Functional Complementation Studies Reveal Different Interaction

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Functional complementation studies reveal different interaction partners of Escherichia coli IscS and human NFS1 Martin Bühning, Martin Friemel, and Silke Leimkühler Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00627 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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Biochemistry

Functional complementation studies reveal different interaction partners of Escherichia coli IscS and human NFS1 Martin Bühning, Martin Friemel and Silke Leimkühler* From the Institute of Biochemistry and Biology, University of Potsdam, D-14476 Potsdam, Germany

*

Corresponding Author

Silke Leimkühler; Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany; Tel.: +49-331977-5603; Fax: +49-331-977-5128; E-mail: [email protected]

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ABBREVIATIONS Acyl carrier protein (ACP) cytosolic iron-sulfur cluster assembly (CIA) iron-sulfur (FeS) ferredoxin (FDX) frataxin (FXN) molybdenum cofactor (Moco) cyclic pyranopterin monophosphate (cPMP) molyptopterin (MPT) cytosine dinucleotide (MCD) bis-MPT guanine dinucleotide (bis-MGD) nitrate reductase (NR) succinate dehydrogenase (SDH) trimethyl-N-oxide reductase (TMAOR)


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ABSTRACT The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways have generally an Lcysteine desulfurase as initial sulfur-mobilizing enzyme in common, which serves as sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron-sulfur (FeS) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human L-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share an amino acid sequence identity of about 60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of FeS clusters. Additionally, NFS1 is also located in lower amounts in the cytosol with a role in Moco biosynthesis and mcm5s2U thiomodifications of nucleosides in tRNA. Conclusively, NFS1 and IscS were shown to have different interaction partners in their respective organism. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in sulfur transfer to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in FeS cluster assembly like IscU, but not with proteins for tRNA thiomodifications or Moco biosynthesis. Further we show, that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was additionally present.


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INTRODUCTION Sulfur containing cofactors and their biological importance were discovered more than a century ago (reviewed in1). The trafficking and delivery of sulfur to these cofactors and nucleosides is in general a highly regulated process, which occurs by complex sulfur relay systems involving numerous proteins that accept and transfer sulfur atoms.2,3 In the cell, Lcysteine is used in most cases as initial sulfur source. The major enzymes involved in the mobilization of sulfur from L-cysteine are L-cysteine desulfurases.3-5 These enzymes catalyze the formation of a persulfide group (R-S-SH) on specific conserved cysteine residues, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like FeS clusters, thiamine, biotin, lipoic acid, Moco, and thiolated nucleosides in tRNA. While all these molecules can be synthesized de novo in bacteria, in eukaryotes solely FeS clusters, lipoic acid, Moco and thionucleosides in tRNA are synthesized de novo.3,6-9 All sulfur transfer pathways have generally an

L-cysteine

desulfurase in common, in

prokaryotes the IscS protein or in eukaryotes NFS1.10-12 L-cysteine desulfurases are pyridoxalphosphate-containing homodimers, which decompose

L-cysteine

to

L-alanine

and sulfane

sulfur.13 The persulfide formed on a conserved cysteine residue can be transferred to acceptor proteins, which are in general specific for each sulfur-containing molecule. IscS in Escherichia coli was shown to interact with a number of acceptor proteins for delivery of sulfur including the involvement of (i) IscU, CyaY, Fdx and IscX for FeS cluster formation, (ii) TusA for either the (c)mnm5s2U34 modifications of tRNA or the biosynthesis of the molybdenum cofactor (Moco), and (iii) ThiI for the synthesis of thiamine or the s4U8 modification of tRNA.3,9 Different binding sites for some of these molecules were mapped on E. coli IscS,14 ensuring either the simultaneous binding or a competitive binding on overlapping binding sites.


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For the assembly of FeS clusters in E. coli the primary sulfur acceptor is the FeS cluster scaffold proteins IscU (Figure 1A).15

Figure 1. Model for the roles of IscS in bacteria and NFS in humans. (A) For FeS cluster assembly in E. coli, IscS interacts with IscU, CyaY, Fdx and IscX. Further, IscS and TusA connect the sulfur transfer pathway for the synthesis of Moco and for (c)mnm5s2U34 modified nucleosides in tRNA. IscS also interacts with ThiI, transferring the sulfur either to the thiamine biosynthetic pathway, or for the synthesis of s4U8 modified nucleosides in tRNA. (B) Localization of Moco biosynthesis, FeS cluster biosynthesis and tRNA thiolation in humans. Shown is a scheme of the biosynthetic pathway for Moco biosynthesis, FeS cluster biosynthesis and tRNA thiolation in humans. In humans, mitochondria present the main compartment for FeS cluster assembly (the CIA pathway is not shown). Here, NFS1 forms a complex with ISD11, ISCU, Frataxin (FXN) and ACP. In mitochondria, NFS1 also transfers the sulfur to TUM1, a protein involved in the τm5s2U34 formation of mitochondrial tRNAs. NFS1 is also present in the cytosol transferring the sulfur to the C-terminal rhodanese like domain of


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MOCS3, a protein shared between Moco biosynthesis and cytosolic mcm5s2U34 tRNA modifications.

Both IscS and IscU form a heterotetrameric complex during FeS cluster assembly, thereby making IscU accessible to receive the persulfide sulfur from IscS.16 The iron source for nascent FeS cluster formation is still under debate, however, CyaY is one of the potential candidates regulating iron entry (Figure 1A).17 During FeS cluster assembly, electrons are required for persulfide reduction, which are provided by Fdx.18-20 After cluster formation on IscU, the two chaperones HscA and HscB catalyze cluster release to carrier proteins such as A-type carriers like IscA in an ATP-dependent manner.21 FeS clusters are important cofactors in the cell which play also a crucial role in the biosynthetic pathways for the biosynthesis of Moco and for the particular thionucleoside modifications s2C32 and ms2i6A37 in tRNA.9 Additionally, IscS interacts with ThiI for sulfur transfer to either thiamine biosynthesis or s4U8 nucleoside thiomodifications in tRNA (Figure 1A).22 In contrast in E. coli, the (c)mnm5s2U thionucleoside modifications of Lys, Gln, and Glu tRNAs are FeS cluster independent but also require the sulfur mobilization by IscS.23 For these thiomodifications, a sulfur-relay system was identified including the sulfur transfer from IscS to the acceptor protein TusA (Figure 1A), which further transfers the sulfur via TusBCD, to TusE and finally to MnmA.23 TusA thereby stimulates the L-cysteine desulfurase activity of IscS about two-fold. To finish (c)mnm5s2U thiomodification, MnmA binds tRNA directly and forms an activated acyl-adenylated U34-intermediate under ATP consumption which receives the persulfide sulfur from Cys199 of MnmA.24-26 Recent investigations showed that TusA has an additional role in Moco biosynthesis as a sulfur transferase 27. It was shown that in E. coli, dithiolene sulfurs of molybdopterin (MPT) are


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formed involving the sulfur transfer from IscS via TusA to the MoaD protein (Figure 1A).27-29 In Moco biosynthesis, the dithiolene group of MPT is formed after insertion of two sulfur atoms to the C1′ and C2′ atoms of cPMP.30 The direct sulfur donor in this reaction is the thiocarboxylate group at the C-terminal glycine of MoaD, present in the MPT synthase complex.31,32 The formation of the thiocarboxylate group on MoaD directly requires MoeB.33,34 In the course of the reaction, MoeB and MoaD form the tetrameric (MoaD–MoeB)2 complex in which an acyladenylate group is formed at the C-terminal glycine of MoaD under ATP consumption.33,34 In its activated form, sulfur is directly transferred from TusA to MoaD–AMP in the (MoaD–MoeB)2 complex.27 In summary, TusA–SSH is shared between Moco biosynthesis and tRNA thiolation by interacting with two sulfur acceptor proteins, namely MoaD for MPT formation and TusD for (c)mnm5s2U34 thiolation.9 In eukaryotes, the mitochondria constitute the main compartment for FeS cluster assembly (Figure 1B).35 In humans, the main proteins required for FeS cluster biosynthesis are NFS1, ISD11, ISCU and frataxin (FXN).36-38 ISD11 thereby is exclusively present in eukaryotes and, while being essential, ISD11was described to stabilize NFS1.39 Additional interaction partners for human NFS1 in mitochondria were shown to be the ferredoxins FDX1 and FDX2 also involved in FeS cluster assembly and the acyl carrier protein (ACP) (Figure 1B).40-42 The crystal structure of the human NFS1/ISD11 proteins in complex with the E. coli ACP protein has been solved recently,42 which showed a fundamentally different architecture as compared to bacterial L-cysteine

desulfurases like E. coli IscS.43 The NFS1 structure revealed that the pair of ISD11

subunits forms the dimeric core of the complex further revealing a solvent-exposed PLP cofactor.42 However, due to a different quaternary structure, intersubunit interactions that line a tunnel to the active site are lost in NFS1. In contrast, NFS1 forms two active sites facing each


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other and allowing ISCU to bind adjacent to one another in the complex. Since following this model the NFS1-NFS1 interactions are weaker as compared to the E. coli homologue, it has been suggested from the structure that the NFS1 monomer first forms a complex with ISD11 and ACP before dimerization occurs. After the synthesis of the FeS cluster on ISCU, by the help of chaperones and carrier proteins the newly formed FeS cluster is further transferred to mitochondrial acceptor proteins in a similar manner as described for E. coli FeS proteins.44 Since FeS clusters are also required for cytosolic proteins, a cytosolic FeS clusters assembly machinery (CIA) exists, which is independent of cytosolic forms of NFS1 and ISCU, but requires a so far unidentified "sulfur compound" from mitochondria (not shown).45 Nevertheless, NFS1 was reported to be present in the cytosol in small amounts playing a role in Moco biosynthesis and cytosolic mcm5s2U34 thiomodifications in tRNA (Figure 1B).46-48 For Moco biosynthesis, cytosolic NFS1 interacts with the MOCS3 protein, the homologue of MoeB in humans (Figure 1B).46,49 MOCS3, however, is a two-domain protein with an N-terminal MoeB-like domain and an additional C-terminal rhodanese-like domain.50 In a similar manner as described for the bacterial proteins, MOCS3 activates the C-terminus of MOCS2A (the MoaD homologue in humans) under ATP consumption, making MOCS2A thereby susceptible for a sulfur atom.50,51 The sulfur for MOCS2A is provided directly by the C-terminal rhodanese-like domain of MOCS3, a protein domain that is distinct from E. coli TusA.52 Together with MOCS2B, MOCS2A then forms the active MPT synthase complex. The interaction and colocalization of NFS1 and MOCS3 was revealed by studies in HeLa cells using Förster resonance energy transfer (FRET), a split-enhanced green fluorescent protein (EGFP) system, immunodetection of fractionated cells and localization studies using confocal fluorescence


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microscopy.46 NFS1 thereby interacts directly with the C-terminal rhodanese-like domain of MOCS3.53 Crucially, MOCS3 is not only involved in Moco biosynthesis, but also in the thiomodification of cytosolic tRNAs.48,54 Besides interacting with MOCS2A, MOCS3 also interacts with URM1 (ubiquitin-related modifier), which acts as a sulfur donor protein involved in the thiolation of mcm5s2U modifications in cytosolic tRNAs Lys, Gln, Glu (Figure 1B).48 Thus, Moco biosynthesis and tRNA thiolation are directly connected in humans by sharing the sulfur delivery pathway composed of NFS1 and MOCS3.9 The pathway for the formation of mcm5s2U34 in cytosolic tRNAs Lys, Glu, Gln, however, is significantly different from the bacterial (c)mnm5s2U34 formation. In humans, it was shown that the proteins MOCS3, URM1, CTU1 and CTU2 are involved in forming the mcm5s2U34 thiomodification.48,55 The pathway of URM1 sulfuration resembles the one of MOCS2A, involving the formation of a C-terminal thiocarboxylate group on URM1 after its activation in complex with MOCS3.9 URM1thiocarboxylate further transfers the sulfur onto U34 of tRNA, aided by the CTU1 and CTU2 proteins under ATP consumption.48 CTU1 is a protein that contains a [4Fe4S] cluster, making mcm5s2U34 modification a FeS cluster dependent pathway in humans.56,57 In total, for human NFS1 distinct roles were suggested in mitochondria and the cytosol. In the mitochondria, the main role of NFS1 is the assembly of FeS clusters in the quaternary complex formed with ISD11, ISCU and FXN.38,58 In the cytosol, in contrast, NFS1 interacts with the C-terminal rhodanese-like domain of MOCS3, providing the sulfur for Moco biosynthesis and mcm5s2U34 tRNA modifications.46 Human NFS1 and E. coli IscS share an amino acid sequence identity of 60% and are believed to be functional orthologues displaying the same role in FeS cluster assembly in each organism. It


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has been shown before that human NFS1 in conjunction with ISD11 can fully complement the growth deficient phenotype of an E. coli iscS mutant strain.53 However, despite the high conservation between orthologues, there are some surprising differences between the two systems. IscS is a highly active enzyme with numerous interaction partners that is, however, fully functional by itself. In contrast, all eukaryotic IscS orthologues including human NFS1 require ISD11 as accessory protein to be functional. ISD11 has been suggested to stabilize NFS1 by preventing its aggregation and degradation.38,59,64 A previous study revealed, however, that E. coli IscS was shown to interact with human ISD11,59 revealing common binding sites besides their evolutionary distance. Since the NFS1 and IscS proteins have different interaction partners in their respective organism, we wanted to dissect in detail whether human NFS1 is fully capable of replacing E. coli IscS in its multiple roles in FeS cluster assembly, Moco biosynthesis and thionucleotide formation for (c)mnm5s2U34, s4U8, s2C32, ms2i6A37. In detail, we quantified the amount of Moco, thionucleosides and the activity of FeS cluster containing proteins after introduction of NFS1 and ISD11 into E. coli ∆iscS cells. In conjunction with interaction studies we show that NFS1 can replace E. coli IscS in its role in FeS cluster assembly, but not in Moco biosynthesis or thiolation of certain tRNAs. However, when we additionally introduced the human MOCS3, MOCS2A and MOCS2B proteins, active molybdoenzymes were obtained. This shows that human NFS1 and E. coli IscS have evolved apart from each other but still share common binding sites for proteins in FeS cluster assembly like IscU, but not with proteins for tRNA thiolation or Moco biosynthesis. Further we show, that in E. coli NFS1 is able to act as sulfur donor for Moco biosynthesis when the specific interaction partner protein MOCS3 from humans is present.


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EXPERIMENTAL PROCEDURES Bacterial Strains, Plasmids, Media, and Growth Conditions. Strains and plasmids used in this study are listed in Table 1. All isogenic BW25113 mutant strains (Keio collection) were obtained from the National BioResource Project (National Institute of Genomics, Japan).60 Table 1. List of used strains and plasmids. Plasmid/Strain Genotype / relevant characterization Plasmid pSL223 thiI gene cloned into NdeI/BamHI sites of pET15b, AmpR pSL209 iscS gene cloned into NcoI/BamHI sites of pET15b, AmpR pJD34 tusA gene cloned into NdeI/BamHI sites of pET11b, AmpR pZM2 truncated NFS1 cloned into XhoI/BamHI of pET15b, expressing N-terminal tagged His6NFS1 ∆ 1-55, AmpR pZM4 ISD11 cloned into NcoI/HindIII of pACYCDuet-1, expressing unmodified ISD11, CmR pSL219 E. coli iscS gene subcloned from pSL209 (NcoI/BamHI in pET15b) into BamHI site of pACYC184 pZM6 ISD11 into the XhoI-BamHI sites of the expression vector pET15b, AmpR pSL206 MOCS2A, MOCS2B, and MOCS3 were PCRamplified and cloned into the NdeI-HindIII site of pET15b, AmpR pMB6 iscU gene cloned into NdeI/BamHI sites of pET15b, AmpR Strains BW25113 (DE3) laclq rrnBT14 lacZWJ16 hsdR514 araBADAH33 rhaBADLD78, (DE3) JW2514 (∆iscS) BW25113 iscS::km, (DE3) (DE3)

Reference 62

29

27

53

53

S. Leimkühler 53

51

62

60, 62

60, 62

When required, the T7 promoter was introduced into E. coli strains BW25113 (wild type) and ∆iscS by using the λDE3 lysogenization kit (Novagen). E. coli cultures were grown in LB


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medium under aerobic or anaerobic conditions at 37°C. When required, 150 µg/mL ampicillin, 25 µg/mL kanamycin or 50 µg/mL chloramphenicol were added to the medium during growth. 15 mM potassium nitrate or 15 mM trimethylamine-N-oxide (TMAO) was added as indicated. Protein expression was induced by the addition of 10 µM IPTG.

Detection

of

aconitase,

succinate

dehydrogenase

(SDH),

fumarase

and

malate

dehydrogenase activities. The enzymatic activity was measured in crude extracts. E. coli strains BW25113 (wild type) and ∆iscS were cultivated under aerobic or anaerobic conditions for 6 h. For SDH the medium was additionally supplemented with 40 mM succinate. For aconitase and malate dehydrogenase activity, cells were lysed by sonification in 50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40 (v/v) (pH 8.0). For SDH activity cells were lysed by sonification in 50 mM MES buffer, 10% (v/v) glycerol, pH 6.5. Aconitase activity was determined in a coupled enzymatic assay monitoring NADPH production at 340 nm from the oxidation of isocitrate produced by isocitrate dehydrogenase. 50 µL cell lysate were incubated for 5 min in 450 µL 50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, 0.5 mM NADP and 0.05 U isocitrate dehydrogenase (pH 8.0). The reaction was started by addition of 500 µL 2.5 mM cis-aconitate in the same buffer. 1 Unit is defined as 1 µmol NADPH formed in 1 min. Aconitase activity was normalized to total protein concentration used in the assay. Malate dehydrogenase activity was detected by activity staining. 25 µg total protein was separated by a 7.5% native PAGE at 4°C. Native polyacrylamide gels were incubated at 37°C in 50 mM Tris-HCl, 5 mg/mL malate, 0.6 mg/mL NAD, 0.5 mg/mL nitro tetrazolium blue, 0.04 mg/mL phenazine methosulfate (pH 8.0) for 5 min. Succinate dehydrogenase (SDH) was assayed in cell lysate that has been incubated in 50 mM Tris-HCl, 4 mM succinate and 1 mM potassium cyanide for 30 min at 30 °C. Activity was


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followed at 600 nm at 30 °C using PMS/DCPIP. SDH activity was normalized to total protein concentration used in the assay.

Quantification of thionucleosides in tRNA. Total tRNA was extracted using the TriFast (peqLab) reagent from cell pellets of E. coli strains BW25113 (wild type) and ∆iscS. Cells were harvested at stationary growth phase by centrifugation and suspended in 25 mL TriReagent. Total RNA was precipitated form the aqueous phase with isopropanol after addition of chloroform. The obtained pellet was washed twice with 75% ethanol and solved in 2x RNA loading dye (95% formamide, 0.025% SDS, 0.025% xylene cyanole, 0.025% bromophenol, 0.5 mM EDTA, pH 8.0). RNA species were separated by 10% Urea-PAGE at 200 V and the bands corresponding to tRNAs were eluted from the gels over night in 50 mM sodium acetate, 150 mM sodium chloride (pH 7.0). tRNAs were precipitated with isopropanol, washed twice with 75% ethanol, resolved in 50 mM sodium acetate, 50 mM zinc acetate (pH 5.3) and the concentration of tRNA was determined spectrophotometrically. An amount of 200 µg tRNA were digested overnight at 37 °C in the same buffer with 4 U P1 nuclease (Sigma) and 4 U fast alkaline phosphate (Sigma) after adjusting the pH to 8.5. The obtained nucleosides were separated on a C18 reversed phase LiChrospher column (4.6x250 nm; particle size 5 µm) at 1 ml/min using 10 mM ammonium phosphate (pH 5.3)/methanol after the protocol reported previously with slight modifications.61 The elution of the nucleosides were followed at 254 nm, 274 nm and 330 nm, the amount quantified by peak integration and normalized to the pseudouridine peak at 254 nm.

Quantification of NR and TMAOR activities. The activity of nitrate reductase (NR) or TMAO reductase (TMAOR) was measured in crude extracts obtained from E. coli strains BW25113 (wild type) and ∆iscS after anaerobic growth for 8 h in the presence of 15 mM potassium nitrate


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or 15 mM TMAO. Cells were harvested at stationary growth phase by centrifugation and resuspended in 50 mM Tris-HCl (pH 7.5) (for NR activities) or 100 mM phosphate buffer (pH 6.5) (for TMAOR activities). Cell lysates were obtained by sonification, transferred into an anaerobic chamber and incubated at 4 °C for at least 3 h. 50 µL of each cell lysate was analyzed for NR or TMAOR activity in a volume of 4 ml containing 0.3 mM benzylviologen, 10 mM KNO3 or 5 mM TMAO in 20 mM Tris-HCl (pH 6.8) or 100 mM phosphate buffer (pH 6.5), respectively. The assay was initiated by injecting sodium dithionite to the anaerobic reaction mixture until OD600 of 0.8-0.9 for reduced benzylviologen was reached. After the addition of crude extract, the oxidation of benzylviologen was recorded at 600 nm for 30 sec. The activity was calculated using the equation U = 0.5 x (∆Abs600/min)/ε600(benzyl viologen)/V, using the extinction coefficient for benzylviologen of 7.4 mmol-1 x cm-1. One Unit is defined as the oxidation of 1 µmol reduced benzylviologen per minute. The activity was normalized to the OD600 of the cells before harvesting. Detection of Moco and cPMP in cell extracts. To determine the total Moco content (as MPT and Moco) 50 ml cultures of E. coli strains BW25113 wild type and ∆iscS were grown anaerobically for 8 h until stationary growth phase in the presence of either 15 mM potassium nitrate or 15 mM TMAO, harvested by centrifugation, and resuspended in 100 mM Tris-HCl, pH 7.2. Cells were lysed by sonification and the cell debris was removed by centrifugation. Samples were oxidized in the presence of acidic iodine at 95 °C for 30 min and excess iodine was removed by the addition of 55 µL of 1% (w/v) ascorbic acid. The pH of the samples was adjusted with 1 M Tris-HCl to pH 8.3. After addition of 40 mM MgCl2 and 1 unit of fast alkaline phosphatase, FormA was obtained. For purification of FormA, the samples were loaded onto a 500 µl QAE ion exchange resin (Sigma) equilibrated in water. The column was washed with 10


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column volumes of water and with 1300 µL 10 mM acetic acid. FormA was eluted with 6 x 500 µL 10 mM acetic acid. The fractions were separated on a C18 reversed phase HPLC column (4.6 x 250 mm ODS Hypersil, particle size 5 µm) equilibrated with 5 mM ammonium acetate, 15% (v/v) methanol at a flow rate of 1 ml/min. Elution of FormA was monitored by an Agilent 1100 series fluorescence detector with an excitation at 383 nm and emission at 450 nm. The total FormA content was normalized to the total protein concentration determined by Bradford. Total cPMP can be converted into its oxidized fluorescent derivate CompoundZ with acidic iodine. CompoundZ was isolated from crude extracts as described above for FormA with slight modifications. Prior to elution from the QAE column, the column was washed with 10 column volumes water and 10 mM acetic acid before CompoundZ was eluted with 5 x 1000 µL 100 mM HCl. The fractions were separated on a C18 reversed phase HPLC column (4.6 x 250 mm ODS Hypersil, particle size 5 µm) equilibrated with 10 mM potassium dihydrogen phosphate (pH 3.0), 1% (v/v) methanol at a flow rate of 1 ml/min. CompoundZ was monitored by its fluorescence with an excitation at 383 nm and emission at 450 nm. The total CompoundZ content was normalized to the total protein concentration determined by Bradford.

Detection of protein-protein interactions IscS,29 NFS1/ISD1153, IscU15, TusA27 and ThiI62 were expressed in ∆iscS(DE3) cells and purified following previously described procedures. To test the complex formation between IscS or NFS1/ISD11 with either TusA, ThiI or IscU, 30 µM of IscS or NFS1/ISD11 was incubated with 60 µM TusA, ThiI or IscU for 25 min at 37 °C. The protein mixture was injected onto a Superdex 200 column connected to an Äkta purifier system, which had been equilibrated in 50 mM Tris-HCl, 100 mM NaCl, 10 mM β-mercaptoethanol (pH


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8.0). Proteins were separated at a flow rate of 1 ml/min and the elution profile was recorded at 280 nm. The proteins in the elution fractions were separated by 15% SDS-PAGE.

Protein concentration quantification. Protein concentrations were determined using the Bradford Reagent Coomassie Plus™ Protein Assay Reagent (Thermo) with bovine serum albumin (BSA) as a standard following manufacture’s instructions.


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Biochemistry

RESULTS Human NFS1 can fully replace the role of E. coli IscS in FeS cluster assembly. It has been shown previously that human NFS1 in conjunction with ISD11 can fully complement the growth deficient phenotype of an E. coli ∆iscS mutant.53 To analyze whether the introduction of NFS1/ISD11 to the E. coli ∆iscS strain is able to functional complement of the role of IscS in FeS cluster assembly, we analyzed the activity of succinate dehydrogenase (SDH) and aconitase as FeS cluster containing enzymes in E. coli.62 E. coli expresses two [4Fe4S] cluster containing aconitases, AcnA and AcnB. Under the aerobic growth conditions we selected for our assay, however, mainly AcnB is measured, while AcnA is only marginally active.62 The results in Figure 2A show that the activity of aconitase can be fully restored in the ∆iscS strain by introduction of E. coli IscS. The additional presence of ISD11 resulted, however, in a 40% reduction of aconitase activity in comparison to the complementation with IscS alone.


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Figure 2. Quantification of aconitase, succinate dehydrogenase and malate dehydrogenase (Mdh) activities in different E. coli strains. The activity of aconitase (A, upper panel), succinate dehydrogense (SDH) (B, upper panel), and malate dehydrogenase (Mdh) (A & B, lower panels) was quantified in the E. coli strain BW25113 (wild type) and ∆iscS strain complemented with either IscS, IscS/ISD11, NFS1, or NFS1/ISD11. A plus symbol (+) indicates overexpression of the respective plasmid-encoded proteins. The strains were grown aerobically (dark grey bars) or anaerobically (light grey bars) in the presence of 10 µM IPTG until mid-exponential phase. For SDH activity the medium was additionally supplemented with 40 mM sodium succinate. (A, upper panel) Aconitase activity was indirectly measured in crude extracts in a coupled enzymatic reaction using the isocitrate dehydrogenase dependent formation of NADPH at 340 nm. The activity was normalized to total protein amount in the reaction. (B, upper panel) SDH activity was obtained in cyanide treated crude extracts following the reduction of PMS/DCPIP at 600 nm. The calculated activity was normalized to total protein amount in the reaction. (A & B, lower panel) 25 µg total protein from the same crude extracts used for aconitase or SDH activity measurements were separated on 7.5% native polyacrylamide gels and stained for malate dehydrogenase (Mdh) activity by detecting formation of formazan at 37 °C. The data are mean values from three independent measurements (± S.D.).


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When NFS1 was introduced into the strain, only 10% of the aconitase activity of the wild type strain was obtained, while the additional presence of ISD11 for active NFS1/ISD11 complex formation resulted in a 30% rescue of aconitase activity. We further tested the same extracts for malate dehydrogenase activity in an in-gel activity stain. Figure 2A shows that the malate dehydrogenase activity was comparable in all strains analyzed. Thus, the activity of a non-FeS cluster requiring enzyme remained mainly unaffected by the introduction of additional genes to the ∆iscS mutant strain under the growth conditions tested for aconitase activity. As second FeS cluster-containing enzyme we analyzed the activity of SDH in aerobically or anaerobically grown cultures in the presence of succinate. Besides a [4Fe4S] cluster, E. coli SDH also contains a [3Fe4S] and a [2Fe2S] cluster. As shown in Figure 2B, SDH had overall lower activity levels under anaerobic conditions, however, the same trend of activities were obtained after the various reconstitutions. Further, the results are consistent with the aconitase activities, showing that IscS can almost fully complement SDH activity, while the additional presence of ISD11 results in a about 20% reduction of the reconstitution efficiency. Further NFS1 alone can only marginally rescue SDH activity to about 10%, while the NFS1/ISD11 complex is able to reconstitute the SDH activity to 55% under aerobic conditions and to 90% under anaerobic conditions. Consistent with the results described above, malate dehydrogenase activity tested in the same extracts grown under aerobic or anaerobic conditions was not affected.

FeS dependent tRNA thiomodifications are synthesized by NFS1/ISD11 in E. coli. The tRNA thiomodifications of s2C32 and ms2i6A37 were shown to be dependent on FeS cluster synthesis.65,66 In E. coli, these tRNA modifications involve TtcA for the synthesis of s2C32 and MiaB for ms2i6A37 modification. TtcA contains one [4Fe4S] cluster, while MiaB contains two


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[4Fe4S] clusters and is a member of the radical S-adenosyl-methionine (SAM) superfamily of proteins. To test whether NFS1/ISD11 can also replace IscS in providing FeS clusters to TtcA and MiaB, the relative amounts of s2C32 and ms2i6A37 were quantified after growth under aerobic and anaerobic conditions. As shown in Figure 3A, IscS or IscS in conjunction with ISD11 were able to fully restore s2C32 formation to the levels detected in the wild-type strain. In comparison, NFS1 in conjunction with ISD11 was able to restore s2C32 levels to about 55% both under aerobic or anaerobic conditions, while no complementation was detected with NFS1 alone.


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Biochemistry

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Figure 3. Quantification of FeS cluster dependent thionucleosides in different E. coli ∆iscS strains. The amount of s2C32 (A) and ms2i6A37 (B) and (C) i6A37 was quantified in the E. coli strain BW25113 (wild type) and ∆iscS strain complemented with IscS, IscS/ISD11, or NFS1, NFS1/ISD11. A plus symbol (+) indicates overexpression of the respective plasmid-encoded protein. The strains were grown aerobically (dark grey bar) or anaerobically (light grey bar) in the presence of 10 µM IPTG. Total tRNAs were separated from other RNA species by 10% Urea-PAGE after phenol/chloroform extraction. Subsequently, tRNAs were hydrolyzed, dephosphorylated and obtained nucleosides were separated by HPLC. The amount of (A) s2C32, (B) ms2i6A37 and (C) i6A37 was quantified at 254 nm and the corresponding peaks were normalized to the internal pseudouridine standard. The data are mean values from three independent measurements (± S.D.). Figure 3B shows that similar results were obtained for the ms2i6A37 thiomodifications of tRNA. Here, also IscS or IscS in conjunction with ISD11 fully restored the cellular ms2i6A37 levels both under aerobic and anaerobic conditions, while NFS1 alone was unable to replace IscS in FeS cluster formation, consistent with the results shown above. NFS1 in complex with ISD11, however, restored the ms2i6A37 levels to 30% under aerobic conditions and to 55% under anaerobic conditions. Measurements for the accumulation of the ms2i6A37 precursor i6A37 (Figure 3C) thereby correlated well with the reduced amounts of ms2i6A37, showing when ms2i6A37 formation is impaired in the cell, the precursor i6A37 accumulated. In conclusion, the NFS1/ISD11 complex was able to partially replace IscS in providing [4Fe4S] clusters for both TtcA and MiaB.

NFS1/ISD11 are unable to rescue FeS cluster independent thiomodifications in tRNA. Besides FeS cluster formation, IscS is further involved in providing the sulfur for the formation of mnm5s2U34 and s4U8 modified uridines of certain tRNAs. Here, the sulfur transfer for thionucleoside formation depends on the direct interaction of the sulfur acceptor proteins TusA and ThiI with IscS, respectively.23,67,68


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Biochemistry

Figure 4A shows the quantified amounts for the mnm5s2U34 thionucleosides after aerobic and anaerobic growth of E. coli. From the results it is obvious that IscS or IscS in conjunction with ISD11 was fully able to restore the mnm5s2U34 levels of the ∆iscS strain, while in cells expressing NFS1 or the NFS1/ISD11 complex no mnm5s2U34 modified nucleosides were detected.


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Figure 4. Quantification of FeS cluster independent thiomodifications in different E. coli ∆iscS strains. The amount of mnm5s2U34 (A) and s4U8 (B) was quantified in the E. coli strain BW25113 (wild type) and ∆iscS strain complemented with IscS, IscS/ISD11, NFS1, or NFS1/ISD11. A plus symbol (+) indicates overexpression of the respective plasmid-encoded protein. The strains were grown aerobically (dark grey bar) or anaerobically (light grey bar) in the presence of 10 µM IPTG. Total tRNAs were separated from other RNA species by 10% Urea-PAGE after phenol/chloroform extraction. Subsequently, tRNAs were hydrolyzed, dephosphorylated and obtained nucleosides were separated by HPLC. The amount of (A) mnm5s2U34 was quantified at 274 nm or (B) of s4U8 at 330 nm. The corresponding peaks were integrated and normalized to the internal pseudouridine standard. The data are mean values from three independent measurements (± S.D.). Similar results were obtained for the quantification of the s4U8 thionucleosides in E. coli strains grown under aerobic and anaerobic conditions (Figure 4B). While IscS and IscS/ISD11 fully restored the s4U8 levels in the ∆iscS strain, no s4U8 thionucleosides were detected in cells complemented with NFS1 or NFS1/ISD11, independent on the presence of oxygen during growth. This implies that NFS1 is unable to transfer the sulfur to either TusA or ThiI in E. coli.

NFS1/ISD11 in conjunction with their specific interaction partner MOCS3 are capable in providing the sulfur for Moco biosynthesis in E. coli. To test whether the NFS1/ISD11 complex can restore molybdoenzyme activity in the E. coli ∆iscS strain, we analyzed the activities of nitrate reductase (NR) and trimethylamine-N-oxide reductase (TMAOR) after introduction of different plasmids into this strain. The activities of both enzymes were compared, since E. coli NR enzymes require FeS clusters for their activities, while the E. coli TMAOR enzyme contains only the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor variant of Moco. We analyzed the activities of both enzymes under anaerobic conditions after the addition of either nitrate or TMAO during growth.


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Biochemistry

As shown in Figures 5A + B, NR and TMAOR activities were fully restored after introduction of either IscS or IscS/ISD11 into the ∆iscS strain. Here, the simultaneous presence of IscS/ISD11, however, reduced the complementation efficiency of NR by 40% and of TMAOR by 10% in comparison to IscS alone.


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Biochemistry

Figure 5. Analysis of the activities of NR, TMAOR, and the amounts of Moco and cPMP in different E. coli ∆iscS strains. NR and TMAOR activities as well as Moco and cPMP content were determined in E. coli strain BW25113 (wild type) and ∆iscS strain complemented with IscS, IscS/ISD11, NFS1, NFS1/ISD11, NFS1/MOCS2A/MOCS2B/MOCS3 or NFS1/ISD11/MOCS2A/MOCS2B/MOCS3. A plus symbol (+) indicates overexpression of the respective plasmid-encoded protein. Cells were grown anaerobically at 37 °C until late stationary phase in the presence of 10 µM IPTG, 15 mM potassium nitrate (black columns) or 15 mM TMAO (white columns). NR activities (A) and TMAOR activities (B) were measured in crude extracts following the oxidation of dithionite-reduced benzylviologen at 600 nm. The activities were normalized to the OD 600 nm of the cells after growth. Total Moco content was quantified in the same cells after growth on (C) nitrate or (D) TMAO. Total Moco was converted into its fluorescent derivate FormA with acidic iodine treatment at 95°C. Sequentially, FormA was isolated from crude extracts and its fluorescence was monitored by excitation at 383 nm and emission at 450 nm on a HPLC. The corresponding peak area was related to the total soluble protein concentration used in the assay. cPMP was meassured in the same cells after growth on (E) nitrate or (F) TMAO. Total cPMP was converted into its fluorescent derivate CompoundZ with acidic iodine treatment. Sequentially, CompoundZ was isolated from crude extracts and its fluorescence was monitored by excitation at 383 nm and emission at 450 nm on a HPLC. The corresponding peak area was related to the total soluble protein concentration used in the assay. All data are mean values from three independent measurements (± S.D.).

When NFS1 in complex with ISD11 was present, NR activities of 20% and TMAOR activities of 10% in comparison to the BW25113 wild type strain were obtained. Additionally, we also tested the complementation efficiency of NFS1/ISD11 in conjunction with the human proteins MOCS3 together with MOCS2A and MOCS2B, forming the MPT synthase complex. While E. coli MoeB and human MOCS3 are homologous proteins they show, however, significant differences in their domain structure with MOCS3 being a two-domain protein with an N-terminal MoeB-like domain and an additional C-terminal rhodanese-like domain onto which NFS1 transfers the sulfur. The results (Figures 5B + C) show that the complementation efficiency was increased when all five proteins, NFS1, ISD11, MOCS2A/MOCS2B, and MOCS3 were present, resulting in a NR activity of 50% and a TMAOR activity of 20% compared to those in the corresponding


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wild type strain. NFS1 without ISD11, however, was partly able to restore NR or TMAOR activities together with MOCS3 and MOCS2A/MOCS2B. In addition to the NR and TMAOR activities, we quantified the amounts of total Moco and of the cyclic pyranopterin monophosphate (cPMP) intermediate in the cell extracts. In Moco biosynthesis, MoaA is a FeS cluster containing protein that harbors two [4Fe4S] clusters and catalyzes the conversion of 5’GTP to cPMP.69 By comparison of the cellular cPMP and Moco levels the activities of MoaA and of MPT synthase can be differentiated. When FeS cluster insertion into MoaA is limiting, both cPMP and Moco would not be produced. For comparison, when active MoaA is produced but sulfur transfer to cPMP for the conversion into MPT is limiting, cPMP consequently accumulates. For the quantification of produced Moco or accumulated cPMP, both molecules were converted into their oxidized fluorescent derivatives FormA and CompoundZ, respectively, purified from crude extracts and separated via HPLC.70,71 The results in Figures 5B + C show the quantification of the relative amounts of total Moco in the respective cell extracts. The results are generally consistent with the quantified NR and TMAOR activities shown in Figures 5A + B, however, some differences are revealed. While the NR activities were fully restored after introduction of IscS into the ∆iscS strain, the total Moco levels were only 70% restored in this cell strain, indicating a preference of Moco insertion into NR under these conditions. Further, total Moco levels were restored to 45% in cells grown on nitrate when NFS1/ISD11 together with MOCS3 and MOCS2A/MOCS2B were present, a number fully consistent with the obtained NR activities. In cells grown on TMAO, however, the Moco levels were restored to 80%, while the TMAOR activity was only restored to 20%. This might imply that the overall Moco produced was preferentially inserted into other molybdoenzymes and not into TMAOR under these conditions. The NFS1/ISD11 complex alone


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Biochemistry

without MOCS3, however, was only marginally able to restore Moco production. Surprisingly, in cells containing NFS1 alone (without ISD11) together with MOCS3 and MOCS2A/MOCS2B, Moco levels of 20% in comparison to the wild type strain were obtained. The quantification of cPMP accumulation in the reconstituted cells strains show that cPMP was accumulated in ∆iscS cells containing the NFS1/ISD11 complex, both in nitrate and TMAO grown cells (Figures 5E + F). This implies that active MoaA was obtained after complementation with the NFS1/ISD11 complex, however, NFS1/ISD11 were only insufficiently able to provide the sulfur for the conversion of cPMP to MPT, consequently resulting in the accumulation of cPMP. Additionally, cPMP also accumulated in cells grown on nitrate and complemented with NFS1/ISD11, MOCS3 and MOCS2A/MOCS2B, in agreement with the only 50% reconstituted NR activities and Moco levels under these conditions. In cells grown with TMAO, however, no cPMP accumulation was detected and Moco levels reached 80% of the levels detected in the wild type strain. The discrepancy of the results are explained by the fact that E. coli produces three NR enzymes which are present at much higher concentrations than TMAOR, so that likely NFS1/ISD11 are only able to provide the FeS clusters for MoaA, but are not fully capable to additionally restore the high demand of sulfur for the MPT synthase reaction. For comparison of the influence of expression of the human proteins MOCS3/MOCS2A and MOCS2B in the ∆iscS strain on tRNA thiolation and FeS cluster biosynthesis, we also tested the levels of aconitase, SDH and thionucleoside formation of mnm5s2U34, s4U8, s2C32 and ms2i6A37. No influence of these additional proteins was observed on the synthesis or other sulfur-containing biomolecules in the cell (supplementary Figures S1 + S2).

The NFS1/ISD11 complex interacts with E. coli IscU, but not with TusA or ThiI.


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The results shown above reveal that the NFS1/ISD11 complex is capable in restoring FeS cluster assembly in E. coli, but is unable to directly provide the sulfur for mnm5s2U and s4U8 tRNA modifications or Moco biosynthesis in E. coli. This might be based on the incapability of the NFS1/ISD11 complex to interact with TusA or ThiI, the proteins that interact with E. coli IscS to transfer the sulfur to the respective pathways.

Figure 6. Analysis of complex formation between NFS1/ISD11 and IscU, TusA or ThiI.


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Biochemistry

Analytical gel filtration analysis is shown of possible complex formation between NFS1/ISD11 and IscU (A), NFS1/ISD11 and TusA (B) or NFS1/ISD11 and ThiI (C), or IscS and IscU (D), IscS and TusA (E), IscS and ThiI (F). Proteins (30 µM NFS1/ISD11 or IscS and 60 µM IscU or TusA or ThiI) were incubated for 20 min at 37 °C, separated via size exclusion chromatography on a Superdex200 column equilibrated in 50 mM Tris-HCl, 100 mM NaCl, 10 mM βmercaptoethanol (pH 8.0). The elution of proteins was followed at 280 nm. Indicated fractions were analyzed for their protein composition by 15% SDS-PAGE.

To analyze the complex formation of NFS1/ISD11 with either E. coli IscU, ThiI or TusA, analytical size exclusion chromatography was performed. Proteins were incubated and analyzed for complex formation using a Superdex 200 column. The results in Figure 6 show that NFS1/ISD11 was able to form a complex with E. coli IscU (Fig. 6D), but not with TusA (Fig. 6E) or ThiI (Fig. 6F). In contrast, IscS readily formed a complex with IscU (Fig. 6A), TusA (Fig. 6B), or ThiI (Fig. 6C). An interaction with NFS1 alone was not tested, due to the intrinsic instability of the purified protein in the absence of ISD11.


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DISCUSSION In this study, we dissected the specific roles of human NFS1 in comparison to E. coli IscS. Different interaction partners were described previously for each protein by a combination of different methods.14 E. coli IscS is thereby a versatile protein with numerous interaction partners.9 The major role of IscS, however, has been implicated in FeS cluster assembly.72 In this process, IscS interacts with the scaffold protein IscU, the regulatory protein for iron entry CyaY, Fdx with a role in electron transfer, and IscX with a suggested role in delivering iron for cluster assembly (Figure 1A).17,73 Interaction sites for each protein have been mapped before and cocrystal structures were solved.14,20 In addition to these main pathways for sulfurtransfer to sulfurcontaining biomolecules or cofactors, additional interaction partners were identified for the E. coli IscS protein, like FdhD the specific chaperone involved in binding and sulfuration of the bisMGD cofactor for formate dehydrogenases or ACP involved in fatty (lipoic) acid biosynthesis.74,75 It remains, however, still elusive how the preference for the specific interaction partner is regulated and which factors direct the sulfur transfer into a specific biomolecule synthesis. Likewise, the human homologue NFS1 has been shown to act similarly in the FeS cluster assembly pathway, thereby forming the quaternary NFS1/ISD11/ISCU/FXN complex (Figure 1B).38,58,76 Previous in vitro studies have shown that the IscU and Fxn proteins from mouse, humans, yeast and the E. coli homologues act interchangeably in FeS cluster assembly, however, some differences exist on the role of frataxin and the bacterial homologue CyaY.17,77 In eukaryotes, a defect in FXN results in severe defects in FeS cluster biogenesis, and in humans, this is associated with Friedreich’s ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in


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Biochemistry

IscS-catalyzed FeS cluster formation.78 However, it was shown that the differences in the role of either frataxin or CyaY are rather directed by the scaffold protein IscU, since a single point mutation in IscU was sufficient to impose a strict dependency of the E. coli proteins on human FXN. Prokaryotic IscU proteins thereby contain a conserved Ile residue, which is substituted by a Met in eukaryotic ISCU proteins.79 The studies in this report confirm the previous investigations and show that NFS1 in conjunction with ISD11 can substitute IscS in its role in FeS cluster biosynthesis. The roles of both proteins in FeS cluster assembly are highly conserved, as visualized by the interchangeable role and interaction of NFS1 with the E. coli IscU protein in this study. NFS1 was able to provide the FeS clusters for the activity of aconitase, SDH, MoaA and NR. The different levels of activity obtained for these proteins in this study might reveal a different FeS cluster reconstitution efficiency of the proteins. Thus, since the activity of NFS1 is lower as compared to E. coli IscS, an overall lower level of FeS clusters might exist in E. coli after reconstitution with the NFS1/ISD11 proteins. This might imply that FeS clusters are inserted into the respective target proteins with a preference for SDH, as revealed by the obtained high SDH activity and lower activities of the other proteins. In contrast, the role of the two L-cysteine desulfurases in other pathways has evolved apart from each other with proposed differences in the interaction sites for each specific partner protein.42 In humans and other eukaryotes, the FeS cluster assembly pathway is located in the mitochondria, clearly separating it from the cytosolic sulfur-dependent pathways like Moco biosynthesis or mcm5s2U34 thiomodifications in tRNA.80,81 Additional interaction partners for human NFS1 in mitochondria were shown to be the ferredoxins FDX1 and FDX2 also involved in FeS cluster assembly and ACP.40-42 Further, NFS1 interacts with the TUM1 protein in mitochondria for τm5s2U34 thiomodifications of mitochondrial tRNAs for Lys, Glu, Gln (Figure 1B).82 In the


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cytosol, however, NFS1 has a role distinct from the CIA pathway for FeS cluster assembly. Here, the main interaction partner was shown to be the MOCS3 protein involved in Moco biosynthesis and cytosolic mcm5s2U34 thionucleoside modifications.46,48,53 It has been shown previously that NFS1 specifically interacts with the C-terminal rhodanese-like domain of MOCS3.53 As additional interaction partner of NFS1, the cytosolic isoform of TUM1 has been described in humans.82 This protein was suggested to act as mediator between NFS1 and MOCS3, facilitating sulfur transfer between both proteins. Our studies clearly reveal differences between E. coli IscS and human NFS1 in their roles for Moco biosynthesis and thiomodifications in tRNA.9 Moco biosynthesis itself is a highly conserved pathway, which is present in most organisms with the exception of the eukaryotic model organism Saccharomyces cerevisiae.83 As revealed by the functional complementation studies described here, NFS1 is clearly able to provide the sulfur for Moco biosynthesis in E. coli, however, the reconstitution is much more productive when its specific interaction partner MOCS3 from humans is additionally present. This reveals in particular the different routes in the sulfur transfer pathways for Moco biosynthesis in E. coli and humans, involving different protein components. NFS1 was shown to interact with the C-terminal rhodanese-like domain of MOCS3, a protein that is not present in E. coli.53 In E. coli, in contrast, the TusA protein was shown to be involved as sulfur transferring protein for Moco biosynthesis and tRNA thiolation.27 The different sulfur transfer routes are also revealed by the inability of human NFS1 to interact with E. coli TusA. Thus, while similar tRNA modifications are synthesized, the routes for sulfur transfer have evolved to utilize different protein components.9 The low reconstitution levels of NFS1 in the absence of MOCS3 show that NFS1 is able to directly transfer the sulfur to E. coli MoaD, a pathway that also has been suggested to occur for E. coli IscS protein in the absence of


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TusA.27 In humans, MOCS3 additionally interacts with the URM1 protein for cytosolic mcm5s2U34 tRNA thiomodifications. Here, the sulfur transfer route involves additionally the CTU1 and CTU2 proteins.55,56 CTU1 thereby was shown to be a FeS cluster containing protein, grouping mcm5s2U34 thiomodifications in humans to an FeS cluster dependent pathway (Figure 1B).57 The crystal structure of the human NFS1/ISD11 proteins in complex with the E. coli ACP protein42 showed a fundamentally different architecture as compared to the E. coli L-cysteine desulfurase IscS.43 Our data, however, show that the NFS1/ISD11 complex despite its different structure, is fully capable to complement IscS in its role for FeS cluster biosynthesis. The thiomodifications of s4U8 and (c)mnm5s2U34 thiomodifications were not synthesized by NFS1/ISD11 due to the inability of the complex to interact with either E. coli ThiI or TusA. Thus, IscS and NFS1 provide different interaction sites for both proteins, which might be blocked by the ISD11 interaction. Since Moco biosynthesis was also restored when MOCS3 was additionally present in E. coli, MOCS3 might also have a different interaction site on NFS1, which is not present on E. coli IscS. While the results in this study emphasize that NFS1 is able to transfer the sulfur to MOCS3 for Moco biosynthesis, an involvement of ISD11 in this reaction still remains unclear. So far, ISD11 has been described as an essential stabilizing factor for mitochondrial NFS1 activity in eukaryotes.39,64 In the absence of ISD11, NFS1 aggregates and conclusively FeS clusters are not formed in mitochondria in humans.39,53, 84, 85 Localization studies showed that ISD11 is mainly located in mitochondria and the nucleus in human cells.64 Thus, the role of ISD11 and its involvement in the interaction of NFS1 and MOCS3 in the cytosol still needs to be revealed. MOCS3 might replace the role of ISD11 as a stabilizing protein to NFS1 in the cytosol. Thus, a


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low activity of NFS1 without its interaction partner ISD11 might be sufficient in the cytosol in humans, where NFS1 mainly interacts with MOCS3. Whether indeed ISD11 is not required for NFS1 activity in the cytosol, however, needs to be clarified in more detail in future studies and was not the scope of this study. Overall, Moco is essential to humans for the activity of a functional sulfite oxidase.86,

87

Sulfite oxidase is involved in the clearance of toxic sulfite and

Moco deficiency results in death in early childhood.88, 89 A deficiency phenotype for cytosolic mcm5s2U34 thiomodifications has not been described in humans yet. A requirement of ISD11 for cytosolic NFS1 and sulfur transfer to MOCS3 would point to a requirement for Moco biosynthesis, thus, a deficiency in ISD11 in humans should have overlapping symptoms with Moco deficiency, a phenotype which has not been described for patients with ISD11 defects so far.84 Future studies are necessary to reveal the interaction site of MOCS3 on NFS1 and to clarify the role of ISD11 in the cytosol.


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AUTHOR INFORMATION

Corresponding Author

*To whom correspondence should be addressed: Silke Leimkühler, Institute of Biochemistry and Biology, Department of Molecular Enzymology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany, Telephone: +49-331-977-5603; Fax: +49-331 977-5128; Email: [email protected]

Funding

The research leading to these results has received funding from the International Max Planck Research School on Multiscale Biosystems to M.B. and S.L. This work was supported by the Deutsche Forschungsgemeinschaft grant LE1171/15-1 and LE1171/11-1 to SL.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

Angelika Lehmann (University of Potsdam) and Natalie Lupilov (Ruhr University Bochum) are thanked for their technical assistance. We thank Dennis Dean (Virginia Tech) for helpful discussions and suggestions.


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Supporting Information Available

Figure S1: Quantification of aconitase, succinate dehydrogenase and malate dehydrogenase (Mdh) activities in different E. coli strains. Figure S2: Quantification of thionucleosides in different E. coli ∆iscS strains.


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Functional complementation studies reveal different interaction partners of Escherichia coli IscS and human NFS1


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Functional Complementation Studies Reveal Different Interaction

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