: trans-translation; KKL-55; tmRNA; tRNA; protein synthesis; elfamycin; antibiotic; EF-Tu
• Research Categories: Chemistry, Biochemistry; Biology, Microbiology; Biology, Molecular

Abstract

trans-Translation is conserved throughout bacteria and is essential in many species. High-throughput screening identified a tetrazole-based trans-translation inhibitor, KKL-55, that has broad-spectrum antibiotic activity. A biotinylated version of KKL-55 pulled down elongation factor thermo-unstable (EF-Tu) from bacterial lysates. Purified EF-Tu bound KKL-55 in vitro with a K d = 2 µM, confirming a high-affinity interaction. An X-ray crystal structure showed that KKL-55 binds in domain 3 of EF-Tu, and mutation of residues in the binding pocket abolished KKL-55 binding. RNA-binding assays in vitro showed that KKL-55 inhibits binding between EF-Tu and transfer-messenger RNA (tmRNA) but not between EF-Tu and tRNA. These data demonstrate a new mechanism for the inhibition of EF-Tu function and suggest that this specific inhibition of EF-Tu•tmRNA binding is a viable target for antibiotic development. IMPORTANCE Elongation factor thermo-unstable (EF-Tu) is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers transfer-messenger RNA (tmRNA)-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.