Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Abstract

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. AcrB of Escherichia coli is a paradigm model of these polyspecific transporters. The molecular determinants and the energetics of the functional rotation mechanism proposed for the export of substrates by this protein have not yet been unveiled. To this aim, we implemented an original protocol that allows mimicking substrate transport in silico. We show that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the surface of the substrate transport channel. This, in turn, allows for a fairly constant hydration of the substrate that facilitates its diffusion.Our findings reveal a new molecular mechanism of transport in polyspecific systems, whereby waters contribute by screening potentially strong substrate-protein interactions. The mechanistic understanding of a fundamental process related to multi-drug transport provided here could help rationalizing the behavior of other polyspecific systems.. CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/182683 doi: bioRxiv preprint first posted online 2 Multi-Drug Resistant (MDR) pathogens represent one of the most pressing health concerns of the XXI Century due to their ability to elude the action of most (in some instances all) antibiotics (1-4). A special family of membrane transport proteins, the so-called efflux pumps, plays a major role in conferring MDR by shuttling a broad spectrum of chemically unrelated cytotoxic molecules out of bacteria (5-9). Polyspecificity and partial overlap among the substrate specificities of different pumps are striking properties of these efflux machineries (10, 11), making them a key survival tool of bacteria.The efflux systems of the Resistance Nodulation-cell Division (RND) superfamily, which span the entire periplasm connecting the inner and the outer membranes, are mainly involved in the onset of MDR in Gram-negative bacteria (5, 12-14). The AcrABZ-TolC efflux pump of Escherichia coli is the paradigm model and the most studied RND efflux pump. It is composed of the outer membrane efflux duct TolC, the inner membrane-anchored adaptor protein AcrA, the small transmembrane protein AcrZ and the inner membrane RND protein AcrB (15). The lattermost is a drug/H + antiporter fuelled by the proton gradient across the inner membrane and involved in the recognition and translocation of a very broad range of compounds (16).The multi-drug recognition capabilities and the postulated efflux mechanism of RND transporters are linked through an intriguing structural puzzle, which raised the question of how these proteins achieve their special features. An important step in this direction was made with the publication of the structure of AcrB ( Figure 1A Successively, a second conformational change from T to O (supposed to be the energyrequi...

Transport Dynamics of MtrD: an RND Multidrug Efflux Pump fromNeisseria gonorrhoeae

“…Notably, the SASAdistal hydrophobic was ≥ 60% hydrated regardless of simulation outcome (Figure 4H-J, Figure S4D-F). Our data support the hypothesis that water mediates substrate movement through the periplasmic cleft 31 .…”

“…In this orientation, we calculated that 43% of MtrDCR103-ERY contacts were contributed by PN2, 43% by PC1, and 14% by the G-Loop (calculated with ChimeraX). Based upon these data and structures of ERY with AcrB, the distal site is thought to be the "second stop" on a ligand's transport pathway (with the proximal site as the "first stop"), and we expected AZY to interact with the distal site when MtrD adopted the Binding conformation 8,10,17,31 . During the subsequent transition from Binding to Extrusion, we then expected AZY to move along PN2 towards the exit of the cleft.…”

“…It has been suggested that water mediates the diffusion of transport substrates through the periplasmic cleft 31 . To elucidate the role of water in substrate transport by MtrD, we calculated the percent hydration of the solvent accessible surface area of AZY (SASAAZY) over the course of the Targeted MD simulations (Figure 4H-J).…”

Structure and mechanism of bacterial tripartite efflux pumps

“…Differences between these two pockets in aromatic, charged, and polar residues are suggested to affect substrate preferences [55]. Water molecules have been found to additionally stabilize substrate binding [51,56] inside the pocket and to contribute to drug binding and transport [57].…”

“…All substrates leave the AcrB protomer via an exit channel (dark red). The latter leads to the funnel from which the substrate then passes through the AcrA-TolC tunnel with facilitation by water molecules [57].…”