Abstract

Many polyubiquitinated proteins are extracted from membranes or complexes by the conserved ATPase Cdc48 (in yeast; p97 or VCP in mammals) before proteasomal degradation. Each Cdc48 hexamer contains two stacked ATPase rings (D1 and D2) and six N-terminal (N) domains. Cdc48 binds various cofactors, including the Ufd1–Npl4 heterodimer. Here, we report structures of the Cdc48–Ufd1–Npl4 complex from Chaetomium thermophilum. Npl4 interacts through its UBX-like domain with a Cdc48 N domain, and it uses two Zn2+-finger domains to anchor the enzymatically inactive Mpr1–Pad1 N-terminal (MPN) domain, homologous to domains found in several isopeptidases, to the top of the D1 ATPase ring. The MPN domain of Npl4 is located above Cdc48’s central pore, a position similar to the MPN domain from deubiquitinase Rpn11 in the proteasome. Our results indicate that Npl4 is unique among Cdc48 cofactors and suggest a mechanism for binding and translocation of polyubiquitinated substrates into the ATPase.

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Acknowledgements

We thank X. Wu and L. Li for assistance with crystallography, D. Finley for critical reading of the manuscript, the SBGrid consortium at Harvard Medical School, and the ICCB Longwood for use of equipment. We thank M. Ebrahim and J. Sotiris at the Rockefeller University Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center for assistance with microscope operation. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the NIH/NIGMS (P41 GM103403). The Pilatus 6 M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Z.J. is supported as a Howard Hughes Medical Institute Fellow of the Damon Runyon Cancer Research Foundation, DRG-2315-18. This research was supported in part by a Helmsley Postdoctoral Fellowship at the Rockefeller University (to K.H.K.), funding from the Blavatnik Family Foundation (to E.N.), a research collaboration with the Waters Corporation (J.R.E.), and NIGMS grants R01GM052586 (to T.A.R.) and T32GM007753 (Harvard/MIT Medical Scientist Training Program). E.N. and T.A.R. are supported as Howard Hughes Medical Institute investigators. We thank P. Carvalho (Oxford University, UK) for providing reagents.

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Author notes

  1. These authors contributed equally: Nicholas O. Bodnar, Kelly H. Kim.

Affiliations

  1. Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    • Nicholas O. Bodnar
    • , Zhejian Ji
    •  & Tom A. Rapoport
  2. Laboratory of Molecular Electron Microscopy, the Rockefeller University, New York, NY, USA

    • Kelly H. Kim
    •  & Thomas Walz
  3. Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA

    • Thomas E. Wales
    •  & John R. Engen
  4. Howard Hughes Medical Institute and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    • Vladimir Svetlov
    •  & Evgeny Nudler

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Contributions

N.O.B. purified proteins, solved the crystal structure, and performed biochemical experiments. K.H.K. performed cryo-EM data collection and analysis. N.O.B. and K.H.K. contributed equally and appear in alphabetical order in the author list. Z.J. purified and tested Cdc48 FFF mutants and performed biochemical experiments. T.E.W. performed hydrogen/deuterium-exchange experiments with supervision from J.R.E. V.S. performed cross-linking mass spectrometry analysis with supervision from E.N. T.W. oversaw the cryo-EM experiments. N.O.B. and T.A.R. wrote the manuscript. T.A.R. supervised the project.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Thomas Walz or Tom A. Rapoport.

Integrated supplementary information

  1. Supplementary Figure 1 Purification of proteins.

    a, Gel-filtration chromatograms for C. thermophilum Cdc48 (blue), Ufd1/Npl4 (UN; green), and Cdc48/Ufd1/Npl4 (Cdc48/UN; red). The elution positions of molecular weight standards are shown in orange. b-d, Samples from the major peaks of the gel-filtration runs in a were subjected to SDS-PAGE and Coomassie blue staining. The single lane on the right in d shows the pooled peak fractions of the Cdc48 complex used for EM. M, molecular weight markers.

  2. Supplementary Figure 2 Image processing of Cdc48 in the presence of ADP.

    a, An area of a cryo-EM image of a vitrified sample is shown with some particles circled. Scale bar: 50 nm. b, Selected 2D class averages obtained with ISAC. Side length of individual averages: 32 nm. c, Initial 3D map obtained with VIPER. d, Image-processing workflow for 3D classification and refinement in RELION-1.4 that yielded density maps at resolutions of 8.9 Å (with no symmetry imposed) and 7.2 Å (with C6 symmetry imposed). See Methods section for details.

  3. Supplementary Figure 3 Image processing of Cdc48 in the presence of ATPγS.

    a, An area of a cryo-EM image of a vitrified sample is shown with some particles circled. Scale bar: 50 nm. b, Selected 2D class averages obtained with ISAC. Side length of individual averages: 25 nm. c, Initial 3D map obtained with VIPER. d, Image-processing workflow for 3D classification and refinement in RELION-1.4 that yielded density maps at resolutions of 10.3 Å (with no symmetry imposed) and 8.2 Å (with C6 symmetry imposed). See Methods section for details.

  4. Supplementary Figure 4 FSC curves and local densities.

    a, FSC curves calculated between independently refined half maps for the six-fold symmetrized density maps of Cdc48 alone in the presence of ADP (blue) or ATPγS (yellow), and for the density maps of the Cdc48/UN complex (no symmetry applied) in the presence of ADP (green) or ATPγS (red). b, Cross-validation FSC curves for the model and map of the Cdc48/UN complex with ATPγS showing no significant overfitting. c, Local resolution map of the Cdc48/cofactor complex structure obtained in the presence of ATPγS. Views are shown for a complete map (left) and a map after removing the front half (right). d, Representative cryo-EM densities with the fit models. ZF1, ZF2, Zn2+ fingers.

  5. Supplementary Figure 5 Comparison of the Cdc48 and Cdc48–UN structures obtained in the presence of ADP and ATP-γS.

    a, The cryo-EM density maps of Cdc48 obtained in the presence of ADP and ATPγS are shown separately (left and middle panels) as well as superimposed (right panel). All maps were low pass-filtered to 8 Å. b, As in a, but for the Cdc48/UN complex. All maps were low pass-filtered to 7 Å. c, The cryo-EM density map of the Cdc48/UN complexes obtained in the presence of ATPγS (left) showed density (blue) for the UBX-like domain of Npl4. An NMR structure of the complex of an N domain and the UBX-like domain (PDB: 2PJH) was docked into this cryo-EM density (inset on the right).

  6. Supplementary Figure 6 Image processing of the Cdc48–UN complex in the presence of ADP.

    a, An area of a cryo-EM image of a vitrified sample is shown with some particles circled. Scale bar: 50 nm. b, Selected 2D class averages obtained with RELION-1.4. Side length of individual averages: 33 nm. c, Initial 3D map obtained with RELION-1.4. d, Image-processing workflow for 3D classification and refinement in RELION-1.4 that yielded a density map at 6.7 Å resolution. Asterisks (*) indicate 3D classes that consist of particles with their N domains in the up-conformation. Daggers (†) indicate 3D classes that consist of particles with an extra density near an N domain that may attribute to the UBX-like domain of Npl4. See Methods section for details.

  7. Supplementary Figure 7 Image processing of the Cdc48–UN complex in the presence of ATP-γS.

    a, An area of a cryo-EM image of a vitrified sample is shown with some particles circled. Scale bar: 50 nm. b, Selected 2D class averages obtained with ISAC. Side length of individual averages: 33 nm. c, Initial 3D map obtained with cryoSPARC. d, Image-processing workflow for 3D classification and refinement in RELION-2 that yielded a density map at 4.3 Å. Asterisks (*) indicate 3D classes that consist of particles with their N domains in the up-conformation. Daggers (†) indicate 3D classes that consist of particles with an extra density near an N domain (only seen with a lower contouring threshold) that likely represents the UBX-like domain of Npl4. See Methods section for details.

  8. Supplementary Figure 8 Analysis of interactions in the Cdc48–UN complex.

    a, The C. thermophilum Cdc48/UN complex was treated with a bifunctional crosslinker and crosslinks were determined by mass spectrometry. See Suppl Data Set 2. b, Residues involved in crosslinks between Cdc48 and Npl4 are mapped onto the structure and shown as red and blue spheres, respectively. c, Purified C. thermophilum UN was treated with increasing concentrations of trypsin and subjected to SDS-PAGE and Coomassie blue staining. N, U: full-length Npl4 and Ufd1. F: a stable fragment of Npl4, identified by mass spectrometry as Npl4 129–602. d, Hydrogen/deuterium (H/D) exchange was performed with Npl4 residues 129–602 or with full-length Ufd1/Npl4. Regions of the Npl4 backbone protected by Ufd1 are colored in turquoise (residues 262–274, 316–327, 360–374, and 428–444) and mapped onto the structure of Npl4 in ribbon representation. The unassigned density from the cryo-EM map (in orange) is predicted to belong to Ufd1. e, Time courses showing relative deuterium exchange protection of the peptides covering regions highlighted in d (turquoise), as well as two peptides that were not protected by Ufd1 (222–237 and 527–545; yellow).

  9. Supplementary Figure 9 Molecular-replacement model and omit map.

    a, Two views of the EM density used as a molecular replacement model to solve the structure of the crystallized Npl4 fragment. The arrow indicates the extra density attributed to Ufd1, which does not reappear in the crystal structure. b, A simulated annealing composite omit map was calculated to assess model bias from the EM density map. The map is displayed at 1.0 σ with a carve of 1.8Å. Shown is the fit of the model into the density of the first Zn2+-finger domain. Arrows indicate residues whose side chains are not well represented in the map due to conformational flexibility. The red sphere indicates the zinc atom. c, As in b, but for the region of the second Zn2+ finger. d, As in b, but for the region of the β-strand finger. e, A second cofactor molecule cannot be fit above the ATPase ring, as there are significant clashes between the two Npl4 molecules (in blue and brown).

  10. Supplementary Figure 10 Characterization of Cdc48 FFF mutants and Npl4 with mutations in the β-strand finger

    . a, The indicated Cdc48 variants were subjected to gel filtration to analyze their oligomeric states. The position of hexamers is indicated. Note that mutation of the central Phe residue (FAF and AAA mutants) affects the migration of Cdc48 in gel filtration. b, Unfolding of poly-ubiquitinated Eos with wild-type Cdc48 and the indicated mutant of the conserved Tyr in the β-strand finger of Npl4. Data are shown as mean ± SD of n=3 technical replicates. c, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated mutations in insert-2, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37ºC) or three days (25ºC). d, Insert-2, in which mutations were made, is shown in purple and the Tyr in the β-strand finger is indicated (C. thermophilum Y472 corresponds to S. cerevisiae Y445).

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https://doi.org/10.1038/s41594-018-0085-x