Published on Tue Oct 05 2021

The membrane-proximal domain of the periplasmic adapter protein plays a role in vetting substrates utilising channels 1 and 2 of RND efflux transporters

Alav, I., Bavro, V. N., Blair, J. M. A.

Active efflux by resistance-nodulation-division (RND) efflux pumps is a major contributor to antibiotic resistance in clinically relevant Gram-negative bacteria. Tripartite RND pumps comprise of an inner membrane RND transporter, a periplasmic adaptor protein (

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Abstract

Active efflux by resistance-nodulation-division (RND) efflux pumps is a major contributor to antibiotic resistance in clinically relevant Gram-negative bacteria. Tripartite RND pumps, such as AcrAB-TolC of Salmonella enterica serovar Typhimurium, comprise of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. Previously, we elucidated binding sites within the PAP AcrA (termed binding boxes) that were important for AcrB-transporter recognition. Here, we have refined the binding box model by identifying the most critical residues involved in PAP-RND binding and show that the corresponding RND-binding residues in the closely related PAP AcrE are also important for AcrB interactions. In addition, our analysis identified a membrane-proximal domain (MPD)-residue in AcrA (K366), that when mutated, differentially affects transport of substrates utilising different AcrB efflux-channels, namely channels 1 and 2, supporting a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket (PBP) of the transporter and substrate vetting. Our model predicts that there is a close interplay between the MPD of the PAP and the RND transporter in the productive export of substrates utilising the PBP.