Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates
Abstract
Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.
Data availability
Atomic coordinates for the crystal structures have been deposited in the Protein Data Bank under accession numbers 7MH6 (EmrE3/L10), 7MGX (EmrE3/L10/methyl viologen), 7SVX (EmrE3/L10/harmane), 7SSU (EmrE3/L10/MeTPP+), 7SV9 (EmrE3/L10/TPP+), 7T00 (EmrE3/L10/benzyltrimethylammonium) and 7SZT (Gdx-Clo/L10). All other data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1 and 5.
-
Structure of EmrE-D3 mutant in complex with monobody L10 and harmaneProtein Data Bank, 7SVX.
-
Structure of EmrE-D3 mutant in complex with monobody L10 and TPPProtein Data Bank, 7SV9.
Article and author information
Author details
Funding
National Institutes of Health (CA194864)
- Shohei Koide
National Science Foundation (CAREER 1845012)
- Randy B Stockbridge
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Kermani et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,490
- views
-
- 425
- downloads
-
- 20
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.
-
- Structural Biology and Molecular Biophysics
The relationship between protein dynamics and function is essential for understanding biological processes and developing effective therapeutics. Functional sites within proteins are critical for activities such as substrate binding, catalysis, and structural changes. Existing computational methods for the predictions of functional residues are trained on sequence, structural, and experimental data, but they do not explicitly model the influence of evolution on protein dynamics. This overlooked contribution is essential as it is known that evolution can fine-tune protein dynamics through compensatory mutations either to improve the proteins’ performance or diversify its function while maintaining the same structural scaffold. To model this critical contribution, we introduce DyNoPy, a computational method that combines residue coevolution analysis with molecular dynamics simulations, revealing hidden correlations between functional sites. DyNoPy constructs a graph model of residue–residue interactions, identifies communities of key residue groups, and annotates critical sites based on their roles. By leveraging the concept of coevolved dynamical couplings—residue pairs with critical dynamical interactions that have been preserved during evolution—DyNoPy offers a powerful method for predicting and analysing protein evolution and dynamics. We demonstrate the effectiveness of DyNoPy on SHV-1 and PDC-3, chromosomally encoded β-lactamases linked to antibiotic resistance, highlighting its potential to inform drug design and address pressing healthcare challenges.