Abstract:The current surge in bacterial multi-drug resistance (MDR) is one of the largest challenges to public health, threatening to render ineffective many therapies we rely on for treatment of serious infections. Understanding different factors that contribute to MDR is hence crucial from the global "one health" perspective. In this contribution, we focus on the prototypical broad-selectivity proton-coupled antiporter EmrE, one of the smallest known ligand transporters that confers resistance to aromatic cations in … Show more
“…Notably, the binding site Trp, W63, which had been shown to be essential in all previous studies with aromatic substrates(85-88), is not required for the transport of non-aromatic substrates by EmrE (3,89). These structural observations are in accord with NMR and computational studies that suggest that EmrE possesses an unusual degree of structural plasticity that might contribute to substrate polyspecificity (79,90). However, it should be emphasized that the observed structural perturbations are limited to the sidechains.…”
Section: J O U R N a L P R E -P R O O Fsupporting
confidence: 85%
“…As a reference, we provide a summary of the scanning mutagenesis studies that have been performed for EmrE in Table 2 . Although too extensive to discuss individually here (see reference ( 77 ) for an in-depth review of EmrE mutagenesis), these studies provided a functional grounding for the interpretation of electron paramagnetic resonance (EPR) distance measurements ( 63 ), models based on the low-resolution EM data with computationally predicted sidechains ( 61 , 78 , 79 ), models derived from NMR chemical shifts and substrate/protein distance restraints ( 80 , 81 ), and ultimately, the high resolution crystal structures, as discussed later ( 3 ). Figure 5 Substrate binding to EmrE.…”
Section: Drug and Antiseptic Exporters (Qac)mentioning
confidence: 99%
“…Note that previous EmrE crystal structures ( 82 ) (Protein Data Bank codes 3B5D , 3B62 , 3B61 ) are low quality and incomplete (only C α atoms are modeled) and experimental maps are unavailable in the Protein Data Bank. These prior structures are broadly considered inadequate for molecular inference ( 3 , 61 , 78 , 79 ) and should not be used.…”
Section: Drug and Antiseptic Exporters (Qac)mentioning
“…Notably, the binding site Trp, W63, which had been shown to be essential in all previous studies with aromatic substrates(85-88), is not required for the transport of non-aromatic substrates by EmrE (3,89). These structural observations are in accord with NMR and computational studies that suggest that EmrE possesses an unusual degree of structural plasticity that might contribute to substrate polyspecificity (79,90). However, it should be emphasized that the observed structural perturbations are limited to the sidechains.…”
Section: J O U R N a L P R E -P R O O Fsupporting
confidence: 85%
“…As a reference, we provide a summary of the scanning mutagenesis studies that have been performed for EmrE in Table 2 . Although too extensive to discuss individually here (see reference ( 77 ) for an in-depth review of EmrE mutagenesis), these studies provided a functional grounding for the interpretation of electron paramagnetic resonance (EPR) distance measurements ( 63 ), models based on the low-resolution EM data with computationally predicted sidechains ( 61 , 78 , 79 ), models derived from NMR chemical shifts and substrate/protein distance restraints ( 80 , 81 ), and ultimately, the high resolution crystal structures, as discussed later ( 3 ). Figure 5 Substrate binding to EmrE.…”
Section: Drug and Antiseptic Exporters (Qac)mentioning
confidence: 99%
“…Note that previous EmrE crystal structures ( 82 ) (Protein Data Bank codes 3B5D , 3B62 , 3B61 ) are low quality and incomplete (only C α atoms are modeled) and experimental maps are unavailable in the Protein Data Bank. These prior structures are broadly considered inadequate for molecular inference ( 3 , 61 , 78 , 79 ) and should not be used.…”
Section: Drug and Antiseptic Exporters (Qac)mentioning
“…Mechanisms to explain the transport promiscuity have been proposed, typically focusing on protein dynamics as a feature that allows it to transport many different substrates (Jurasz et al, 2021;Robinson et al, 2017). However, the structural basis for substrate binding is unknown, and for many years, structural information was limited to low-resolution models without loops or sidechains (Fleishman et al, 2006;Ubarretxena-Belandia et al, 2003), impeding a full description of the molecular mechanism.…”
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.
“…Thermodynamic and kinetic information could be extracted via statistical analyses. Biologically relevant processes generally happen at time scales of μs, ms, and even longer, which are inaccessible in modern computational modeling. − Enhanced sampling techniques are often employed to deal with the time-scale problem. ,− Modifications of the Hamiltonian of the simulated system are added to accelerate the phase space exploration, and post-simulation analyses are used to recover the statistics in the original unperturbed ensemble. − The Hamiltonian or model employed to describe the system under investigation determines the accuracy of the simulation outcome. − Although detailed descriptions such as quantum mechanics (QM) calculations are accurate, their practical use is quite limited due to high computational costs. − To obtain sufficient data for post-processing analyses, the approximated mean-field molecular mechanics (MM) force fields are widely employed in molecular dynamics (MD) simulations of biomolecular systems. − …”
Describing, understanding, and designing complex interaction networks within macromolecular systems remain challenging in modern chemical research. Host− guest systems, despite their relative simplicity in both the structural feature and interaction patterns, still pose problems in theoretical modeling. The barrel-shaped supramolecular container cucurbit[8]uril (CB8) shows promising functionalities in various areas, e.g., catalysis and molecular recognition. It can stably coordinate a series of structurally diverse guests with high affinities. In this work, we examine the binding of seven commonly abused drugs to the CB8 host, aiming at providing a general picture of CB8−guest binding. Extensive sampling of the configurational space of these host−guest systems is performed, and the binding pathway and interaction patterns of CB8−guest complexes are investigated. A thorough comparison of widely used fixed-charge models for drug-like molecules is presented. Iterative refitting of the atomic charges suggests significant conformation dependence of charge generation. The initial model generated at the original conformation could be inaccurate for new conformations explored during conformational search, and the newly fitted charge set improves the prediction−experiment correlation significantly. Our investigations of the configurational space of CB8−drug complexes suggest that the host−guest interactions are more complex than expected. Despite the structural simplicities of these molecules, the conformational fluctuations of the host and the guest molecules and orientations of functional groups lead to the existence of an ensemble of binding modes. The insights of the binding thermodynamics, performance of fixed-charge models, and binding patterns of the CB8−guest systems are useful for studying and elucidating the binding mechanism of other host−guest complexes.
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