The p97/VCP adaptor UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions

Abstract

p97, also known as valosin-containing protein, is an essential cytosolic AAA+ (ATPases associated with diverse cellular activities) hexamer that unfolds substrate polypeptides to support protein homeostasis and macromolecular disassembly. Distinct sets of p97 adaptors guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adaptor localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. Here we identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact human p97-UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second (D2) AAA+ domain. Together, these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis and comparisons to other adaptors further reveal how adaptors containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.

Fig. 1. Cryo-EM structures of p97–UBXD1 closed and open states.

a, Domain schematics of UBXD1 and p97 (not to scale) showing reported interactions (solid lines) between conserved domains^,– and the UBX–NTD interaction previously reported to not occur for UBXD1 (dashed line)^,,. b, AlphaFold model of UBXD1 showing structured regions (H1/H2, VIM, H4, PUB, helical lariat and UBX) colored as in a. c, Steady-state ATPase activity (y axis, normalized to activity at 0 nM UBXD1) of p97 at increasing concentrations of UBXD1 (x axis), resulting in a calculated IC₅₀ of 25 nM. Data are from n = 3 independent experiments, each with three technical replicates. Data are presented as mean values from each independent experiment. d, Representative 2D class averages following the initial classification of the full p97–UBXD1 dataset, showing the p97 hexamer and no additional density for UBXD1. Scale bar, 100 Å. e,f, Final cryo-EM reconstructions of p97–UBXD1^closed (e) and p97–UBXD1^open (f) states with top-view 2D projections showing UBX–PUB density (*) and open p97 ring (arrow) compared to cartoon depictions of the corresponding complexes (top row); cryo-EM density maps (p97–UBXD1^open is a composite map; see Methods), colored to show the p97 hexamer (light and dark blue, with protomers labeled P1–P6) and UBXD1 density for the VIM (brown), UBX (yellow) and lariat (orange) domains (bottom row). The 8 Å separation between protomers P1 and P6 is indicated for p97–UBXD1^open. g, Low-pass filtered maps and fitted models of p97–UBXD1^closed (left) and p97–UBXD1^open (right) exhibiting low-resolution density for the PUB domain (gray). Source data

Fig. 2. UBXD1-mediated p97 hexamer remodeling.

a,b, The p97 hexamer and rotated side view of the seam protomers P1 and P6 for p97–UBXD1^closed (a) and p97–UBXD1^open (b) structures, colored according to Cα r.m.s.d. values relative to the p97^ADP symmetric state (PDB 5FTK, aligned to P3 and P4). The largest changes (>15 Å, magenta with wider tubes) are identified for α12′ of P1 (labeled) in the open state, intermediate changes (∼10 Å, red) for P1 and P6 with rotations of the NTDs relative to p97^ADP shown and small to no changes for the remaining regions (<5 Å, white); α5′ of P6, which disappears in the open state, is labeled in a. c, Distribution of states for the ADP and ATPγS superstoichiometric datasets (2:1 UBXD1:p97 monomer) and the ADP substoichiometric dataset (1:6 UBXD1:p97 monomer). For each dataset, all particles after 2D classification were subjected to 3D classification using the same references. Classes corresponding to junk particles were excluded and the proportions of the remaining classes were plotted. d, Schematic of p97 remodeling in various nucleotide-bound and UBXD1-bound states. Source data

Fig. 3. Interactions by conserved VIM, UBX and PUB domains of UBXD1 across the p97 hexamer.

a, Sharpened map of the P1 NTD (dark blue) and the VIM helix (brown) from p97–UBXD1^closed. b, Corresponding model showing VIM helix interactions with the NTD, colored as in a, with labeled interacting residues. c, Sharpened map of the P6 NTD (light blue) and UBX domain (yellow) from p97–UBXD1^closed. d,e, Corresponding model of the UBX and NTD showing a conserved orientation of the S3/S4 loop (arrow) (d) and non-canonical structural elements Uα2, Uα3 and Uβ0 (e), colored as in c. The N-terminal (Nn) and C-terminal (Nc) lobes of the NTD are indicated. f, Overlay of PUB domains from p97–UBXD1-PUB_in (gray) and p97–UBXD1-PUB_out (white), aligned to the UBX (yellow) domain, showing 46° rotation of the PUB domain position. g,h, Low-pass filtered map and model of p97–UBXD1-PUB_in depicting PUB domain contact with p97 and model for C-terminal HbYX tail interaction from the adjacent P5 protomer (arrow) (g) and bottom view of the hexamer map with ‘out’ (white) and ‘in’ (gray) positions of the PUB (h). i, Cartoon of p97–UBXD1^closed depicting UBXD1 interactions across three p97 protomers (P1–VIM, P6–UBX and P5–PUB) through canonical p97-interacting domains.

Fig. 4. p97 remodeling interactions by UBXD1 helical lariat and VIM–H4.

a, Closed state map (from p97–UBXD1^meta) showing density for the UBXD1 helical lariat (orange) and UBX (yellow) encircling the P6 NTD with Lα2, Lα3 and Lα4 interacting along the P6–P1 interprotomer interface. b, Expanded view showing Lα3 and Lα4 (orange) contacts with P6 across the NTD, D1 and D2, including putative electrostatic interactions (dashed lines). c, View of Lα2 interactions involving hydrophobic packing into the NTD using F292 and F293. d, View of the P6–P1 interface showing key contacts by Lα3 with the D1 α12 helix of protomer P1. e, View of Lα3 and Lα4 intra-lariat contacts (between R313, R318 and E326) and contacts with D2 (by L317 and T319), stabilizing the helical lariat. f, Unsharpened map of p97–UBXD1^H4, showing density for H4 (green) adjacent to the VIM (brown) and along the P6–P1 interface. Shown below is an expanded view of the VIM–H4 sequence, featuring only a short, seven-amino acid linker connecting the two helices. g, Modeled view (see Extended Data Fig. 7c) of helix H4 interacting across the D2 domains at the P1–P6 interface from p97–UBXD1^H4 (P1, dark blue; P6, light blue), overlaid (by alignment of the P1 D2 large subdomain) with p97–UBXD1^closed (gray, showing conformational changes at the P6–P1 interface including displacement of P6 helix α5′ (red) and large rotation of P1 α12′).

Fig. 5. Analysis of the helical lariat and VIM–H4 as conserved p97-remodeling motifs.

a, Domain schematics of UBXD1, ASPL and SVIP (not to scale). b,c, Overlay of the UBX–helical lariat of ASPL (residues 318–495 from PDB 5IFS, colored as in a) and UBXD1 (residues 270–441 from the p97–UBXD1^closed model, in white) (b) and the VIM–‘H4-like’ region of SVIP (AlphaFold model, colored as in a) and UBXD1 (residues 50–93 from the AlphaFold model, in white) (c). d, Steady-state ATPase activity of p97 as a function of ASPL-C or SVIP concentration (normalized to activity at 0 nM adaptor). Data are from n = 3 independent experiments, each with three technical replicates. Data are presented as mean values from each independent experiment. Calculated IC₅₀ values are also shown. Source data

Fig. 6. Mutational analysis of UBXD1.

a, Schematic of UBXD1 mutants tested. b, Steady-state ATPase activity of p97 as a function of UBXD1 protein concentration for WT (wild type), LX (lariat mutant: E299R/R302E/R307E/E312R) or H4X (helix H4 sequence scramble) (normalized to activity at 0 nM UBXD1). Dashed lines represent the minimal activity (or maximal UBXD1 inhibition) obtained from the corresponding curve fit. Data are from n = 3 independent experiments, each with three technical replicates. Data are presented as mean values from each independent experiment. c, As in b, but for UBXD1-N (residues 1–133), UBXD1-C (residues 94–end) or an equimolar combination of UBXD1-N and UBXD1-C. Data are from n = 3 independent experiments, each with three technical replicates. Data are presented as mean values from each independent experiment. d Calculated IC₅₀ values for ATPase inhibition by UBXD1 mutants. Data are from n = 3 independent experiments, each with three technical replicates. Error bars, 95% CI; ND, not determined. Source data

Fig. 7. Structural analysis of p97–UBXD1 mutant complexes and model for p97 hexamer remodeling through UBXD1 domain interactions.

a, Table of ADP-bound p97–UBXD1 cryo-EM datasets (WT, LX and H4X) and corresponding UBXD1 domains observed as densities in the reconstructions. b, Unsharpened map and fitted model of the VIM–H4-bound P1 protomer from p97–UBXD1^LX (Extended Data Fig. 9d), colored as in Fig. 1. c, First and last frames of the 3D variability analysis output for p97–UBXD1^LX showing P6 D2 density (*) but no VIM–H4 in one end state (top) and no P6 D2 in the other when VIM–H4 density is present (bottom). d, P1–P6 interprotomer distances (based on centroid positions) for the D1 and D2 domains of p97^ADP (PDB 5FTK), p97–UBXD1^closed, p97–UBXD1^LX, p97–UBXD1^H4X and p97–UBXD1^open. Dashed lines represent the minimal distances observed in p97^ADP. A schematic representing the distances calculated is shown (right). e, Model of p97–UBXD1 interactions and structural remodeling of the hexamer. State I, side view of p97^ADP (PDB 5FTK), colored as in Fig. 1. NTDs are shaded for clarity. State II, p97–UBXD1^VIM, in which the VIM initially associates with the NTD of P1. The position of the D2 small subdomain is illustrated by α12′ and an adjacent helix. State III, the p97–UBXD1^closed state, in which the UBX, PUB and helical lariat contact P1, P5 and P6, resulting in the disruption of D1–D1 contacts at the P1–P6 interface. State IV, the p97–UBXD1^H4 state, in which H4 is positioned on top of the D2 domain of P1, causing it to rotate upward and displacing a helix from the D2 domain of P6. State V, the p97–UBXD1^open state, in which P6 and P1 have completely separated and all protomers are arranged into a shallow right-handed helix. f, Summary of p97–UBXD1 interactions identified in this study. Source data

Extended Data Fig. 1. Biochemical and cryo-EM analysis of the p97–UBXD1 interaction.

(a) SEC traces of p97–UBXD1 samples. Fractions in the shaded range were analyzed by SDS–PAGE. No p97 monomer peak was observed with UBXD1 incubation. Data are representative of three independently performed experiments with similar results. (b) Coomassie Brilliant Blue-stained SDS–PAGE gels of fractions from SEC runs in (a). Data are representative of three independently performed experiments with similar results. (c) Representative micrograph of the p97–UBXD1^WT•ADP dataset (scale bar equals 100 nm). (d) Representative 2D class averages of the p97–UBXD1^WT•ADP dataset (scale bar equals 100 Å). No p97 monomers were identified during 2D classification. (e) Processing workflow for structures obtained from the p97–UBXD1^WT•ADP dataset. Class 1 corresponds to p97–UBXD1^closed, class 2 to p97–UBXD1^open, and class 3 to p97–UBXD1^VIM. Masks used for the P1 and P6 focused classification and masked local refinement of p97–UBXD1^open are shown in transparent blue and yellow, respectively. (f) Overlay of all protomers from p97–UBXD1^closed (blue) with a protomer in the ADP-bound, down NTD conformation (pink, PDB 5FTK) and a protomer in the ATPγS-bound, up NTD conformation (green, PDB 5FTN), aligned by the D1 large subdomain (residues 211-368). For all protomers, the NTDs are colored, and the D1 and D2 domains are white. (g) As in (f), but depicting protomers from p97–UBXD1^open (blue). (h) Nucleotide densities for representative D1 and D2 pockets in p97–UBXD1^closed and p97–UBXD1^open. (i) Representative additional density in NTD corresponding to a VIM helix (unsharpened map of P4 in p97–UBXD1^open). Source data

Extended Data Fig. 2. Cryo-EM densities and resolution estimation from the ADP-bound p97–UBXD1^WT dataset (superstoichiometric).

(a to j) Fourier shell correlation (FSC) curves, particle orientation distribution plots, and sharpened density maps colored by local resolution (0.143 cutoff) for (a) p97–UBXD1^closed, (b) p97–UBXD1^open (consensus map), (c) p97–UBXD1^open P1 focus, (d) p97–UBXD1^open P6 focus, (e) p97–UBXD1^VIM, (f) p97–UBXD1^meta, (g) p97–UBXD1^para, (h) p97–UBXD1-PUB_out, (i) p97–UBXD1-PUB_in, and (j) p97–UBXD1^H4. (k) Map-model FSC curves for p97–UBXD1^closed, p97–UBXD1^open (composite map), p97–UBXD1^VIM, p97–UBXD1^meta, p97–UBXD1^para, p97–UBXD1-PUB_in, and p97–UBXD1^H4. Displayed model resolutions were determined using the masked maps.

Extended Data Fig. 3. Additional configurations of p97–UBXD1 complexes.

(a to c) Cartoons, top view projections of sharpened maps showing UBX/PUB density (*), and sharpened maps of (a) p97–UBXD1^VIM, (b) p97–UBXD1^meta, and (c) p97–UBXD1^para (scale bar equals 100 Å). In p97–UBXD1^VIM, the VIM density is depicted as a difference map of p97–UBXD1^VIM and a map generated from a model without VIM helices.

Extended Data Fig. 4. Hexamer remodeling in p97–UBXD1^closed and p97–UBXD1^open.

(a) Overlay of protomers P1 (dark blue) and P6 (light blue) from p97–UBXD1^closed, aligned to protomers P3 and P4 from PDB 5FTK. P1 and P6 protomers from 5FTK are shown in gray. (b) As in (a), but depicting p97–UBXD1^open protomers. (c) Side-by-side view of individual protomers aligned based on position in the p97–UBXD1^open hexamer, showing vertical displacement along the pseudo-C6 symmetry axis. (d) Overlay of the D1 (left) and D2 (right) AAA+ domains of P1 for p97^ADP, p97–UBXD1^closed, and p97–UBXD1^open, aligned to the large subdomains and colored as indicated. ADP is shown with conserved Walker A/B (green and purple, respectively) and trans-acting (P6) Arg finger residues indicated. The large rotation of the D2 small subdomain, exemplified by α12′, is shown (relative to p97^ADP) for the closed (1) and open (2) states. (e) Unsharpened map of the D2 domains of protomers P1 and P6 of p97–UBXD1^open, overlaid with the D2 domain from p97^ADP on P6, showing lack of density for helix α5′ (gray, encircled) normally contacting the counterclockwise D2 domain. The D2 domain of P1 of the open state is shown for clarity. (f) Top view overlay of the D1 (left) and D2 (right) domains for the P6-P1 pair in the three states and aligned to P1 to show relative rotations of P6, colored as indicated. Rotations shown are from p97^ADP to p97–UBXD1^open and determined from centroid positions of the D1 and D2 domains.

Extended Data Fig. 5. Cryo-EM analysis of p97–UBXD1 with ATPγS and with substoichiometric UBXD1.

(a) Processing workflow for structures obtained from the p97–UBXD1^WT•ATPγS dataset. Class 1 resembles p97–UBXD1^closed, class 2 is identical to ATPγS-bound p97 (PDB 5FTN), and class 3 resembles p97–UBXD1^VIM. (b) (Top row) sharpened maps of class 1-3 refinements. (Bottom row) p97–UBXD1^closed model overlaid with filtered map, unsharpened map, ATPγS-bound p97 hexamer with NTDs in the up state (PDB 5FTN) overlaid with the class 2 unsharpened map, and p97–UBXD1^VIM model overlaid with the class 3 unsharpened map, respectively. All maps are colored as in Fig. 1. (c) Representative nucleotide densities for class 1-3 refinements (sharpened maps), showing clear γ-phosphate and Mg²⁺ density. The nucleotide and surrounding binding pocket from PDB 5FTN are shown for clarity. (d) Processing workflow for structures from the p97–UBXD1^WT•ADP substoichiometric dataset. Class 1 resembles p97–UBXD1^open, while classes 2 and 3 resemble unbound p97 hexamers in the ADP state. (e) Top and side views of class 1 (unsharpened) from (d), colored by p97 protomer. (f) Transparent top view of class 1 from (d), overlaid with p97 protomers from the p97–UBXD1^open model.

Extended Data Fig. 6. VIM, UBX, and PUB structural alignments and validation.

(a) Overlay of NTD-VIM from p97–UBXD1^closed (colored) and gp78 (PDB 3TIW, white). (b) Map and model of the NTD and VIM from p97–UBXD1^closed, colored as in (a). (c) Overlay of NTD-UBX from p97–UBXD1^closed (colored) and UBXD7 (PDB 5X4L, white). (d) Map and model of the NTD and UBX from p97–UBXD1^closed, colored as in (c). (e) Unsharpened, zoned map and model of UBX and PUB from p97–UBXD1-PUB_out. (f) Unsharpened, zoned map and model of UBX and PUB from p97–UBXD1-PUB_in. (g) Model of the UBX (yellow), PUB (gray), and UBX-PUB linker (light blue) from p97–UBXD1^closed. (h) Unsharpened map of p97–UBXD1-PUB_out.The VIM and helical lariat are colored the same as their corresponding protomers for clarity. (i) Unsharpened map of p97–UBXD1-PUB_in. The VIM and helical lariat are colored the same as their corresponding protomers for clarity. (j) Filtered map of p97–UBXD1-PUB_out, colored as in (d), showing weak density connecting the PUB and P5 CT tail. (k) As in (j), but for p97–UBXD1-PUB_in.

Extended Data Fig. 7. Validation of the helical lariat, UPCDC30245 binding, and additional structural features of p97–UBXD1^H4 and p97–UBXD1^open.

(a) Sharpened map and model of Lα2-4, connecting strands of the helical lariat, and adjacent regions of P6 of p97–UBXD1^meta, colored as in Fig. 1. (b) Overlay of P6 (blue) and Lα3-4 (orange) from p97–UBXD1^closed with a p97 protomer (white) bound to UPCDC30245 (yellow) (PDB 5FTJ), aligned by the D2 domain (residues 483-763). (c) View of H4 and surrounding p97 density in p97–UBXD1^H4 (left: unsharpened, right: sharpened) overlaid with the model for this state. (d) Sharpened map and model of the D2 domain of protomer P6 of p97–UBXD1^H4, showing lack of density for α5′ (encircled). (e) Unsharpened map of protomer P1 of p97–UBXD1^open. Density putatively corresponding to H4 is colored in green.

Extended Data Fig. 8. Conservation of UBXD1, ASPL, and SVIP sequences.

(a) Alignment of UBX-lariat sequences from UBXD1 (residues 270-441) and ASPL (residues 318-495). Structural elements in the UBXD1 sequence are indicated above. Cov = covariance relative to the human sequence, Pid = percent identity relative to the human sequence. (b) Alignment of VIM–H4 sequences from UBXD1 (residues 50-93) and SVIP (residues 18-64). Structural elements in the UBXD1 sequence are indicated above.

Extended Data Fig. 9. Biochemical and cryo-EM analysis of lariat and H4 mutations.

(a) Residues mutated in Lα3 of the UBXD1 helical lariat, shown on p97–UBXD1^closed. (b) SEC traces of UBXD1 mutants alone or incubated with p97 and ADP, showing a left shift in peak elution volume for p97 samples with UBXD1. Fractions in the shaded range were analyzed by SDS–PAGE. Data are representative of three independently performed experiments with similar results. (c) Coomassie Brilliant Blue-stained SDS–PAGE gels of fractions from SEC runs in (b). Data are representative of three independently performed experiments with similar results. (d) Cryo-EM processing workflow for the p97–UBXD1^LX•ADP dataset. Class 1 is identical to p97–UBXD1^VIM; class 2 has VIM and H4 density. (e) FSC curve and particle orientation distribution plot for p97–UBXD1^LX. (f) Sharpened density map colored by local resolution (0.143 cutoff) of p97–UBXD1^LX. (g) Map-model FSC for p97–UBXD1^LX. Displayed resolution was determined using the masked map. (h) Cryo-EM processing workflow for the p97–UBXD1^H4X•ADP dataset. Class 1 resembles p97–UBXD1^VIM; classes 2 and 3 resemble p97–UBXD1^closed. (i) Unsharpened map of class 1 from p97–UBXD1^LX•ADP dataset. (j) Unsharpened map of class 1 from p97–UBXD1^H4X•ADP. (k) Top: unsharpened map of p97–UBXD1^LX. Note lack of density for the D2 domain of the protomer clockwise from the VIM-H4 bound protomer. Bottom: filtered map, colored only by p97 density, confirming that density for the aforementioned D2 domain is present. (l) Top: sharpened map of p97–UBXD1^H4X, colored as in Fig. 1. Bottom: filtered map, confirming that density for the PUB domain is present. (m) Positions of calculated centroids for D1 (residues 210-458, magenta spheres) and D2 domains (residues 483-763, green spheres) of P1 and P6, for p97^ADP and p97–UBXD1^open. Source data

Extended Data Fig. 10. Alignment of UBXD1 sequences.

Multiple sequence alignment of UBXD1 homologs from Homo sapiens, Mus musculus, Xenopus tropicalis, Gallus gallus, Rattus norvegicus, Bos taurus, Pan troglodytes, and Danio rerio. Structural elements and residue ranges (in the human sequence) are marked above. Cov = covariance relative to the human sequence, Pid = percent identity relative to the human sequence.