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• W2300286959 abstract "Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism. Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism. The relentless rise in multidrug resistance exerts devastating human and economic tolls, raising the specter of a public health crisis (1Higgins C.F. Multiple molecular mechanisms for multidrug resistance transporters.Nature. 2007; 446: 749-757Crossref PubMed Scopus (722) Google Scholar, 2Fischbach M.A. Walsh C.T. Antibiotics for emerging pathogens.Science. 2009; 325: 1089-1093Crossref PubMed Scopus (1335) Google Scholar). A major mechanism underlying the rampant multidrug resistance is through integral membrane proteins named “multidrug transporters,” which can remove a plethora of therapeutic drugs from the cell (1Higgins C.F. Multiple molecular mechanisms for multidrug resistance transporters.Nature. 2007; 446: 749-757Crossref PubMed Scopus (722) Google Scholar). The spread of multidrug resistance, at a rate unprecedented in the past several decades, is outpacing drug discovery and accelerating (2Fischbach M.A. Walsh C.T. Antibiotics for emerging pathogens.Science. 2009; 325: 1089-1093Crossref PubMed Scopus (1335) Google Scholar). As such, we may remain on the losing side of the war against multidrug resistance until we understand how multidrug transporters export drugs and how they can be countervailed. Multidrug and toxic compound extrusion (MATE) 3The abbreviations used are: MATEmultidrug and toxic compound extrusionTPPtetraphenylphosphoniumR6Grhodamine 6G. proteins constitute a ubiquitous family of multidrug transporters and couple the efflux of structurally dissimilar, typically cationic compounds to the influx of Na+ or H+ (3Brown M.H. Paulsen I.T. Skurray R.A. The multidrug efflux protein NorM is a prototype of a new family of transporters.Mol. Microbiol. 1999; 31: 394-395Crossref PubMed Scopus (294) Google Scholar, 4Omote H. Hiasa M. Matsumoto T. Otsuka M. Moriyama Y. The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations.Trends Pharmacol. Sci. 2006; 27: 587-593Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 5Kuroda T. Tsuchiya T. Multidrug efflux transporters in the MATE family.Biochim. Biophys. Acta. 2009; 1794: 763-768Crossref PubMed Scopus (244) Google Scholar). The known MATE transporters can be separated into the NorM, DinF (DNA damage-inducible protein F) and eukaryotic subfamilies based on their amino acid sequence similarity (3Brown M.H. Paulsen I.T. Skurray R.A. The multidrug efflux protein NorM is a prototype of a new family of transporters.Mol. Microbiol. 1999; 31: 394-395Crossref PubMed Scopus (294) Google Scholar). MATE transporters are appealing targets for tackling multidrug resistance because they can extrude a wide variety of antibiotic, anticancer, and diabetic drugs across the cell membrane (4Omote H. Hiasa M. Matsumoto T. Otsuka M. Moriyama Y. The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations.Trends Pharmacol. Sci. 2006; 27: 587-593Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 5Kuroda T. Tsuchiya T. Multidrug efflux transporters in the MATE family.Biochim. Biophys. Acta. 2009; 1794: 763-768Crossref PubMed Scopus (244) Google Scholar). multidrug and toxic compound extrusion tetraphenylphosphonium rhodamine 6G. Over the past 6 years, the x-ray structures of Na+-coupled NorM from Vibrio cholera and Neiserria gonorrheae (NorM-VC and NorM-NG), and H+-coupled DinF from Pyrococcus furiosus and Bacillus halodurans (PfMATE and DinF-BH) have been reported, revealing the transporter architecture and providing important insights into the underlying transport mechanisms (6He X. Szewczyk P. Karyakin A. Evin M. Hong W.X. Zhang Q. Chang G. Structure of a cation-bound multidrug and toxic compound extrusion transporter.Nature. 2010; 467: 991-994Crossref PubMed Scopus (214) Google Scholar, 7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar, 8Tanaka Y. Hipolito C.J. Maturana A.D. Ito K. Kuroda T. Higuchi T. Katoh T. Kato H.E. Hattori M. Kumazaki K. Tsukazaki T. Ishitani R. Suga H. Nureki O. Structural basis for the drug extrusion mechanism by a MATE multidrug transporter.Nature. 2013; 496: 247-251Crossref PubMed Scopus (194) Google Scholar, 9Lu M. Radchenko M. Symersky J. Nie R. Guo Y. Structural insights into H+-coupled multidrug extrusion by a MATE transporter.Nat. Struct. Mol. Biol. 2013; 20: 1310-1317Crossref PubMed Scopus (78) Google Scholar, 10Radchenko M. Symersky J. Nie R. Lu M. Structural basis for the blockade of MATE multidrug efflux pumps.Nat. Commun. 2015; 67995Crossref PubMed Scopus (71) Google Scholar). Nevertheless, these findings highlight new areas of uncertainty that need to be addressed experimentally (11Lu M. Structures of multidrug and toxic compound extrusion transporters and their mechanistic implications.Channels. 2016; 10: 88-100Crossref PubMed Scopus (39) Google Scholar). Particularly, in all of the published MATE structures, the interface between the amino (N) and carboxyl (C) domains always opens into the extracellular milieu, i.e. adopting the extracellular facing conformations. Therefore, the molecular basis for the interconversion between the extracellular and intracellular facing conformations, which lies at the heart of the transport mechanism, remains poorly understood. Previously we deduced the intracellular facing models of MATE transporters and posited how they undergo conformational changes during drug export (9Lu M. Radchenko M. Symersky J. Nie R. Guo Y. Structural insights into H+-coupled multidrug extrusion by a MATE transporter.Nat. Struct. Mol. Biol. 2013; 20: 1310-1317Crossref PubMed Scopus (78) Google Scholar). Here we utilized mutational, cross-linking, and biochemical methods to elucidate such structural changes in NorM-NG. On the basis of our new biochemical data and available crystal structures, we uncovered the movement of transmembrane helices during transport in NorM-NG, shedding new light on how MATE transporters switch between their extracellular and intracellular facing states. Our findings take our understanding of the MATE-mediated multidrug efflux to new depths and lend fresh hope for thwarting multidrug resistance. Full-length NorM-NG was expressed with a decahistidine tag at the C terminus using a modified pET15b vector (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar). Mutations were introduced into the norM-NG gene by using the QuikChange method (Agilent Technologies) and were confirmed by DNA sequencing. Vector-specific primers (5′-GCTAGTTATTGCTCAGCGG-3′ and 5′-TAATACGACTCACTATAGGG-3′), as well as NorM-NG-specific primers (5′-ATCGCCAAGGAAAAATTCTTCC-3′ and 5′-CCATTGTCGCCACGCCGCAACC-3′), were used. The membrane expression levels of the NorM-NG mutants discussed in this work were similar to that of the wild-type protein, based on the Western blot using an antibody against the His tag. Poorly expressed and/or unstable NorM-NG mutants had been removed from further study. For the Western blot, the antibody (Qiagen, catalog no. 34460) was diluted 2,500-fold before being mixed with the transfer membranes, and each sample examined on the SDS-PAGE gel was derived from cell membranes isolated from 80 μg of Escherichia coli BL21 (DE3) ΔacrABΔmacABΔyojHI cells expressing the NorM-NG variants. The NorM-NG variants were expressed and purified as follows. Briefly, E. coli BL21 (DE3) ΔacrABΔmacABΔyojHI cells transformed with the protein expression vectors were grown in Luria-Bertani (LB) medium to an optical density of 0.5 at 600 nm and induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside at 30 °C for 4 h. The cells were harvested by centrifugation and ruptured by multiple passages through a precooled French pressure cell. All the protein purification experiments were performed at 4 °C. Membranes were collected by ultracentrifugation and extracted with 1% (w/v) n-dodecyl-β-maltoside (Anatrace) in 20 mm HEPES-NaOH, pH7.5, 100 mm NaCl, 20% (v/v) glycerol, and 1 mm tris(2-carboxyethyl)phosphine. The soluble fraction was equilibrated with nickel-nitrilotriacetic acid resin in 20 mm HEPES-NaOH, pH 7.5, 100 mm NaCl, 25% glycerol, 0.05% n-dodecyl-β-maltoside, and 1 mm tris(2-carboxyethyl)phosphine for >3 h. Protein was eluted using the same buffer supplemented with 450 mm imidazole. The eluted protein sample was promptly buffer-exchanged into 20 mm HEPES-NaOH, pH 7.5, 0 or 100 mm NaCl, 20% glycerol, 0.05% n-dodecyl-β-maltoside by using gel filtration chromatography. The expression levels of NorM-NG variants were low, which may explain why the well characterized MATE transporters confer only modest levels of cellular resistance against drugs (4Omote H. Hiasa M. Matsumoto T. Otsuka M. Moriyama Y. The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations.Trends Pharmacol. Sci. 2006; 27: 587-593Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 5Kuroda T. Tsuchiya T. Multidrug efflux transporters in the MATE family.Biochim. Biophys. Acta. 2009; 1794: 763-768Crossref PubMed Scopus (244) Google Scholar). Purified NorM-NG variants at 10 μm were incubated with 50 or 200 μm CuSO4 and 100 or 400 μm 1,10-phenanthroline in 20 mm HEPES, pH 7.5, 0 or 100 mm NaCl, 0 or 1 mm tetraphenylphosphonium (TPP), 20% glycerol, and 0.05% n-dodecyl-β-maltoside buffer for 10 min at 22 °C (12Kubo Y. Konishi S. Kawabe T. Nada S. Yamaguchi A. Proximity of periplasmic lops in the metal-tetracycline/H+ antiporter of Escherichia coli observed on site-directed chemical cross-linking.J. Biol. Chem. 2000; 275: 5270-5274Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 13Brocke L. Bendahan A. Grunewald M. Kanner BI. Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.J. Biol. Chem. 2002; 277: 3985-3992Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14Ryan R.M. Mitrovic A.D. Vandenberg R.J. The chloride permeation pathway of a glutamate transporter and its proximity to the glutamate translocation pathway.J. Biol. Chem. 2004; 279: 20742-20751Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 15Tao Z. Zhang Y.W. Agyiri A. Rudnick G. Ligand effects on cross-linking support a conformational mechanism for serotonin transport.J. Biol. Chem. 2009; 284: 33807-33814Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 16Reyes N. Ginter C. Boudker O. Transport mechanism of a bacterial homologue of glutamate transporters.Nature. 2009; 462: 880-885Crossref PubMed Scopus (338) Google Scholar, 17Valdés R. Shinde U. Landfear S.M. Cysteine cross-linking defines the extracellular gate for the Leishmania denovani nucleoside transporter 1.1 (LdNT1.1).J. Biol. Chem. 2012; 287: 44036-44045Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). 20 mm N-(5-fluoresceinyl)maleimide was then added to the samples and left for 10 min at 22 °C in the dark. Some samples were additionally treated with 10 mm DTT, whereas all samples were incubated in the dark for an additional 5 min at 22 °C. Prior to the nonreducing SDS-PAGE analysis, 100 mm N-ethylmaleimide was added to all the samples and incubated for 10 min at 22 °C. The gels were visualized by using both Coomassie staining and UV illumination. For studies of the membrane-embedded proteins, the crude E. coli membranes were isolated by ultracentrifugation; washed in buffers containing 20 mm HEPES, pH 7.5, 100 mm NaCl, and 20% glycerol; and cross-linked as in detergents and analyzed by using nonreducing SDS-PAGE and Western blot (16Reyes N. Ginter C. Boudker O. Transport mechanism of a bacterial homologue of glutamate transporters.Nature. 2009; 462: 880-885Crossref PubMed Scopus (338) Google Scholar). The drug export activities of NorM-NG variants were evaluated based on their ability to confer cellular resistance against cytotoxic chemicals (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar, 10Radchenko M. Symersky J. Nie R. Lu M. Structural basis for the blockade of MATE multidrug efflux pumps.Nat. Commun. 2015; 67995Crossref PubMed Scopus (71) Google Scholar). Drug susceptibility experiments were conducted in LB medium, with each assay repeated at least three times. Briefly, the exponential phase bacterial culture from freshly transformed E. coli BL21 (DE3) ΔacrABΔmacABΔyojHI cells was diluted to 1 × 104 colony-forming units/ml with LB broth containing 0.3 mm isopropyl β-d-1-thiogalactopyranoside and 200 μg/ml ampicillin, with or without 250 μm CuSO4, 500 μm 1,10-phenanthroline, and 250 μm DTT at each drug concentration. The culture was then incubated at 30 °C with shaking, and the bacterial growth was monitored after 10 h. Assays were performed in 96-well plates, and the optical density at 595 nm was measured using a microplate reader (Tecan GENios Plus). We defined the minimal inhibitory concentration as the lowest concentration of antimicrobial compounds that precludes the growth of E. coli under our experimental conditions (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar). Cultures of the E. coli BL21 (DE3) ΔacrABΔmacABΔyojHI cells expressing NorM-NG variants were grown to an A600 nm of 0.5 and induced with 1 mm isopropyl β-d-1-thiogalactopyranoside at 30 °C for 4 h. The cells were harvested; washed three times with 100 mm Tris-HCl, pH 7.0; resuspended in the same buffer to an A600 nm of 1.0/ml. Rhodamine 6G (R6G) or ethidium was added to the cells at a final concentration of 4 μg/ml, with or without the inclusion of 300 μm CuSO4, 600 μm 1,10-phenanthroline, and 750 μm DTT. After incubation on ice for 10 min, a 1-ml sample was withdrawn every 2.5 min, and cells were harvested by centrifugation and washed twice with 100 mm Tris-HCl, pH 7.0. The R6G or ethidium fluorescence was then measured with the excitation and emission wavelengths of 485 and 590 nm, respectively (10Radchenko M. Symersky J. Nie R. Lu M. Structural basis for the blockade of MATE multidrug efflux pumps.Nat. Commun. 2015; 67995Crossref PubMed Scopus (71) Google Scholar). In this work, we aimed to use our intracellular facing model of NorM-NG as a guide for site-directed mutagenesis (9Lu M. Radchenko M. Symersky J. Nie R. Guo Y. Structural insights into H+-coupled multidrug extrusion by a MATE transporter.Nat. Struct. Mol. Biol. 2013; 20: 1310-1317Crossref PubMed Scopus (78) Google Scholar) and then “freeze” NorM-NG in its intracellular facing conformation by utilizing disulfide cross-linking (16Reyes N. Ginter C. Boudker O. Transport mechanism of a bacterial homologue of glutamate transporters.Nature. 2009; 462: 880-885Crossref PubMed Scopus (338) Google Scholar). To accomplish this aim, we first attempted to remove all the endogenous cysteines in NorM-NG and then to carry out paired cysteine substitutions to generate mutants wherein disulfide bonds can be formed in the intracellular facing conformation but not the extracellular facing conformation. NorM-NG has four endogenous cysteines: Cys202 (TM6), Cys381 (TM10), Cys407 (TM11), and Cys444 (TM12), most of which are removed from the substrate- and Na+-binding sites and not conserved even in the NorM branch (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar). Therefore, the replacement of these cysteines by isosteric serine or valine, we reasoned, would be unlikely to affect the protein folding or transport function. Much to our amazement, although the single NorM-NG mutants C202S and C381S were both expressed to the same level as that of the wild-type protein, the replacement of Cys407 or Cys444 in NorM-NG, by serine or valine, completely abolished the protein expression (data not shown). To overcome this difficulty, we aligned the amino acid sequence of NorM-NG with those of >30 NorM orthologues. Based on the sequence alignment, we substituted Cys407 and Cys444 in NorM-NG individually with the equivalent residues found in other NorM orthologues and then examined the membrane expression and solution behaviors of those single mutants by using Western blot and gel filtration chromatography, respectively (18Li X. Dang S. Yan C. Gong X. Wang J. Shi Y. Structure of a presinilin family intramembrane aspartate protease.Nature. 2013; 493: 56-61Crossref PubMed Scopus (163) Google Scholar). Well behaved single mutations were then selected and combined to generate double, triple, and finally quadruple mutants (data not shown). After screening >40 different NorM-NG mutants, we identified a quadruple mutant, C202S/C381S/C407F/C444R, hereafter termed “Cys-less NorM-NG,” that was expressed to a similar level to that of NorM-NG and exhibited good solution behavior. Based on the drug resistance and accumulation assays (see below), we found that the Cys-less NorM-NG retained the multidrug efflux activity, suggesting that the quadruple mutation had not disrupted the transporter structure or function and thus was suitable for further mutagenesis and functional studies. Previously we hypothesized that NorM-NG switches from its extracellular to intracellular facing conformation upon substrate release and Na+ binding (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar). Our studies further predicted that in the intracellular facing NorM-NG, the extracellular portions of TM1 and TM2 are in close proximity to those of TM8 and TM7, respectively (9Lu M. Radchenko M. Symersky J. Nie R. Guo Y. Structural insights into H+-coupled multidrug extrusion by a MATE transporter.Nat. Struct. Mol. Biol. 2013; 20: 1310-1317Crossref PubMed Scopus (78) Google Scholar). If our prediction is correct, once we replace the amino acids envisioned to be close enough in the intracellular facing NorM-NG with cysteines, we may be able to employ disulfide cross-linking to “stitch” together the extracellular portions of TM1 and TM8, TM2 and TM7, or TM2 and TM8. Along this line of reasoning, we identified 14 pairs of amino acids in NorM-NG whose Cα positions are far apart in the extracellular facing structure (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar), but close to each other in the intracellular facing model (Fig. 1). The intracellular facing model of NorM-NG was constructed by rotating the C domain as a rigid body in the extracellular facing structure by 20° relative to the N domain (9Lu M. Radchenko M. Symersky J. Nie R. Guo Y. Structural insights into H+-coupled multidrug extrusion by a MATE transporter.Nat. Struct. Mol. Biol. 2013; 20: 1310-1317Crossref PubMed Scopus (78) Google Scholar). Among the 14 pairs of amino acids, 8 are located within TM1 and TM8, 5 are within TM2 and TM7, and 1 is within TM2 and TM8. Notably, most of these amino acids are removed from the substrate- and Na+-binding sites (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar), thereby diminishing the possibility of interfering with substrate- and/or Na+-binding and hence giving rise to inactive proteins upon cysteine substitutions. We then replaced these amino acids in the Cys-less NorM-NG with cysteines singularly or in pairs, yielding a total of 28 and 14 of what we denoted “mono-” and “dicysteine” mutants, respectively. Among the 14 dicysteine mutants, only 7 were expressed to similar levels to that of NorM-NG (Fig. 2) and were well folded based on their profiles on gel filtration chromatography (data not shown). Importantly, these 7 mutants retained the ability to extrude different drugs (see below) and were therefore suitable for further investigation.FIGURE 2Western blot analysis of the NorM-NG variants. Western blot analysis of NorM-NG variants in membrane preparations was performed by using an antibody against the His tag. This analysis suggested that all of the NorM-NG variants investigated in this work were expressed at similar levels in the E. coli membrane. The positions of two molecular weight markers are also indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We first asked whether the seven dicysteine mutants, namely T42C/L289C, A45C/V285C (TM1 and TM8), L58C/V269C, L58C/F270C, A62C/F265C, A62C/S266C (TM2 and TM7), and A57C/Q284C (TM2 and TM8) can form intramolecular disulfide bonds in vitro. We expressed and purified these dicysteine mutants in the presence of Na+. We then incubated these proteins with an oxidizing agent (copper phenanthroline) and performed nonreducing SDS-PAGE analysis (Fig. 3A). We observed that for five mutants, T42C/L289C, A45C/V285C, L58C/F270C, A62C/S266C, and A57C/Q284C, copper phenanthroline treatment yielded distinct protein species with markedly higher electrophoretic mobility. Moreover, the faster migrating protein species contained no free cysteine thiols, because they could no longer react with a thiol-reactive fluorophore, N-(5-fluoresceinyl)maleimide (Fig. 3B). Notably, the electrophoretic mobility shift unlikely resulted from protein aggregation and/or denaturation induced by copper phenanthroline, because the five dicysteine mutants remained monomeric and well folded after the copper phenanthroline treatment, as judged by their behavior on gel filtration chromatography (data not shown). Indeed, the changes in electrophoretic mobility could be completely reversed by incubating the protein samples subsequently with the reducing agent DTT (see below), arguing that the gel mobility shift was due to oxidative cross-linking of the paired cysteines, and the disulfide bonds in a well folded transporter were readily reduced by DTT. Moreover, the cross-linking of the five NorM-NG mutants depended on the presence of copper phenanthroline, because neither the gel mobility shift nor the loss of free cysteine thiols could be detected for the untreated protein samples even after prolonged storage (data not shown). By contrast, copper phenanthroline treatment failed to alter the electrophoretic mobility of another two dicysteine mutants L58C/V269C and A62C/F265C (Fig. 3A). Additionally, these two mutants retained the free cysteine thiols even after the exposure to copper phenanthroline (Fig. 3B). Because NorM-NG is a monomer in solution (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar, 10Radchenko M. Symersky J. Nie R. Lu M. Structural basis for the blockade of MATE multidrug efflux pumps.Nat. Commun. 2015; 67995Crossref PubMed Scopus (71) Google Scholar), our data were consistent with the five cysteine pairs: T42C/L289C, A45C/V285C, L58C/F270C, A62C/S266C, and A57C/Q284C, each forming an intramolecular disulfide bond. The formation of such disulfide bonds likely prevented complete unfolding of the protein under nonreducing and yet denaturing conditions and thus substantially altered the electrophoretic mobility of the transporter (13Brocke L. Bendahan A. Grunewald M. Kanner BI. Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.J. Biol. Chem. 2002; 277: 3985-3992Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 16Reyes N. Ginter C. Boudker O. Transport mechanism of a bacterial homologue of glutamate transporters.Nature. 2009; 462: 880-885Crossref PubMed Scopus (338) Google Scholar). Significantly, under our experimental conditions, the five detergent-purified dicysteine mutants could be converted into the faster migrating species almost completely, further attesting to the validity of our experimental design and the intracellular facing model (9Lu M. Radchenko M. Symersky J. Nie R. Guo Y. Structural insights into H+-coupled multidrug extrusion by a MATE transporter.Nat. Struct. Mol. Biol. 2013; 20: 1310-1317Crossref PubMed Scopus (78) Google Scholar). In comparison with the five shifted dicysteine mutants, copper phenanthroline had no measurable effect on the gel mobility of the wild-type or Cys-less NorM-NG or the single mutant D41A (Fig. 3A). D41A exhibited severely crippled transport function (7Lu M. Symersky J. Radchenko M. Koide A. Guo Y. Nie R. Koide S. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2099-2104Crossref PubMed Scopus (107) Google Scholar, 10Radchenko M. Symersky J. Nie R. Lu M. Structural basis for the blockade of MATE multidrug efflux pumps.Nat. Commun. 2015; 67995Crossref PubMed Scopus (71) Google Scholar) and was used as a negative control in our functional assays. Because both NorM-NG and D41A bear the four endogenous cysteines, our data implied that under our experimental conditions, copper phenanthroline did not cause the formation of intramolecular disulfide bond(s) among the endogenous cysteines or intermolecular disulfide bond(s) between different NorM-NG molecules. Nonetheless, the Cys-less NorM-NG vastly simplified the interpretation of our experimental data (e.g. the detection of free cysteine thiols; Fig. 3B) and also ruled out the possibility of forming disulfide bond(s) between the endogenous and newly introduced cysteines. Therefore, we deemed the Cys-less NorM-NG better suited for our cross-linking studies than the wild-type transporter. Unlike the five cross-linkable dicysteine mutants, the electrophoretic mobility of the corresponding monocysteine mutants was not affected by the copper phenanthroline and/or DTT treatment (Fig. 4). Additionally, the 10 monocysteine mutants retained their free cysteine thiols even after the copper phenanthroline treatment, in marked contrast to the dicysteine mutants (Fig. 3B), indicating that under our experimental conditions, copper phenanthroline catalyzed the oxidation of the paired cysteines rather than the single cysteines in NorM-NG. We further examined the impact of substrates on the cross-linking. We found that TPP, a substrate of NorM-NG (7Lu M. Syme" @default.
• W2300286959 created "2016-06-24" @default.
• W2300286959 creator A5034402015 @default.
• W2300286959 creator A5040048565 @default.
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• W2300286959 date "2016-04-01" @default.
• W2300286959 modified "2023-10-12" @default.
• W2300286959 title "Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux" @default.
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