Quaternary Structure of Fur Proteins, a New ... - ACS Publications

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Quaternary Structure of Fur Proteins, a New Subfamily of Tetrameric Proteins Julien Pérard,†,‡,§ Jacques Covès,∥,⊥,# Mathieu Castellan,∥,⊥,# Charles Solard,∥,⊥,# Myriam Savard,∥,⊥,# Roger Miras,†,‡,§ Sandra Galop,†,‡,§ Luca Signor,∥,⊥,# Serge Crouzy,†,‡,§ Isabelle Michaud-Soret,*,†,‡,§ and Eve de Rosny*,∥,⊥,# †

CNRS, Laboratoire de Chimie et Biologie des Métaux (LCBM), UMR 5249, CNRS-CEA-UJF, F-38054 Grenoble, France CEA, LCBM, F-38054 Grenoble, France § Univ. Grenoble Alpes, LCBM, F-38054 Grenoble, France ∥ Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France ⊥ CNRS, IBS, F-38044 Grenoble, France # CEA, IBS, F-38044 Grenoble, France ‡

S Supporting Information *

ABSTRACT: The ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of “in solution” techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity.

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pylori,16 and Vibrio cholerae17], it is an attractive target for the development of new antimicrobial drugs.18 In the more common regulatory mechanism, the affinity of Fur for target promoter regions increases upon binding of ferrous iron, leading to repression or activation of the transcription of the downstream genes. However, other regulatory mechanisms exist, and in some strains like H. pylori and Campylobacter jejuni, Fur is able to bind specific target promoters in its apo form.19−21 Four crystal structures of Fur bond to zinc have been determined so far, Fur from P. aeruginosa (PaFur),22 H. pylori (HpFur),23 C. jejuni (CjFur),20 and V. cholerae (VcFur).24 Very recently, structures of Fur from Magnetospirillum gryphiswaldense (MgFur) were released. They correspond to

he ferric uptake regulator (Fur) is ubiquitous in Gramnegative bacteria1 but also present in Gram-positive organisms such as Bacillus subtilis.2 While first described for its critical role in iron homeostasis, proteomic and transcriptomic studies have revealed >100 genes regulated by Fur, making it a master regulator. For example, transcriptomic studies in Escherichia coli allowed identification of up to 196 genes whose expression was dependent on Fur.3,4 A more recent study revealed 81 genes directly regulated by Fur in E. coli. Among them, 47% are not related to iron transport and regulation but to energy metabolism, DNA synthesis, or nutriment search.5 In many pathogenic strains, such as Pseudomonas aeruginosa,6−8 Yersinia pestis,9,10 Francisella tularensis,11 and E. coli,12 Fur also contributes to virulence by its capacity to regulate virulence determinants, directly or indirectly.13 Furthermore, because Fur mutations decrease the virulence of various pathogens [P. aeruginosa,14 F. tularensis (personal communication), Staphylococcus aureus,15 Helicobacter © XXXX American Chemical Society

Received: September 29, 2015 Revised: January 6, 2016

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DOI: 10.1021/acs.biochem.5b01061 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 1. Amino acid sequence alignment of Fur proteins. Sequence alignment of P. aeruginosa (Pa), H. pylori (Hp), V. cholerae (Vc), C. jejuni (Cj), M. gryphiswaldense (MgFur), E. coli (Ec), F. tularensis (Ft), Y. pestis (Yp), and Legionella pneumophila (Lp) generated by Clustal Omega. Residues involved in metal-binding sites 1−3 are colored orange, green, and red, respectively (only for Fur with known X-ray structures). Residues revealed by PISA27 to be involved in PaFur tetramerization are highlighted in gray. The DNA-interacting lysine 77 of EcFur is framed in red. Conserved arginines 18 and 56, shown to interact with DNA in Zur28 and MgFur,25 are framed in blue. Secondary structure elements from the X-ray structure of PaFur (PDB entry 1MZB) are shown above the alignment, where H stands for α-helix (α1−α5) and B for β-strand (β1−β5). The asterisk indicates the first residue of the N-terminal DNA-binding domain of PaFur.

conformational changes leading to DNA binding.23,31 The position and nature of three residues, H32, E80, and E100 (the numbering corresponds to PaFur), are highly conserved. The other ligands belong to a short and highly conserved region, although their position and number vary. The third site, “site 3”, is formed by conserved histidines, aspartic acids, and glutamic acids located in the dimerization domain. Analysis of the sequences (Figure 1) shows that those residues are also found in EcFur, FtFur, and YpFur, yet in FtFur, the last conserved histidine at the C-terminal extremity is absent and replaced by Y124. The diversity of the Fur proteins is notably reflected by the differences in geometries and coordination modes of the metal-binding sites. A recent review summarizes the nomenclature and the structural properties of these sites.1 In vivo, Fur specifically binds different promoter sequences with various stoichiometries, depending on the strain and on the nucleotide sequence. However, they are all able to bind the inverted repeat consensus sequence GATAATGATAATCATTATC.32,33 This sequence, also called the 19 bp Fur box, is able to accept two dimers of Fur, docked on opposite sides of the DNA. Even if ferrous iron is the physiologically sensed metal, it has been shown that PaFur, YpFur, and EcFur can be activated in vitro by other divalent ions such as Zn2+, Co2+, Ni2+, and Mn2+.9,10,34−36 Those metal ions are easier to handle than Fe2+,

an inactive form and to activated forms bound to Mn2+, free and in complex with DNA.25 With regard to Fur from E. coli (EcFur), the dimerization domain was characterized by nuclear magnetic resonance (NMR) and the structure of the DNAbinding domain was obtained by X-ray crystallography.26 Despite low degrees of sequence homology, ranging from 24 to 49%, the metal-bound crystallized Fur proteins exhibit very similar overall structures with a V-shaped conformation (named “closed form”) formed by the two N-terminal winged-helix DNA-binding domains and the two dimerization domains. Each subunit contains two or three metal-binding sites that were called sites 1−3. “Site 1” is a structural site that contains one or two CXXC motifs, as exemplified by EcFur or HpFur, respectively (Figure 1). This site is very well conserved in Fur proteins, although not present in PaFur and MgFur that do not contain CXXC motifs. It was shown to be essential for dimerization in EcFur29 and HpFur,30 and its affinity for zinc is very high as an EDTA concentration of hundreds of millimolar is needed to remove the metal. “Site 2” is proposed to be the regulatory site that binds iron under physiological conditions. It is positioned in the hinge region, between the DNA-binding and dimerization domains, and involves histidine and glutamic acid residues from the two domains. It was shown to be essential for triggering the B


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Biochemistry

HNO3 overnight at room temperature. Briefly, 60 μL of each protein at 125 μM (2 mg/mL) was incubated overnight at room temperature with 530 μL of 10% HNO3; 5.4 mL of pure water was then added before measurement. The results were expressed in micrograms per liter and converted into micromolar for each element. Cysteine Alkylation and Mass Spectrometry Analysis. Alkylation of FtFur was performed as previously described.31 Briefly, proteins were buffer-exchanged into 100 mM Tris-HCl (pH 7.5) and 100 mM NaCl with or without 50 mM EDTA. Protein solutions were diluted to a final concentration of 40 μM in the appropriate buffer (with or without EDTA). A 3 mM iodoacetamide solution (Sigma-Aldrich) was freshly prepared in the same buffer. The alkylation reaction was performed by addition of 10 equiv of iodoacetamide in the dark at room temperature. Aliquots of 20 μL (1 nmol of protein) were taken at various times and immediately frozen in liquid nitrogen to stop the reaction. Iodoacetamide was eliminated by bufferexchange into 100 mM Tris-HCl (pH 7.5) and 100 mM NaCl. Electrospray ionization (ESI) time-of-flight (TOF) mass spectrometry analysis was performed on a 6210 mass spectrometer coupled with a HPLC system (1100 series, Agilent Technologies). All solvents used were HPLC grade; the HPLC mobile phases were as follows: phase A, 95% H2O, 5% ACN, and 0.03% TFA; phase B, 95% ACN, 5% H2O, and 0.03% TFA. Protein samples (5 μM dilution in mobile phase A) were desalted online on a C8 reverse phase microcolumn (Zorbax 300SB-C8, 5 μm, 5 mm × 0.3 mm, Agilent Technologies) for 3 min at 100 μL/min with 100% mobile phase A and then eluted at 50 μL/min with 70% mobile phase B. MS acquisition was conducted in positive ion mode in the m/z 300−3000 range, and the data were processed with MassHunter (version B.02.00, Agilent Technologies). The mass spectrometer was calibrated with tuning mix (ESI-L, Agilent Technologies). Activity Assay. The ability of metal-substituted Fur to bind DNA as a function of the nature of the divalent metal ions was investigated by using the previously described nuclease protection assay.18 Typically, 10 μL of a reaction mixture containing 2 μM Fur and 100 ng of pDT10 DNA vector in BTP buffer [20 mM BisTrisPropane (pH 7.5), 100 mM KCl, and 5 mM MgCl2 in ultrapure water (chromasolv grade, Fluka)] were incubated for 20 min at room temperature in the presence or absence of metal ions. An additional incubation of 1 h at 37 °C was then conducted after the addition of 2 units of HinfI restriction enzyme (NEB). Negative controls were performed the same way in the presence of EDTA. The reaction was stopped by addition of loading buffer containing EDTA, and the digestion products were immediately analyzed on 1% agarose gels in 1× Tris-acetate buffer (1× TAE buffer). Electrophoretic Mobility Shift Assays. Two DNA duplexes of 25 bp were prepared, one containing the specific 19 bp Fur box consensus sequence (5′-GGGGATAATGATAATCATTATCGGG-3′) and another containing an unspecific sequence (5′-GGGGATACTGATAGTCCTGATCGGG-3′) as described by Gonzales de Peredo et al.31 For annealing, 25 nM complementary oligonucleotides were mixed in 20 mM TrisHCl (pH 8.0), 150 mM NaCl, and ultrapure water. The mixture was heated at 95 °C for 5 min and rapidly cooled on ice. The formation of DNA duplexes was confirmed by electrophoresis on a 10% acrylamide gel in 1× TAE buffer [40 mM Tris acetate (pH 8.2) and 1 mM EDTA]. DNA radiolabeling was performed by incubating 20 nM DNA for

because of the stability of their divalent state, and are commonly used for in vitro studies. Because of its involvement in pathogenicity, Fur constitutes a promising target for antimicrobial strategies. We have performed the biochemical characterization of this protein in four pathogenic strains: P. aeruginosa, F. tularensis, Y. pestis, and Legionella pneumophila. The systematic analysis of the quaternary structures by size exclusion chromatography multiangle laser light scattering (SEC-MALLS) revealed that PaFur, FtFur, and LpFur do not form dimers in solution but are tetramers. The shape of FtFur and PaFur was further investigated by small-angle X-ray scattering (SAXS), confirming the tetrameric organization of the two proteins. Our data therefore demonstrate that Fur proteins can be classified into two subfamilies, according to their quaternary structure.

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MATERIALS AND METHODS Cloning, Protein Expression, and Purification. The genes encoding PaFur, YpFur, LpFur, and FtFur proteins were amplified by polymerase chain reaction (PCR) from genomic DNA of P. aeruginosa PAO1, Y. pestis CO92, L. pneumophila Paris, or F. tularensis FSC198. For subcloning, the sense and antisense primers were designed to contain NdeI and XhoI restriction sites, respectively. The primer sequences are shown in the Supporting Information (Table S1). PCR-amplified fragments were inserted into a pCR-Blunt II-TOPO vector (Invitrogen) prior to sequencing and subcloning into pET30c to obtain a pET30c-Fur expression vector. For each protein expression, 1 L of LB supplemented with kanamycin was inoculated with 10 mL of an overnight culture containing E. coli BL21(DE3) transformed with the corresponding pET30c-Fur vector. Cells were grown at 37 °C to an absorbance of 3 at 600 nm prior to addition of 500 mL of fresh LB/kanamycin medium. Fur expression was then induced by addition of 1 mM isopropyl thio-β-galactopyranoside (IPTG) and incubation for an additional ∼16 h under agitation at 18 °C. Because of their specific properties, individual purification procedures were developed for each Fur protein. EcFur was purified as previously described,29 and the purification of YpFur followed a similar protocol. Purifications of PaFur and LpFur were performed using size exclusion and DEAE-Sepharose columns. For FtFur purification, an additional step on a butylSepharose-FF column was necessary. All the details of each purification protocol are described in the Supporting Information. Purification steps were performed at 4 °C unless specified. Chromatographic fractions were tested for the presence of proteins by recording the absorbance at 280 nm. The presence of Fur was detected by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) analysis. All the chromatography media were purchased from GE-Healthcare, and the complete antiprotease cocktail was from Roche. After purification, proteins were concentrated by ultracentifugation, flash-frozen in liquid nitrogen, and stored at −80 °C until further use. ICP-AES. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Shimadzu ICP 9000 instrument with Mini plasma Torch in axial reading mode) was used to measure the zinc content. Standard solutions of zinc and ytterbium for atomic absorption spectroscopy (Sigma-Aldrich) were used for quantification [calibration curve between 10 and 500 μg/L with 1% HNO3 (Fluka)]. Ytterbium was used as an internal standard to prevent calibration drift and fluidic perturbation. Samples were routinely incubated in 10% C


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Biochemistry 30 min at 37 °C in the presence of 1 unit of T4 polynucleotide kinase (NEB) and 1 μL of γATP at 1 mCi/mmol. Labeled DNA was diluted 10 times in 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl, desalted on a G25 mini spin column, and stored at −20 °C. EMSA experiments were performed with freshly prepared radiolabeled DNA complexes; 250 pM DNA were added to different concentrations of protein in a binding buffer [20 mM BisTrisPropane (pH 8.5), 100 mM KCl, 3 mM MgCl2, 10 μM CoCl2, 5% (v/v) glycerol, and 0.01% Triton X-100]. After incubation for 10−30 min at room temperature, 10 μL of reaction mixtures was loaded on a prerun 10% polyacrylamide (29/1) gel. The gel was prerun for 30 min at 100 V in TA buffer [40 mM Tris acetate (pH 8.2)] supplemented with 100 μM CoCl2 and then run in TA buffer supplemented with 10 μM CoCl2. Mobility shifts were revealed by exposing the gels on a storage phosphor screen (GE healthcare) for 1−12 h. Band intensities were quantified with a cyclone phosphoimager (PerkinElmer). Cross-Linking Experiments. Cross-linking experiments were performed in the presence and absence of DNA. The specific and unspecific DNA duplexes (see Electrophoretic Mobility Shift Assays) were generated by incubation for 10 min at 95 °C prior to being slowly cooled at room temperature. The amine-specific homobifunctional cross-linker bis(sulfosuccinimidyl)suberate (BS3, Thermo Scientific) was used to cross-link the Fur proteins. The assays were performed at room temperature. Typically, 10 μL of a solution containing 0.3 nmol of Fur [in 50 mM HEPES (pH 7.5) and 50 mM NaCl] was incubated for 20 min in the presence of 2 equiv of metal ion prior to addition of 2 equiv of DNA or heparin for 20 min. A 50-fold molar excess of a BS3 solution was then added. After 30 min, the reaction was quenched by addition of TrisHCl (pH 8.0) to a final concentration of 250 μM and analyzed via a 15% SDS−PAGE gel. Native Molecular Mass Determination. Each purified Fur protein was loaded onto an analytical HiLoad 10/30 Superdex 75 column previously calibrated with conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and blue dextran (dead volume). Protein elution was detected by recording the absorbance at 280 nm. The column was equilibrated either with 50 mM Tris-HCl (pH 8.0) and 100 mM NaCl (PaFur, EcFur, and LpFur) or with 100 mM CAPS (pH 10) and 100 mM NaCl (YpFur). The molecular masses of native Fur proteins were determined according to the calibration curve, while the molecular mass of each Fur subunit was calculated from the corresponding amino acid content. Alternatively, 20 μL of Fur protein at 2 mg mL−1 was loaded on an analytical Superdex-S200 increase (GE Healthcare) preequilibrated with 20 mM Tris-HCl (pH 8.8), 150 mM NaCl, and 0.5 mM TCEP and connected to an in-line multiangle laser light scattering (MALLS) spectrometer (DAWN HELEOS II, Wyatt Instruments). Analytical size exclusion chromatography was performed at a rate of 0.5 mL min−1. An in-line refractive index detector (Optirex, Wyatt Instruments) was used to follow the differential refractive index relative to the solvent. After baseline subtraction of the buffer solution, all samples presented a single peak that allowed calculation of absolute molecular masses with the Debye model using ASTRA version 5.3.4.20 (Wyatt Instruments) and a theoretical dn/dc value of 0.185 mL g−1. The final values correspond to the average of three independent experiments. Small-Angle X-ray Scattering Experiments. Before each experiment, all samples were extemporaneously filtered on a

SEC Superdex 200 instrument (GE Healthcare) equilibrated in BTP buffer. SAXS data were collected at the European Synchrotron Radiation Facility (Grenoble, France) on beamline BM29 BioSAXS. The scattering profiles were measured at several protein concentrations: 1.25, 2.5, 5, and 10 mg/mL. Data were processed using standard procedures with the ATSAS version 2.5.1 suite of programs.37 The ab initio determination of the molecular shape of the proteins was conducted as previously described.38 Briefly, the radius of gyration (Rg), the forward intensity at zero angle [I(0)], Porod volumes, and a Kratky plot were determined using the Guinier approximation and PRIMUS programs.39 To build ab initio models, 49 independent DAMMIF40 models were calculated in slow mode with the pseudo chain option and analyzed using DAMAVER.41 CRYSOL42 was used to generate the theoretical scattering curves from the tetrameric structure of PaFur generated by PISA27 and from the PDB coordinates of the known structures of Fur proteins. Docking of the tetrameric Xray structure into the measured SAXS envelope was generated by SUPCOMB.43 Modeling of EcFur. A full-length model of the EcFur dimer “closed active form” was previously obtained using the VcFUR dimer structure (PDB entry 2W57), the closest in sequence homology (77% identity), with MODELLER and refined with the molecular mechanics CHARMM EEF1 force field in implicit solvent.18 This 148-amino acid model was necessary for SAXS refinement. A shortened model from residue 1 to 133 is accessible via http://www.modelarchive.org/doi:10.5452/maaq91z. The EcFur “inactive dimeric form” model was obtained from the “closed active form” model by rigid body modeling with BUNCH 08.44 BUNCH 08 was used in automatic mode with the N-terminal (1−81) and C-terminal (90−131) domains fixed. The flexible linker1 (82−89) and linker2 regions (132− 148) were generated by ab initio modeling with BUNCH 08. Amino acids in the linker regions were represented as spheres around their Cα atom. Several independent models were calculated with BUNCH 08. The best models fitting experimental SAXS curve were selected using a Normalized Spatial Discrepancy (NSD) score determined using DAMAVER41 and SUPCOMB.43 Missing atoms in the linker regions were first built from internal coordinates. Then NOE restraints as found experimentally by NMR26 were introduced: 11 restraints between backbone H atoms and 5 restraints between H atoms from methyl groups, per monomer. Finally, the model was energy minimized with these constraints using the ABNR algorithm down to a gradient of 0.1 kcal mol−1 Å−1.

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RESULTS AND DISCUSSION Purification of Fur from Pathogenic Strains and Analysis of Their Metal Content Reveal Different Behaviors. A specific behavior of each Fur protein was observed right from the purification as different protocols were necessary to obtain homogeneous samples (see Materials and Methods and Supporting Information). The oligomerization states of the Fur proteins evaluated by gel filtration also exhibited differences. At pH 8.0, EcFur, PaFur, and LpFur eluted as a single peak, while YpFur was very heterogeneous. However, a reasonably homogeneous sample was obtained by increasing the pH to 10.0 (Figure S1). Such pH dependence of the oligomerization state was previously reported for EcFur.29,45 The aggregation was also shown to be dependent on salt concentration, with EcFur being predominantly a dimer D


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Biochemistry

Figure 2. Metal ion-dependent activation of Fur from Y. pestis (YpFur), P. aeruginosa (PaFur), and F. tularensis (FtFur). Activation was followed by a nuclease protection assay with the pDT10 plasmid. The indicated numbers (eq) correspond to the concentration ratios between metal ion and Fur. In this assay, active Fur binds to a f ur-specific sequence that overlaps one of the four hinf I restriction sites on a pDT10 plasmid (framed scheme). It results in the production of three restriction fragments, compared to the four expected in the absence of nuclease protection.18 The line with EDTA shows the digestion pattern obtained with inactive Fur.

at high ionic forces.29 The elution volumes of EcFur at pH 8.0 and YpFur at pH 10.0 were compatible with a classical dimeric organization corresponding to a molar mass of ∼35 kDa (Figure S1). In contrast, PaFur and LpFur were eluted at significantly smaller elution volumes, suggesting that their oligomeric states were higher than dimers. The metal content was measured in the purified proteins by ICP-AES for EcFur, YpFur, PaFur, and LpFur and by ICP-MS for FtFur. Zinc was detected in EcFur, YpFur, and FtFur at 0.8, 0.2, and 1.0 equiv per subunit, respectively. PaFur and LpFur were found in apo forms. The presence of 1 equiv of zinc in FtFur indicates the existence of a high-affinity site, like the socalled “site 1”. This site, notably identified in EcFur, HpFur, and CjFur,20,30 involves one CXXC motif in EcFur and two CXXC motifs in HpFur or CjFur (Figure 1). To confirm the involvement of a “site 1” in FtFur, alkylation with iodoacetamide was performed. The time course of alkylation in the presence of iodoacetamide was monitored by mass spectrometry, with or without EDTA (Figure S2 and Table S2). In the apo form after incubation with EDTA, four cysteines were modified very quickly. Without EDTA, the cysteines reacted at a much slower rate, indicating that they were involved in Zn binding. This protection of cysteines by zinc, previously observed with HpFur and EcFur,30 confirms the presence of a high-affinity site corresponding to “site 1” in FtFur. Among the panel of studied proteins, YpFur and LpFur also contain one or two conserved CXXC motifs. Their low or null zinc content after purification may arise from a partial oxidation of the cysteines. Such an oxidation was reported for EcFur29 and shown to prevent zinc binding. It was also observed in the VcFur structure.24 To check this hypothesis, YpFur and LpFur were incubated with 5 mM reducing agent TCEP and 1 equiv of zinc per monomer. The low-affinity bound zinc ions were eliminated by gel filtration. Under these conditions, ICP-MS measurements showed that dimeric YpFur and tetrameric LpFur contained 0.8 and 0.6 equiv of zinc per monomer, respectively, consistent with the presence of a high-affinity “site 1” in the two proteins, and indicating that some of the cysteine ligands were oxidized at the

end of the purification. The absence of zinc in PaFur and the presence of zinc at ratios inferior to 1 in the protein subunits containing “S1 site” motifs suggest that, at the end of the purification, all the proteins are inactive, with an empty regulatory “S2 site”. The observed differences in oligomeric states raised the question of the ability of these Fur proteins to bind DNA specifically; it was addressed in vitro by nuclease protection assays. Fur Proteins Are Activated in Vitro with Different Efficacies and Specificities by Divalent Metal Ions. The efficiency of binding of Fur to DNA was tested as a function of the nature and concentration of divalent metal ions by nuclease protection assays (Figure 2). Regardless of the initial metal content of the Fur preparations, in the absence of added metal ions all the proteins were inactive as purified, confirming the absence of metal ion in their regulatory site (Figure 2 for FtFur and not shown for PaFur and YpFur). The digestion profiles show that addition of metal ions activated PaFur, YpFur, and FtFur with both different efficacies and specificities. PaFur was activated by Co2+ and Mn2+, with cobalt being the most efficient. As previously mentioned,36 zinc was not an activator in the assay, even though the X-ray structure demonstrates that it is able to bind the regulatory site.22 YpFur and FtFur were both activated by the three metal ions, as found with EcFur.34,35,46 It is consistent with the high degree of sequence homology between YpFur and EcFur. For YpFur, the activation efficiencies decreased in the following order: Zn2+ > Co2+ > Mn2+. In the case of FtFur, Co2+ was the most efficient while Zn2+ and Mn2+ gave the same type of response. LpFur was found unable to be activated by any of the metals tested (data not shown). Therefore, it was not integrated into the DNA binding experiments described hereafter. Because all the proteins, except LpFur, were able to specifically bind DNA in a metal-dependent manner, they likely represent relevant biological forms. The close analysis of their oligomeric state was therefore an interesting issue. Fur Proteins Can Be Classified into Two Groups: The Dimers and the Tetramers. The quaternary structures of Fur E


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Biochemistry

because a 2-fold axis symmetry of the monomeric subunit, which constitutes the asymmetric unit, generates a dimer. It was also proposed to behave as a dimer in solution,51 but this result was based on the study of a truncated form containing only the last 73 C-terminal residues of the dimerization domain. The absence of the dimeric form of PaFur in solution raised the question of the relevance of the dimer in the crystal structure. PaFur in the Crystal State Is Compatible with a Tetrameric Organization. To test the compatibility of the crystal structure with the tetrameric organization determined in this study, the PDB coordinates (1MZB) were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA)27 server. Remarkably, the given “most probable multimeric state” was a tetramer made of a dimer of dimers. Figure 4 shows that this structure is rather compact and exhibits an intertwining of the DNA-binding domains. The

proteins were investigated by size exclusion chromatography coupled to a multiangle laser light scatter and refractometer (SEC-MALLS-RI). As shown in Figure 3 and Table 1, the Fur

Figure 3. SEC-MALLS-RI analysis of Fur proteins from Y. pestis (YpFur), P. aeruginosa (PaFur), F. tularensis, (FtFur), E. coli (EcFur), and L. pneumophila (LpFur). Elution profiles from the refractive index detector (arbitrary unit) are represented in different colors for each sample, together with the calculated molecular masses.

proteins exhibit different oligomeric states with EcFur and YpFur being dimers and PaFur, FtFur, and LpFur being tetramers. The stability of PaFur and FtFur tetramers was tested under different buffer conditions, i.e., in the presence (0.1−1 M) or absence of NaCl, in the presence of 100 mM EDTA, and in the presence of 1 mM TCEP as a reducing agent. Under all these conditions, they remained tetrameric (data not shown). Addition of 4 equiv of Mn2+ ions had no impact on the quaternary structure of FtFur and induced some aggregation on PaFur with the appearance of high-molecular mass species (data not shown). The absence of dimer formation upon addition of metal shows that binding of metal to PaFur and FtFur does not trigger the dissociation of the tetramers. To date, Fur and Fur-like proteins, such as Zur,28 Nur,47 Mur,48 and PerR,49 have been considered as dimers in their apo and metal-bound forms, although other oligomeric states have been described. For EcFur29 and HpFur,30 monomeric forms were detected by gel filtration under oxidizing conditions. This conformation was linked to the presence of disulfide bridges involving cysteines of the structural metal-binding “site 1”. Addition of a reducing agent and zinc to oxidized EcFur and HpFur monomers induced dimerization of these proteins. The quaternary structure of FurA from the cyanobacterium Anabaena sp. PCC 7120 was also shown to be dependent on the redox states of cysteines.50 In the absence of DTT, a majority of trimers were detected by atomic force microscopy as well as some dimers and monomers. Addition of a reducing agent induces rearrangement of the trimer to protein monomers and a major fraction of FurA dimer.50 This study unambiguously reveals that PaFur, FtFur, and LpFur constitute very stable tetramers in solution. Pohl et al.22 described PaFur as a dimer on the basis of the X-ray structure

Figure 4. Tetrameric structure of Fur from P. aeruginosa. The structure was generated by PISA (http://www.ebi.ac.uk/pdbe/pisa)27 from PDB entry 1MZB. It shows a dimer of dimers with one dimer formed by the subunits colored green (A) and lemon (B) and the other by the subunits colored magenta (D) and light pink (C). The DNA-binding domains are intertwined. In this structure, the metal sites are not represented for the sake of clarity.

buried area corresponding to the dimer−dimer interface (between subunits AB and CD in Figure 4) is 2120 Å2, and the solvation free energy gain upon formation of the interface is −11.5 kcal/mol. For comparison, the buried interface between the two subunits in a dimer (between subunits A and B or C and D) is 1327 Å2 and the solvation free energy −21.1 kcal/ mol. Analysis of the interactions that stabilize the tetramer revealed 16 residues, 14 belonging to the DNA-binding domain (Figure 1 and Figure S3). The interactions between the two dimers mainly rely on hydrogen bonds involving atoms of the backbone, although the side chains of E36, R69, D73, H76, R18, and R56 also contribute. In addition to the two functionally important zinc ions, two extra Zn2+ ions per

Table 1. Molecular Masses and Oligomeric States of Fur Proteins at pH 8.0a subunit molecular mass (kDa) native molecular mass (kDa)c oligomeric stated

b

EcFur

YpFur

LpFur

PaFur

FtFur

16.8 33.3 ± 0.8 2.0

16.7 33.0 ± 0.6 2.0

15.4 57.2 ± 0.4 3.7

15.2 60.0 ± 0.5 3.9

16.1 64.1 ± 0.8 4.0

a c

Except for YpFur analyzed at pH 10 because of the existence of various oligomeric states at pH 8.0. bCalculated from the amino acid content. Determined by SEC-MALLS (in kilodaltons). dMWnative/MWsubunit. F


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Biochemistry

profiles and the resulting pair distance distribution patterns (Figure 5) show that the shapes of the proteins are different. EcFur and FtFur display skewed pair distance distribution patterns with a well-defined maximum at a distance inferior to Dmax/2 indicative of a rather elongated particle, whereas PaFur has a maximum at Dmax/2 that is more typical of globular proteins.42 The Porod volumes show that the sizes of PaFur and FtFur are different from that of EcFur (Table 2), consistent with FtFur and PaFur being higher-order oligomers than EcFur. The Dmax value of 82 Å obtained with EcFur is in agreement with the diameter of 80 Å observed by AFM on the negatively stained free protein.45 YpFur and metal-bound PaFur were also tested on SAXS but were poorly monodisperse, and the data could not be accurately interpreted. Altogether, SAXS experiments confirm that the inactive forms of EcFur is a dimer and FtFur and PaFur are tetramers and show that the three proteins have different envelope shapes with EcFur and FtFur being more elongated than PaFur. Three structures of related proteins in their inactive form have been published so far: PerR, a Fur-like protein responding to hydrogen peroxide, CjFur, and MgFur. Their conformations are very different. In inactive PerR, the DNA-binding domains are far away, leading to an “open form” (Figure S5). In CjFur and MgFur, the DNA-binding domains exhibit an overall V-shaped conformation comparable to that of the active proteins. However, the orientation of the DNA-binding and dimerization domains differs, due to structural differences in the hinge region. This rotation results in a different positioning of the “β1β2” sheet motif either “inside” when pointing toward the interior of the V-shape of the dimer or “outside” when pointing toward the exterior (Figure 6). In inactive CjFur, an ∼180° rotation is observed for both DNA-binding domains, resulting in two “β1β2 outside” motif. In inactive MgFur, a dissymmetry appears between the two subunits with one “β1β2 inside” motif and one “β1β2 outside” motif, whereas they are both “β1β2 inside” motifs in the active form. These differences show that although the conformations of the active closed forms of Fur are similar, at least in the X-ray structures, the inactive conformations vary in the orientation of their DNA-binding domains (Figure 6). The PaFur Tetrameric X-ray Structure Fits the SAXS Envelope of the Protein in Solution. To compare the shape of the protein in crystal with that of the protein in solution, a SAXS scattering profile was simulated with the PISA coordinates,27 using CRYSOL.42 The comparison was possible between only the inactive form in solution and the metal-bound form in crystallo, because addition of metal ions to PaFur resulted in partial aggregation and prevented the acquisition of accurate SAXS data. As shown in Figure 7, the simulated and measured scattering curves display similar profiles, although with some differences for Q values higher than 0.1 A−1. The sizes and shapes of the two proteins are therefore comparable, although subtle differences remain in the folding. These differences may originate from the difference in metalation of the two samples or from the absence of hydration in the model built with the PISA coordinates. Nevertheless, docking the high-resolution structure into the ab initio bead model illustrates the good agreement between the structure of PaFur in crystallo and in solution (Figure 7) with a χ2 value of ∼4.5. Besides, the similarity between inactive and zinc-bound envelopes of PaFur indicates that metal ion binding does not trigger important conformational changes. This observation also suggests that the DNA-binding domains are in the closed

subunit located between symmetry-related dimers were observed in the X-ray structure.22 Because they are coordinated to unconserved residues in the Fur sequences, they were proposed to be important for only crystal packing. Among them, one is found at the interface of two subunits from different dimers in the tetramer [subunits A−B and C−D (Figure S3)]. It involves H76 and D73 of one subunit and E36 of the other subunit. Considering the four subunits, four extra zinc ions are therefore present in the tetramer. To check the importance of this zinc in the stabilization of the quaternary structure, a H76A mutant was produced. However, it displayed a retention time upon gel filtration chromatography similar to that of the tetrameric wild-type protein (data not shown). This is consistent with the results obtained on the EDTA-treated metal free PaFur that constantly showed a tetramer, demonstrating that the interfacing zinc is not responsible for the tetramerization. Among the residues involved in the stabilization of the tetramer, five are not conserved in EcFur and YpFur, namely, A63, D73, H76, A77, and V93 (Figure S3). They are all interacting with conserved residues and give the following associations: A63/R56, D73−H76/E36 (via a zinc atom), A77/ G75, and V93/A52. V93 is the only residue that is not present in the dimers but is conserved in all the tetramers (Figure 1). In the PaFur structure, V93 forms a hydrogen bond with A52 (Figure S3). This interaction involves atoms of the backbone and not of the side chains; it is therefore very unlikely that it drives the formation of the tetramer. The specificity of the dimer−dimer interaction more likely relies on the tertiary structures of the dimers that generate interacting areas at the surface rather than a specific conserved sequence. To know if the tetrameric organization determined by PISA from the X-ray data is relevant to the structure in solution, SAXS experiments were performed to gather information about the shape of the tetrameric PaFur in solution. EcFur, PaFur, and FtFur Exhibit Different SAXS Envelopes. Because of the inaccessibility of the DNA-binding domains, the tetrameric structure of PaFur proposed by PISA obviously represents an inactive form that cannot bind DNA. The relevance of the X-ray structure compared to the protein in solution was therefore explored by SAXS analysis. FtFur, the other characterized tetramer, was added to this study. EcFur was also added as a control of a dimeric form. The quality of the Fur samples was verified by analysis of the Guinier and Kratky plots (Figure S4). The linearity of the Guinier plots confirms that the samples are monodisperse, and the bellshaped curves, with a well-defined maximum, of the Kratky plots confirm that the proteins are folded. Moreover, the calculated molecular masses are in agreement with those determined by MALLS (Tables 1 and 2). The SAXS scattering Table 2. In Solution Structural Parameters Determined by SAXSa sample

Rg (Å)

Dmax (Å)

Porod volume (Å3)

MW (kDa)b

EcFur PaFur FtFur

25.6 ± 0.8 27.1 ± 0.4 27.2 ± 0.5

82.0 ± 0.8 80.1 ± 0.7 84.3 ± 3.0

60.0 ± 3.3 99.5 ± 3.4 119.5 ± 5.2

32.5 ± 1.3 58.5 ± 1.9 61.1 ± 0.5

a

The SAXS data are shown as mean values and standard deviations of four experiments at different protein concentrations (the individual data are listed in Table S3). bCalculated from the extrapolated scattering intensity at zero angle (I0); BSA was used for calibration. G


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Biochemistry

Figure 5. Small-angle X-ray scattering (SAXS) analysis of Fur from P. aeruginosa (PaFur), F. tularensis (FtFur), and E. coli (EcFur). The averaged scattering curves (A) and the pair distance distribution function (B) are represented with different colors for each sample. The ab initio models (C) were determined as described in Materials and Methods.

the theoretical curves calculated from the three proteins crystallized in their inactive form: CjFur, PerR, and MgFur. The three proteins exhibit different orientations of their β1β2 domains, as discussed above (Figure 6 and Figure S5). The poor fits between the experimental and calculated curves, with high χ2 values, revealed significant differences in overall shapes (Table S4). The experimental scattering data were then used to build a high-resolution model structure with BUNCH 08. This program performs modeling of multidomain proteins against SAXS data using a combined rigid body and ab initio modeling approach and allows determination of the three-dimensional domain structure of proteins when the structures of individual domains are available. Such a strategy was successfully used for other systems.53,54 The EcFur high-resolution model corresponds to an inactive “closed conformation” with a “β1β2 outside” orientation of the DNA-binding domains, as shown in Figure S5. By construction, this structure respects the NOE distances measured experimentally by NMR.26 The calculated scattering and pair distribution function curves fit the experimental curves with a good χ2 of 2.5 (Figure 8). FtFur and PaFur Bind the 19 bp Fur Box DNA. The ability of FtFur and PaFur to specifically bind the 19 bp DNA Fur box was tested by an electrophoretic mobility shift assay in the presence of cobalt. For both proteins, the results show a single shifted band whose intensity depends on the protein concentration (Figure 9). Negative controls were performed with unspecific DNA sequence (Figure S6).31 These results demonstrate that DNA binding is dependent on metalation and is specific to the 19 bp Fur box. The apparent dissociation constants determined from the titrations are 3 and 9 nM for PaFur and FtFur, respectively. They are comparable to the affinities previously described for EcFur31,46 and HpFur.23

Figure 6. Comparison of active and inactive structures of Fur proteins. X-ray structures of CjFur (4ETS) from L2 to K154 and MgFur in its active (4RAZ) and inactive (4RAY) forms from M1 to L134 show different conformations of the “β1β2” sheet motifs in FUR proteins. The dimers were built with missing atoms and residues added using internal coordinates with CHARMM. Harmonic restraints were applied to heavy atoms present in the X-ray structures with a force constant of 5 kcal mol−1 Å−2, and the coordinates of the dimers were energy minimized down to a gradient of 0.1 kcal mol−1 Å−1. The three proteins are shown in the same orientation after superimposition of their α5 helices (S104−H118 in MgFur). The β1β2 sheet motifs either “inside” or “outside” the V-shape of the dimers are colored red. Figures were created with VMD.52

form in inactive PaFur, as described for inactive CjFur20 and MgFur.25 Inactive EcFur Exhibits a “Closed Conformation” with a “β1β2 Outside” Orientation of the DNA-Binding Domains. The structural information about inactive EcFur mainly relies on NMR data of the dimerization domain, X-ray crystallography of the DNA-binding domain, and cross-linking experiments.26 However, the relative orientation of the DNAbinding domains and the dimerization domains is not known. To better characterize the overall conformation of inactive EcFur, the experimental scattering curve was first compared to H


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Biochemistry

Figure 7. Comparison of experimental and theoretical SAXS data of Fur from P. aeruginosa. (A) Experimental scattering curves of inactive PaFur in solution (black) and calculated scattering curve of Zn-bound PaFur in crystallo (red). The scattering curve was generated with CRYSOL,42 on the basis of the structure produced by PISA with the coordinates of PDB entry 1MZB. (B) Pair distance distribution functions of the two forms. (C) Docking of tetrameric Zn-bound PaFur structure into the SAXS envelope, determined by ab initio modeling of inactive PaFur in solution.

Figure 8. Comparison of experimental SAXS data and the computed model of inactive EcFur. (A) Experimental scattering curve of inactive EcFur in solution (black) and calculated scattering curve generated by CRYSOL42 (red) on the basis of the refined model of EcFur. (B) Pair distribution function of the two forms. (C) Docking of the EcFur model into the SAXS envelope determined by ab initio modeling of apo-EcFur in solution with SUPCOMB.

PaFur Is Tetrameric in Solution and Evolves toward a Dimer in the Presence of DNA. The effect of DNA binding on the quaternary structure of Fur proteins was tested by crosslinking experiments, with the specific 19 bp Fur box sequence and with an unspecific sequence. The cross-linked species were analyzed by electrophoresis under denaturing conditions. YpFur was taken as a member of the group of dimers and PaFur as a member of the group of tetramers. The wellcharacterized EcFur was also tested as a control of a dimeric

organization. The cross-linker agent BS3, with its 11.4 Å spacer arm, was chosen because it was long enough to allow crosslinking between lysines of different subunits. Similar results were obtained with YpFur and EcFur, which is consistent with their high degree of sequence identity [85% (see Table S5)]. In the presence of the cross-linking agent, two main bands were detected: one corresponding to the size of one subunit, as obtained under the untreated condition, and the other I


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Biochemistry

binding, EcFur and YpFur were incubated with an unspecific DNA sequence, in the presence of activating metals (Co2+ or Mn2+). This results in a pattern identical to that observed in the absence of DNA. Unexpectedly, zinc that activates YpFur in the nuclease protection assay has no impact on cross-linking, as shown by the similar intensity of the bands corresponding to monomers in the presence of specific or unspecific DNA. The cause of this behavior remains unclear. PaFur was tested under the same conditions as EcFur and YpFur, with different results. Addition of BS3 led to a pattern with a weak band at ∼35 kDa corresponding to cross-linked dimers and two intense bands close to 70 and 100 kDa assigned to tetramers and higher-molecular mass species, respectively. This pattern constitutes a further indication that PaFur exists as a tetramer in solution. Addition of metal ions had a dramatic impact on PaFur, resulting in the formation of very heavy crosslinked oligomers. Most of those oligomers were too large to enter the resolving gel, as revealed by the low intensity of the bands especially under the Co2+ and Mn2+ conditions. Addition of DNA results in a significant increase in the amount of crosslinked dimers along with a decrease in the amounts of the oligomeric forms. This suggests that PaFur is able to dimerize in the presence of DNA. However, the same pattern is obtained under all conditions that include DNA, regardless of the added metal ion and/or the DNA sequence. The observed dimerization of PaFur is therefore not dependent on the DNA sequence, even if the EMSA results, described above, confirm that PaFur specifically binds the 19 bp DNA Fur box with high affinity. Heparin was used as a mimic of the DNA backbone but does not induce the dimerization observed with DNA (Figure S7). While little information about the oligomerization of apo-Fur in solution is available, the quaternary structure and the stoichiometry of Fur bound to DNA have been more widely studied. In the case of EcFur,55 Mur from Rhizobium leguminosarum,48 BsFur,32 and HpFur,19 it was found that two dimers are able to bind the cognate target DNA sequences. Moreover, the recent crystal structure of Zur bound to DNA28 revealed two dimers docked on opposite sides of the DNA. Binding to DNA was shown to be cooperative.28 It points out a multistep process with one dimer binding after the other. It should be noted that FurA from Cyanobacterium Anabaena derogates from this rule as it was shown to bind its DNA target sequence as a monomer prior to sequential binding of other subunits resulting in dimeric, trimeric, and tetrameric complexes.56 Even if its regulatory site is occupied by zinc, the tetrameric structure of PaFur proposed by PISA represents a form that cannot bind DNA because of the inaccessibility of the DNAbinding domains. Therefore, the dissociation into two dimers is a necessary step in the activation process. In this regard, the cross-linking experiments, described here, confirm the modification of the quaternary structure toward dimerization, in the presence of DNA. However, this modification is dependent neither on metal binding nor on the nucleotide sequence. It is also not driven by the negative charges of the phosphates, as shown by the results with heparin. Therefore, the dissociation of the tetramer is very likely principally driven by the physicochemical nature of nitrogenous bases. Two conserved arginines that were shown to interact with DNA in Zur (R28 and R65) and MgFur (R20 and R57) are involved via their side chains in the stabilization of the PaFur tetramer (R18 and R56, respectively).28 R28 in Zur and R20 in MgFur are bound to a

Figure 9. DNA binding analysis of Fur from P. aeruginoa (PaFur) and F. tularensis (FtFur) by an EMSA. Radiolabeled 19 bp Fur box duplexes were incubated with different concentrations of PaFur or FtFur, in the presence of 10 μM cobalt, and loaded onto a native 6% polyacrylamide gel. The arrows correspond to migration of the free DNA. The apparent dissociation constants Kd (9 and 3 nM for FtFur and PaFur, respectively) correspond to 50% complex.

corresponding to the size of two covalently bound subunits (Figure 10).

Figure 10. Quaternary structure of Fur from Y. pestis (Yp), E. coli (Ec), and P. aeruginosa (Pa) analyzed by cross-linking with BS3. Crosslinked products were generated by incubation of 20 μM protein with (+) or without (−) 40 μM metal ion and with (+) or without (−) 40 μM DNA corresponding to 19 bp Fur box sequence (s) or to nonspecific DNA [ns (see Materials and Methods)]. After addition of 1 mM BS3, the reaction products were analyzed by SDS−PAGE (10% acrylamide). M, D, and T correspond to the expected positions of the monomers, dimers, and tetramers, respectively. The gels shown here are representative of a minimum of three experiments.

Higher-molecular mass complexes were also produced although to a lesser extent. The addition of BS3 in the presence of metal activators did not significantly modify the intensity of the monomer and dimer bands. However, further addition of the 19 bp Fur box induced a decrease in the level of the cross-linked dimers. Because BS3 reacts with primary amine groups, this decrease may be attributed to the protection of lysine(s) upon DNA binding. In that sense, K77 had been proposed to interact with DNA in EcFur31 and is conserved in YpFur but not in PaFur (Figure 1). To confirm that the diminution of the amount of cross-linked dimers is due to DNA J


Article

Biochemistry Scheme 1. Mechanistic Hypothesis of DNA-Induced Tetramer Dissociation of PaFur into Dimersa

a

Secondary structures of the proteins are highlighted in cartoon representation: subunits A (lime), B (green), C (violet), and D (magenta). Zinc atoms are represented as grey spheres. Figures were created with VMD52 (see Supplementary Materials and Methods).

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phosphate of the minor groove via an electrostatic interaction, while R65 and R57 make hydrogen bonds with a nitrogenous base. It is therefore conceivable that those conserved residues participate in the switch between the tetramer and dimers via a competition between their interacting residues partner and the DNA helix.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01061. Additional figures, tables, and materials and methods (PDF)

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CONCLUSION

Fur proteins are ubiquitous in Gram-negative bacteria and have been widely studied for their crucial role as master metalloregulators in physiological processes. However, most of the previously reported studies describe in vivo analysis of their function. Biochemical characterizations are much less developed and cover only a smaller number of strains.1,57 Here we report the purification and characterization of three Fur proteins from pathogenic strains, FtFur, YpFur, and LpFur. We added to this series PaFur, which has been the subject of several studies but has been biochemically characterized very little, and EcFur, the best characterized in term of biochemistry but with a lack of structural knowledge. The determination of the SAXS envelope combined with molecular modeling allowed the proposal of a structural model of EcFur in its inactive form: a “closed conformation” with a “β1β2 outside” orientation of the DNA-binding domains is found. The data collected for PaFur allow us to propose a scheme for its activation (Scheme 1). In this scheme, PaFur is stored as a tetramer, in a form unable to bind DNA. Its full activation requires two events: dissociation into the dimers and metal binding. However, the structure generated by PISA shows that metal binding can occur in the tetrameric form, and the crosslinking data show that the dissociation of the tetramer is not dependent on the metalation state of the protein. This suggests that those two events are necessary but independent. The results also show for the first time that several Fur proteins are stable tetramers in solution and therefore derogate from the dogma of Fur and Fur-like proteins being only dimers. Our work therefore reveals that Fur proteins can be divided into two groups according to their quaternary structure, the group of dimers with EcFur and YpFur and the group of tetramers with PaFur, FtFur, and LpFur. This classification probably also corresponds to a functional classification, because the tetramers need a supplementary step for activation, as compared to the dimers.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

J.P. designed and performed protein expression and purification of FtFur and EcFur as well as all the MALLS, SAXS, EMSA, and ICP-AES experiments. J.P. also participated in the analysis of the data. J.C. was involved in the design of the experiments and analysis of the data and contributed to the writing of the paper. M.C., C.S., and M.S. performed protein expression and purification, cross-linking, activity assay, and analytical SEC experiments on EcFur, PaFur, FtFur, and YpFur. R.M. was involved in protein expression and MALLS experiments on FtFur. S.G. performed cloning, protein expression, and purification as well as cysteine alkylation of FtFur. L.S. performed the MALDI-TOF experiments. S.C. generated the molecular model of PaFur bound to DNA. I.M.-S. was involved in the design of the experiments and analysis of the data and contributed to the writing of the paper. E.d.R. performed the cloning of the genes encoding PaFur, YpFur, and LpFur. E.d.R. was also involved in the design of the experiments and the analysis of the data and contributed to the writing of the paper. Funding

This work was supported by the French national research agency (ANR-11-BS07-0007 PepSiFUR), the LabEx Arcane, and the CEA. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Josiane Arnaud for metal quantification by ICP-MS, David Cobessi for his help in using PISA, and Isabelle PetitHaertlein for her technical participation at the beginning of the project. We also thank Adam Round and Petra Pernot for their help on the BioSAXS BM29 beamline at the ESRF and K


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Biochemistry

(14) Hassett, D. J., Sokol, P. A., Howell, M. L., Ma, J. F., Schweizer, H. T., Ochsner, U., and Vasil, M. L. (1996) Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities. J. Bacteriol. 178, 3996−4003. (15) Horsburgh, M. J., Ingham, E., and Foster, S. J. (2001) Staphylococcus aureus, fur is an interactive regulator with PerR, contributes to virulence, and Is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis. Journal of bacteriology 183, 468−475. (16) Pich, O. Q., and Merrell, D. S. (2013) The ferric uptake regulator of Helicobacter pylori: a critical player in the battle for iron and colonization of the stomach. Future Microbiol. 8, 725−738. (17) Mey, A. R., Wyckoff, E. E., Kanukurthy, V., Fisher, C. R., and Payne, S. M. (2005) Iron and fur regulation in Vibrio cholerae and the role of fur in virulence. Infect. Immun. 73, 8167−8178. (18) Cisse, C., Mathieu, S. V., Abeih, M. B., Flanagan, L., Vitale, S., Catty, P., Boturyn, D., Michaud-Soret, I., and Crouzy, S. (2014) Inhibition of the Ferric Uptake Regulator by Peptides Derived from Anti-FUR Peptide Aptamers: Coupled Theoretical and Experimental Approaches. ACS Chem. Biol. 9, 2779−2786. (19) Agriesti, F., Roncarati, D., Musiani, F., Del Campo, C., Iurlaro, M., Sparla, F., Ciurli, S., Danielli, A., and Scarlato, V. (2014) FeONFeOFF: the Helicobacter pylori Fur regulator commutates ironresponsive transcription by discriminative readout of opposed DNA grooves. Nucleic Acids Res. 42, 3138−3151. (20) Butcher, J., Sarvan, S., Brunzelle, J. S., Couture, J. F., and Stintzi, A. (2012) Structure and regulon of Campylobacter jejuni ferric uptake regulator Fur define apo-Fur regulation. Proc. Natl. Acad. Sci. U. S. A. 109, 10047−10052. (21) Delany, I., Spohn, G., Rappuoli, R., and Scarlato, V. (2003) An anti-repression Fur operator upstream of the promoter is required for iron-mediated transcriptional autoregulation in Helicobacter pylori. Mol. Microbiol. 50, 1329−1338. (22) Pohl, E., Haller, J. C., Mijovilovich, A., Meyer-Klaucke, W., Garman, E., and Vasil, M. L. (2003) Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator. Mol. Microbiol. 47, 903−915. (23) Dian, C., Vitale, S., Leonard, G. A., Bahlawane, C., Fauquant, C., Leduc, D., Muller, C., de Reuse, H., Michaud-Soret, I., and Terradot, L. (2011) The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites. Mol. Microbiol. 79, 1260−1275. (24) Sheikh, M. A., and Taylor, G. L. (2009) Crystal structure of the Vibrio cholerae ferric uptake regulator (Fur) reveals insights into metal co-ordination. Mol. Microbiol. 72, 1208−1220. (25) Deng, Z., Wang, Q., Liu, Z., Zhang, M., Machado, A. C., Chiu, T. P., Feng, C., Zhang, Q., Yu, L., Qi, L., Zheng, J., Wang, X., Huo, X., Qi, X., Li, X., Wu, W., Rohs, R., Li, Y., and Chen, Z. (2015) Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator. Nat. Commun. 6, 7642. (26) Pecqueur, L., D’Autreaux, B., Dupuy, J., Nicolet, Y., Jacquamet, L., Brutscher, B., Michaud-Soret, I., and Bersch, B. (2006) Structural changes of Escherichia coli ferric uptake regulator during metaldependent dimerization and activation explored by NMR and X-ray crystallography. J. Biol. Chem. 281, 21286−21295. (27) Krissinel, E., and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774−797. (28) Gilston, B. A., Wang, S., Marcus, M. D., Canalizo-Hernandez, M. A., Swindell, E. P., Xue, Y., Mondragon, A., and O’Halloran, T. V. (2014) Structural and mechanistic basis of zinc regulation across the E. coli Zur regulon. PLoS Biol. 12, e1001987. (29) D’Autreaux, B., Pecqueur, L., Gonzalez de Peredo, A., Diederix, R. E., Caux-Thang, C., Tabet, L., Bersch, B., Forest, E., and MichaudSoret, I. (2007) Reversible redox- and zinc-dependent dimerization of the Escherichia coli fur protein. Biochemistry 46, 1329−1342.

Mohamed Ould Abeih and Aynur Ahmadova for fruitful discussions and the iRTSV SEC-MALLS-RI-QUELS platform.

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ABBREVIATIONS Fur, ferric uptake regulator; ESI-TOF MS, electrospray ionization time-of-flight mass spectrometry; SAXS, small-angle X-ray scattering; EMSA, electrophoretic mobility shift assay; SEC-MALLS, size exclusion chromatography multiangle laser light scattering; ICP-AES, inductively coupled plasma-atomic emission spectrometry; ICP-MS, inductively coupled plasma mass spectrometry; BS3, bis(sulfosuccinimidyl) suberate; MOPS, 3-(N-morpholino)propanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; ICT, trypsin−chymotrypsin inhibitor; IPTG, isopropyl β-D-1-thiogalactopyranoside; PMSF, phenylmethanesulfonyl fluoride; DEAE, diethylaminoethyl; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; PDB, Protein Data Bank.

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REFERENCES

(1) Fillat, M. F. (2014) The FUR (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators. Arch. Biochem. Biophys. 546, 41−52. (2) Baichoo, N., Wang, T., Ye, R., and Helmann, J. D. (2002) Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon. Mol. Microbiol. 45, 1613−1629. (3) McHugh, J. P., Rodriguez-Quinones, F., Abdul-Tehrani, H., Svistunenko, D. A., Poole, R. K., Cooper, C. E., and Andrews, S. C. (2003) Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis. J. Biol. Chem. 278, 29478− 29486. (4) Abed, N., Bickle, M., Mari, B., Schapira, M., Sanjuan-Espana, R., Robbe Sermesant, K., Moncorge, O., Mouradian-Garcia, S., Barbry, P., Rudkin, B. B., Fauvarque, M. O., Michaud-Soret, I., and Colas, P. (2007) A comparative analysis of perturbations caused by a gene knock-out, a dominant negative allele, and a set of peptide aptamers. Mol. Cell. Proteomics 6, 2110−2121. (5) Seo, S. W., Kim, D., Latif, H., O’Brien, E. J., Szubin, R., and Palsson, B. O. (2014) Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli. Nat. Commun. 5, 4910. (6) Cornelis, P., Matthijs, S., and Van Oeffelen, L. (2009) Iron uptake regulation in Pseudomonas aeruginosa. BioMetals 22, 15−22. (7) Vasil, M. L., and Ochsner, U. A. (1999) The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol. Microbiol. 34, 399−413. (8) Balasubramanian, D., Schneper, L., Kumari, H., and Mathee, K. (2013) A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res. 41, 1−20. (9) Gao, H., Zhou, D., Li, Y., Guo, Z., Han, Y., Song, Y., Zhai, J., Du, Z., Wang, X., Lu, J., and Yang, R. (2008) The iron-responsive Fur regulon in Yersinia pestis. Journal of bacteriology 190, 3063−3075. (10) Zhou, D., Qin, L., Han, Y., Qiu, J., Chen, Z., Li, B., Song, Y., Wang, J., Guo, Z., Zhai, J., Du, Z., Wang, X., and Yang, R. (2006) Global analysis of iron assimilation and fur regulation in Yersinia pestis. FEMS Microbiol. Lett. 258, 9−17. (11) Olakanmi, O., Gunn, J. S., Su, S., Soni, S., Hassett, D. J., and Britigan, B. E. (2010) Gallium disrupts iron uptake by intracellular and extracellular Francisella strains and exhibits therapeutic efficacy in a murine pulmonary infection model. Antimicrob. Agents Chemother. 54, 244−253. (12) Huja, S., Oren, Y., Biran, D., Meyer, S., Dobrindt, U., Bernhard, J., Becher, D., Hecker, M., Sorek, R., and Ron, E. Z. (2014) Fur is the master regulator of the extraintestinal pathogenic Escherichia coli response to serum. mBio 5, e01460-14. (13) Litwin, C. M., and Calderwood, S. B. (1993) Role of iron in regulation of virulence genes. Clin. Microbiol. Rev. 6, 137−149. L


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Biochemistry

Crystal structure of the apo-PerR-Zn protein from Bacillus subtilis. Mol. Microbiol. 61, 1211−1219. (50) Lostao, A., Peleato, M. L., Gomez-Moreno, C., and Fillat, M. F. (2010) Oligomerization properties of FurA from the cyanobacterium Anabaena sp. PCC 7120: direct visualization by in situ atomic force microscopy under different redox conditions. Biochim. Biophys. Acta, Proteins Proteomics 1804, 1723−1729. (51) Bai, E., Rosell, F. I., Lige, B., Mauk, M. R., Lelj-Garolla, B., Moore, G. R., and Mauk, A. G. (2006) Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa. Biochem. J. 400, 385−392. (52) Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: visual molecular dynamics. J. Mol. Graphics 14, 27−38. (53) Kikhney, A. G., and Svergun, D. I. (2015) A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Lett. 589, 2570−2577. (54) Schneidman-Duhovny, D., Kim, S. J., and Sali, A. (2012) Integrative structural modeling with small angle X-ray scattering profiles. BMC Struct. Biol. 12, 17. (55) Lavrrar, J. L., Christoffersen, C. A., and McIntosh, M. A. (2002) Fur-DNA interactions at the bidirectional fepDGC-entS promoter region in Escherichia coli. J. Mol. Biol. 322, 983−995. (56) Pallares, M. C., Marcuello, C., Botello-Morte, L., Gonzalez, A., Fillat, M. F., and Lostao, A. (2014) Sequential binding of FurA from Anabaena sp. PCC 7120 to iron boxes: exploring regulation at the nanoscale. Biochim. Biophys. Acta, Proteins Proteomics 1844, 623−631. (57) Lee, J. W., and Helmann, J. D. (2007) Functional specialization within the Fur family of metalloregulators. BioMetals 20, 485−499.

(30) Vitale, S., Fauquant, C., Lascoux, D., Schauer, K., Saint-Pierre, C., and Michaud-Soret, I. (2009) A ZnS(4) structural zinc site in the Helicobacter pylori ferric uptake regulator. Biochemistry 48, 5582−5591. (31) Gonzalez de Peredo, A., Saint-Pierre, C., Latour, J. M., MichaudSoret, I., and Forest, E. (2001) Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor. J. Mol. Biol. 310, 83−91. (32) Baichoo, N., and Helmann, J. D. (2002) Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence. Journal of bacteriology 184, 5826−5832. (33) Escolar, L., Perez-Martin, J., and de Lorenzo, V. (1998) Binding of the fur (ferric uptake regulator) repressor of Escherichia coli to arrays of the GATAAT sequence. J. Mol. Biol. 283, 537−547. (34) Bagg, A., and Neilands, J. B. (1987) Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry 26, 5471−5477. (35) Mills, S. A., and Marletta, M. A. (2005) Metal binding characteristics and role of iron oxidation in the ferric uptake regulator from Escherichia coli. Biochemistry 44, 13553−13559. (36) Ochsner, U. A., Vasil, A. I., and Vasil, M. L. (1995) Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. J. Bacteriol. 177, 7194−7201. (37) Petoukhov, M. V., Franke, D., Shkumatov, A. V., Tria, G., Kikhney, A. G., Gajda, M., Gorba, C., Mertens, H. D., Konarev, P. V., and Svergun, D. I. (2012) New developments in the program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45, 342− 350. (38) Perard, J., Leyrat, C., Baudin, F., Drouet, E., and Jamin, M. (2013) Structure of the full-length HCV IRES in solution. Nat. Commun. 4, 1612. (39) Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J., and Svergun, D. I. (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277−1282. (40) Franke, D., and Svergun, D. I. (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Crystallogr. 42, 342−346. (41) Volkov, V. V., and Svergun, D. I. (2003) Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860−864. (42) Svergun, D., Barberato, C., and Koch, M. H. J. (1995) CRYSOL - A program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768− 773. (43) Kozin, M. B., and Svergun, D. I. (2001) Automated matching of high- and low-resolution structural models. J. Appl. Crystallogr. 34, 33− 41. (44) Petoukhov, M. V., and Svergun, D. I. (2005) Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys. J. 89, 1237−1250. (45) Le Cam, E., Frechon, D., Barray, M., Fourcade, A., and Delain, E. (1994) Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes. Proc. Natl. Acad. Sci. U. S. A. 91, 11816−11820. (46) Althaus, E. W., Outten, C. E., Olson, K. E., Cao, H., and O’Halloran, T. V. (1999) The ferric uptake regulation (Fur) repressor is a zinc metalloprotein. Biochemistry 38, 6559−6569. (47) An, Y. J., Ahn, B. E., Han, A. R., Kim, H. M., Chung, K. M., Shin, J. H., Cho, Y. B., Roe, J. H., and Cha, S. S. (2009) Structural basis for the specialization of Nur, a nickel-specific Fur homolog, in metal sensing and DNA recognition. Nucleic Acids Res. 37, 3442−3451. (48) Bellini, P., and Hemmings, A. M. (2006) In vitro characterization of a bacterial manganese uptake regulator of the fur superfamily. Biochemistry 45, 2686−2698. (49) Traore, D. A., El Ghazouani, A., Ilango, S., Dupuy, J., Jacquamet, L., Ferrer, J. L., Caux-Thang, C., Duarte, V., and Latour, J. M. (2006) M


Quaternary Structure of Fur Proteins, a New ... - ACS Publications

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