Function and Crystal Structure of the Dimeric P-Loop Atpase CFD1 Coordinating an Exposed [4Fe-4S] Cluster for Transfer to Apoproteins

Function and crystal structure of the dimeric P-loop ATPase CFD1 coordinating an exposed [4Fe-4S] cluster for transfer to apoproteins

Oliver Stehlinga, Jae-Hun Jeoungb, Sven A. Freiberta,c, Viktoria D. Paula, Sebastian Bänfera, Brigitte Niggemeyera, Ralf Rössera, Holger Dobbekb, and Roland Lilla,c,1

aInstitut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, 35033 Marburg, Germany; bInstitut für Biologie, Strukturbiologie/Biochemie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany; and cZentrum für Synthetische Mikrobiologie SynMikro, Offensive for the Development of Scientific and Economic Excellence of the State of Hesse (LOEWE), 35043 Marburg, Germany

Edited by Elizabeth Anne Craig, University of Wisconsin, Madison, WI, and approved August 13, 2018 (received for review May 4, 2018) Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires leased from the scaffold and transiently bound by transfer pro- complex machineries in mitochondria and cytosol. Initially, Fe-S teins. Finally, dedicated targeting factors assist the insertion of clusters are assembled on dedicated scaffold proteins and then are the cluster into specific apoproteins. trafficked to target apoproteins. Within the cytosolic Fe-S protein Insights into the composition and function of the CIA ma- assembly (CIA) machinery, the conserved P-loop nucleoside triphos- chinery were initially obtained in yeast. Two homologous P-loop phatase Nbp35 performs a scaffold function. In yeast, Nbp35 ATPases termed “Cfd1” and “Nbp35” were shown to serve as a cooperates with the related Cfd1, which is evolutionary less scaffold complex that de novo assembles a [4Fe-4S] cluster (12– conserved and is absent in plants. Here, we investigated the 18). This synthesis reaction requires the electron transport chain potential scaffold function of human CFD1 (NUBP2) in CFD1- composed of the diflavin oxidoreductase Tah18 and the Fe-S 55 depleted HeLa cells by measuring Fe-S enzyme activities or Fe in- protein Dre2 (19, 20). Trafficking of the [4Fe-4S] cluster and corporation into Fe-S target proteins. We show that CFD1, in com- its insertion into apoproteins are accomplished by the iron-only plex with NBP35 (NUBP1), performs a crucial role in the maturation hydrogenase-like Fe-S protein Nar1 (21, 22) and the CIA- of all tested cytosolic and nuclear Fe-S proteins, including essential targeting complex (CTC) composed of the WD40 protein Cia1, ones involved in protein translation and DNA maintenance. CFD1 the DUF59 protein Cia2, and the HEAT repeat family protein also matures iron regulatory protein 1 and thus is critical for cellular Mms19 (23–25). A further, highly specialized function is exe- iron homeostasis. To better understand the scaffold function of cuted by the adapter complex Yae1-Lto1 that facilitates [4Fe-4S] Chaetomium ther- CFD1-NBP35, we resolved the crystal structure of cluster insertion into the ABC protein Rli1 (26). Functional mophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 analyses revealed an additional requirement of the cytosolic protein. Importantly, two ctCfd1 monomers coordinate a bridging monothiol glutaredoxins Grx3 and Grx4 for cytosolic-nuclear Fe-S [4Fe-4S] cluster via two conserved cysteine residues. The surface- protein biogenesis, but the step in the maturation pathway at exposed topology of the cluster is ideally suited for both de novo which they act is currently unknown (27). assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed Significance by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaf- Eukaryotic iron-sulfur (Fe-S) proteins play essential roles in fold components during Fe-S cluster assembly and/or release. energy conversion, antiviral defense, protein translation, genome integrity, and iron homeostasis. Assembly of the CIA machinery | NUBP1-NUBP2 | NBP35 | iron-sulfur protein | metallo-cofactors is assisted by complex machineries involving iron homeostasis more than 30 known components. The initial phase of Fe-S protein maturation in the human cytosol is poorly studied thus far, with the P-loop nucleoside triphosphatase NBP35 being the roteins harboring iron-sulfur (Fe-S) cofactors participate in only known assembly factor. Here, we identified and charac- numerous essential cellular processes including respiration, P terized human CFD1 as an indispensable complex partner of nucleotide and amino acid metabolism, genome maintenance, NBP35 in cytosolic Fe-S protein assembly (CIA). The crystal ribosome function, antiviral response, and iron homeostasis. The

structure of fungal holo-Cfd1 showed a surface-exposed [4Fe-4S] BIOCHEMISTRY synthesis of these simple metallo-cofactors and their target- cluster. Its shared, surface-exposed coordination by two specific insertion into apoproteins follows a complex pathway Cfd1 monomers has important mechanistic implications for the mediated by conserved assembly systems in mitochondria and – ATP-dependent de novo cluster assembly and subsequent cytosol (1 4). The mitochondrial Fe-S cluster (ISC) assembly transfer to apoproteins via downstream CIA components. machinery includes the cysteine desulfurase complex NFS1-

ISD11 that provides the sulfur required for the assembly of Author contributions: O.S., J.-H.J., S.A.F., H.D., and R.L. designed research; O.S., J.-H.J., both mitochondrial and extramitochondrial Fe-S clusters (5, 6). S.A.F., V.D.P., S.B., B.N., and R.R. performed research; O.S., J.-H.J., S.A.F., V.D.P., S.B., H.D., An export system with the mitochondrial inner membrane ATP- and R.L. analyzed data; and O.S., J.-H.J., S.A.F., H.D., and R.L. wrote the paper. binding cassette (ABC) transporter Atm1 (human ABCB7) The authors declare no conflict of interest. transports an ill-defined sulfur-containing compound from mi- This article is a PNAS Direct Submission. tochondria to the cytosolic Fe-S protein assembly (CIA) ma- Published under the PNAS license. chinery that catalyzes maturation of both cytosolic and nuclear Data deposition: The atomic coordinates and structure factors reported in this paper have Fe-S proteins (7–9). Although the ISC and CIA systems are not been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 6G2G). evolutionarily related, they share some common mechanistic 1To whom correspondence should be addressed. Email: [email protected]. principles during biogenesis (10, 11). First, dedicated assembly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. factors catalyze the de novo formation of an Fe-S cluster on a 1073/pnas.1807762115/-/DCSupplemental. scaffold complex. Second, the newly made Fe-S cluster is re- Published online September 10, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1807762115 PNAS | vol. 115 | no. 39 | E9085–E9094 Downloaded by guest on September 26, 2021 In human cells, the CIA pathway is less well characterized, Depletion of Human CFD1 Affects both Fe-S Cluster Assembly on especially in its initial steps where only NBP35 [also termed IRP1 and Cellular Iron Homeostasis. We tested the effect of CFD1 “NUBP1” (28)] has been functionally defined as a CIA factor depletion on the maturation of iron regulatory protein 1 (IRP1), (29). So far, the human homologs of yeast Tah18, Dre2, and a bifunctional cytosolic protein with a critical sensory role in iron Cfd1, termed “NDOR1,”“CIAPIN1,” and “CFD1” (NUBP2), metabolism (2, 44). Assembly of its [4Fe-4S] cluster and con- respectively, have not been assigned a function in Fe-S protein version to cytosolic aconitase (cytAco) was shown previously to biogenesis in vivo. Work performed in vitro and by yeast com- require NBP35, IOP1, and the specialized CIA-targeting factor plementation has suggested that human NDOR1 and CIAPIN1 CIA2A (29, 36, 38). The Fe-S cluster is lost under iron deficiency have a biochemical function similar to that of their homologs in or upon Fe-S protein biogenesis defects, which induce IRP1 to bind to mRNA stem–loop structures called “iron-responsive el- yeast (20, 30). Interestingly, plants lack a homolog of Cfd1, and ” plant Nbp35 has been shown to fulfill its scaffold function in- ements (IREs), thereby posttranscriptionally regulating the dependently of this partner (31). Trypanosomes, on the other expression of various proteins involved in cellular iron homeostasis. We assessed cytAco activity in cytosolic fractions almost hand, seem to rely on both Nbp35 and Cfd1 for cytosolic Fe-S devoid of mitochondrial contaminations as indicated by cytosolic protein biogenesis (32). In higher eukaryotes NBP35 and CFD1 lactate dehydrogenase (LDH) and mitochondrial citrate synthase have been implicated in the organization of microtubules, cen- – (CS) enzyme activities (SI Appendix,Fig.S3A and B). RNAi- trosomes, and cilia (33 35), but it remains unclear whether this mediated depletion of CFD1 resulted in a time-dependent de- role is direct or indirect. The second part of the human CIA cline of cytAco activity by up to 70% (Fig. 1B, Lower and SI pathway is much better characterized than the initial phase. Cell Appendix, Fig. S3C), in line with earlier findings in HEK293 cells biological and biochemical studies have identified essential roles (45). Further, IRP1 protein levels decreased substantially (Fig. 1B, of the Nar1 homolog IOP1, the CTC components CIAO1- Upper and SI Appendix, Fig. S3D), presumably due to impaired – CIA2B-MMS19, and CIA2A (25, 36 41). The latter four com- IRP1 protein stability as a consequence of defective Fe-S cluster ponents, in particular, were shown to assist several target-specific assembly (38, 46). Consistent with the cytosolic location of CFD1, Fe-S cluster-insertion routes by transient binding to their dedi- its depletion did not affect activities or protein levels of the two cated Fe-S apoproteins (SI Appendix, Fig. S1) (9, 25, 38, 42). mitochondrial Fe-S enzymes aconitase (mtAco) and succinate Nbp35 and Cfd1 belong to the SIMIBI (signal recognition, dehydrogenase (SDH) (SI Appendix,Fig.S3E–H). MinD and BioD) (43) family of P-loop nucleoside triphospha- To directly determine the assembly of Fe-S clusters on IRP1, tases (NTPases), forming a subgroup with several metal-binding we used a newly developed 55Fe radiolabeling assay with and -trafficking proteins. The yeast Nbp35-Cfd1 complex pos- IRP1 fused to a tripartite EGFP-TEV-FLAG tag (SI Appendix, sesses ATPase activity in vitro (18), and mutation of the Walker Fig. S4A) (47). HeLa cells were RNAi-depleted for CFD1, or for nucleotide-binding motifs impairs de novo [4Fe-4S] cluster as- mitochondrial NFS1 and cytosolic NBP35 as controls, by two sembly on this complex in vivo (13). Site-directed mutagenesis consecutive rounds of transfection (6, 29). During the second revealed cluster coordination via two essential cysteine ligands round, IRP1 reporter or control plasmids were included. Cells present in the conserved C-terminal CPxC motifs of both pro- were then grown in the presence of 55Fe-loaded transferrin for 3 d teins. Consistent with a scaffold function, Fe-S cluster binding is and were lysed, and the IRP1-EGFP-TEV-FLAG fusion protein labile, thus facilitating rapid transfer to apoproteins (14). In was immunoprecipitated by an anti-FLAG affinity matrix. To de- contrast, a second, functionally essential [4Fe-4S] cluster is stably crease the background radioactivity from beads, IRP1-EGFP was bound at the N terminus of Nbp35. released by tobacco etch virus (TEV)-protease cleavage, and IRP1- To better characterize the initial phase of the human CIA associated radioactivity and fluorescence were determined. The 55 pathway, we depleted human CFD1 by RNAi in HeLa cells and ratio of Fe-to-EGFP fluorescence in the TEV eluates reflected investigated the protein’s potential function in cytosolic and the ratio of mature IRP1 to total IRP1-EGFP and hence was in- nuclear Fe-S protein biogenesis. To gain mechanistic insights dependent of variations in the IRP1-EGFP levels among different samples (SI Appendix,Fig.S4B). Depletion of CFD1 or of into the scaffold function of Cfd1-Nbp35, we determined the 55 crystal structure of holo-Cfd1 from the thermostable fungus NFS1 and NBP35 resulted in a substantial decrease in Fe-loaded Chaetomium thermophilum as a model P-loop NTPase. Our re- IRP1-EGFP fusion protein per total IRP1-EGFP (Fig. 1C and SI sults document a crucial function of human CFD1 for biogenesis Appendix,Fig.S4C). In line with the degradation of apo-IRP1, the amount of immunoprecipitated 55Fe per total input lysate was of all tested cytosolic-nuclear Fe-S proteins and provide impor- similarly diminished by up to 85%, clearly showing the Fe-S tant structural clues about how these P-loop NTPases execute maturation defect. The 55Fe content in cell lysates hardly varied their scaffold function. among the various samples, excluding iron availability as a reason Results for the observed effects (SI Appendix, Fig. S4 D and E). Together, the results demonstrate that CFD1 is required for Fe-S cluster Human CFD1 Is an Essential Cytosolic Protein. We first determined insertion into IRP1, similar to the function of NFS1 and NBP35. the subcellular localization of human CFD1 by immunofluores- Since CFD1 deficiency lowered the cytAco activity of IRP1, we cence and digitonin-based cell fractionation. Immunofluores- examined whether, vice versa, the IRE-binding capacity of IRP1 cence showed a cytoplasmic distribution (SI Appendix, Fig. S2A). was increased. IRP1 and its iron-regulated counterpart IRP2 co- Fractionation indicated colocalization of CFD1 with cytosolic ordinately regulate cellular iron homeostasis (48, 49). We used tubulin but not with the mitochondrial intermembrane space RNA EMSA (REMSA) to determine the mRNA-binding capac- protein MIA40 (Fig. 1A). RNAi-mediated depletion of CFD1 by ities of both IRP1 and IRP2 using a [γ-32P]CTP-labeled ferritin three consecutive rounds of transfection at a 3-d interval with IRE probe (38). Since both protein-RNA complexes run similarly four individual siRNAs or a pool of these siRNAs resulted in a on native gels, we performed supershifts by attaching specific decline of CFD1 mRNA and protein by more than 75% and a antibodies to one of the IRPs, allowing analysis of the other. substantial loss of the immunofluorescence signal, verifying the Depletion of CFD1 for 9 d by RNAi increased the IRE-binding specificity of the CFD1 immunodetection (Fig. 1A and SI Ap- capacity of residual cellular IRP1 more than threefold (Fig. 1D, pendix, Fig. S2 A–C). Knockdown of CFD1 was accompanied by Lower and SI Appendix,Fig.S5A). For this evaluation, we used the a substantial time-dependent decrease in cell count and total proportion of intrinsic [Fig. 1D, Upper,noβ-mercaptoethanol protein yield (SI Appendix, Fig. S2 D and E), indicating a vital (β-ME)] to maximal (with β-ME) IRP1 binding activity to ac- cellular function of CFD1 in HeLa cells. count for the two outcomes associated with IRP1 assembly defects,

E9086 | www.pnas.org/cgi/doi/10.1073/pnas.1807762115 Stehling et al. Downloaded by guest on September 26, 2021 A D

CFD1 no α β-ME α-Tubulin -IRP2 supershift with MIA40 β-ME

Lysate Cytosol Organelles 400 Scr [%] siCFD1 bound 300 IRE B - 200 IRP1 [CTP] 100 -

β-Actin P 32

Ratio of IRP1- 0 Growth time [days] 3 6 9 100 E 50 0.3% β-ME Scr α-IRP1 supershift

Enzyme activity Enzyme siCFD1 cytAco / LDH [%] cytAco 0

Growth time [days] 3 6 9 [%] (Transfection) (1st) (2nd) (3rd) 100 IRE bound C 50

100 [CTP]- -

IRP2- Scr

P siCFD1 32 0 IRP1 [%] 50 Growth time [days] 3 6 9 Fe- 55 0 F TFR1 H-FT β-Actin β-Actin Recovery of 55Fe after IP: per purified IRP1-EGFP 150 (maturation status of IRP1) per total lysate 100 (cellular amount of mature IRP1)

Level [%] 50 Scr siCFD1 0 Tf-FITC TFR1 H-FT

Fig. 1. Depletion of human CFD1 both impairs Fe-S cluster assembly on IRP1 and affects cellular iron regulation. HeLa cells were transfected at 3-d intervals with no siRNA (Mock), scrambled siRNAs (Scr), or a pool of CFD1-directed siRNAs (siCFD1). After each round of transfection cells were fractionated using 0.01% digitonin. (A) Total-cell lysates as well as cytosol and organelle fractions obtained after three transfections were analyzed for the indicated proteins by immunoblotting. (B) After each round of transfection total-cell lysates were immunoblotted for IRP1 and β-actin (Upper), and cytosolic fractions were analyzed for enzymatic activities of cytAco relative to LDH (Lower). (C) HeLa cells were depleted of NFS1, NBP35, or CFD1 for a total of 6 d by RNAi. At the second round of transfection at day 3, cells received plasmids encoding either an IRP1-EGFP-TEV-FLAG fusion protein or an EGFP-TEV-FLAG reference protein (EGFP only) and were grown for another 3 d in the presence of 55Fe-labeled transferrin. FLAG-tagged proteins were immunopurified, and associated radioactivity was determined relative to either recovered IRP1-EGFP fluorescence (filled bars) or total lysate protein (hatched bars). (D, Upper) IRP1 binding (IRP2 supershift method) to 32P- labeled IRE (32P-[CTP] IRE) of human ferritin mRNA in the presence or absence of 1.7% β-mercaptoethanol (β-ME) was quantified by phosphorimaging of a native gel. (Lower) The ratio of IRP1-bound 32P-IRE probe in the absence and presence of β-ME is an inverse measure for the Fe-S cluster maturation status of IRP1. (E)IRE- binding of IRP2 (IRP1 supershift) was probed as in D (Upper) and quantified (Lower), but in the presence of 0.3% β-ME. (F) After the third round of transfection, total-cell lysates were analyzed for TFR1, H-FT, and β-actin by immunoblotting. (Upper) Representative blots. (Lower) Protein levels were quantitated relative to β-actin. Further, TFR1 expression was functionally assessed by estimating cellular binding of Tf-FITC relative to total cellular protein. All values were normalized to mock-transfected cells (set to 100% and indicated by dashed lines) and are expressed as mean ± SD (n ≥ 3). sh, shRNA; si, siRNA.

namely the increase in IRP1’s intrinsic IRE-binding activity and heavy chain (H-FT) were slightly decreased (Fig. 1F and SI BIOCHEMISTRY the decrease in β-ME–induced maximal IRE binding explained Appendix, Fig. S5D). Both observations are fully explained by the by lower cellular levels of the protein (Fig. 1B). Depletion of impaired maturation of IRP1, although lower mRNA binding of CFD1 also affected IRP2, resulting in a 40% drop of its IRE- IRP2 might partially compensate for elevated IRP1 binding, binding activity (Fig. 1E and SI Appendix, Fig. S5B). This effect particularly to ferritin IREs (50). This compensatory mechanism has also been observed during NBP35 depletion (29) and might might explain why we did not observe changes in cellular 55Fe be caused by higher cytosolic iron levels resulting from increased content during the earlier phase (days 3–6) of CFD1 deficiency IRP1 activity. In turn, such a situation leads to increased iron- (SI Appendix, Fig. S4 D and E). Taken together, the enzyme dependent IRP2 degradation (45). activity, immunoblotting, 55Fe-incorporation, and IRE-binding The alteration of IRP1/2 activities during CFD1 depletion assays uniformly document that depletion of human CFD1 re- impacted cellular iron regulation by altering transferrin receptor sults in impaired Fe-S cluster insertion into IRP1, thus affecting (TfR) and ferritin protein levels. After 9 d of CFD1 depletion we cellular iron homeostasis. found a significant increase in cellular binding of fluorescently labeled holo-transferrin (Tf-FITC) and an elevation of TfR Depletion of CFD1 Impairs Maturation of Cytosolic Fe-S Proteins. We levels (Fig. 1F and SI Appendix, Fig. S5 C and D), which is next asked whether CFD1 depletion exerts a general effect on reminiscent of an iron-deficiency condition (+DFO samples in SI cytosolic Fe-S protein biogenesis. Since IRP1 is specifically ma- Appendix, Fig. S5C). Consistently, the protein levels of ferritin tured by the CIA2A pathway, we examined several Fe-S proteins

Stehling et al. PNAS | vol. 115 | no. 39 | E9087 Downloaded by guest on September 26, 2021 known to be assembled along the CIAO1-CIA2B-MMS19– A dependent route (SI Appendix,Fig.S1) (25, 38). A clinically rel- evant example is dihydropyrimidine dehydrogenase (DPYD) (51), [14C]-DHT an Fe-S enzyme catalyzing the reduction of pyrimidines and hence detoxifying some of their derivatives such as the anticancer drug 5- fluorouracil (52). Maturation of DPYD was determined based on the enzyme’s activity to convert [4-14C]-thymine into [4-14C]- 14 [ C]-T dihydrothymine ([4-14C]-DHT), which were separated by TLC (47). CFD1 depletion was associated with a 90% decrease in Growth [days] 3 6 9 DPYD activity (Fig. 2A and SI Appendix,Fig.S6A)andasimilar but less severe loss of DPYD protein, presumably due to degradation of its apo form (Fig. 2B and SI Appendix,Fig.S6B). Both 100 effects were consistent with a crucial function of CFD1 in Fe-S cluster assembly on DPYD. 50 A second CTC-dependent Fe-S target protein with two “ Scr [4Fe-4S] clusters is ABCE1 (also known as RNase L inhibitor Enzyme activity Enzyme DPYD / LDH [%] / LDH DPYD siCFD1 RLI”), a cytosolic ABC protein involved in ribosome biogenesis 0 and recycling (SI Appendix, Fig. S1) (25, 38, 53–55). To directly Growth [days] 3 6 9 follow its Fe-S maturation, we analyzed the incorporation of 55Fe into an EGFP-ABCE1-TEV-FLAG fusion protein. Knockdown B of CFD1, NFS1, or NBP35 resulted in a substantial decrease in 55Fe binding to EGFP-ABCE1, by up to 75% (Fig. 2C and SI DPYD Appendix, Fig. S6C), although cellular iron availability was not β-Actin decreased in the various depleted cells (SI Appendix, Fig. S6 D and E). Immunoblotting revealed that, in contrast to IRP1 and DPYD, ABCE1 protein levels declined only slightly upon RNAi- 100 mediated depletion of NFS1, NBP35, or CFD1, suggesting that apo-ABCE1 is relatively stable (SI Appendix, Fig. S6 F and G). In contrast to DPYD, Fe-S cluster insertion into glutamine 50 phosphoribosylpyrophosphate amidotransferase (GPAT), a tet-

Protein level Scr rameric enzyme catalyzing the first step of purine nucleotide siCFD1 -actin [%] / β -actin DPYD 0 synthesis (56), is mainly dependent on CIAO1-CIA2B but does Growth [days] 3 6 9 not require MMS19 (25, 38, 57). GPAT is translated as an inactive proenzyme and becomes matured by consecutive in- C corporation of a [4Fe-4S] cluster and subsequent removal of an N-terminal undecamer propeptide. Since cluster insertion criti- Fe) /

(55 100 cally determines protein stability (29, 56), the protein level of GPAT provides a direct measure of Fe-S cluster insertion into the enzyme. Depletion of CFD1 by RNAi resulted in a profound 50 time-dependent decrease in GPAT protein by up to 60% (Fig. 2D and SI Appendix, Fig. S6H), suggesting that CFD1 is also 0 crucial for GPAT maturation. In summary, human CFD1 is re- mature ABCE1 total ABCE1 (EGFP) [%] quired for Fe-S cluster assembly, not only on IRP1 but also on DPYD, ABCE1, and GPAT, and thus may act as a general CIA factor that supports both the CTC and CIA2A pathways. D Depletion of CFD1 Impairs Maturation of Nuclear Fe-S Proteins. Previous work has shown that the CIA system also plays a crit- GPAT ical role in the biogenesis of multiple nuclear target Fe-S pro- β-Actin teins, including those involved in various aspects of genome maintenance (25, 38, 39, 42). A typical example is the catalytic subunit of DNA polymerase δ, POLD1, a homolog of yeast Pol3 100 (58). Insertion of its C-terminal [4Fe-4S] cluster in human cells strictly requires CIAO1 and MMS19 but not CIA2B (38). De-

β -actin [%] pletion of CFD1 led to a decrease in POLD1 protein levels by 50 more than 50% (Fig. 3A and SI Appendix, Fig. S7A), consistent Protein level Scr with lower apoprotein stability due to inefficient Fe-S cluster siCFD1 GPAT / GPAT 0 assembly (25, 38). Growth [days] 3 6 9 Similar results were obtained for the DNA base-excision repair (BER) enzyme NTHL1, a glycosylase/AP-lyase related to Fig. 2. Depletion of CFD1 impairs the maturation of cytosolic Fe-S proteins. HeLa cells were depleted for CFD1 as in Fig. 1, and DPYD, ABCE1, and GPAT were analyzed for enzyme activity, 55Fe incorporation, or protein levels. (A, Upper) DPYD-dependent formation of [4-14C]-dihydrothymine ([4-14C]-DHT) pleted of NFS1, NBP35, or CFD1 by RNAi, and incorporation of 55Fe into an from [4-14C]-thymine ([4-14C]-T) was determined by TLC and subsequent EGFP-ABCE1-TEV-FLAG fusion protein was determined as in Fig. 1. (D) GPAT autoradiography. (Lower) Specific DPYD enzyme activity was calculated levels were examined and quantified as in B. Representative autoradio- from the proportion of [4-14C]-DHT converted from [4-14C]-T and is pre- graphs and blots are shown. All values were normalized to mock-transfected sented relative to LDH activity. (B) DPYD and β-actin levels were examined by cells (set to 100% and indicated by dashed lines) and are expressed as immunoblotting (Upper) and quantified (Lower). (C) HeLa cells were de- mean ± SD (n ≥ 3). scr, scrambled siRNAs; sh, shRNA; si, siRNA.

E9088 | www.pnas.org/cgi/doi/10.1073/pnas.1807762115 Stehling et al. Downloaded by guest on September 26, 2021 A incorporation into NTHL1 by at least 40% but did not lower cellular iron content (Fig. 3C and SI Appendix,Fig.S7C–E). These results demonstrate that CFD1, like NFS1, contributes to Fe-S POLD1 cluster assembly on the BER enzyme. Taken together, both im- β-Actin munoblotting and 55Fe incorporation studies assign a critical role to human CFD1 in the maturation of essential nuclear Fe-S proteins.

100 Human CFD1 Interacts with NBP35 and IOP1 and Is Required for Maturation of IOP1. The general involvement of human CFD1 in cytosolic and nuclear Fe-S protein assembly is shared with the 50 CIA components NBP35 and IOP1 (29, 36). To test whether the Scr three proteins interact physically, various tagged versions were Protein level siCFD1 coexpressed in HeLa cells, and potential protein interactions

POLD1 / β -actin [%] 0 were analyzed by coprecipitation. Strep-tagged CFD1 copurified Growth [days] 3 6 9 both endogenous and myc-tagged NBP35 as well as endogenous and HA-tagged IOP1 (Fig. 4A, Middle and Top). Vice versa, B affinity purification of myc-tagged NBP35 recovered both endogenous and Strep-tagged CFD1 as well as endogenous and NTHL1 HA-tagged IOP1 (Fig. 4A, Bottom and Top). Likewise, HA- tagged IOP1 precipitated both Strep-tagged and endogenous β-Actin CFD1 and NBP35, but at lower efficiencies (Fig. 4A, Bottom and Middle). All pulldowns were specific, because no interaction was observed in coprecipitations in which the proteins did not carry 120 specific tags (Fig. 4A, right lanes). Consistent with the physical interaction of CFD1 and NBP35, 80

-actin [%] RNAi-mediated depletion of CFD1 resulted in a slight decrease β in NBP35 protein (Fig. 4B and SI Appendix, Fig. S8A). Con- 40 versely, shRNA-mediated knockdown of NBP35 impaired cel- Protein level lular CFD1 levels, suggesting a mutual stabilization of the two

NTHL1 / 0 CIA factors. CFD1 deficiency also caused a substantial loss of 369 369369369369 369 369 36 Growth [days] the [4Fe-4S] cluster-containing CIA component IOP1 (Fig. 4C siRNA and SI Appendix, Fig. S8B). We therefore determined the maturation of IOP1 by 55Fe incorporation into an IOP1-EGFP- TEV-FLAG reporter protein. Depletion of CFD1, NFS1, or 55 C NBP35 substantially decreased Fe-S cluster assembly on the

Fe) / IOP1 fusion protein but did not alter cellular iron content (Fig.

(55 100 4D and SI Appendix, Fig. S8 C–E). Collectively, these results indicated that human CFD1 physically interacts with the known 50 CIA proteins NBP35 and IOP1 and is required for the maturation of IOP1. These findings characterize CFD1 as a central component of the early phase of the CIA pathway. 0 mature NTHL1 total NTHL1 (EGFP) total NTHL1 [%] The Crystal Structure of C. thermophilum Cfd1 Reveals a Homodimer with a Surface-Exposed Bridging [4Fe-4S] Cluster. Better mechanistic understanding of the eukaryotic CIA scaffold complex function will benefit from structural insights into the 3D architecture of Fig. 3. Depletion of CFD1 impairs assembly on the nuclear Fe-S proteins. these proteins, in particular the coordination of the transiently HeLa cells were RNAi-depleted for CFD1 or for the indicated ISC or CIA bound [4Fe-4S] cluster. We succeeded in crystallizing Cfd1 from factors as in Fig. 1. (A and B) POLD1 (A) and NTHL1 (B) as well as β-actin as a the thermophilic fungus C. thermophilum (ct), which has a higher control were examined by immunoblotting (Upper) and quantified (Lower). stability than Saccharomyces cerevisiae or human counterparts The immunoblots show the last round of transfection only. (C) Incorporation (13, 18). The crystal structure [2.57-Å resolution; Protein Data

55 BIOCHEMISTRY of Fe into a NTHL1-EGFP-TEV-FLAG fusion protein was determined as in Bank (PDB) ID code 6G2G] showed a ctCfd1 homodimer with a Fig. 1. Representative blots are shown. All values were normalized to mock- transfected cells (set to 100% and indicated by the dashed line) and are bound [4Fe-4S] cluster (Fig. 5A, SI Appendix, Table S1, and expressed as mean ± SD (n ≥ 3). scr, scrambled siRNAs; sh, shRNA; si, siRNA. Movie S1). The fold of each subunit was dominated by an eight- stranded β-sheet flanked on both sides by α-helices, as typically found in MinD-type NTPases including other metal-binding the bacterial [4Fe-4S] cluster-containing endonuclease III (59). proteins (SI Appendix, Table S2). The electron density and a NTHL1 is enzymatically inactive in murine tissues lacking the strong anomalous scattering signal within the loop regions con- mitochondrial ISC factor frataxin (56) and is physically associ- necting two antiparallel β-strands indicated the presence of a ated with the CTC complex (25, 38). Knockdown of CFD1 or of subunit-bridging [4Fe-4S] cluster at the protein surface (SI Ap- various ISC or CIA factors resulted in an up to 80% loss of pendix, Fig. S9A). Coordination of the cluster was mediated by NTHL1 protein, indicating that the apoprotein is highly sensitive cysteine residues C199 and C202 of the conserved CPxC motif of to degradation upon impaired cofactor insertion (Fig. 3B and SI each subunit (Fig. 5A). Corresponding residues of S. cerevisiae Appendix,Fig.S7B). Notably, siRNA-mediated ablation of CIA2A Cfd1 and the related Nbp35 previously have been found to be did not affect NTHL1 protein levels, in line with the dedicated essential for both cell viability and Fe-S cluster binding, and CIA2A function in IRP1 maturation. Using a NTHL1-EGFP-TEV- hence a bridging Fe-S cluster coordination had been proposed FLAG fusion protein, we directly assessed Fe-S cluster assembly by (13). Two other conserved, but nonessential Cys residues (cor- 55Fe radiolabeling. Depletion of CFD1 or NFS1 decreased 55Fe responding to ctCfd1 C180 and C205) were not needed for Fe-S

Stehling et al. PNAS | vol. 115 | no. 39 | E9089 Downloaded by guest on September 26, 2021 A Input Pull-Down cluster binding to S. cerevisiae Cfd1 or Nbp35. Our crystal IOP1-HA3 + + + – + + + + + – structure of ctCfd1 located these additional Cys residues as ei- NBP35-DsRed2 – – + – – – – – + – NBP35-myc + + – + + + + + – + ther distant from the cluster coordination site (C180) or pointing CFD1-DsRed2 – + – – – – – + – – away from the metal center (C205). CFD1-Strep + – + + + + + – + + The surface-exposed location of the [4Fe-4S] cluster is ideally IOP1-HA 3 * suited for its facile de novo assembly and dislocation. In- IOP1endo terestingly, modeling of the structure of mitochondrial ctInd1 NBP35-DsRed2 (human NUBPL), a Cfd1- and Nbp35-related P-loop NTPase NBP35-myc implicated in [4Fe-4S] cluster delivery to respiratory complex I NBP35endo (60), revealed a putative Fe-S cluster-binding site at a CPxC CFD1-DsRed2 motif and loop location similar to that of ctCfd1 (SI Appendix, CFD1-Strep Fig. S9B and Table S3). Likewise, the crystal structure of an CFD1endo archaeal Cfd1-like protein of unknown function (AF_226 of + Archaeoglobus fulgidus) contained a bound Zn2 ion at the corresponding CPxC motif (PDB ID code 3KB1) (SI Appendix, Fig. S9B and Table S2). In contrast, the Fe-S center of ctCfd1 was located on the opposite side (relative to the Walker motif) of the B metal-binding site of other members of metal-binding P-loop NBP35 NTPases, e.g., NifH binding a [4Fe-4S] cluster for electron + CFD1 transfer or the Ni2 enzyme maturases CooC1 and UreG (Fig. 5 β-Actin B and C and SI Appendix, Fig. S9B and Table S2) (61–63). This is reflected by the different locations of the metal-coordinating 100 residues within these different proteins of similar structure (SI Appendix, Fig. S9C). Apparently, versatile solutions exist for β -actin [%] 50 metal cofactor binding and delivery within this class of proteins. Scr The nucleotide-binding P-loop in our ctCfd1 crystal structure Protein level siCFD1 accommodated a sulfate ion and was located opposite the bound

NBP35 / 0 100 [4Fe-4S] cluster (Fig. 5). Since sulfate and phosphate ions often bind at the same position in proteins, we used the structure of A. fulgidus AF_226 with bound ADP as a template for modeling

actin [%] 50 nucleotides into the ctCfd1 structure. ADP could be fitted into a β - preformed pocket of ctCfd1 without steric clashes with the

Protein level β shNBP35 -phosphate located at the site of sulfate. Two locations for the CFD1 / 0 purine ring were possible in negatively charged environments (SI Growth [days] 3 6 9 Appendix, Fig. S9D). The negative charges of the β-phosphate of ADP were bound to the P-loop lysine K21 within the Walker A motif, while the signature lysine K16 was directed outwards to- C ward its symmetry mate (SI Appendix, Fig. S9E). The strictly IOP1 conserved glutamine Q166 was located exactly opposite the nu- β-Actin cleotide sugar (Movie S1). Since ATP and GTP could be modeled equally well into the ctCfd1 nucleotide-binding pocket (SI Appendix,Fig.S9F), we ex- 100 perimentally tested the nucleotide specificity of ctCfd1 (and of

-actin [%] ctNbp35) by equilibrium titrations using microscale thermopho- β 50 resis (MST). CtCfd1 exhibited a 10-fold higher affinity to ATP Scr Protein level siCFD1 than to GTP, while it showed no significant binding to CTP (Fig. IOP1 / 0 6A and SI Appendix,Fig.S10A). Surprisingly, when we determined Growth [days] 3 6 9 the affinities of ctNbp35 to nucleotides, the opposite specificity for D the two purine nucleotides was observed. As expected, ctCfd1 and ctNbp35 formed a tight complex with a Kd of 0.6 μM as measured

Fe) / 100 by MST (Fig. 6B and SI Appendix,Fig.S10B). This complex (55 showed similar affinities for both purine nucleotides, clearly dis- tinguishing their properties from the yeast counterparts, which 50 bind only ATP (Fig. 6A and SI Appendix,Fig.S10C) (18). At variance, human CFD1 and NBP35 showed similar affinities to

mature IOP1 0 total IOP1 (EGFP) [%] both ATP and GTP, but they bound to CTP only weakly (SI Appendix,Fig.S11). Apparently, the selectivity of Cfd1 and Nbp35 for ATP or GTP is not conserved in different species.

Fig. 4. Human CFD1 interacts physically with NBP35 and IOP1 and is required for Fe-S cluster assembly on IOP1. (A) HeLa cells were cotransfected depleted for CFD1 or NBP35 as in Fig. 1 and were analyzed for CFD1, NBP35,

with plasmids encoding human IOP1 fused to a C-terminal HA-tag (HA3), or β-actin levels as indicated. (C) HeLa cells were depleted for CFD1 and human NBP35 fused to a C-terminal DsRed2- or myc-tag, or human analyzed for IOP1 and β-actin levels. (D) HeLa cells were depleted for NFS1, CFD1 fused to a C-terminal DsRed2- or Strep-tag, as indicated. After 3 d of NBP35, or CFD1, and incorporation of 55Fe into a IOP1-EGFP-TEV-FLAG fusion tissue culture, cleared lysates were subjected to affinity purification by Strep- protein was determined as in Fig. 1. Representative blots are shown. All Tactin, anti-myc, or anti-HA resins. Cleared lysate (50 μg protein, Input) and values were normalized to mock-transfected cells (set to 100% and indicated bead-associated material (one-third of total precipitate) were immunos- by the dashed lines) and are expressed as mean ± SD (n ≥ 3). scr, scrambled tained for endogenous or tagged IOP1, NBP35, and CFD1. (B) HeLa cells were siRNAs; sh, shRNA; si, siRNA.

E9090 | www.pnas.org/cgi/doi/10.1073/pnas.1807762115 Stehling et al. Downloaded by guest on September 26, 2021 Discussion In this study, we present cell biological and ultrastructural findings that define the essential role of the human P-loop ATPase CFD1 (NUBP1) in the assembly of Fe-S clusters on cytosolic and nuclear target proteins. We localized human CFD1 in the cytosol as an interaction partner of its close relative NBP35 (NUBP1), a P-loop NTPase indispensable for the assembly of cytosolic and nuclear Fe-S proteins in yeast, plants, trypanosomes, and humans (12, 29, 31, 32). CFD1 also interacted with the known CIA component IOP1, suggesting an early function in the CIA pathway. This task is in line with and extends earlier studies in yeast, where the Cfd1-Nbp35 heteromeric complex has been assigned a scaffold function in the CIA system during the initial assembly of a [4Fe-4S] cluster (12–15, 17, 18). Our 3D crystal structure of a fungal Cfd1 dimer with a bound [4Fe-4S] cluster provides a first molecular view of how the scaffold function might be executed. The cellular role of CFD1- NBP35 appears to be conserved from yeast to man, but the green eukaryotic lineage was shown to lack a cytosolic CFD1 ortholog (31). Apparently, plant NBP35 can take over the CFD1 function, presumably by acting in a homomeric fashion. To examine whether CFD1 performs a general or specialized function within the human CIA system, we used a variety of approaches. For instance, we measured the activities of Fe-S enzymes and followed the stability of various Fe-S proteins upon depletion of CFD1. As a direct test of de novo Fe-S cluster insertion into a given target apoprotein, we employed a radiolabeling assay with 55Fe. Collectively, we found that all cytosolic and nuclear Fe-S reporter proteins tested in this study required CFD1 for their maturation, revealing a general rather than specialized task of this P-loop NTPase within the branched human CIA system. CFD1 therefore acts early in the CIA pathway and joins the CIA factors NBP35 and IOP1, which are also involved in the maturation of all cytosolic and nuclear [4Fe-4S] proteins including essential proteins required for translation (ABCE1) (26, 54, 64, 65) or nucleotide metabolism and DNA maintenance (GPAT, DPYD, POLD1, and NTHL1) (51, 56, 66, 67). Moreover, CFD1 supports the maturation of cytosolic IRP1 and hence is critically involved in cellular iron regulation (45). Our results revealed that both CFD1 and NBP35 support each branch of the late CIA pathway in HeLa cells (SI Appendix, Fig. S1) (9). Mass spectrometric analyses have suggested that the last common CIA component before branching of the CIA pathway is IOP1 (38), a protein carrying two [4Fe-4S] clusters of unknown but essential function (22). IOP1 has been shown to be involved in cluster insertion into IRP1, and its yeast ortholog Nar1 is required for maturation of Fe-S proteins dependent on CTC (CIAO1-CIA2B-MMS19) (21, 25, 36). However, vice versa, the CTC components are not critically required for Fe-S cluster insertion into Nar1 (17). In our study, we found that human

CFD1 and NBP35 interact physically with IOP1 and support BIOCHEMISTRY 55Fe incorporation into this protein. These results sketch a se- quence of events in which CFD1 and NBP35 cooperate to allow Fe-S cluster assembly on IOP1, which in turn assists both the CIA2A- and CTC-dependent targeting pathways of Fe-S cluster insertion into dedicated target proteins. Using Cfd1 from the thermotolerant fungus C. thermophilum, we determined its crystal structure with a bound [4Fe-4S] cluster. While the observed dimeric architecture and the nucleotide- Fig. 5. Crystal structure of C. thermophilum Cfd1. (A) Cartoon presentation binding regions are similar overall to other P-loop NTPases of the crystal structure of dimeric ctCfd1. The left monomer is colored from such as NifH (SI Appendix, Fig. S9B and Table S2) (43, 61), the blue at the N terminus to red at the C terminus. The right monomer is shown in blue and red for α-helices and β-strands, respectively. A sulfate ion near the P-loop is depicted as spheres. A [4Fe-4S] cluster at the dimer interface is indicated by the anomalous scattering contribution of iron at 1.73-Å nitrogenase Fe-protein NifH (SI Appendix, Table S2). A subunit of ctCfd1 wavelength (blue meshed map contoured at 8 σ). Conserved cysteine resi- (cyan) with iron ions shown as pink spheres is superposed onto a subunit of dues are highlighted as sticks with carbon in cyan and sulfur in gold. Stars the Fe-protein (PDB ID code 4WZB, green) with irons shown as light blue indicate residues belonging to the symmetry mate. Dashed lines indicate spheres. (C) Schematic comparison of the relative arrangement of Fe-S regions with weak electron density. (B) Superposition of ctCfd1 and bacterial clusters and ATP in ctCfd1 and NifH.

Stehling et al. PNAS | vol. 115 | no. 39 | E9091 Downloaded by guest on September 26, 2021 The function of yeast Cfd1 and Nbp35 NTPases in cytosolic ABctCfd1 0,4 ctNbp35 2 Fe-S protein assembly is critically dependent on their Walker A ctCfd1/ctNbp35 0,3 nucleotide-binding motifs (13, 18). This raises the questions (i) ] ] 1 - -1 which nucleotides are used by these proteins and (ii) which 0,2 1 [µM [µM function may be assisted by the putative nucleoside triphospha- 0,1 tase hydrolysis. Yeast Nbp35 (but not Cfd1) has been shown to Binding constant nd nd Binding constant ′ ′ ′ 0,0 0 bind the ATP-derivative 2 /3 -O-(N -methylanthraniloyl)-ATP Nucleotide ATP GTP CTP Binding (mantATP) and to exert slow ATPase activity when present in partner homodimeric (Nbp35)2 or heterodimeric (Nbp35-Cfd1) complexes (18). In the present study, we found differential binding Fig. 6. C. thermophilum Cfd1 and Nbp35 form a complex with differential specificities of ctCfd1 and ctNbp35 for (nonmodified) ATP and binding preferences for ATP and GTP. Purified recombinant ctCfd1 and ctNbp35 were labeled with the fluorescent dye NT 647 either individually or GTP by thermophoresis. CtCfd1 exhibited a 10-fold higher as complex and were analyzed for ligand binding by thermophoresis. (A) affinity to ATP than to GTP, whereas ctNbp35 showed the

Binding constants (1/Kd) for the interaction between labeled ctNbp35, opposite behavior. The ctCfd1-ctNbp35 complex bound both ctCfd1, or the ctCfd1-ctNbp35 complex and the three nucleotides ATP, GTP, purines equally well. Surprisingly, human CFD1 and NBP35 did or CTP. (B) Binding constants (1/Kd) for labeled ctNbp35 or ctCfd1 and their not display any preference for one of the purines. Apparently, ± ≥ nonlabeled protein partners. Values are given as mean SD (n 3). nd, these proteins do not exhibit a clear selectivity for one of the two nondetectable. purines. This may explain why both ATP and GTP could be modeled well into our ctCfd1 crystal structure. ctCfd1 dimer forms a new structural subclass which binds a Mutation of the Walker motifs of yeast Cfd1 and Nbp35 se- bridging [4Fe-4S] cluster at a surface-exposed CPxC motif. This verely interferes with in vivo Fe-S cluster loading onto these functionally essential motif is also found in Nbp35 and is arranged proteins (13). It is unknown at which step of Cfd1-Nbp35 in a loop structure. In the ctCfd1 dimer, the two loops are held function the bound nucleotides are hydrolyzed and whether the together by stacking interactions of the conserved Phe166, two subunits communicate in this step. Hydrolysis may be used which has been shown to be crucial for Cfd1-Nbp35 homo- and either for Fe-S cluster assembly or for subsequent cluster dislo- heterocomplex formation and for Fe-S cluster binding in vivo (14). cation. Both steps could be accompanied by conformational Hence, the crystal structure of homodimeric ctCfd1 may serve as a changes including movements of the dimer subunits against each paradigm for the putative scaffold function of the heterocomplex. other, as proposed for CooC1 (73) and related SIMIBI proteins Likewise, ctCfd1 is structurally similar to the mitochondrial (74). In both CooC1 and NifH the nucleotide phosphate groups member of the Cfd1-Nbp35 family termed “Ind1” (NUBPL) and point toward the metal-binding sites, and hydrolysis may evoke to bacterial ApbC (SI Appendix, Fig. S9 B and C)(60,68).Both conformational changes that are transduced to the metal-binding proteins transiently bind [4Fe-4S] clusters and are involved in the site via the switch regions (61, 73). In contrast, in ctCfd1 the maturation of respiratory complex I and bacterial TcuB, re- pyrophosphate group points away from the metal center, sug- spectively. The ctCfd1 structural subclass is also represented by gesting that ATP hydrolysis per se is unlikely to influence the archaeal AF_226 (PDB ID code 3KB1). The dimer contains a coordination of the [4Fe-4S] cluster. Consistently, the addition of bridging Zn ion instead of an Fe-S cluster at the CPxC motifs and ATP or GTP does not alter the electron paramagnetic resonance thus may serve as a Zn chaperone. However, since Zn ions have a signal of a reconstituted Cfd1-Nbp35 holocomplex (13), and Fe-S tendency to associate with Fe-S cluster-binding sites, a more likely cluster transfer to target apoproteins can occur independently of scenario is that AF_226 also binds and transfers Fe-S clusters. added ATP. Consistent with this idea, homologous archaeal proteins can During the preparation of this paper, another group has complement bacterial apbC deletion mutants and bind Fe-S reported that neither CFD1 nor NBP35 is required for human clusters (69). Collectively, the Cfd1-Nbp35 subclass of P-loop cytosolic-nuclear Fe-S protein biogenesis (75). In line with the NTPases appears to use their conserved CPxC loop motif for findings presented here, the authors show that depletion of either transient binding and transfer of [4Fe-4S] clusters. CFD1 or NBP35 by RNAi destabilized the other interaction The MRP-MinD subfamily of P-loop NTPases containing partner, but in marked contrast to our previous and present Cfd1 and Nbp35 encompasses a number of other archaeal and studies (29), no effects on Fe-S proteins were found. Instead of bacterial members that are involved in metal center assembly, CFD1-NBP35 functioning as a scaffold complex, these authors but their primary sequences are homologous to Cfd1-Nbp35 only attribute an equivalent role to cytosolic ISCU1, suggesting that it within the Walker region. For example, CooC1 assists the maturation of the Ni-containing carbon monoxide dehydrogenase assembles an Fe-S cluster with the help of a cytosolic NFS1 ver- + (CODH) (70, 71). Two monomers transiently bind a Ni2 ion in sion. This scenario is not supported by previous complementation a bridging fashion and presumably transfer it to CODH in an studies showing that neither cytosolic NFS1 nor cytosolic ISCU1 ATP-dependent fashion. Likewise, HypB and UreG act as could assist human cytosolic Fe-S protein biogenesis in the ab- + metallo-chaperones to facilitate insertion of their bound Ni2 sence of the mitochondrial versions of these proteins (6, 76). – ions into [Ni-Fe]-hydrogenase and -urease, respectively (63, 72). Similar findings have been made in yeast (77 79). Therefore, a Similarly, the Fe-protein NifH of bacterial nitrogenase binds a function of ISCU1 as an alternative to the CFD1-NBP35 scaffold bridging [4Fe-4S] cluster between two monomers but fulfils two seems unlikely to us based on the available data. Rather, our different roles: It is involved in nitrogenase assembly by assisting current findings for CFD1 and earlier results on NBP35 demon- molybdenum and homocitrate transfer to a precursor, and it is strate the general role of these P-loop NTPases in cytosolic- required for electron transfer from its [4Fe-4S] cluster to the nuclear Fe-S protein biogenesis. This view is further supported molybdenum cofactor during the catalytic cycle of nitrogenase by the conserved function of these proteins in yeast, plants, and (61). Interestingly, the metal cofactors in all these proteins are trypanosomes (12, 29, 31, 32). Finally, in perfect agreement with bound at rather different locations and binding motifs compared earlier biochemical studies on yeast Cfd1-Nbp35, our 3D structure with ctCfd1 and AF_226 (SI Appendix, Fig. S9 B and C), in- of ctCfd1 with its surface-exposed [4Fe-4S] cluster provides a dicating that this class of proteins has developed several distinct molecular explanation of how CFD1 and NBP35 may execute solutions for metal cofactor binding and transfer. their scaffold function (12–15, 17).

E9092 | www.pnas.org/cgi/doi/10.1073/pnas.1807762115 Stehling et al. Downloaded by guest on September 26, 2021 Materials and Methods whole-cell lysates by analyzing the conversion of [4-14C]-thymine into 14 Plasmids, cDNA, and siRNAs. Plasmids encoding human CFD1 (NUBP2, clone [4- C]-dihydrothymine using TLC (25, 38). IRE RNA-binding capacities of IRP1 and IRAUp969A1022D6) or human NBP35 (NUBP1, clone IMAGp958F152017Q2) of IRP2 were examined by REMSA-related native gel electrophoresis (38, 81). – μ · were obtained from Deutsches Resourcenzentrum für Genomforschung. For immunoblotting 40 50 g of total cell lysate was applied to Tris glycine Genomic DNA of C. thermophilum was a kind gift of E. Hurt, Ruprecht-Karls- gradient gels (6–16%). Protein–protein interactions were analyzed by University, Heidelberg and was used for cloning of ctCfd1. For bacterial coimmunoprecipitation and subsequent immunostaining (38). TfR expres- expression of ctNbp35 a codon-optimized version was synthesized by sion was evaluated by a fluorometric approach (29). The incorporation of GenScript. Supporting information about sources and cloning of plasmids 55Fe into Fe-S proteins has been described in detail (47). Additional in- used in this study is provided online (SI Appendix). formation including data presentation is provided online (SI Appendix). A set of four ON-TARGETplus siRNAs (SI Appendix, Table S4) directed against the mRNA of human CFD1 was purchased from Dharmacon (Thermo Biochemical Methods for Analyses of Purified Proteins. Nucleotide affinity Scientific Life Sciences). Nontargeting (scrambled) siRNAs, siRNAs directed measurements were performed by MST using a Monolith NT.115 (NanoTemper against human CIAO1, CIA2A, CIA2B, and MMS19 (25, 38, 80), and shRNAs Technologies GmbH) at 21 °C. Purified recombinant ctNbp35 or ctCfd1 was directed against mRNAs of human NFS1 and NBP35 have been described (6, 29). labeled with the dye NT 647 and titrated with increasing amounts of ATP, GTP, or CTP up to 0.5 mM. At least six independent MST experiments were recorded Antibodies. Specific rabbit antisera against human CFD1, NBP35, and at 680 nm and analyzed using Origin 8G software. IOP1 were raised using recombinant full-length proteins. Further antibodies For crystallization and structure determination of ctCfd1, Fe-S clusters were used in this study are described online (SI Appendix). chemically reconstituted under anaerobic conditions. The protein was con- centrated to 20 mg/mL, and crystallization occurred in 0.1 M Na·Hepes (pH 7.0) Tissue Culture and Cell Transfection. HeLa cells were cultured and transfected and 1.5 M ammonium sulfate. Diffraction data were collected on BL14.1 at the as described (SI Appendix) (25, 29). Usually, a total of 660 pmol siRNA (final BESSY II electron storage ring. The crystal structure of ctCfd1 was determined at concentration 2.6 μM) or 12.5–15 μg of shRNA vectors were applied per 2.57 Å using molecular replacement combined with single anomalous disper- transfection. Plasmids encoding fusion proteins were used at amounts of sion phasing based on the anomalous scattering of Fe and is deposited in the 7.5–12.5 μg. Vector-based gene expression was improved by the addition of Protein Data Bank database (PDB ID code 6G2G). 3–5 μg of the pVA-RNAI plasmid (SI Appendix).

ACKNOWLEDGMENTS. We thank the Protein Spectroscopy and Protein Methods for Analyses of Tissue-Culture Samples. Gene expression in HeLa cells Biochemistry Core Facility of Philipps-Universität Marburg. This work re- was analyzed by qRT-PCR in a Stratagene MxPro device (Agilent Technolo- ceived financial support from Deutsche Forschungsgemeinschaft (Sonderfor- gies) with mRNA extracted from snap-frozen samples (38). Primer sequences schungsbereich SFB 593, Koselleck Grant, Priority Program SPP 1927), and for CFD1 and HPRT1 (hypoxanthine phosphoribosyltransferase 1) expression the Offensive for the Development of Scientific and Economic Excellence analyses are given in SI Appendix, Table S5. HeLa cells were harvested by Program of the State of Hesse (LOEWE) and received networking support trypsinization, permeabilized using 0.01% digitonin, fractionated, and an- from the Cooperation in Science and Technology (COST) Action Cellular Bi- alyzed for aconitase, succinate dehydrogenase, citrate synthase, and lactate ology to Molecular Aspects Contract CA15133 (to R.L.). H.D. received funding dehydrogenase activities (25, 29). DPYD enzyme activity was determined in from the Deutsche Forschungsgemeinschaft SPP 1927 (Grant DO 785/7-1).

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