Iron Is a Physiological Ligand of SecA-like Metal Binding Domains

The ATPase SecA is required for translocation of most proteins across the cytoplasmic membrane in bacteria. In Escherichia coli, SecA contains a small metal binding domain (MBD) at its extreme C-terminus that is widely conserved in other bacterial species and is required for its interaction with SecB. The MBD is thought to coordinate Zn via a conserved cysteinecontaining motif. Here, we investigated the metal binding properties of two E. coli proteins that contain SecA-like MBDs: YecA and YchJ. Both proteins copurify with metal, predominantly zinc. However, both proteins also copurify with significant amounts of iron. In YecA, iron binding is mediated by the MBD. Re-evaluation of the metal-binding properties of SecA indicate that: (i) SecA copurifies with stoichiometric amounts of iron; (ii) binding is mediated by the MBD; (iii) the MBD binds to iron with equal or greater affinity than to zinc; and (iv) the affinity for iron (but not for zinc) is mediated by a highly conserved serine in the metal-binding motif. Taken together, our results suggest that iron is a physiological ligand


INTRODUCTION
Translocation of proteins across the cytoplasmic membrane is carried out by the Sec machinery (1,2). The central component of this machinery is a channel in the cytoplasmic membrane, which is composed of the proteins SecY, -E and -G in bacteria (3). The translocation of a subset of proteins through SecYEG requires the activity of the ATPase SecA (4,5). The SecA proteins of many bacterial species contain a small metal binding domain (MBD) at its extreme Cterminus (6), and in species that contain an MBD, its sequence is highly conserved ( figure 1A). The MBD binds to a single Zn 2+ ion, which is coordinated by three conserved cysteines and a histidine with the motif CXCX 8 C(H/C) (figure 1A) (6,7). In Escherichia coli, the MBD mediates the interaction of SecA with SecB (7,8), a chaperone that assists in the translocation of a subset of SecA substrate proteins (9). In addition, the MBD contains several conserved residues that are not obviously involved in binding to Zn 2+ or SecB (6,10).
In this work, we investigated the metal binding properties of two E. coli proteins that contain SecA-like MBDs: YecA and YchJ ( Figure  1A) (11,12). YecA contains an N-terminal UPF0149 domain and a C-terminal MBD. YchJ contains N-and C-terminal MBDs, which flank a UPF0225 domain. We purified YecA and YchJ and identified the copurifying metals. Although both proteins copurified predominantly with zinc, mass spectrometry and absorbance spectroscopy indicated that a significant fraction of the proteins copurified with iron. NMR and EPR spectroscopy indicated that YecA binds to iron via the MBD. These results prompted us to re-examine the metal-binding properties of the MBD of SecA.
SecA copurified with iron in stoichiometric amounts, and a peptide consisting of the MBD displayed a binding preference for iron. These results indicate that iron is a physiological ligand of SecA-like MBDs.

RESULTS
Mass spectrometry analysis of metals that co-purify with YecA and YchJ. To investigate the metal-binding properties of the MBDs of YecA and YchJ, we purified fusion proteins between YecA or YchJ and hexahistidine-tagged SUMO from Saccharomyces cerevisiae and determined the amount of copurifying manganese, iron, cobalt, copper and zinc by ICP-MS (figure 1B). YecA co-purified with zinc at a stoichiometry of 0.84 ± 0.03, and YchJ co-purified with zinc at a stoichiometry of 1.30 ± 0.17. The amount of copurifying zinc was consistent with the number of MBDs in each protein. Both proteins also copurified with lower, but detectable, amounts of copper (0.17 ± 0.11 for YecA; 0.02 ± 0.01 for YchJ) and iron (0.04 ± 0.03 for YecA; 0.05 ± 0.04 for YchJ).
Absorbance spectroscopy of purified YecA and YchJ. During purification, the colour of the YecA-and YchJ-bound Ni-NTA columns was yellow. If TCEP was included in the buffers used for purification of YecA, the Ni-NTA column remained yellow after elution of the bound protein (supporting figure S1A), and the EDTA eluate contained a large amount of iron (supporting figure S1B). Purified YecA and YchJ were pale yellow in colour and gradually became colourless with incubation at 4°C-typically over a period of one to several hours. YecA absorbed light with a peak at ~330 nm, and YchJ absorbed light with a peak at ~340 nm (figure 2A). Dialysis of YecA against buffer containing EDTA resulted in a decrease in absorbance at 330 nm (figure 2B), and the addition of an equimolar concentration of FeSO 4 restored absorbance of YecA at 330 nm (figure 2B), suggesting that the yellow colour of the purified YecA protein was due to coordination of Fe 2+ (or potentially Fe 3+ ). Dialysis of YchJ against EDTA resulted in aggregation. Consequently, we did not investigate YchJ further.
EPR analysis of Fe 3+ binding by YecA. To confirm that YecA can bind iron, we investigated the interaction of YecA with Fe 3+ using EPR spectroscopy. (Fe 2+ is not detectable by EPR.) The addition of a stoichiometric amount of purified metal-free YecA to a solution of Fe 3+ (figure 2C, blue line) resulted in a new feature at 0.6145 T that is ~g = 4 (figure 2C, red line), indicating that there was a change in the electronic environment of the iron and indicating that YecA can bind to Fe 3+ .
Identification of the iron-coordinating domain in YecA by NMR spectroscopy. To determine which domain was responsible for binding to iron, we purified 15 N-and 13 C-labelled YecA and investigated its structure using NMR spectroscopy. We could assign the resonances for the C-terminal 20 amino acids in the TROSY spectrum of the protein (excluding Pro-206 and Pro-208, which do not contain an N-H bond) (figure 3A). Several amino acids, including Arg-203, Asp-204, Asp-205, Leu-220 and His-221, produced two resonances, suggesting that the MBD in the metal-free protein exists in two distinct conformations (figure 3A). Addition of FeSO 4 resulted in the broadening and flattening of most of the resonances from amino acids in the MBD due to the paramagnetic properties of iron, but it affected very few of the resonances from the N-terminal ~200 amino acids ( figure 3B). This result suggested that the MBD of YecA binds iron.
Co-purification of SecA-biotin with iron. The similarity of the MBDs of YecA and YchJ to the MBD of SecA suggested that SecA might also bind iron ( figure 1A). Purification of SecA normally involves overproduction of the protein, which could lead to competition between metal ligands in vivo, followed by multiple purification steps, which could lead to metal exchange in vitro. To avoid these issues, we produced SecA from a chromosomally encoded, IPTG-inducible copy of the secA gene. The SecA produced by these strains contained a C-terminal tag that caused it to be biotinylated (SecA-biotin) (13) and allowed us to purify it using streptavidin-coated sepharose beads. SDS-PAGE indicated that the purified protein samples contained only two proteins: SecA and unconjugated streptavidin (supporting figure  S2). ICP-OES indicated that SecA copurified with stoichiometric amounts of iron and lower amounts of zinc (figure 4A), suggesting that SecA binds preferentially to iron in vivo.
EPR analysis of Fe 3+ binding by the SecA MBD. To demonstrate that the SecA MBD can bind to iron, we investigated the effect of the MBD on the EPR spectrum of Fe 3+ . The addition of a metal-free synthetic peptide consisting of the C-terminal 27 amino acids of SecA (SecA-MBD) caused a large increase in the EPR signal of Fe 3+ at 1.2155 T (~g = 2) and altered the shape of the EPR spectrum ( figure 4B). SecA-MBD also inhibited the formation of iron oxides in solutions containing FeSO 4 (supporting figure S3). These results indicated that SecA-MBD can bind iron.
1 H-NMR analysis of metal binding by SecA-MBD. We investigated binding of the SecA-MBD peptide to zinc and iron using 1 H-NMR. Consistent with previous studies (6), the addition of ZnSO 4 to SecA-MBD resulted in multiple changes in its 1 H-NMR spectrum including the appearance of resonances in the 8.5-9.5 ppm region, indicative of the formation of secondary structure (figure 5A, blue and green traces). The addition of FeSO 4 resulted in a broadening and flattening of most of the 1 H resonances (figure 5A, red trace). This effect was due to binding to iron since FeSO 4 did not affect the spectra of control peptides that do not bind iron.
We next investigated the ability of zinc to compete with iron for binding to SecA-MBD. To this end, we monitored the resonances of 1 H signals from the valine methyl groups, which displayed differences distinctive of its metalbound state (figure 5B). The dissociation rate of Zn 2+ from the MBD is relatively rapid (half-life of minutes) (7). However, the addition of ZnSO 4 to SecA-MBD that had been pre-incubated with FeSO 4 did not cause a detectable change in the 1 H-NMR spectrum, even after ~40 minutes of incubation (figure 5C), suggesting that SecA-MBD binds preferentially to iron.
Role of conserved serine in metal preference. The SecA MBD contains a strongly conserved serine residue, Ser-890 (figure 1A) (6), which is also conserved in the MBDs of both YecA and YchJ (supporting figure S4). It is unlikely that Ser-890 is directly involved in the interaction of the MBD with SecB since it is not located on the SecB-interaction surface (10). Furthermore, Ser-890 is conserved in the Nterminal MBD of YchJ, which lacks the amino acid residues required for binding to SecB. However, SecA-MBD containing an alanine substitution at this position (SecA-MBD S890A ) exchanged iron for zinc at a detectable rate (figure 5D), suggesting that Ser-890 is important for the folding of the MBD and/or coordination of the metal.
Binding affinity of SecA-MBD peptides for Zn 2+ . To determine whether the alanine substitution caused a general defect in metal binding, we determined the affinity of SecA-MBD and SecA-MBD S890A for Zn 2+ using isothermal titration calorimetry (ITC). The affinity of the wild-type SecA-MBD for Zn 2+ (36.0 ± 11.6 nM; supporting figure S5A) was not significantly different from that of SecA-MBD S890A (46.0 ± 9.0 nM; supporting figure S5B), suggesting that the substitution does not affect its affinity for Zn 2+ . We could not determine the affinity for SecA-MBD for iron due to the interfering heat exchange caused by aerobic oxidation of Fe 2+ and because binding of SecA-MBD to Fe 3+ did not cause a detectable heat exchange. Nonetheless, these results, taken together with the 1 H-NMR experiments, suggested that the serine-to-alanine substitution specifically affects the affinity of SecA-MBD S890A for iron.

DISCUSSION
Our results indicate that iron is a physiological ligand of SecA-like MBDs. Analysis of the metal content of purified YecA and YchJ by mass spectrometry and absorbance spectroscopy indicate that both proteins copurify with significant amounts of iron. Indeed, SecA itself copurified with iron. EPR experiments indicated that both YecA and the SecA-MBD peptide bind to iron, and structural analysis of YecA by NMR indicates that it binds to iron via its C-terminal MBD. Finally, 1 H-NMR and ITC experiments suggest that the SecA MBD displays a binding preference for iron and that this specificity is determined in part by a conserved serine.
Previous studies suggested that zinc is the physiological ligand of the SecA MBD because it co-purifies with Zn 2+ (7). However, many ironbinding proteins have a high intrinsic affinity for zinc (14), and unlike iron, zinc is stable under aerobic conditions. Purification of SecA typically takes several hours, and without extraordinary effort, zinc typically contaminates water, salts and glassware used to make purification buffers in significant amounts (15). For example, ICP-MS analysis suggested that our purification buffers typically contained ~300 nM zinc. It is therefore possible that iron is displaced by zinc during purification. Other factors, such as the presence of reducing agents and overproduction of the protein, could exacerbate this issue.
The strong conservation of Ser-890 in SecA-like MBDs suggests that these MBDs normally also bind to iron in vivo. Although Ser-890 is not involved in coordinating the Zn 2+ ion in structures of the MBD (6,10), its hydroxyl group points inward toward the metal-binding site. These structures also suggest that the tetrahedral coordination of zinc by Cys-886, -888 and -897 and His-898 is strained (6). It is possible that octahedral coordination of iron by these residues and Ser-890 could relieve this strain (16). Indeed, copper, which can be coordinated octahedrally, copurifies in significant amounts with YecA and YchJ and stabilises binding to SecB to a greater extent than Zn 2+ (7,14). However, it is unlikely that copper is a physiological ligand since it is not normally found in the cytoplasm (14).
Finally, the affinity of the SecA MBD peptide for Zn 2+ suggests that zinc is not a physiological ligand. ITC indicated that SecA-MBD binds to Zn 2+ with a K D of ~40 nM, and competition experiments suggested that it binds to iron with a similar or higher affinity. These affinities are consistent with cytoplasmic ironbinding proteins and the cytoplasmic concentration of iron (50-100 nM) but not that of zinc (pM) (14,16,17).

EXPERIMENTAL PROCEDURES
Chemicals and media. All chemicals were purchased from Fisher or Sigma-Aldrich unless indicated. Synthetic peptides were synthesised by Severn Biotech (Kidderminster, UK) or using an in-house synthesiser. The quality of the peptides was checked using MALDI mass spectrometry. 100X EDTA-free protease inhibitor cocktail was purchased from Pierce (Thermo-Fisher). Cells were grown using LB medium (18). Where indicated, IPTG was added to the culture medium. Where required, kanamycin (30 μ g/ml) was added to the growth medium.
Purification of YecA and YchJ. The yecA and ychJ genes from Escherichia coli K-12 were fused in-frame to the 3′ end of the gene encoding SUMO from S. cerevisiae in plasmid pCA528 and purified as described previously (19). A detailed description of the purification of YecA and YchJ, can be found in the supporting information.
Metal ion analysis. The metal ion content of purified YecA and YchJ was determined using ICP-MS (School of GEES, University of Birmingham). The 5 kDa MWCO concentrator filtrate (Sartorius, Göttingen, Germany) was used to control for the amount of unbound metal in the protein samples. The zinc and iron ion content of the EDTA eluate from the Ni-NTA column after purification in the presence of 1 mM TCEP was determined using ICP-OES (School of GEES).
Absorbance spectroscopy. The absorbance spectra of 200 μ l of 600-800 μ M purified YecA or YchJ in buffer 1 (20 mM potassium HEPES, pH 7.5, 100 mM potassium acetate, 10 mM magnesium acetate) were determined from 300-600 nm using a CLARIOstar plate reader (BMG Labtech) using UV-clear flat bottomed 96-well plates (Greiner). The absorbance spectrum for the buffer alone was subtracted from that of the purified protein, and the absorbance was normalised to the concentration of the protein in the sample.
EPR spectroscopy. EPR samples were suspended in 50 μ l buffer containing 30% glycerol. For YecA, samples contained 0.85 mM FeCl 3 or 0.6 mM YecA and FeCl 3 . For SecA-MBD, samples contained 0.5 mM FeSO 4 , which had been left to oxidize aerobically, alone or with 0.5 mM SecA-MBD. The EPR spectra of the samples was determined as described in the supporting information. The resultant echodetected field swept profiles were normalised for plotting, taking account of differences in video gain, concentration and numbers of averages.
NMR backbone assignment of YecA. The 1 H, 15 N, and 13 C resonances of the YecA backbone were assigned using BEST TROSY versions of HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, HNCO and HN(CA)CO (20-26). All spectra were acquired using a Bruker 900 MHz spectrometer equipped with a 4-channel AVANCE III HD console and a 5mm TCI z-PFG cryogenic probe. A detailed description of the experimental conditions can be found in the supporting information. Nonuniformed sampled data were reconstructed using the compressed sensing algorithm with MDDNMR (27) and processed using nmrPipe (28). Spectra were analysed in Sparky (29).
Determination of metal content of SecAbiotin. 100 ml cultures of DRH839 (MC4100 Δ secA λ -p trc -secA-biotin..SpecR) (13) were grown in 10 μ M or 100 μ M IPTG to OD 600 ~ 1. Cells were lysed using cell disruption, and lysates were incubated with 100 μ l Streptactin-sepharose (IBA Lifesciences, Göttingen, Germany) for 15 minutes. The beads were washed four times with 30 ml buffer (50 mM potassium HEPES, pH 7.5, 100 mM potassium acetate, 10 mM magnesium acetate, 0.1% nonidet P40). Metal was eluted from the beads by incubating with 10 mM HEPES, pH 7.5, 50 mM EDTA) at 55˚C for 30 minutes, and the zinc and iron content was determined using ICP-OES. The amount of bound protein was determined by boiling in SDS sample buffer and analysing using Bradford reagent (BioRad, Hercules, CA). The eluted protein was resolved on a BioRad 15% TGX gel.
1 H-NMR of SecA-MBD. All spectra were obtained at 298 K on a Bruker 900 MHz spectrometer equipped with a cryogenically cooled 5mm TCI probe using excitation sculpting for water suppression on a sample in 90% H 2 O/10% D 2 O. Sequence specific assignments were completed using a TOCSY experiment in 90% H 2 O/10% D 2 O using a DIPSI2 spin-lock with a mixing time of 65 ms, 32 transients and collecting 512 increments with a spectral width of 10 ppm in both dimensions. 1D data sets comprised 16 transients, 32000 data points and a spectral width of 16 ppm. All data were processed using Topspin 3.2.6 software using an exponential window function with a line broadening of 1 Hz.
ITC. ITC measurements were carried out in a MicroCal VP-ITC calorimeter (Piscataway, NJ, USA). All solutions were centrifuged for 5 min at 13,000 rpm and then thoroughly degassed under vacuum for 5 min with gentle stirring immediately before use. 0.1mM ZnSO 4 was titrated into a solution of the indicated peptide (0.01 mM) in the sample cell (Vo = 1.4037 ml). Titrations consisted of a preliminary 2 µl injection followed by 50 6 µl injections of 12 s duration with 700 s between each injection. All experiments were carried out at 25˚C with an initial reference power of 10 µcal/s. The raw data were analysed with Origin 7.0 using one-binding site model and were corrected for the heat of dilution of the metal ion in the absence of peptide.

SUPPLEMENTAL METHODS
Purification of YecA and YchJ. For identification of the copurifying metals and absorbance spectroscopy, BL21(DE3) cells containing the SUMO-YecA and SUMO-YchJ plasmids were grown to OD 600 1.0 at 37°C. Cells were then shifted to 25°C and grown overnight in the presence of 1 mM IPTG. Cells were lysed in buffer 1 (20 mM potassium HEPES, pH 7.5, 100 mM potassium acetate 10 mM magnesium acetate) containing protease inhibitor by cell disruption. Where indicated, TCEP was added at a concentration of 1 mM. Lysates were passed over a 1 ml His-Trap HF column (GE Healthcare). The bound protein was washed with 15 ml buffer 1 containing 500 mM potassium acetate and 50 mM imidazole) and 15 ml buffer 1 containing 50 mM imidazole and was eluted using buffer 1 containing 500 mM imidazole. The eluted protein was dialysed against buffer 1 to remove the imidazole and concentrated using concentrators with a 5 kDa cutoff (Vivaspin).
For HSQC-NMR analysis of YecA, cells containing the YecA plasmid were grown in M9 minimal media containing 15 NH 4 Cl, 1 H 7 -13 C 6 -glucose (2 g/l) and trace minerals. Cultures were grown to OD 600 0.8 at 37°C, shifted to 18°C and induced overnight with 1mM IPTG. Cells were lysed by cell disruption and purified using a 1 ml His-Trap column as described above. After elution, the protein was treated with purified hexahistine-tagged Ulp1 from S. cerevisiae to remove the SUMO tag and dialysed overnight against buffer 1 containing 5 mM β -mercaptoethanol to remove the imidazole. The SUMO tag and Ulp1 protease were removed from the purified protein by passing the cut protein over a 1 ml His-Trap column. Purified YecA was then concentrated using anion exchange chromatography, and the eluate was dialyzed against buffer 1 containing 5 mM β -mercaptoethanol and 1 mM EDTA. NMR backbone assignment of YecA. Assignment spectra were acquired on 0.5 mM 15 N, 13  All experiments were performed at 298 K and acquired with a spectral width of 14 ppm in 1 H , collecting 1024 real data points, and 30 ppm in 15 N, collecting 92 increments. The centre of the spectra was set to 4.698 ppm in the 1 H and 118 ppm in 15 N. All spectra were acquired collecting 128 increments in the 13 C dimension using a non-uniform sampling scheme. The HN(CO)CACB and HNCACB experiments were acquired using 64 scans per increment, a spectral width of 76 ppm in the 13 C direction with the centre around 43.665 ppm. The HNCA and the HN(CO)CA experiments were acquired using 32 scans per increment, a 13 C spectral width of 30 ppm with the centre of the spectra set to 55.9 ppm. The HN(CA)CO and HNCO experiments were acquired using 32 scans per increment, a 13 C spectral width of 16 ppm centred around 176.2 ppm.
EPR spectroscopy. Measurements were taken with a Bruker Elexsys E580 spectrometer with an ER 5106QT-2w cylindrical resonator operating at 34 GHz, i.e. Q-band. Quartz tubes with 3 mm O.D. were used. Experiments were carried out at 10 K using a cryogen free variable temperature cryostat (from Cryogenic Limited). Echo-detected field sweeps were carried out by sweeping from 0.1 to 1.7 T with 4000 points using a Hahn echo sequence where the π pulse length was 32 ns and the time between pulses was 400 ns. The power level was determined by observing the maximum echo and was around 0.38 mW which is indicative of a high-spin system. The shot repetition time was set at 100 μ s with 50 shots per point which was sufficient for the iron, though caused some saturation of the Mn 2+ contaminant peak.