Crystal structures of multidrug efflux pump MexB bound with high-molecular-mass compounds.
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ABSTRACT:
SUBMITTER: Sakurai K
PROVIDER: S-EPMC6416280 | biostudies-literature | 2019 Mar
REPOSITORIES: biostudies-literature
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Project description:Transporters of the resistance-nodulation-cell division (RND) superfamily of proteins are the dominant multidrug efflux power of Gram-negative bacteria. The major RND efflux pump of Pseudomonas aeruginosa is MexAB-OprM, in which the inner membrane transporter MexB is responsible for the recognition and binding of compounds. The high importance of this pump in clinical antibiotic resistance made it a subject of intense investigations and a promising target for the discovery of efflux pump inhibitors. This study is focused on a series of peptidomimetic compounds developed as effective inhibitors of MexAB-OprM. We performed multi-copy molecular dynamics simulations, machine-learning (ML) analyses, and site-directed mutagenesis of MexB to investigate interactions of MexB with representatives of efflux avoiders, substrates, and inhibitors. The analysis of both direct and water-mediated protein-ligand interactions revealed characteristic patterns for each class, highlighting significant differences between them. We found that efflux avoiders poorly interact with the access binding site of MexB, and inhibition engages amino acid residues that are not directly involved in binding and transport of substrates. In agreement, machine-learning models selected different residues predictive of MexB substrates and inhibitors. The differences in interactions were further validated by site-directed mutagenesis. We conclude that the substrate translocation and inhibition pathways of MexB split at the interface (between the main putative binding sites) and at the deep binding pocket and that interactions outside of the hydrophobic patch contribute to the inhibition of MexB. This molecular-level information could help in the rational design of new inhibitors and antibiotics less susceptible to the efflux mechanism. IMPORTANCE Multidrug transporters recognize and expel from cells a broad range of ligands including their own inhibitors. The difference between the substrate translocation and inhibition routes remains unclear. In this study, machine learning and computational and experimental approaches were used to understand dynamics of MexB interactions with its ligands. Our results show that some ligands engage a certain combination of polar and charged residues in MexB binding sites to be effectively expelled into the exit funnel, whereas others engage aromatic and hydrophobic residues that slow down or hinder the next step in the transporter cycle. These findings suggest that all MexB ligands fit into this substrate-inhibitor spectrum depending on their physico-chemical structures and properties.
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Project description:The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.
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Project description:The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.
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Project description:Antibiotic-resistant strains of the Gram-negative pathogen Acinetobacter baumannii have emerged as a significant global health threat. One successful therapeutic option to treat bacterial infections has been to target the bacterial ribosome. However, in many cases, multidrug efflux pumps within the bacterium recognize and extrude these clinically important antibiotics designed to inhibit the protein synthesis function of the bacterial ribosome. Thus, multidrug efflux within A. baumannii and other highly drug-resistant strains is a major cause of failure of drug-based treatments of infectious diseases. We here report the first structures of the Acinetobacter drug efflux (Ade)J pump in the presence of the antibiotic eravacycline, using single-particle cryo-electron microscopy (cryo-EM). We also describe cryo-EM structures of the eravacycline-bound forms of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. Our data indicate that the AdeJ pump primarily uses hydrophobic interactions to bind eravacycline, while the 70S ribosome utilizes electrostatic interactions to bind this drug. Our work here highlights how an antibiotic can bind multiple bacterial targets through different mechanisms and potentially enables drug optimization by taking advantage of these different modes of ligand binding. IMPORTANCE Acinetobacter baumannii has developed into a highly antibiotic-resistant Gram-negative pathogen. The prevalent AdeJ multidrug efflux pump mediates resistance to different classes of antibiotics known to inhibit the function of the 70S ribosome. Here, we report the first structures of the A. baumannii AdeJ pump, both in the absence and presence of eravacycline. We also describe structures of the A. baumannii ribosome bound by this antibiotic. Our results indicate that AdeJ and the ribosome use very distinct binding modes for drug recognition. Our work will ultimately enable structure-based drug discovery to combat antibiotic-resistant A. baumannii infection.
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Project description:Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A direct observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer.Multidrug efflux pumps significantly contribute for bacteria resistance to antibiotics. Here the authors present the structure of Campylobacter jejuni CmeB pump combined with functional FRET assays to propose a transport mechanism where each CmeB protomers is functionally independent from the trimer.
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