ABSTRACT
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 Zn2+ via a conserved cysteine-containing 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 of SecA-like MBDs.
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 C-terminus (6), and in species that contain an MBD, its sequence is highly conserved (figure 1A). The MBD binds to a single Zn2+ ion, which is coordinated by three conserved cysteines and a histidine with the motif CXCX8C(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 Zn2+ 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 FeSO4 restored absorbance of YecA at 330 nm (figure 2B), suggesting that the yellow colour of the purified YecA protein was due to coordination of Fe2+ (or potentially Fe3+). Dialysis of YchJ against EDTA resulted in aggregation. Consequently, we did not investigate YchJ further.
EPR analysis of Fe3+ binding by YecA
To confirm that YecA can bind iron, we investigated the interaction of YecA with Fe3+ using EPR spectroscopy. (Fe2+ is not detectable by EPR.) The addition of a stoichiometric amount of purified metal-free YecA to a solution of Fe3+ (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 Fe3+.
Identification of the iron-coordinating domain in YecA by NMR spectroscopy
To determine which domain was responsible for binding to iron, we purified 15N- and 13C-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 FeSO4 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 Fe3+ 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 Fe3+. 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 Fe3+ 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 FeSO4 (supporting figure S3). These results indicated that SecA-MBD can bind iron.
1H-NMR analysis of metal binding by SecA-MBD
We investigated binding of the SecA-MBD peptide to zinc and iron using 1H-NMR. Consistent with previous studies (6), the addition of ZnSO4 to SecA-MBD resulted in multiple changes in its 1H-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 FeSO4 resulted in a broadening and flattening of most of the 1H resonances (figure 5A, red trace). This effect was due to binding to iron since FeSO4 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 1H signals from the valine methyl groups, which displayed differences distinctive of its metal-bound state (figure 5B). The dissociation rate of Zn2+ from the MBD is relatively rapid (half-life of minutes) (7). However, the addition of ZnSO4 to SecA-MBD that had been pre-incubated with FeSO4 did not cause a detectable change in the 1H-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 N-terminal 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-MBDS890A) 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 Zn2+
To determine whether the alanine substitution caused a general defect in metal binding, we determined the affinity of SecA-MBD and SecA-MBDS890A for Zn2+ using isothermal titration calorimetry (ITC). The affinity of the wild-type SecA-MBD for Zn2+ (36.0 ± 11.6 nM; supporting figure S5A) was not significantly different from that of SecA-MBDS890A (46.0 ± 9.0 nM; supporting figure S5B), suggesting that the substitution does not affect its affinity for Zn2+. We could not determine the affinity for SecA-MBD for iron due to the interfering heat exchange caused by aerobic oxidation of Fe2+ and because binding of SecA-MBD to Fe3+ did not cause a detectable heat exchange. Nonetheless, these results, taken together with the 1H-NMR experiments, suggested that the serine-to-alanine substitution specifically affects the affinity of SecA-MBDS890A 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, 1H-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 Zn2+ (7). However, many iron-binding 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 Zn2+ 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 Zn2+ (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 Zn2+ suggests that zinc is not a physiological ligand. ITC indicated that SecA-MBD binds to Zn2+ with a KD of ~40 nM, and competition experiments suggested that it binds to iron with a similar or higher affinity. These affinities are consistent with cytoplasmic iron-binding 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 μg/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 μl of 600-800 μM 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 μl buffer containing 30% glycerol. For YecA, samples contained 0.85 mM FeCl3 or 0.6 mM YecA and FeCl3. For SecA-MBD, samples contained 0.5 mM FeSO4, 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 echo-detected 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 1H, 15N, and 13C 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 SecA-biotin
100 ml cultures of DRH839 (MC4100 ΔsecA λ-ptrc-secA-biotfn..SpecR) (13) were grown in 10 μM or 100 μM IPTG to OD600 ~ 1. Cells were lysed using cell disruption, and lysates were incubated with 100 μl 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.
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% H2O/10% D2O. Sequence specific assignments were completed using a TOCSY experiment in 90% H2O/10% D2O 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 ZnSO4 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.
CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest with the contents of this article.
ACKNOWLEDGEMENTS
We thank J. Cole, J. Green, A. Peacock and O. Daubney for advice and assistance. We thank Drs S. Baker, M. Thompson, H. El Mkami and A. Shah for technical assistance and members of the Henderson, Lund and Grainger labs for insightful discussions. TCS was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) Midlands Integrated Integrative Biosciences Training Partnership (MIBTP). MA was funded by Jouf University. DH and MJ were funded by BBSRC grant BB/L019434/1. TK was funded by BBSRC grant BB/P009840/1. J.E.L. thanks the Royal Society for a University Research Fellowship and the Wellcome Trust for the Q-band EPR spectrometer (099149/Z/12/Z). NMR work was supported by the Wellcome Trust (099185/Z/12/Z), and we thank HWB-NMR at the University of Birmingham for providing open access to their Wellcome Trust-funded 900 MHz spectrometer.
ABBREVIATIONS
- BEST
- band-selective short transient excitation
- DIPSI
- decoupling in the presence of scalar interactions
- DTT
- dithiothreitol
- EDTA
- ethylene diamine tetra-acetic acid
- EPR
- electron paramagnetic resonance
- HSQC
- heteronuclear single quantum coherence
- ICP
- inductively coupled plasma
- IPTG
- isopropyl-β-thiogalactoside
- ITC
- isothermal titration calorimetry
- MS
- mass spectrometry
- NMR
- nuclear magnetic resonance
- NTA
- nitrilotriacetic acid
- OES
- optical emission spectrometry
- SUMO
- small ubiquitin-like modifier
- TCEP
- tris(2-carboxyethyl)phosphine
- TOCSY
- total correlated spectroscopy
- TROSY
- transverse relaxation optimised spectroscopy
- UPF
- unidentified protein function
- UV
- ultraviolet