Structural and functional analysis of the promiscuous AcrB and AdeB efflux pumps suggests different drug binding mechanisms

Alina Ornik-Cha,Giuliano Malloci,Klaas M Pos,Julia Wilhelm,Hanno Sjuts,Attilio V Vargiu,Julian Reitz,Jessica Kobylka,Anja Seybert,Achilleas S Frangakis

doi:10.1038/s41467-021-27146-2

Alina Ornik-Cha, Giuliano Malloci + Show 8 more

Open Access

https://doi.org/10.1038/s41467-021-27146-2

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Abstract

Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H+/drug antiporter module that recognizes structurally diverse substances, including antibiotics. Here, we show the 3.5 Å structure of subunit AdeB from the Acinetobacter baumannii AdeABC efflux pump solved by single-particle cryo-electron microscopy. The AdeB trimer adopts mainly a resting state with all protomers in a conformation devoid of transport channels or antibiotic binding sites. However, 10% of the protomers adopt a state where three transport channels lead to the closed substrate (deep) binding pocket. A comparison between drug binding of AdeB and Escherichia coli AcrB is made via activity analysis of 20 AdeB variants, selected on basis of side chain interactions with antibiotics observed in the AcrB periplasmic domain X-ray co-structures with fusidic acid (2.3 Å), doxycycline (2.1 Å) and levofloxacin (2.7 Å). AdeABC, compared to AcrAB-TolC, confers higher resistance to E. coli towards polyaromatic compounds and lower resistance towards antibiotic compounds.

Highlights

Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell divisiontype tripartite efflux pumps
AdeB from A. baumannii was produced in E. coli C43(DE3) ΔacrAB and purified by immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography (SEC)
After classification of all protomers, we found that approximately 10% of the protomers adopt an intermediate state, i. e. 30% of particles adopt a trimeric arrangement of two O protomers together with a previously uncharacterized conformation

Summary

Introduction

Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell divisiontype tripartite efflux pumps These include a H+/drug antiporter module that recognizes structurally diverse substances, including antibiotics. The Gram-negative opportunistic pathogen Acinetobacter baumannii exhibits a high level of multidrug resistance (MDR) to drugs including carbapenems and the last-resort antibiotics tigecycline and colistin[1]. The superfamily of resistance-nodulation-cell division (RND) efflux pumps plays a key role in intrinsic MDR in Gram-negative bacteria. These tripartite complexes comprise an RND transporter in the inner membrane that acts as a secondary active H +/drug antiporter extruding a vast spectrum of structurally unrelated drugs through a periplasmic membrane fusion protein channel connected to an outer membrane channel factor across the outer membrane[4]. PN1, PN2, PC1 and PC2, while the funnel domain consists of the DN and DC subdomains[5]

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