Nathan E. Thomas's research while affiliated with University of Wisconsin–Madison and other places

Publications (13)

Preprint
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Small multidrug resistance (SMR) transporters perform coupled antiport of protons and toxic substrates, contributing to antibiotic resistance through efflux of these compounds from the bacterial cytoplasm. Extensive biophysical studies of the molecular transport mechanism of the E. coli SMR transporter EmrE indicate that it should also be capable o...
Article
Full-text available
Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transpor...
Article
Full-text available
The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport....
Preprint
Full-text available
Transport stoichiometry provides insight into the mechanism and function of ion-coupled transporters, but measuring transport stoichiometry is time-consuming and technically difficult. With the increasing evidence that many ion-coupled transporters employ multiple transport stoichiometries under different conditions, improved methods to determine t...
Article
Full-text available
Secondary active transporters couple the transport of an ion species down its concentration gradient to the uphill transport of another substrate. Despite the importance of secondary active transport to multidrug resistance, metabolite transport, and nutrient acquisition, among other biological processes, the microscopic steps of the coupling mecha...
Article
Full-text available
Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alter...
Article
Full-text available
Ion-coupled transporters must regulate access of ions and substrates into and out of the binding site to actively transport substrates and minimize dissipative leak of ions. Within the single-site alternating access model, competitive substrate binding forms the foundation of ion-coupled antiport. Strict competition between substrates leads to stoi...
Article
EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton...
Preprint
EmrE is a small multidrug resistance transporter found in E. coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug....

Citations

... After the 1950s, the dosing, formulation, and administration methods of anti-microbial were expanded. [1,2] Additionally, the mechanism of action of anti-microbial was re-investigated in detail. Nevertheless, meanwhile the start of the 1990s and the 2000s, anti-microbial resistance has increased to importance. ...
... In addition to revealing the binding mode of the guanidinyl headgroup, the structure of Gdx-Clo with octylguanidinium showed that hydrophobic repacking of residues lining one side of the binding pocket opens a portal from the substrate binding site to the membrane interior, accommodating the substrate's long alkyl tail. In addition, a model of an EmrE mutant with reduced conformational exchange dynamics, S64V, computed from extensive NMR measurements, was also reported recently (Shcherbakov et al., 2021). ...
... In contrast, a large inward-facing F 4 -TPP + gradient reverses the net transport direction, indicating that protons are driven out of the liposomes against their concentration gradient. This reversal of current is indicative of coupled transport 35,36 and demonstrates that F 4 -TPP + is antiported by EmrE. Although the timescale of transport differs between transporters, a similar reversal of current is observed for proton/guanidinium antiport by the EmrE homolog Gdx 36 . ...
... Biochemical and biophysical data have shown that the transport process of EmrE is highly complex. For example, in addition to acting as a proton-coupled antiporter, EmrE can also function as a proton-coupled symporter or uncoupled uniporter under different conditions [15][16][17][18][19] . Elucidating the mechanism of membrane transport by EmrE requires atomicresolution structural information for multiple states of the protein, as well as dynamics information about the protein and the ligands throughout the transport cycle. ...
... Extensive structural assessment of EmrE has shown that it actively works as a dimer exchanging two protons for one drug molecule per cycle (37,39,40,54,65,66). It displays an alternating access mechanism, having at least two conformations (dual-topology) in the inner membrane which includes the inward plus outward-facing forms with both the cytoplasm or periplasm having access to the drug-binding sites (37,39,40,54,(65)(66)(67)(68)(69). The interconversion of these forms is promoted by the drug/proton binding and aids in its efflux activity. ...
... EmrE has been studied in great breadth and depth. Full mutagenic scans coupled with growth assays (Amadi et al., 2010;Gutman et al., 2003;Mordoch et al., 1999;Wu et al., 2019), functional assays with reconstituted transporter (reviewed in Schuldiner, 2009), and EPR and NMR spectroscopy experiments Amadi et al., 2010;Banigan et al., 2015;Dastvan et al., 2016;Leninger et al., 2019;Thomas et al., 2018 have all revealed detailed information about the positions that contribute to substrate binding and conformational change, even as the structural details were lacking. Our structure corroborates many of the specific predictions regarding sidechains that contribute to the binding pocket, including the importance of W63 for aromatic packing with the substrate (Elbaz et al., 2005) and the cross-subunit engagement of Y60 (Vermaas et al., 2018). ...
... EmrE also provides resistance to biocides from these substrate classes with long alkyl tails, such as benzalkonium and cetyltrimethylammonium, which are found in common household antiseptics. 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. ...