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

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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 presum- ably 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 homeo- stasis. 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).
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