Identification of Elongation Factor G as the Conserved Cellular Target of Argyrin B

Beat Nyfeler,Zuncai Wang,Leon O Murphy,Ruth E Caughlan,Ralph Riedl,Gejing Deng,Philipp Krastel,David Estoppey,Deborah Palestrant,Swann Gaulis,Michael Salcius,Charles R Dean,Adriana K Jones,John A Tallarico,Ervan Hauy,N Rao Movva,Gregory A Michaud,Dominic Hoepfner,Christopher J Wilson,Saskia M Brachmann,Thomas Aust,Gregory Mcallister,John R Walker,Christian Studer,S Whitney Barnes,Lewis Whitehead,Robert K Yu ,C.a Kirby ,Angela Woods

doi:10.1371/journal.pone.0042657

Abstract

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.

Highlights

Natural products constitute a major resource for the identification of bioactive molecules
Argyrin B inhibits bacterial elongation factor G We first confirmed the activity of argyrin B against P. aeruginosa
To exclude the possibility that selection of fusA mutations using argyrin B might reflect an indirect resistance mechanism rather than direct interaction of argyrin with this target protein, we tested if purified P. aeruginosa elongation factor G1 (EF-G1) binds to argyrin B

Summary

Introduction

Natural products constitute a major resource for the identification of bioactive molecules. The argyrins are natural peptides produced by myxobacteria and actinomycetes that have an intriguing antibacterial spectrum of activity [1,2,3]. This includes the intrinsically drug resistant organism Pseudomonas aeruginosa, but not other Gram negatives tested, such as Escherichia coli or Salmonella typhimurium, unless the cells are compromised in their outer membrane permeability barrier, presumably allowing access to the intracellular target [1,2]. To shed more light on the cellular target of this interesting class of natural products, we employed bacterial and yeast mutant selection and whole genome sequencing to identify the target of argyrin B and explored whether the mechanism of action is conserved in mammalian cells

Results

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Discussion

Experimental Procedures