Cryo-EM structures of human p97 double hexamer capture potentiated ATPase-competent state

The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes for disaggregation and degradation. The tandem ATPase domains (D1 and D2) of the p97/Cdc48 hexamer form stacked rings. p97/Cdc48 can unfold substrates by threading them through the central pore. The pore loops critical for substrate unfolding are, however, not well-ordered in substrate-free p97/Cdc48 conformations. How p97/Cdc48 organizes its pore loops for substrate engagement is unclear. Here we show that p97/Cdc48 can form double hexamers (DH) connected through the D2 ring. Cryo-EM structures of p97 DH reveal an ATPase-competent conformation with ordered pore loops. The C-terminal extension (CTE) links neighboring D2s in each hexamer and expands the central pore of the D2 ring. Mutations of Cdc48 CTE abolish substrate unfolding. We propose that the p97/Cdc48 DH captures a potentiated state poised for substrate engagement.


Fig
. S1 EM analysis of human p97-SPRTN complex. a Domains and motifs of human SPRTN. b Binding between SPRTN and p97. Beads bound to MBP or MBP-SPRTN were incubated with p97 and washed. Proteins bound to beads were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. c The gel filtration profiles of the p97-SPRTN complex with or without ATP on the Superose 6 column. d Negative-stain EM micrograph of the p97-SPRTN complex. Particles likely represent p97 double hexamers (DH) are boxed with red dashed lines. e Representative raw cryo-EM micrograph of the p97-SPRTN complex after motion correction by RELION. The DH particles in side views are boxed with red dashed lines. f Representative 2D class averages. g Workflow of cryo-EM data processing to obtain the 3D reconstructions of p97 single hexamer (SH) and DH. h Distribution of particle orientations in the last round of structural refinement of DH conformers I (right) and II (left). i Representative cryo-EM density segments of the EM map highlighting key structural elements of p97 DH with a contour level at 0.020 (5s).

Fig. S2
Cryo-EM density of the bound nucleotides in p97 DHs. a The gold-standard Fourier shell correlation (FSC) curves for the overall cryo-EM map (blue) and the final refined model versus map (red) of p97 DH conformer I. b FSC curves for model versus half map 1 (working, red) or half map 2 (free, green) of p97 DH conformer I. c The gold-standard FSC curve for the overall cryo-EM map (blue) and the final refined model versus map (red) of p97 DH conformer II. d FSC curves for the model versus half map 1 (working, red) or half map 2 (free, green) and the model versus final refined map (black) of p97 DH conformer II. e ATPγS overlaid with its EM density in the D1 ring of DH conformer I. The contour levels in this figure are set at 0.020 (5s). f ADP overlaid with its EM density in the D2 ring of DH conformer I. g ATPγS overlaid with its EM density in the D1 ring of DH conformer II. h ATPγS overlaid with its EM density in the D2 ring of DH conformer II. i p97 DH I fitted into EM maps (sideview; left) and ribbon diagram of the D2 ring pore loops (top view; right). The angles between the two interacting protomers from the two SHs along the central axis are indicated. j The DH interface and the C-terminal extension (CTE) of DH I fitted into the EM map. FSC curves for the model versus half map 1 (working, red) or half map 2 (free, green) and the model versus final refined map (black) of p97 SH conformer II. e The cryo-EM map and final model for conformer II with each protomer colored differently. f The cryo-EM density of the nucleotides in the D1 and D2 domains of p97 SH conformer II. g (Left panel) The gold-standard FSC curves (left) for the overall cryo-EM map (blue) and final refined model (red) of p97 SH conformer III. (Right panel) FSC curves for the model versus half map 1 (working, red) or half map 2 (free, green) and the model versus final refined map (black) of p97 SH conformer III. h The cryo-EM map and final model for conformer III with each protomer colored differently. i The cryo-EM density of the nucleotides in the D1 and D2 domains of p97 SH conformer III.

Fig. S6 Interactions involving the C-terminal extension (CTE) of p97 single hexamer (SH) and double hexamer (DH). a
The cryo-EM density of ɑ9 and R766 in the SH conformer I. b The cryo-EM density of ɑ9 and CTE in the DH conformer II. The contour levels for the SH and DH are set at 0.010 (2s) and 0.020 (5s), respectively. c Sequence alignment of the CTE-binding α6 helix (left) and the CTE (right). The sequences were aligned using Clustal Omega and numbered according to human p97. d Superimposition of the ATP-binding pocket of the D1 domain from p97 SH I and DH I and II. Color schemes for each conformer are indicated below.

Fig. S7 The pore loops are better defined in the p97 double hexamer (DH). a,b
Cryo-EM density of the pore loops in the D1 and D2 rings of p97 SH II (a) and DH I (b). The contour levels for the SH and DH are set at 0.010 (2s) and 0.020 (5s), respectively.

Fig. S8 Structural comparison of p97 DH and substrate-bound Cdc48. a
The side (left) and bottom (right) views of the superimposed cartoon diagrams of p97 DH conformer II (green) and substrate-bound Cdc48 (cyan; PDB code: 6OPC). The Sc Shp1 UBX domain is colored magenta. ɑ9 helices of p97 and Cdc48 are colored yellow and light purple, respectively. The substrate is shown in surface drawing and colored by elements. b The staircase distribution of the D1 and D2 pore loops in substrate-bound Cdc48. A, B, C subunits are colored green, cyan and yellow, respectively. The substrate is shown in surface drawing and colored by elements. c The symmetric and planar distribution of pore loops in the substrate-free conformation of p97 DH II. A, B, C subunits are colored green, cyan and yellow, respectively. d Superimposed cartoon diagrams of a single p97 protomer (green) and all six protomers (each colored differently) of Cdc48 (PDB code: 6OPC), with their D1 domain aligned and boxed with a red rectangle. e Superimposed cartoon diagrams of a single p97 protomer (green) and all six protomers (each colored differently) of Cdc48 (PDB code: 6OPC), with their D2 domain aligned and boxed with a red rectangle.

Fig. S9
The C-terminal extension and α9 strengthen the D2 ring. a Gel filtration profiles of the p97 ND1 fragment (residues 1-462) without (Apo) or with different nucleotides on the Superose 6 column. b Gel filtration profiles of the p97 D1D2 fragment (residues 210-806) without (Apo) or with different nucleotides on the Superose 6 column. c Gel filtration profiles of p97 D2 (residues 463-806) wild type (WT) and the indicated mutants without nucleotides on the Superose 6 column. d Coomassie stained SDS-PAGE of p97 D2 WT and mutants from gel filtration fractions in (c). e Gel filtration profiles of p97 D2 E578A and its mutants with ATP on the Superose 6 column. f Relative ATPase activities of p97 D2 WT and E578A at indicated concentrations. The activities are normalized to that of D2 WT at 5 nM concentration. Mean ± SEM (n = 3 independent experiments).