PMC

IC₅₀ values (µM) for p97 inhibitors against p97-p47 mixtures for (A) DBeQ, (B) ML240, (C) ML241, and (D) NMS-873.

(A) Structures of DBeQ, ML240, ML241, and NMS-873. (B) to (E) IC₅₀ (µM) of WT, D1-E305Q, D1-K251A, D2-E578Q, D2-K524A, and ND1L for (B) DBeQ, (C) ML240, (D) ML241, and (E) NMS-873 with 200 µM ATP or 875 µM ATP.

(A) ATPase activities of WT p97 and the A232E disease mutant (with Walker B mutations) indicated that the elevated ATPase activity originated from D2. (B) Inhibition of ATPase activity of WT p97 and eight disease mutants by ML240 and ML241.

(A, B) SPR sensorgrams for ADP (0–16.67 µM, 3-fold dilutions) binding to the ND1 (A) and ND1L (B) domains of p97. Response data were fit to a one-site kinetic model (orange lines) to obtain kinetic binding parameters. (C) SPR sensorgrams for ADP (0–150 µM, 3-fold dilutions), binding to full-length p97. Data were fit to a two-site kinetic binding model to obtain K_D values for the two ATPase domains in p97.

(A) Fluorescence anisotropy measurements for BODIPY-FL-ATP binding to WT p97 and Walker A mutants. (B) Binding of the BODIPY-FL-ATP probe in the presence of the D2 specific inhibitors, ML240 and ML241. Data were determined in triplicate, and errors in binding affinities shown are standard errors of the mean of the fit.

(A) Human p97 mutants and variants generated in this study. (B) ATPase activities of WT p97, ND1, ND1L, and LD2 domains with 0.01% Triton X-100 at 600 µM ATP. (C, D) The linker region (a.a. 459–480; SNPSALRETVVEVPQVTWEDIG) stabilized the D1 domain. Differential scanning fluorimetry revealed enhanced stability of the ND1 domain with the linker peptide (i.e., a 13 °C higher melting temperature). Melting temperatures: ND1 (Tm 41.9 °C, 55.7 °C), ND1L (Tm 55.5 °C) and full-length p97 (Tm 59.7 °C, 66.0 °C).

(A) ATPase activities of WT and Walker A and B mutants for D1 (D1-K251A; D1-E305Q) and D2 (D2-K524A; D2-E578Q), with or without 0.01% Triton X-100 at 200 µM ATP. Blue lettering indicates the ATPase active domain in each protein. Red lettering indicates the Walker A mutant. Green lettering indicates the Walker B mutant. (B) and (C) Michaelis-Menten plots of ATP hydrolysis for WT and mutant p97. From (D) to (F), black font indicates the ATPase active domain in each protein. (D) WT p97 hydrolyzes 7.48 ± 0.01 ATP molecules per minute per monomer (turnover number, k_cat, min⁻¹). The Walker A mutation of D2 (D2-K524A) decreases k_cat 22-fold. (E) The apparent Michaelis-Menten constant, K_m, of WT p97 is 287 ± 10 µM. The Walker B mutation of D2 (D2-E578Q) decreases K_m 50-fold. (F) The catalytic efficiency (k_cat/K_m) for WT p97 is 0.026 min⁻¹ µM⁻¹. A 2-fold decrease for the Walker A mutation of D1 (D1-K251A), a 3-fold increase for the Walker B mutation of D1 (D1-E305Q), a 15-fold decrease for the Walker A mutation of D2 (D2-K524A), and a 14-fold increase for the Walker B mutation of D2 (D2-E578Q) together suggest that D1 is a catalytically competent ATPase, when D2 is able to bind to nucleotides.