The cytosolic iron–sulfur (Fe–S) assembly (CIA) pathway is required for the insertion of Fe–S clusters into cytosolic and nuclear client proteins, including many DNA replication and repair factors. The molecular mechanisms of client protein recognition and Fe–S cluster transfer remain unknown. Here, we report crystal structures of the CIA targeting complex (CTC), revealing that its CIAO2B subunit is centrally located and bridges CIAO1 and the client adaptor protein MMS19. Cryo-EM reconstructions of human CTC bound either to the DNA replication factor primase or to the DNA helicase DNA2, combined with biochemical, biophysical and yeast complementation assays, reveal an evolutionarily conserved, bipartite client recognition mode facilitated by CIAO1 and the structural flexibility of the MMS19 subunit. Unexpectedly, the primase Fe–S cluster is located ~70 Å away from the CTC reactive cysteine, implicating conformational dynamics of the CTC or additional maturation factors in the mechanism of Fe–S cluster transfer.
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The atomic coordinates and structure factors reported in this study have been deposited in the Protein Data Bank (PDB) under accession codes 6TBN (CIAO1–CIAO2B complex), 6TBL (MMS19CTD–CIAO1–CIAO2B complex) and 6TC0 (MMS19–CIAO1–CIAO2B complex). The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-11016, EMD-11017 and EMD-11018 (CTC–primase), and EMD-11019, EMD-11020 and EMD-11021 (CTC–DNA2). Source data for Fig. 1f, Fig. 3, Extended Data Fig. 2b and Extended Data Fig. 6a are available with the paper online.
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We thank S. Cavadini and A. Schenk for help with cryo-EM data collection and processing and for advice about sample optimization. We are particularly grateful to the FMI core facilities: A. Graff Meyer and C. Genoud (electron microscopy); H. Gut, J. Keusch and G. Kempf (X-ray crystallography); and V. Iesmantavicius, D. Hess and J. Seebacher (mass spectrometry). Part of this work was performed at beamlines X06DA and X10SA of the Swiss Light Source (Paul Scherrer Institute). We thank the staff at both beamlines for assistance with X-ray data collection; W. Chazin (Vanderbilt University) for providing the plasmids for recombinant expression of human primase in bacteria and purified primase protein for initial studies; A. Potenza for help with protein expression in the early stage of the project; R. Bunker for help and advice with crystallographic data collection and processing; K. Shimada, M. Hauer and I. Deshpande for sharing protocols and advice on yeast experiments; K. Gari and D. Odermatt for sharing plasmids and helpful discussions; M. Jinek for advice on crystallographic data collection, processing and model building, and critical reading of the manuscript; and J. Reinert and F. Bleichert for comments on the manuscript. S.A.K. was supported by a long-term postdoctoral fellowship from the European Molecular Biology Organization (EMBO, ALTF 871–2014). This work was funded by the Swiss National Science Foundation through Sinergia grant number CRSII3_160734 and by the European Research Council under the European Union’s Horizon 2020 Research and Innovation program, grant number 666068, to N.H.T.
The authors declare no competing interests.
Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Coomassie-stained SDS-PAGE of purified Drosophila melanogaster CIAO1-CIAO2B complex. b, Surface representation of CIAO1 colored according to amino acid conservation across species based on the alignment in e. c, Surface representation of CIAO1 colored according to electrostatic potential from -3 to +3 kBT/e. d, Strep pull-down assay from Hi5 insect cells expressing Strep-CIAO2B and His-CIAO1 wt or indicated mutants. e, CIAO1 sequence alignment used to map evolutionary conservation in b. Secondary structure elements of human CIAO1 are indicated above the alignment. Numbering is relative to the human sequence. f, Yeast drop assay of CIAO1 point mutations located at the top face of the β-propeller. A Gal-CIA1 strain was transformed with either empty vector, wt human CIAO1, or a CIAO1 point mutant; a dilution series of each culture was spotted on agar plates in the presence of glucose or galactose.
a, Coomassie-stained SDS-PAGE gels of purified MMS19CTD-CIAO2B-CIAO1 and MMS19-CIAO2B-CIAO1 complexes. b, Pull-down assay from HEK293 cells using Flag-tagged human MMS19 wt and mutants, and myc-tagged human CIAO2B/CIAO1. WCE (right) and eluted proteins (IP:Flag, left) were analyzed by Western blot. Corresponding to Fig. 1f. c, Yeast drop assay of MMS19 mutations. A MMS19 knockout strain was transformed with either empty galactose-inducible p415GAL1 vector, p415GAL1 expressing wt MMS19, or p415GAL1 vectors expressing MMS19 mutants. A dilution series of each culture was spotted on agar plates in the presence or absence of galactose and 20 mM HU. d, Lysine residues implicated as targets for ubiquitination by MAGE-F1-NSE1 are shown in green. e, Close-up view of the CIAO2B-CIAO2B dimer interface. Potential Fe-S cluster-coordinating residues H85, C86, and H119 are shown as sticks. Uncropped blot images for panel b are available as source data.
a, Size exclusion chromatography profile of assembled CTC-primase complex. b, Coomassie-stained SDS-PAGE of CTC-primase complex. c, Negative stain 2D class averages of the CTC-primase complex. d, Cryo-EM micrograph of CTC-primase sample. e, Representative 2D class averages of the CTC-primase data set. Scale bar = 100 Å. f, Overview of data processing and classification scheme. g, Angular distribution of particle orientations in the reconstruction. h, FSC plot for half-maps of the reconstruction, 0.143 FSC criterion is indicated.
a, Size exclusion chromatography profile of assembled CTC-DNA2 complex. b, Coomassie-stained SDS-PAGE of CTC-DNA2 complex. c, Negative stain 2D class averages of the CTC-DNA2 complex. d, Cryo-EM micrograph of CTC-DNA2 sample. e, Representative 2D class averages of the CTC-DNA2 data set. Scale bar = 100 Å. f, Overview of data processing and classification scheme. g, Angular distribution of particle orientations in the reconstruction. h, FSC plot for half-maps of the reconstruction, 0.143 FSC criterion is indicated.
Extended Data Fig. 6 Pull-down assays in mammalian cells pinpoint blade 3 in CIAO1 as the main interaction site for recruitment of client proteins.
a, Co-expression and co-IP of Flag-MMS19 with myc-CIAO2B, wild-type or mutant myc-CIAO1, and either myc-PriL/S or myc-DNA2 in HEK293 cells. b, Location of mutated amino acid residues around blade 3 of CIAO1. The inset shows the location of the conserved and charged patches in blade 3 of CIAO1. Uncropped blot images for panel a are available as source data.
Extended Data Fig. 7 Compositional dynamics of the CTC and its potential role in Fe-S cluster transfer.
a, Compositional dynamics of the CTC might not only be required for maturation of different client proteins (maturation of certain cytosolic proteins by the core complex, and of DNA metabolism proteins by the complete CTC), but also play a role in Fe-S cluster coordination and transfer through CTC dimerization. b, Model of Fe-S cluster transfer and client protein binding by the CTC. See Supplementary Note 1.
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Kassube, S.A., Thomä, N.H. Structural insights into Fe–S protein biogenesis by the CIA targeting complex. Nat Struct Mol Biol 27, 735–742 (2020). https://doi.org/10.1038/s41594-020-0454-0