Fig 1: UBXD1 forms a ternary complex with p97 and a model substrate adapter Ufd1:Npl4.a Michaelis–Menten kinetics of p97 ATP hydrolysis without (gray) and with (blue) UBXD1 (2 µM) (n = 3 independent experiments. Data are represented as mean ± SD), showing that vmax is lower in combination with UBXD1. b Relative ATPase activity of p97 in presence of increasing UBXD1 concentrations (n = 3 independent experiments. Data are represented as mean ± SD). c Effect of the UBXD1-VIMx mutation on inhibition by UBXD1 (n = 3 independent experiments. Data are represented as mean ± SD. p97 ? p97 + UBXD1-wt, p = 0.040; p97 ? p97 + UBXD1-VIMx, p = 0.189; p97 + UBXD1 wt ? p97 + UBXD1-VIMx, p = 0.014). Note that the inhibition of p97 ATPase activity by UBXD1-wt is relieved by VIMx mutation. d Control measurements with chemical inhibitors of p97: CB5083 and NMS853 (n = 3 independent experiments. Data are represented as mean ± SD. p97 ? p97 + CB5083, p = 0.002; p97 ? p97 + NMS853, p = 0.0004; p97 + CB5083 ? p97 + NMS853, p = 0.282). e In presence of Ufd1:Npl4, a much lower inhibitory activity of UBXD1 on p97 ATPase activity is observed (n = 3 independent experiments. Data are represented as mean ± SD. p97 ? p97 + UBXD1, p = 0.0002; p97 ? p97 + Ufd1:Npl4, p = 0.331; p97 ? p97 + UBXD1 + Ufd1:Npl4, p = 0.011; p97 + UBXD1 ? p97 + Ufd1:Npl4, p = 0.010; p97 + UBXD1 ? p97 + UBXD1 + Ufd1:Npl4, p = 0.0004; p97 + Ufd1-Npl4 ? p97 + UBXD1 + Ufd1:Npl4, p = 0.256). Data c-e was analyzed by a Shapiro–Wilk-Test followed by a two-sided Welch-Test. n.s. indicates not significant, *p < 0.05, **p < 0.01, ***p < 0.001. f Substrate Adapter (Ufd1:Npl4) and UBXD1-TwinStrep can bind p97-fl simultaneously but compete for p97-?D2-C. Purified proteins were pulled down with StrepTactin beads (n = 1 independent experiments). Ufd1:Npl4 co-precipitate with UBXD1-TwinStrep dependent on p97-fl and only compete at increasing concentrations for p97-?D2-C. g UBXD1 does not inhibit p97:Ufd1:Npl4 mediated protein unfolding. p97 (500 nM hexamer), UBXD1 (2 µM), Ufd1:Npl4 (2 µM) were incubated with a polyubiquitylated Eos-based model substrate protein in the presence of ATP (3 mM). Loss of Eos fluorescence reflects substrate unfolding (n = 1 independent experiments). Unfolding was dependent on Ufd1:Npl4 and was not affected by the presence of UBXD1. h 1H-15N-HSQC titration of 15N-labeled UBXD1-PUB (400 µM) and HR23b-UBL (0 µM black, 1300 µM red). Shifting of signals indicate binding. Signals with the most pronounced chemical shift perturbation are labeled. i Structural model of the p97-C:UBXD1-PUB:HR23b-UBL complex docked with HADDOCK based on NMR chemical shift perturbations. (UBXD1-PUB in blue, p97-C in orange, HR23b-UBL in gray, residues used as docking restraints in magenta). Source data are provided as a source data file.