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• W3036794625 abstract "The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum. Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr–ICE complex. The addition of EDTA to the Irr–ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr–ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29–mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr–ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29. The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum. Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr–ICE complex. The addition of EDTA to the Irr–ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr–ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29–mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr–ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29. Iron is a necessary micronutrient for all organisms, as it plays a key role in cellular functions, acting as a chemical catalyst in addition to influencing electron transport, oxygen transport, and mitochondrial energy generation (1Aisen P. Enns C. Wessling-Resnick M. Chemistry and biology of eukaryotic iron metabolism.Int. J. Biochem. Cell Biol. 2001; 33 (11470229): 940-95910.1016/S1357-2725(01)00063-2Crossref PubMed Scopus (603) Google Scholar). However, excessive intracellular iron is cytotoxic; this is because of its ability to convert molecular oxygen into reactive oxygen species (ROS) that can cause damage to cellular components. Because of this, iron homeostasis is strictly regulated, and iron acquisition, storage, and consumption are balanced to maintain a constant cellular iron level (2Wang J. Pantopoulos K. Regulation of cellular iron metabolism.Biochem. J. 2011; 434 (21348856): 365-38110.1042/BJ20101825Crossref PubMed Scopus (667) Google Scholar). Defects in this regulation lead to severe diseases, such as anemia, pica, liver cirrhosis, and neurological deficits. Despite its biological importance, the molecular control mechanisms of iron homeostasis are still undiscovered, and functional characterization of the essential regulatory proteins is also still quite limited. To investigate the molecular mechanisms of cellular iron homeostasis, Bradyrhizobium japonicum, a nitrogen-fixing bacterium that exists as an endosymbiont in root nodule of plants, is used as the typical model system. In this species of bacteria, various types of iron-containing proteins or heme, which is an iron-protoporphyrin IX complex, are used to support nitrogen fixation. The nitrogenase complex, which comprises more than 10% of cellular proteins in nitrogen-fixing bacteria, such as some cyanobacteria and azotobacteraceae, includes 30–34 iron ions per molecule (3Eady R.R. Structure−function relationships of alternative nitrogenases.Chem. Rev. 1996; 96 (11848850): 3013-303010.1021/cr950057hCrossref PubMed Scopus (693) Google Scholar). B. japonicum is known to possess a global regulator protein, known as the iron response regulator (Irr), which regulates iron homeostasis and associated metabolic processes (4Rudolph G. Semini G. Hauser F. Lindemann A. Friberg M. Hennecke H. Fischer H.M. The iron control element, acting in positive and negative control of iron-regulated Bradyrhizobium japonicum genes, is a target for the Irr protein.J. Bacteriol. 2006; 188 (16385063): 733-74410.1128/JB.188.2.733-744.2006Crossref PubMed Scopus (62) Google Scholar, 5Yang J. Sangwan I. Lindemann A. Hauser F. Hennecke H. Fischer H.M. O'Brian M.R. Bradyrhizobium japonicum senses iron through the status of haem to regulate iron homeostasis and metabolism.Mol. Microbiol. 2006; 60 (16573691): 427-43710.1111/j.1365-2958.2006.05101.xCrossref PubMed Scopus (78) Google Scholar, 6Ishikawa H. Nakagaki M. Bamba A. Uchida T. Hori H. O'Brian M.R. Iwai K. Ishimori K. Unusual heme binding in the bacterial iron response regulator protein: spectral characterization of heme binding to the heme regulatory motif.Biochemistry. 2011; 50 (21192735): 1016-102210.1021/bi101895rCrossref PubMed Scopus (37) Google Scholar). When cells are grown under iron-limiting conditions, Irr functions as a transcriptional repressor of the hemB gene, which encodes one of the heme biosynthetic enzymes, δ-aminoleuvulinic acid dehydratase; this prevents accumulation of a cytotoxic heme precursor, protoporphyrin IX. Under iron-replete conditions, Irr is thought to degrade, allowing hemB transcription to resume and promoting heme biosynthesis (7Kobayashi K. Nakagaki M. Ishikawa H. Iwai K. O'Brian M.R. Ishimori K. Redox-dependent dynamics in heme-bound bacterial Iron response regulator (Irr) protein.Biochemistry. 2016; 55 (27379473): 4047-405410.1021/acs.biochem.6b00512Crossref PubMed Scopus (14) Google Scholar, 8Qi Z. Hamza I. O'Brian M.R. Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein.Proc. Nat. Acad. Sci. U S A. 1999; 96 (10557272): 13056-1306110.1073/pnas.96.23.13056Crossref PubMed Scopus (145) Google Scholar, 9Yang J. Ishimori K. O'Brian M.R. Two heme binding sites are involved in the regulated degradation of the bacterial iron response regulator (Irr) protein.J. Biol. Chem. 2005; 280 (15613477): 7671-767610.1074/jbc.M411664200Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Using a yeast one-hybrid system, the in vivo interaction of Irr with the iron control element (ICE), which is an incomplete inversed repeat cis-performing AT-rich DNA region, has been reported (10Botello-Morte L. Pellicer S. Sein-Echaluce V.C. Contreras L.M. Neira J.L. Abian O. Velazquez-Campoy A. Peleato M.L. Fillat M.F. Bes M.T. Cysteine mutational studies provide insight into a thiol-based redox switch mechanism of metal and DNA binding in FurA from Anabaena sp. PCC 7120.Antioxid. Redox. Signal. 2016; 24 (26414804): 173-18510.1089/ars.2014.6175Crossref PubMed Google Scholar). This motif is located upstream of Irr- and other iron-regulated genes (4Rudolph G. Semini G. Hauser F. Lindemann A. Friberg M. Hennecke H. Fischer H.M. The iron control element, acting in positive and negative control of iron-regulated Bradyrhizobium japonicum genes, is a target for the Irr protein.J. Bacteriol. 2006; 188 (16385063): 733-74410.1128/JB.188.2.733-744.2006Crossref PubMed Scopus (62) Google Scholar, 11Rodionov D.A. Gelfand M.S. Todd J.D. Curson A.R. Johnston A.W. Computational reconstruction of iron- and manganese-responsive transcriptional networks in alpha-proteobacteria.PLoS Comput. Biol. 2006; 2 (17173478): e16310.1371/journal.pcbi.0020163Crossref PubMed Scopus (121) Google Scholar), and two B. japonicum genes that contain ICE-like motifs within their promoter regions were previously shown to be negatively regulated by Irr (4Rudolph G. Semini G. Hauser F. Lindemann A. Friberg M. Hennecke H. Fischer H.M. The iron control element, acting in positive and negative control of iron-regulated Bradyrhizobium japonicum genes, is a target for the Irr protein.J. Bacteriol. 2006; 188 (16385063): 733-74410.1128/JB.188.2.733-744.2006Crossref PubMed Scopus (62) Google Scholar, 11Rodionov D.A. Gelfand M.S. Todd J.D. Curson A.R. Johnston A.W. Computational reconstruction of iron- and manganese-responsive transcriptional networks in alpha-proteobacteria.PLoS Comput. Biol. 2006; 2 (17173478): e16310.1371/journal.pcbi.0020163Crossref PubMed Scopus (121) Google Scholar, 12Sangwan I. Small S.K. O'Brian M.R. The Bradyrhizobium japonicum Irr protein is a transcriptional repressor with high-affinity DNA-binding activity.J. Bacteriol. 2008; 190 (18539736): 5172-517710.1128/JB.00495-08Crossref PubMed Scopus (23) Google Scholar). By occupying the promoter-binding site in iron-limited cells, Irr represses gene transcription (12Sangwan I. Small S.K. O'Brian M.R. The Bradyrhizobium japonicum Irr protein is a transcriptional repressor with high-affinity DNA-binding activity.J. Bacteriol. 2008; 190 (18539736): 5172-517710.1128/JB.00495-08Crossref PubMed Scopus (23) Google Scholar). The mRNA transcripts of Irr-regulated genes as well as the promoter occupancy by Irr were only seen under iron-depleted conditions, indicating that the interactions between Irr and the ICE-like motif depend on the iron status of the cells. Interestingly, interactions between Irr and the ICE-like motif are also dependent on manganese (Mn2+) (12Sangwan I. Small S.K. O'Brian M.R. The Bradyrhizobium japonicum Irr protein is a transcriptional repressor with high-affinity DNA-binding activity.J. Bacteriol. 2008; 190 (18539736): 5172-517710.1128/JB.00495-08Crossref PubMed Scopus (23) Google Scholar). In media supplemented with Mn2+, genes including ICE-like motifs were strongly regulated by iron, whereas this iron-dependent regulation was lost under low-manganese conditions. Thus, Mn2+ contributes to the expression of genes including ICE-like motifs, suggesting that the binding of Irr to the ICE-like motifs requires Mn2+ binding to Irr. Despite this evidence, Mn2+ binding to purified Irr protein has not yet been confirmed. To detect the iron status of the intracellular environment, Irr has been found to utilize heme as a regulatory molecule (8Qi Z. Hamza I. O'Brian M.R. Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein.Proc. Nat. Acad. Sci. U S A. 1999; 96 (10557272): 13056-1306110.1073/pnas.96.23.13056Crossref PubMed Scopus (145) Google Scholar). Irr degrades in iron-replete cells, restarting transcription of the genes it was repressing; this degradation is thought to be induced by the direct binding of heme. Irr has two heme binding sites, the first of which is Cys29 in the heme-regulatory motif (HRM), which contains a short consensus sequence composed of Cys and Pro and is found in many heme-regulated proteins, such as heme-regulated inhibitor kinases (13Chen J.-J. Londo I.M. Regulation of protein synthesis by heme-regulated eIF-2a kinase.Trends Biochem. Sci. 1995; 20: 105-10810.1016/S0968-0004(00)88975-6Abstract Full Text PDF PubMed Scopus (265) Google Scholar), heme activator proteins (14Zhang L. Guarente L. Heme binds to a short sequence that serves a regulatory function in diverse proteins.EMBO J. 1995; 14 (7835342): 313-32010.1002/j.1460-2075.1995.tb07005.xCrossref PubMed Scopus (253) Google Scholar), and circadian factors (15Yang J. Kim K.D. Lucas A. Drahos K.E. Santos C.S. Mury S.P. Capelluto D.G. Finkielstein C.V. A novel heme-regulatory motif mediates heme-dependent degradation of the circadian factor period 2.Mol. Cell. Biol. 2008; 28 (18505821): 4697-471110.1128/MCB.00236-08Crossref PubMed Scopus (81) Google Scholar). The other heme binding site is in the His-cluster region far from the HRM region. Heme binding to these sites in Irr induces the oxidative modification of the protein moiety, leading to Irr degradation (9Yang J. Ishimori K. O'Brian M.R. Two heme binding sites are involved in the regulated degradation of the bacterial iron response regulator (Irr) protein.J. Biol. Chem. 2005; 280 (15613477): 7671-767610.1074/jbc.M411664200Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). It should be noted here that heme binding to Irr and the successive oxidative modification are inhibited in the presence of Mn2+, and that the addition of Mn2+ to heme-bound Irr releases the heme from the protein (16Puri S. Hohle T.H. O'Brian M.R. Control of bacterial iron homeostasis by manganese.Proc. Natl. Acad. Sci. U S A. 2010; 107 (20498065): 10691-1069510.1073/pnas.1002342107Crossref PubMed Scopus (47) Google Scholar). In a heme-deficient mutant strain, ΔhemAH, the addition of Mn2+ suppressed the degradation of Irr under the moderated heme concentrations (200 nm); however, cells grown under conditions with large excesses of heme (5 mm) did not accumulate Irr in the presence of Mn2+, suggesting that the effectiveness of Mn2+ on preventing Irr degradation depends on the heme concentration (16Puri S. Hohle T.H. O'Brian M.R. Control of bacterial iron homeostasis by manganese.Proc. Natl. Acad. Sci. U S A. 2010; 107 (20498065): 10691-1069510.1073/pnas.1002342107Crossref PubMed Scopus (47) Google Scholar). Therefore, Mn2+ is likely a competitive inhibitor of heme binding to the Irr protein; however, the binding site of Mn2+ in Irr has not yet been determined. In this paper, we confirmed that the binding of Mn2+ is essential for Irr to bind to the ICE-like motif, using purified proteins and a fluorophore-labeled ICE-like oligomer DNA. The Mn2+ binding site was found to be in the His-cluster region of Irr, which overlaps one of the heme binding sites in Irr. Heme binding to Irr likely induces the release of Mn2+ from the binding site in the His-cluster region, dissociating Irr from the ICE-like motif. In addition, the conformational changes associated with heme binding to another heme binding site, Cys29 in the HRM region located near the ICE binding site, also inhibit binding to ICE. Although heme bound to Irr would trigger oxidative modification of the protein, which has been thought to be essential for the dissociation of Irr from the ICE-like motif, the heme-induced oxidative modification would be initiated after the dissociation of Irr from the target gene to protect the target gene from the ROS produced by heme-bound Irr. Because of this, the oxidative modification and protein degradation would inhibit the rebinding of Irr to the target gene and/or the formation of cytotoxic free heme. Previous studies have reported that the promoter occupancy for genes including ICE-like motifs was suppressed under Mn2+-limited conditions, suggesting that Mn2+ binding is essential for the binding of ICE to Irr (16Puri S. Hohle T.H. O'Brian M.R. Control of bacterial iron homeostasis by manganese.Proc. Natl. Acad. Sci. U S A. 2010; 107 (20498065): 10691-1069510.1073/pnas.1002342107Crossref PubMed Scopus (47) Google Scholar). To confirm this, a 6-carboxyfluorescein-modified DNA oligomer (FAM-ICE) was prepared as a model for the ICE-like motif, and the formation of the complex between Irr and FAM-ICE was monitored using fluorescence anisotropy. In the presence of Mn2+, fluorescence anisotropy of FAM-ICE was increased by the addition of Irr, corresponding to complex formation (Fig. 1, green). In the absence of Mn2+, this increase was much smaller than that in the presence of Mn2+ (Fig. 1, orange), indicating that binding of the ICE-like motif to Mn2+-free Irr was nonspecific and Mn2+ is essential for Irr to bind to the ICE-like motif. The Mn2+-mediated binding of Irr to ICE was also confirmed by the addition of a metal chelator, EDTA, to the Irr-ICE complex. When EDTA was added to the Irr-ICE complex, fluorescence anisotropy of FAM-ICE was decreased (Fig. 2A), indicating that removal of Mn2+ led to dissociation of Irr from FAM-ICE. The inductively coupled plasma-optical emission spectrometer (ICP-OES) measurements confirmed the complete dissociation of Mn2+ from Irr after the EDTA treatment (Table 1). Supporting this, in the presence of FAM-ICE, the addition of Mn2+ to Mn2+-free Irr enhanced the fluorescence anisotropy, indicating that Mn2+ restored the binding ability of Irr (Fig. 2B). This type of essential role for metal binding in the binding activity to target DNA for transcriptional regulators is one of the characteristics of metal-responsive transcriptional regulators, including Fur. The apo (metal-free) state of Magnetospirillum gryphiswaldense MSR-1 Fur (MgFur) is unable to bind to the target DNA site; however, metal binding results in the restoration of this activity (17Deng 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. et al.Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator.Nat. Commun. 2015; 6 (26134419): 764210.1038/ncomms8642Crossref PubMed Scopus (77) Google Scholar, 18Perard J. Coves J. Castellan M. Solard C. Savard M. Miras R. Galop S. Signor L. Crouzy S. Michaud-Soret I. de Rosny E. Quaternary structure of Fur proteins, a new subfamily of tetrameric proteins.Biochemistry. 2016; 55 (26886069): 1503-151510.1021/acs.biochem.5b01061Crossref PubMed Scopus (18) Google Scholar). The binding of two metal ions to apo MgFur stabilizes the hinge conformation associated with conversion of the DNA binding domain into its DNA binding state.Table 1Mn2+ binding property of IrrProteinMn2+ content (mol/monomer)Holo-Irr1.82 ± 0.32Apo-Irr<0.1Heme–Irr complex<0.1C29A2.08 ± 0.23H63A<0.1H117-119A0.44 ± 0.07 Open table in a new tab To determine the Mn2+-binding affinity of Irr, competition experiments with a fluorimetric dye, Mag-fura-2 (19Golynskiy M.V. Gunderson W.A. Hendrich M.P. Cohen S.M. Metal binding studies and EPR spectroscopy of the manganese transport regulator MntR.Biochemistry. 2006; 45 (17176058): 15359-1537210.1021/bi0607406Crossref PubMed Scopus (87) Google Scholar), were carried out. However, the competition experiments required excess amounts of Mn2+-free Irr to that of the dye, and rather higher concentrations of Mn2+-free Irr (>10 μm) resulted in less accurate estimation of Kd(Mn2+), varying from the nm to µm range because of the structural instability of Irr under the high-concentration range. The Mn2+ binding affinity of Irr seemed rather high compared with those of other Fur family proteins, such as Escherichia coli Fur (EcFur) [Kd(Mn2+), 24 μm] (20Mills S.A. Marletta M.A. Metal binding characteristics and role of iron oxidation in the ferric uptake regulator from Escherichia coli.Biochemistry. 2005; 44 (16216078): 13553-1355910.1021/bi0507579Crossref PubMed Scopus (88) Google Scholar) and manganese transport regulator (MntR) [Kd(Mn2+), 50 and 160 μm] (19Golynskiy M.V. Gunderson W.A. Hendrich M.P. Cohen S.M. Metal binding studies and EPR spectroscopy of the manganese transport regulator MntR.Biochemistry. 2006; 45 (17176058): 15359-1537210.1021/bi0607406Crossref PubMed Scopus (87) Google Scholar), which would be close to the heme binding affinity of Irr [Kd(heme), 1 and 80 nm for two heme binding sites] (21Qi Z. O'Brian M.R. Interaction between the bacterial iron response regulator and ferrochelatase mediates genetic control of heme biosynthesis.Mol. Cell. 2002; 9 (11804594): 155-16210.1016/S1097-2765(01)00431-2Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The comparable Mn2+ affinity to the heme binding affinity of Irr was supported by the competitive binding between heme and Mn2+. As previously reported (16Puri S. Hohle T.H. O'Brian M.R. Control of bacterial iron homeostasis by manganese.Proc. Natl. Acad. Sci. U S A. 2010; 107 (20498065): 10691-1069510.1073/pnas.1002342107Crossref PubMed Scopus (47) Google Scholar), the addition of 20 μm Mn2+ to 4 μm heme-bound Irr completely displaced heme from Irr. The metal binding in Fur superfamily proteins also affects oligomerization status to regulate the binding affinity to target DNA (22D'Autréaux B. Pecqueur L. Gonzalez de Peredo A. Diederix R.E.M. Caux-Thang C. Tabet L. Bersch B. Forest E. Michaud-Soret I. Reversible redox- and zinc-dependent dimerization of the Escherichia coli Fur protein.Biochemistry. 2007; 46 (17260962): 1329-134210.1021/bi061636rCrossref PubMed Scopus (37) Google Scholar). We examined the oligomerization status of Irr upon the binding of Mn2+ using size-exclusion column chromatography. Fig. 3A shows chromatograms for Irr in the absence and presence of Mn2+; a major peak appeared at 14.7 ml, corresponding to an estimated molecular mass of 40 kDa (Fig. 3B). As the calculated molecular mass of monomeric Irr is 18 kDa, this result indicates that the Mn2+-bound and Mn2+-free Irr forms dimers. Irr, therefore, forms a stable dimeric state, which is similar to that seen in the group including EcFur (23Pecqueur L. D'Autreaux B. Dupuy J. Nicolet Y. Jacquamet L. Brutscher B. Michaud-Soret I. Bersch B. Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and X-ray crystallography.J. Biol. Chem. 2006; 281 (16690618): 21286-2129510.1074/jbc.M601278200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and Helicobacter pylori Fur (HpFur) (24Dian C. Vitale S. Leonard G.A. Bahlawane C. Fauquant C. Leduc D. Muller C. de Reuse H. Michaud-Soret I. Terradot L. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites.Mol. Microbiol. 2011; 79 (21208302): 1260-127510.1111/j.1365-2958.2010.07517.xCrossref PubMed Scopus (95) Google Scholar). In the chromatogram of Mn2+-free Irr, two additional peaks around 12.4 and 17.3 ml were observed, indicating the appearance of high- and low-molecular-mass species of Irr in the solution without Mn2+. In the presence of EDTA, Mn2+-free Irr was unstable and gradually formed precipitates under the concentrated conditions. The appearance of the high-molecular-mass species suggested the formation of oligomers by partially denatured Irr during gel chromatography. On the other hand, the reasons for the appearance of the low-molecular-mass species are unclear. Because the apparent molecular mass of the low-molecular-mass species was less than that of monomeric Irr, the interaction between partially denatured Irr and gels in column may retard the moving of the protein, resulting in the slow-moving species in the chromatogram. That the longer incubation with EDTA before applying the column enhanced the intensity of the low-molecular mass species also supports the gradual denaturation of Irr in the absence of Mn2+. A trough around 18 ml also would be because of perturbations of the column conditions induced by nonspecific interactions between partially denatured Irr and gels in the column. The Fur superfamily has two metal binding sites (25Pohl E. Haller J.C. Mijovilovich A. Meyer-Klaucke W. Garman E. Vasil M.L. Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator.Mol. Microbiol. 2003; 47 (12581348): 903-91510.1046/j.1365-2958.2003.03337.xCrossref PubMed Scopus (261) Google Scholar). Site 1 is found in the C-terminal dimerization domain, and site 2 is found in the histidine-rich hinge region, known as the His-cluster region, between the DNA binding and dimerization domains. To confirm the metal binding to purified Irr, we utilized ICP-OES, which showed that Irr incubated with 0.1 mm MnCl2 contained 1.82 ± 0.32 mol Mn/mol protein (Table 1). Other metals were present at less than 0.1 mol metal/mol protein. Although we found that Irr as isolated bound Zn2+, the binding affinity of Zn2+ to Irr was much lower than that of Mn2+, and incubation of purified Irr in a solution containing 0.1 mm MnCl2 completely replaced Zn2+ with Mn2+. The functional significance of the binding of Zn2+ in Irr as isolated is unclear, and considering that the expression level of Irr and the ICE binding depended only on Mn2+, not on Zn2+, in B. japonicum (16Puri S. Hohle T.H. O'Brian M.R. Control of bacterial iron homeostasis by manganese.Proc. Natl. Acad. Sci. U S A. 2010; 107 (20498065): 10691-1069510.1073/pnas.1002342107Crossref PubMed Scopus (47) Google Scholar), and overexpressed Bacillus subtilis Fur (BsFur) incorporated Zn2+ into the metal binding site (26Bsat N. Helmann J.D. Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo.J. Bacteriol. 1999; 181 (10400588): 4299-430710.1128/JB.181.14.4299-4307.1999Crossref PubMed Google Scholar), the Mn2+ binding sites of newly synthesized Irr in cells was occupied by Zn2+ because of the overexpression of Irr and the abundance of Zn2+ in E. coli cells (27Outten C.E. O'Halloran T.V. Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis.Science. 2001; 292 (11397910): 2488-249210.1126/science.1060331Crossref PubMed Scopus (1158) Google Scholar). MgFur, one of the Fur proteins possessing two metal binding sites, similarly has two metal binding sites; site 1 is in the dimerization domain (green), and site 2 is in the histidine-rich hinge region (gray) near the DNA binding domain (blue) (Fig. 4). The amino acid residues His33, Glu81, His90, and Glu101, which constitute the histidine-rich hinge region in MgFur, are conserved in Irr (17Deng 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. et al.Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator.Nat. Commun. 2015; 6 (26134419): 764210.1038/ncomms8642Crossref PubMed Scopus (77) Google Scholar), corresponding to His63, Asp111, His119, and Asp130, respectively. Most of the amino acid residues forming these two metal binding sites are conserved across all Fur superfamily proteins. For Bacillus subtilis PerR (BsPerR), which is one of the Fur superfamily proteins and an iron-dependent peroxide sensor, functional characterization using mutant proteins (24Dian C. Vitale S. Leonard G.A. Bahlawane C. Fauquant C. Leduc D. Muller C. de Reuse H. Michaud-Soret I. Terradot L. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites.Mol. Microbiol. 2011; 79 (21208302): 1260-127510.1111/j.1365-2958.2010.07517.xCrossref PubMed Scopus (95) Google Scholar, 28Lee J.-W. Helmann J.D. Functional specialization within the Fur family of metalloregulators.Biometals. 2007; 20 (17216355): 485-49910.1007/s10534-006-9070-7Crossref PubMed Scopus (328) Google Scholar), crystal structures (29Jacquamet L. Traoré D.A.K. Ferrer J.L. Proux O. Testemale D. Hazemann J.L. Nazarenko E. El Ghazouani A. Caux-Thang C. Duarte V. Latour J.M. Structural characterization of the active form of PerR: insights into the metal-induced activation of PerR and Fur proteins for DNA binding.Mol. Microbiol. 2009; 73 (19508285): 20-3110.1111/j.1365-2958.2009.06753.xCrossref PubMed Scopus (87) Google Scholar), and in silico study of free energy calculations, including molecular dynamics (30Ahmad R. Brandsdal B.O. Michaud-Soret I. Willassen N.P. Ferric uptake regulator protein: binding free energy calculations and per-residue free energy decomposition.Proteins. 2009; 75 (18831042): 373-38610.1002/prot.22247Crossref PubMed Scopus (34) Google Scholar), revealed that metal occupancy at site 2 is necessary for the conformational changes required for binding to the operator sequence of genes. Although the metal binding sites in Irr have not yet been identified, the consensus amino acid sequence for site 2 in BsPerR is completely conserved in this protein, suggesting that Mn2+ binds to site 2 in a similar manner in Irr. To confirm Mn2+ binding to site 2 in Irr, we mutated amino acid residues constituting this site (H63A and H117-119A). These His-cluster mutants were quite unstable at higher concentrations (>10 μm), and, as illustrated in Fig. 3A, high- and low-molecular-weight species were detected in the gel chromatogram, as found for Mn2+-free Irr. Longer incubation of these mutants under high concentrations before applying the column resulted in aggregation and precipitation. In the His117-119A mutant, we detected substantial amounts of high-molecular-weight species, probably because of the aggregation of the partially denatured proteins. The destabilized structures of these mutants also suggest that the His-cluster region is the keystone for the protein structure of Irr. On the other hand, no aggregation or precipitat" @default.
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