Structural and Functional Studies of NirC from Salmonella typhimurium

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Structural and Functional Studies of NirC from Salmonella typhimurium € †, Susana Andrade†,{, Adriana Rycovska-Blume*,1, Wei Lu †,{ Klaus Fendler*, Oliver Einsle *Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Frankfurt am Main, Germany † Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Freiburg im Breisgau, Germany { BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Freiburg im Breisgau, Germany 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Production and Isolation of StNirC 2.1 Expression constructs for functional and structural studies 2.2 Overproduction of StNirC 2.3 Isolation and reconstitution of StNirC for SSM-based electrophysiology 3. Structural Analysis of StNirC 3.1 Structure determination 3.2 Properties of StNirC 4. Functional Assays for StNirC 4.1 Electrophysiological analysis in a PLB 4.2 SSM-based electrophysiology 4.3 H+ transport of StNirC in native membranes 5. Conclusions Acknowledgments References

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Abstract NirC is a pentameric transport system for monovalent anions that is expressed in the context of assimilatory nitrite reductase NirBD in a wide variety of enterobacterial species. A NirC pentamer contains individual pores in each protomer that mediate the passage of at least the nitrite ðNO2
Þ and nitrate ðNO3
Þ anions. As a member of the formate/nitrite transporter family of membrane transport proteins, NirC shares a range of structural and functional features with the formate channel FocA and the hydrosulfide channel AsrD (HSC). NirC from the enteropathogen Salmonella typhimurium has been studied by X-ray crystallography, proton uptake assays, and different electrophysiological techniques, and the picture that has emerged shows a fast and

Methods in Enzymology ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2014.12.034

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versatile transport system for nitrite that doubles as a defense system during the enteric life of the bacterium. Structural and functional assays are described, which shed light on the transport mechanism of this important molecular machine.

ABBREVIATIONS BTS 1,3-bis-[tris(hydroxymethyl)methylamino]-propane DDM n-dodecyl-β-D-maltoside His-tag polyhistidine tag IPTG isopropyl-β-D-thiogalactoside LB Luria–Bertani growth media Ni-NTA nickel-nitrilotriacetic acid PLB planar lipid bilayer S. typhimurium “Salmonella typhimurium,” precisely Salmonella enterica serovar typhimurium SDS–PAGE sodium dodecyl sulfate–polyacrylamide electrophoresis SSM solid-supported membrane TEV Tobacco Etch Virus

1. INTRODUCTION The nitrite anion, NO2
, is a central intermediate in the biogeochemical cycle of nitrogen (Einsle & Kroneck, 2004). Bacteria utilize various redox conversions of nitrite for energy conservation, including the oxidation to nitrate in nitrification, the reduction to nitric oxide in denitrification, and the reduction to ammonium in ammonification. Alternatively, nitrite can serve as a substrate for the assimilation of nitrogen into biomolecules, a process that invariably also starts from the most reduced form of the element, the ammonium cation, NH4 +. The six-electron reduction of nitrite to ammonium proceeds according to NO2
+ 6e
+ 8H + ! NH4 + + 2H2 O: It exists as an assimilatory and a dissimilatory variant, and in both cases, the reaction is catalyzed by fundamentally different biocatalysts. While dissimilatory nitrite reductase (Nrf ) is a pentaheme cytochrome c enzyme that couples nitrite reduction directly or indirectly to the oxidation of menaquinol in the membrane and the generation of a proton motive force (Einsle, 2011; Einsle et al., 1999), the assimilatory variant uses a siroheme cofactor. Assimilatory nitrite reductase is a NirBD heterodimer encoded in the enterobacterium Salmonella enterica serovar typhimurium in a

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nirBDCcysG gene cluster together with accessory genes for the synthesis of siroheme from uroporphyrinogen III and the open reading frame nirC (Peakman et al., 1990). The NirC gene product was later identified to be a specific transport system for nitrite (Clegg, Yu, Griffiths, & Cole, 2002; Jia, Tovell, Clegg, Trimmer, & Cole, 2009). NirC is widely distributed among bacterial species. A special situation exists in enterobacteria where the utility of NirC originates from a positive side effect in the defense against the host’s immune systems (Das, Lahiri, Lahiri, & Chakravortty, 2009). NirC is an integral membrane protein belonging to the formate/nitrite transporter (FNT) family of integral membrane proteins (Saier et al., 1999). The family further comprises the formate channel FocA (Delomenie et al., 2007; Suppmann & Sawers, 1994), the formate uptake permease FdhC (White & Ferry, 1992), and a hydrosulfide channel termed HSC or AsrD (Czyzewski & Wang, 2012). They have been characterized as channels for monovalent anions with a remarkably broad-substrate spectrum (L€ u et al., 2012, 2013), and they can function both in export or import of their cargo. Structures of FocA revealed a topological homology to the tetrameric aqua- and glyceroporins, in spite of the absence of any detectable sequence homology (L€ u et al., 2011; Waight, Love, & Wang, 2010; Wang et al., 2009). Although members of this permease family are termed “channels,” their coupling to the proton motive force is by no means clear. Based on its physiological function, the related HSC hydrosulfite channel, for example, has been proposed to function as a proton/hydrosulfide exchanger. Also, the coupling of the NirC nitrite transport to the proton motive force is still controversially discussed (L€ u et al., 2012; Rycovska, Hatahet, Fendler, & Michel, 2012). It seems likely that these transporters optionally work as channels or high turnover proton-coupled transport systems.

2. PRODUCTION AND ISOLATION OF StNirC As an integral membrane protein from a bacterial origin, NirC from S. typhimurium (StNirC) was obtained through recombinant production in Escherichia coli as a heterologous host. Membranes were isolated, and the protein was solubilized from the lipid bilayer using n-dodecyl-β-D-maltoside for solid-supported membrane (SSM) electrophysiology (Rycovska et al., 2012) and n-octyl-β-D-glucopyranoside for structure determination and planar lipid bilayer (PLB) electrophysiology (L€ u et al., 2012). The difference in individual protocols is due to the fact that the experiments described in this

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chapter originate from the independent efforts from two groups, and relevant variations are pointed out in the text.

2.1 Expression constructs for functional and structural studies StNirC was cloned from genomic DNA and inserted either into the T7-based pET21a vector (Novagen), adding a C-terminal hexahistidine affinity tag, or a pTTQ18-derived, tac promoter-based vector (Surade et al., 2006), with an N-terminal hexahistidine tag with a cleavage site for Tobacco Etch Virus (TEV) protease and a C-terminal StrepTag(II). Although the presence of a polyhistidine tag (His-tag) at the N-terminus could have a detrimental effect on production yields of integral membrane proteins with a periplasmic location of the N-terminus, the presence of a His-tag at the N- or C-terminus of StNirC led to similar production levels affirming cytoplasmic location of both ends (Rycovska et al., 2012). As a representative protocol, the procedures used to produce StNirC for SSM-based electrophysiological studies are detailed in the following (Rycovska et al., 2012). For structure solution and PLB electrophysiology, a slightly different route was taken, as described elsewhere (L€ u, Schwarzer, et al., 2012), but key steps were retained with the most notable differences found in the expression constructs described above.

2.2 Overproduction of StNirC For acridine orange uptake assays and SSM electrophysiology, StNirC was produced along the following steps: 1. Transform E. coli BL21(DE3) with 1–2 μL of plasmid DNA, use 200 μL of SOC-recovery mixture to inoculate 150 mL of modified Luria–Bertani medium (LBGlc) (0.5% (w/v) yeast extract, 1% (w/v) tryptone, 1% (w/v) NaCl) supplemented with 0.5% (w/v) glucose and the appropriate antibiotic, and incubate overnight at 37 °C under agitation. Transformation was carried out by electroporation (Hengen, 1995). 2. Early on the following day, use the overnight preculture to inoculate 12 L of prewarmed LBGlc in six 5-L baffled Erlenmeyer flasks with 25 mL of inoculum. Incubate the culture at 37 °C for 2.5 h under 120 rpm agitation. 3. Induce the protein production at an A600 of 0.4–0.6 by addition of 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) and incubate under

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agitation at 37 °C for 30 min, followed by slow cooling to 25 °C over the following 3 h. Harvest the cells by centrifugation at 4800  g, for 20 min at 4 °C. Transfer cell sediment into a fresh Falcon tube with a spatula and freeze in liquid nitrogen for storage at
80 °C until use. To prepare total membranes, thaw the cell pellet on ice and resuspended in 300 mL of breaking buffer (50 mM Tris/HCl, 150 mM NaCl, 5% (v/v) glycerol, 10 mM EDTA, adjusted with HCl to pH 7.4), add one tablet of “complete” protease inhibitor cocktail (Roche). Disrupt cells at 13,000 psi of pressure, using an M-110L Microfluidizer (Microfluidics). Remove unbroken cells by centrifugation (15,000  g, 20 min, 4 °C) and transfer the supernatant into clean, high-speed centrifugation tubes. Sediment membrane fragments by ultracentrifugation (140,000  g, 1.5 h, 4 °C). Transfer membrane sediment into a glass potter homogenizer, add 40 mL of storage buffer (50 mM Tris/HCl, 250 mM NaCl, 15% (v/v) glycerol, adjusted with HCl to pH 7.4), and homogenize the membrane fragments. Determine the total protein content, adjust the final protein concentration to 20 mg mL
1 with storage buffer, dispense membranes into 5–10 mL aliquots, freeze in liquid nitrogen, and store at
80 °C until use.

2.3 Isolation and reconstitution of StNirC for SSM-based electrophysiology StNirC was isolated from the membranes by a three-step procedure, starting with affinity chromatography on nickel-nitrilotriacetic acid (Ni-NTA) agarose. In the second step, we removed the His-tag by TEV protease cleavage and subsequently, the free His-tag oligopeptide by a second Ni-NTA affinity purification. To remove the TEV protease, Strep-tag affinity chromatography was used as a third step. A typical purification from 5 L of culture yielded approximately 0.75 mg of StNirC devoid of the N-terminal hexahistidine tag. 1. Thaw a membrane aliquot (as prepared in Section 2.2, Steps 5–10) on ice, dilute the twofold with SNi binding buffer (50 mM Tris/HCl, pH 8, 250 mM NaCl, 6% (v/v) glycerol), add dropwise n-dodecyl-β-Dmaltoside (DDM;Glycon Biochemicals) to a final concentration of

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1.2% (w/v), and incubate for 1 h at 4 °C with slow inverting. Separate insoluble membrane material by ultracentrifugation (180,000  g, 30 min, 4 °C). To prepare the detergent stock, weigh the required amount of detergent and dissolve it in small amount (2 mL) of SNi binding buffer. To bind the target proteins, mix the supernatant with the equilibrated Ni-NTA agarose beads (Qiagen, 0.5 mL per 200 mg of total protein) and incubate for 1 h at 4 °C. Quantitatively load the mixture on a gravity column, wash with 20 column volumes (CV) of WNi buffer (50 mM Tris/HCl, pH 7.4, 0.4 M NaCl, 5% (v/v) glycerol, 25 mM imidazole, and 0.02% (w/v) DDM), and elute the protein with 5–10 CV of elution buffer (20 mM Tris/HCl, pH 7.4, 350 mM NaCl, 200 mM imidazole, pH 8, and 0.02% (w/v) DDM). To remove the His-tag, add ProTEV (TEV; Promega) protease to the eluted protein (10 U mL
1 of eluted protein) and dialyze against D buffer (20 mM Tris/HCl, pH 8, 350 mM NaCl, 0.02% (w/v) DDM) overnight at 4 °C. To remove the free His-tag oligopeptide and the contaminating proteins nonspecifically binding to Ni-NTA beads, mix the protein cleavage mixture with the equilibrated Ni-NTA agarose beads (0.5 mL per 200 mg of total proteins) and incubate for 1 h at 4 °C. Quantitatively load the mixture on a gravity column and collect the flow-through. To remove free TEV protease, mix the flow-through from Step 4 with 0.5 mL of equilibrated Streptactin Sepharose beads (IBA) and incubate under gentle agitation for 30 min at 4 °C. Quantitatively load the mixture on a gravity column, wash with 20 CV of WStre buffer (20 mM Tris/ HCl, pH 8, 350 mM NaCl, 0.02% (w/v) DDM) and elute with 5 CV of WStre buffer supplemented with 15 mM D-desthiobiotine (Sigma). Analyze all fractions on SDS–PAGE gels by Western blotting and the final fraction by size-exclusion chromatography and determine the total protein content.

3. STRUCTURAL ANALYSIS OF StNirC NirC is a member of the FNT family of integral membrane proteins, and for another member of this family, the formate channel FocA, threedimensional structures were available for the paralogs from E. coli (Wang et al., 2009), Vibrio cholerae (Waight et al., 2010), and S. typhimurium (L€ u et al., 2011). In spite of a clear overall homology, however, FocA channels

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exhibit a complex, pH-dependent change in transport mode and direction that indicates additional levels of regulation that do not seem to be relevant for NirC (L€ u et al., 2013). Also, FocA proteins show a broad range of transported cargo molecules that span the entire range of organic acids generated in the metabolic pathway where the channel plays a role, the anaerobic mixed-acid fermentation of glucose (L€ u, Du, et al., 2012). This functionality is at least in part related to conformational changes of the extramembranous N-terminal helix of FocA (L€ u et al., 2011), and possibly also to a conformationally flexible loop region located directly at a constriction of the transmembrane pore (Waight et al., 2010). The function of NirC did not indicate a requirement for such features, and a major goal of the structure determination was to analyze how the corresponding regions of the structure are constructed.

3.1 Structure determination Recombinant production of StNirC in E. coli as a heterologous host yielded sufficient amounts of pure protein to initiate large-scale crystallization trials supported by automated liquid handling systems and nanoliter dropsetting robots. The construct used for crystallization differed from the full-length protein by a truncation of the 17 C-terminal residues. The protein was isolated by Ni-affinity chromatography, followed by an additional step of sizeexclusion chromatography on Sepharose S200 to obtain a monodisperse preparation. Here, all steps were carried out using A¨KTA prime plus or ¨ KTA pure chromatography systems (GE Healthcare). The affinity tag A was not cleaved off for crystallization. 3.1.1 Crystallization of StNirC Solubilized NirC was crystallized by the sitting drop vapor diffusion method at a concentration of 8 mg mL
1, using drops of 2 μL of protein solution mixed with 2 μL of a reservoir buffer containing 27% (w/v) of polyethylene glycol 100 and 0.1 M of sodium citrate buffer at a pH of 4.5 with the addition of 0.4 μL of the detergent cyclohexylbutanoyl-N-hydroxyethylglucamide (C-HEGA 10) directly to the crystallization drop as an additive. The final pH of the drop was 5.0, and large single crystals appeared within 2–3 days at 4 °C. 3.1.2 Diffraction data collection and processing Crystals of StNirC were dried out for several minutes in the crystallization drop by opening up the sealed crystallization compartment, until the

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concentration increase due to vapor evaporation made the resulting, concentrated buffer a suitable cryoprotectant. Diffraction data were collected at beam line X06SA at the Swiss Light Source (Paul-Scherrer-Institute, Villigen, CH) using a Pilatus 6M direct photon counting detector (Dectris). Data were indexed, integrated, and scaled using XDS (Kabsch, 2010) and XPREP (Bruker). 3.1.3 Structure solution and refinement Due to the availability of three-dimensional structures for another subfamily of the FNR family of anion channels, the formate channel/transporter FocA (L€ u et al., 2011; Waight et al., 2010; Wang et al., 2009), the crystallographic phase problem for NirC was solved by molecular replacement, using the structural model for S. typhimurium FocA (PDB ID 3Q7K) as a search model for MOLREP (Vagin & Teplyakov, 2010). Model building and insertion of the correct amino acid sequence was carried out in COOT (Emsley, Lohkamp, Scott, & Cowtan, 2010), and the resulting model was refined to a resolution of 2.4 A˚ by maximum-likelihood positional refinement with REFMAC5 (Murshudov et al., 2011). The final model for StNirC was deposited with the Protein Data Bank at http://www.pdb.org with the Accession No. 4FC4.

3.2 Properties of StNirC The tertiary structure of NirC is similar to the one of FocA (Fig. 1), but even more so to the hydrosulfide channel AsrD (HSC) (Czyzewski & Wang, 2012). The topology that FNT family proteins generally share with aquaand glyceroporins is fully retained, in particular, with respect to the interruptions in helices 2 and 5 that generate extended loops with a central role in forming the selectivity pore (L€ u et al., 2013). Contrary to the FocA structures, where the N-terminal helix of the peptide displayed significant structural flexibility related to a pH-dependent gating mechanism, this secondary structure element was found to tightly adhere to the NirC monomer, stabilized through a series of hydrogen bonds and salt bridge interactions (L€ u et al., 2011; L€ u, Schwarzer, et al., 2012). As in FocA, the selectivity filter in the center of the pore of each protomer consists of two rings of hydrophobic residues that form the boundaries of a central, hydrophobic vestibule. The proposed mechanism of transport thus includes a transient protonation of the anionic cargo molecule when approaching either the outer or inner constriction. Only in the uncharged, protonated form, the cargo can then enter and pass the central vestibule, to be deprotonated after passing the

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Figure 1 Three-dimensional structure of StNirC. (A) Top view of the NirC pentamer from the extracellular side. Each protomer contains a conducting channel, while the central pore of the assembly is likely closed by two to three embedded phospholipid molecules of the outer leaflet. (B) Side view of the pentamer (cytoplasmic side down), with one protomer colored from blue (different gray shades in the print version) at the N-terminus to red (dark gray in the print version) at the C-terminus. (C) Stereo representation of the NirC protomer with the conduction pore shown as a dotted surface. The selectivity filter at the center of the pore is largely constructed by the loop regions interrupting the broken transmembrane helices 2 (light green (light gray in the print version)) and 5 (orange (gray in the print version)).

second hydrophobic constriction. This model accommodates two distinct functional modes of transport that have previously been described for FNT proteins (Clegg et al., 2002; Suppmann & Sawers, 1994). When functioning as a passive channel, a proton is retained around the selectivity filter, passing along an isolated chain of hydrogen bonds connecting residues T81 and H197 and a single, coordinated water molecule within the membrane. The proton can thus accompany a cargo anion through the pore and then return to its starting position via this chain of H-bonds (L€ u et al., 2013; L€ u, Schwarzer, et al., 2012). Ion translocation is dependent on the presence

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of the proton that, if lost, must be replenished from either side of the membrane. Alternatively, a major difference in proton concentration on either side of the membrane can also lead to a systematic cotransport of the cargo anion with the proton. At a 1:1 stoichiometry, this process would be electroneutral, but it would constitute a secondary active transport, with anion translocation driven by the proton gradient. Alternative passive and active translocation of anions was postulated earlier for FocA proteins, where the change of transport mode corresponds to a metabolic switch in formate utilization from extracytoplasmatic oxidation to CO2 at the periplasmic formate dehydrogenase to cytoplasmic disproportionation to H2 and CO2 by the formate:hydrogen lyase complex (Sawers, 2005). For NirC, this complex dual functionality was not described earlier ( Jia et al., 2009), but no functional studies on isolated protein had been carried out.

4. FUNCTIONAL ASSAYS FOR StNirC The actual mechanism of FNT proteins directly relates to the question what methods are suitable for its analysis. While fast-conducting channels can ideally be studied by direct current measurements in reconstituted systems, label-free functional assays for secondary active transporters and uniporters are notoriously difficult. They not only require a quantitative assessment of substrate transport but also the establishment of a “driving” electrochemical gradient. This is only possible in a compartmentalized system, such as a cell, membrane vesicle, proteoliposome, or a PLB. In addition, the turnover of a transporter is typically five to six orders of magnitude lower than that of ion channels, making the detection of transport a challenge and electrophysiological assessment of single transporter currents impossible. An additional complication arises for electrophysiological techniques, because apart from a few rare exceptions, bacterial transporters cannot be investigated using microelectrode voltage clamp or patch clamp methods due to the small size of bacteria and because bacterial transporters are often difficult to produce in mammalian cells. In the following, we describe three techniques that were successfully applied for the functional analysis of StNirC: (1) Electrophysiological recording of StNirC integrated into PLBs. (2) Electrophysiological recording of StNirC in proteoliposomes adsorbed to a SSM. (3) Recording of pH in inverted membrane vesicles using a pH-sensitive dye.

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4.1 Electrophysiological analysis in a PLB PLBs provide a highly versatile system for the analysis of ion currents through integral membrane proteins under maximal experimental control. The lipid bilayer is painted across a small bore separating two reaction chambers commonly termed the cis and trans chambers. The formation of a bilayer can be monitored by following the current profile when a small voltage is applied from cis to trans, and the quality of the bilayer is then reflected by its capacitance. In the setup with a vertical bilayer, both the cis and trans chambers are directly accessible, and even after formation of a bilayer, careful buffer exchange is possible. The downside of the method is the relatively small area of the planar bilayer that only allows for the inclusion of a limited number of protein molecules. The method is consequently best suited for rapidly translocating systems that commonly are passive channels (Knol et al., 1996). In all studies with StNirC reconstituted into PLBs, a full-length construct of the protein was used. 4.1.1 Preparation of proteoliposomes and reconstitution into planar bilayers For all assays, liposomes were generated from E. coli polar lipid extract (Avanti) following published procedures (Knol et al., 1996). 1. Wash lipids and L-α-phosphatidylcholine (Avanti; 25 mg mL
1) twice with 100% ethanol and resuspend dried lipid layer in 20 mM Tris/HCl buffer at pH 8.8, containing 450 mM NaCl at a volume ratio of 3:1. 2. Homogenize the mixture by sonication (5 min), then extrude several times through a membrane with a pore size of 200 nm until the lipid mixture clears up. Dilute the resulting liposomes to 4 mg mL
1 with same buffer. 3. Destabilize liposomes by addition of 48 mM (final concentration) of dodecyl-β-D-maltopyranoside (DDM). Add full-length StNirC in 20 mM Tris/HCl buffer at pH 8.0, 450 mM NaCl buffer solubilized in DDM at a protein:lipid ratio of 1:50 (w/w). To investigate singlechannel currents, prepare proteoliposomes at a protein:lipid ratio of 1:5. 4. Remove DDM by adding 0.2 g mL
1 of Biobeads SM-2 (Bio-Rad) for 2 h at room temperature. 5. Collect proteoliposomes by centrifugation at 100,000  g for 40 min, resuspend in the same buffer to a final concentration of 4 mg mL
1, and freeze in liquid nitrogen until further use.

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6. To fuse the proteoliposomes to a PLB, add 2 μL of the proteoliposome solution close to the bilayer to the cis chamber of Planar Lipid Bilayer Workstation (Warner/Harvard). 7. Clamp the system at a holding potential of 100 mV and confirm the insertion of protein by the appearance of single-channel events. 4.1.2 Measurement of macroscopic currents Electrophysiological recordings were carried out on a Planar Lipid Bilayer Workstation (Harvard Apparatus) with an Axon Digidata 1440A digitizer (Molecular Devices) and a Bilayer Clamp BC-535 amplifier (Warner Instruments). The software pClamp 10 (Molecular Devices) was used for signal processing. 1. To show the substrate dependence, both the cis and trans chambers contain 10 mM histidine and 20 mM sodium nitrite at a final pH of 7.9, and then a gradient for nitrite is created by addition of 30.4 μL of 8 M sodium nitrite to the cis side, yielding 100 mM of nitrite in cis and 20 mM in trans chamber. 2. To record macroscopic currents from a larger number of individual StNirC channels, apply holding potentials ranging from
70 to +70 mV in steps of 10 mV. 3. To vary pH, add 0.7N HCl stepwise to the trans side and derive the resulting pH value from a calibration curve determined separately. 4.1.3 Single-channel recordings Single-channel events could be recorded when only one or two pentamers of NirC inserted into the bilayer. All recordings were made in the same buffer with a holding potential of 100 mV or 150 mV. Variations in nitrite concentrations were symmetric, meaning that equivalent amounts of sodium nitrite were added to both the cis and trans chambers. 4.1.4 Conclusions The reconstitution of NirC into PLBs was straightforward and yielded macroscopic currents that reversed at the potentials predicted by Nernst’s equation (Fig. 2A). In line with the findings from the three-dimensional structure, where the N-terminal helices of each protomer were adhering tightly to the protein rather than showing significant flexibility (L€ u, Schwarzer, et al., 2012), macroscopic currents for NirC did not show a pH-dependent gating behavior (Fig. 2B). Contrary to the complex dual role of FocA as an export channel and import transporter (L€ u et al., 2013), NirC

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Figure 2 Electrophysiological characterization of StNirC in a PLB. Solubilized protein was reconstituted in a planar bilayer of 200 μm diameter. (A) Current–voltage diagram of macroscopic current measurements at two different nitrite gradients. The observed reversal potentials match the expectations from Nernst's equation. (B) Voltage-clamp studies of NirC at pH 7.9 (red) (gray in the print version) and pH 4.0 (black) show that StNirC is not gated in a pH-dependent manner. However, the curves show a shift in reversal potential. (C) The channel exhibits bursts of fast gating, as well as a slower gating process. In the transport model, the fast process could correspond to functional opening and closing of the constrictions in the substrate channel, while a longer closed phase is entered upon loss of a proton that is required to transiently neutralize the anionic cargo.

is thought to act as a bidirectional permease for monovalent anions at all times, so that a regulation by (external) pH is not required. However, currents measured at different pH values lead to a characteristic shift in reversal potential, indicating that protons are involved in NirC function. This was assembled into a functional model, wherein a proton resides within the membrane and helps the anionic cargo to cross a hydrophobic barrier through transient protonation (L€ u et al., 2013). Also, this finding is relevant in light of the results from SSM electrophysiology and fluorescence dequenching (see Sections 4.2 and 4.3). Using lower protein:lipid ratios during the formation of proteoliposomes, planar lipid membranes with only one or two NirC pentamers

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could be obtained that were suitable for single-channel recordings (Fig. 2C). Here, the observed conductance for single channels was in the low pA range, providing evidence that the protein acted as a fast ion channel. Gating of currents was observed on two levels, with bursts of fast gating, interrupted by slower gating events on a longer timescale (Fig. 2C). This was interpreted within the existing mechanistic model in that the fast gating reflects the transient opening events of the selectivity filter that must be short in order to avoid the passage of water or the uncoupling of existing ion gradients. In contrast, the slow gating was suggested to represent the loss of a proton to either side of the membrane that then needs to be replenished before anions can again pass the protein (L€ u et al., 2013; L€ u, Schwarzer, et al., 2012).

4.2 SSM-based electrophysiology In SSM-based electrophysiology, proteoliposomes are adsorbed to an SSM (Fig. 3A) and activated using a rapid concentration jump of a transported species. The resulting charge translocation is measured by capacitive coupling via the proteoliposome/SSM contact area. The adsorption of multiple proteoliposomes allows a large number of transporters to be immobilized on the electrode in a simple, spontaneous process. Therefore, also low-turnover systems such as transporters generate large electrical currents (0.5–10 nA) and can be readily investigated (Schulz, Garcia-Celma, & Fendler, 2008). 4.2.1 Setup of the SSM and recording of transient currents A detailed description of the technique and the experimental setup, including the gold electrode, the SSM cuvette, and the fluid handling is given in Bazzone et al. (2013) and Schulz et al. (2008). 1. The SSM is formed on a microstructured gold electrode of 1 mm diameter on a glass sensor chip. On the gold surface, an octadecanethiol monolayer is chemisorbed. Then a solution of diphytanoylphosphatidylcholine solution in n-decane is added and the sensor chip is mounted in an acrylic glass cuvette, allowing for rapid solution exchange. Rinsing with aqueous solution then forms an SSM, with a specific conductance and capacitance typical for a free-standing lipid bilayer. 2. The proteoliposome suspension (30 μL) is added to the SSM via the fluid outlet of the cuvette. During a waiting time of approximately 1 h, the proteoliposomes spontaneously adsorb to the SSM.

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Figure 3 Charge transport by StNirC investigated by SSM-based electrophysiology. (A) Schematic representation of a proteoliposome containing StNirC adsorbed to the planar lipid surface of the SSM. A KNO2 concentration jump generates an inflow of negatively charged NO2
ions. Coupling to H+ is still controversial. (B) Transient currents using StNirC proteoliposomes after a NO2
concentration jump at different NO2
concentrations (in mM, see numbers in the figure). Thin red line (grey in print version) is the reconstructed transporter current. (C) NO2
concentration dependence of the peak currents. Square symbols (black line is a guide to the eye): averaged peak currents (n ¼ 3, error bars indicate SD) obtained at different NO2
concentrations using proteoliposomes (■) and control liposomes (¼no StNirC, □). Red (gray in the print version) bullets (red (gray in the print version) line is a hyperbolic fit to the data): peak currents generated by StNirC (proteoliposomes—empty liposomes). Adapted from Rycovska et al. (2012) with permission from Elsevier.

3. The gold electrode is connected to a Keithly 427 current amplifier (gain 109–1010 V A
1, low-pass filter rise time 10 ms) and a reference Ag/AgCl electrode in the outlet pathway of the cuvette. Solution exchange in the cuvette is effected via a fluid handling system consisting of computer-controlled electromagnetic valves and pressurized solution containers. The entire system is mounted in a Faraday cage.

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4. Electrophysiological measurements were performed using rapid substrate addition ðNO2
Þ. The solution exchange protocol consisted of three phases (nonactivating–activating–nonactivating solution) of 0.5 s duration. The activating solution contained substrate (KNO2) at a defined concentration, while the nonactivating solution contained nontransported KCl only. Total salt concentration was kept constant by adding the appropriate amount of KCl. The activating solution contained x mM KNO2 plus 100–x mM KCl, the nonactivating solution 100 mM KCl, in both cases in 100 mM KPi (NaOH) buffer at pH 7.0. Approximately 0.55 s after the start of the solution exchange protocol, the activating solution reached the SSM with immobilized StNirC proteoliposomes, generating a negative transient current.

4.2.2 Electrophysiological characterization of purified StNirC in proteoliposomes 1. Negative transient currents were observed after a NO2
concentration jump (Fig. 3B), in agreement with NO2
transport into the proteoliposomes. The currents were clearly dependent on the NO2
concentrations (Fig. 3C). 2. The transient currents decayed with a rapid phase (time constant ca. 10 ms) and a slow phase (ca. 50 ms). Here, the rapid phase corresponded to a fast charge displacement after solution exchange and is most probably an artifact generated by the interaction of the anion NO2
with the SSM. Anions interact strongly with lipid head groups, generating transient currents on the SSM (Garcia-Celma, Hatahet, Kunz, & Fendler, 2007). Indeed, artifact currents detected in control measurements using protein-free control liposomes amounted to 30% of the total signal at 100 mM KNO2 (Fig. 3C). The slow decay phase was assigned to charging of the proteoliposomes due to the stationary transport activity of the transporter (Schulz et al., 2008), as proposed previously (Ganea, Pourcher, Leblanc, & Fendler, 2001). 3. Using the electrical properties of the SSM, the original transport current could be reconstructed from the measured transient current (Mager, Rimon, Padan, & Fendler, 2011). The resulting, reconstructed current is shown for the highest concentration (100 mM) of substrate anions. A transient current followed by a phase of stationary current is obtained. The stationary current represents the continuous transport activity of StNirC.

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4. Figure 3C shows the substrate dependency of NirC transport. To correct for artifacts, the currents were determined with StNirC proteoliposomes (signal, filled squares) and with proteoliposomes reconstituted with StPutP (control, open squares). The corrected peak currents, signal minus control, are shown in red (gray in the print version) and were evaluated using a hyperbolic function. The half-saturation concentration Km determined for NO2
was 2.9 mM. In fact, the corrected current at 100 mM substrate was approximately of the size of the stationary current determined by the reconstruction procedure (Fig. 3B). Therefore, we conclude that the transient phases of the reconstructed currents are most probably artifact currents, while the stationary currents represent the activity of StNirC. 4.2.3 Interpretation The SSM measurements demonstrate that application of a NO2
gradient to StNirC proteoliposomes generates the displacement of negative charges into the liposomes. Interestingly, NO2
as well as NO3
are transported, although NO3
has a reduced turnover (ca. 50% of NO2
) or a different stoichiometry and a fourfold lower substrate affinity as for NO2
(Rycovska et al., 2012). These data are consistent with the assumption that StNirC acts as a NO2
and NO3
channel or an uniporter, in line with the finding that other FNT proteins show a relatively broad spectrum of cargo molecules (L€ u, Du, et al., 2012). They may, however, also represent NO2
=H + antiport activity (or even symport with a nNO2
=H + (n > 1) stoichiometry). Comparing the size of the signals, 0.3 nA for the corrected currents, with other transporters measured with the same technique, a turnover of 10–100 s
1 was estimated, placing NirC transport rates in the range of transporters (Rycovska et al., 2012) although channel function cannot be excluded on the basis of our SSM experiments.

4.3 H+ transport of StNirC in native membranes The electrophysiological characterization of membrane proteins translocating charged substrates provides an indispensable wealth of information on the kinetics of transport and mechanistic details. However, potentiometric measurements could not resolve stoichiometry of transported ions. Demand to determine the stoichiometry of transported ions might be overcome by employing a second method. A number of methods are available to directly or indirectly measure the translocation of ions, for example, by

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employing fluorescent or radioactive probes. To examine whether StNirC mediates H+-coupled NO2
transport, we prepared inside-out membrane vesicles and determined the H+ transport by acridin orange assay. 4.3.1 Preparation of inside-out membrane vesicles The inside-out (everted) membrane vesicles were prepared according to Rosen (1986). 1. Overproduce StNirC as described in Section 2.2, Steps 1–3. Induce the protein production by the addition of 0.5 mM IPTG and incubate at 37 °C for 2–3 h. The overproduction of membrane proteins frequently results in cytoplasmic accumulation of inactive aggregated proteins, which in the case of StNirC could amount to up to 55% of its total production at 37 °C. Since aggregated protein does not affect functional assays based on membrane vesicles, this was not taken as a disadvantage in the present procedure. 2. Harvest the cells by centrifugation 4800  g for 20 min at 4 °C. 3. Wash the cell pellet once with ice-cold deionized water and then with ice-cold TCS buffer (10 mM Tris/Cl, pH 7.4, 0.14 M choline chloride, 0.25 M sucrose, 5 mM MgCl2). 4. Suspend the cell pellet in a minimal volume (30–35 mL) of TCS buffer supplemented with 0.1 U mL
1 of benzoase (Novagen). Keep on ice. 5. Break cells by a single passage through a French pressure cell (Aminco) at 10,000 psi. 6. Separate cell debris by centrifugation (5000  g, 10 min, 4 °C) and transfer the supernatant into clean high-speed centrifugation tubes. To increase the yield of inside-out vesicles, unbroken cells could be resuspended in fresh TCS buffer with benzoase and passed through the French pressure cell one more time. 7. Sediment the inside-out vesicles out of supernatant by centrifugation (100,000  g, 1 h, 4 °C). 8. Suspend membrane vesicle pellet in TCS buffer using a glass potter homogenizer. Determine the total protein concentration and dispense membrane vesicles into aliquots (0.5–1 mL). Freeze aliquots in liquid nitrogen and store at
80 °C. 4.3.2 NO22/H+ antiport fluorescent assay Antiport activity was determined using the method described by Rosen (1986). The assay is based on monitoring the changes in ΔpH that are induced by activation of proton pumping of the respiratory chain or by

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the H+ antiport activity of transporters (Fig. 4A). The fluorescence of the pH-sensitive, lipophilic dye acridine orange is quenched by acidification and dequenched by alkalization of the environment. 1. Prepare fresh BTS-CS buffer (10 mM 1,3-bis-[tris(hydroxymethyl) methylamino]-propane (BTS), pH 6–9, 0.14 M choline chloride, 0.25 M sucrose, 5 mM MgCl2) supplemented with 2.5 μM acridine orange (Eastman Organic Chemicals), 1 M sodium D/L-lactate (Fluka), and 1 M KNO2. Thaw one aliquot of inside-out membrane vesicles on ice. Set the fluorescence spectrophotometer to time-scan, with an

H+

B

H+

Lactate +

H

H+

Pyruvate +

H

H+ H+

+

H

Lactate

1800 1500

NO280 mM 70 mM 60 mM 50 mM 40 mM 30 mM 20 mM 10 mM 5 mM

0% Quenching

1200 900 600

Dequenching

NO2−

Fluorescence (a.u.)

A

100% Quenching

300 0

0

100

200

300

400

Time (s)

C

100 80 1/Dequenching (100x)

% Dequenching

120

60 40 20 0

0

2.6 1.8

0.8

0.00

0.06 0.10 1/KNO2 (mM −1)

10 20 30 40 50 60 70 80 90

KNO2 (mM)


Figure 4 StNirC shows NO2 =H antiport activity in inside-out membrane vesicles. (A) Schematic presentation of inside-out membrane vesicle containing overproduced target protein and other cytoplasm-facing E. coli membrane proteins oriented towards the outside of the membrane vesicles. (B) Changes in ΔpH were monitored by acridine orange, a ΔpH sensitive fluorescent dye. The respiratory reaction connected to proton pumping into the vesicles was initiated by addition of 10 mM Na-DL-lactate (full arrow). Acidification of vesicular lumen was monitored by the fluorescence quenching until a steady-state level of ΔpH (100% quenching) was reached. The H+ antiport activity (fluorescence dequenching) was assayed by addition (open arrow) of 5–70 mM KNO2. (C) The NO2
=H + antiport (% dequenching) as a function of NO2
concentration fitted to the Hill equation and plotted in a Lineweaver–Burk plot. Adapted from Rycovska et al. (2012) with permission from Elsevier. +

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2.

3.

4.

5.

Adriana Rycovska-Blume et al.

excitation wavelength of 430 nm, an emission wavelength of 530 nm, 10-nm slit width, and a time range of 400 s. Pipet BTS-CS buffer with acridine orange (1.5 mL final reaction volume) into a cuvette, add inside-out vesicles (150–180 mg of total protein), and record fluorescence for at least 30 s. The initial fluorescence is taken as 0% quenching (Fig. 4B). While recording, add 10 mM of sodium D/L-lactate to activate proton pumping by the respiratory chain and monitor fluorescence quenching until a steady state is achieved (100% quenching, Fig. 4B). The time required for this may vary with the quality of membrane vesicle preparation and the pH of reaction. Add 5–80 mM KNO2 and record the increase of fluorescence (dequenching) until a new steady state is obtained (x% dequenching). Figure 4B shows the substrate dependency. To test for an establishing proton gradient or the effect of membrane potential on protein activity, add 5 μM valinomycin or 0.1 μM of nigericin. Plot the percentage of dequenching against the KNO2 concentrations (Fig. 4C) and determine apparent Km and vmax from a hyperbolic or sigmoidal fit. The concentrations of ions producing half-maximal dequenching are a good approximation of the apparent Km values (Schuldiner & Fishkes, 1978).

5. CONCLUSIONS The different functional assays described above yield a somewhat contradictory picture for StNirC function. It seems clear that transport is electrogenic and associated with the displacement of negative charge over the membrane, which argues for the NO2
anion as transported species. StNirC could, therefore, be a NO2
uniporter or channel. On the other hand, experimental evidence strongly suggests coupling of nitrite transport to the proton motive force. In which way this coupling is realized is still a matter of discussion (L€ u, Schwarzer, et al., 2012; Rycovska et al., 2012), but the existing mechanistic model suggests the direct involvement of a proton within the membrane as a means to neutralize the charge of the anion before crossing a hydrophobic barrier (L€ u et al., 2013). This mechanism readily accommodates various possibilities for sym- and antiport of protons and anions, but it does not imply that in the physiological context of the intact cell all these possibilities will in fact be realized. Functional characterization

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as described above in combination with structural information has good chances of solving this puzzle in the near future.

ACKNOWLEDGMENTS This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (SFB 807 to K. F.; Ei 520/3 and Ei 520/6 to O. E.; An 676/3 to S.L.A.) and the European Union (E-MeP).

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