Extended Data Fig. 4: Effect of the inhibitor-sensitizing Asp-to-Cys mutation on the AAA proteins VPS4B and FIGL1.
From: A chemical genetics approach to examine the functions of AAA proteins
a, SDS–PAGE analysis of purified recombinant human wild type (WT) and mutant (D135C) HS-VPS4B constructs, and wild type and mutant (D402C) FIGL1 constructs (Coomassie blue staining). b, Percentage steady-state ATPase activity of FIGL1 and HS-VPS4B (WT) in the presence of ASPIR-1 (5 µM, 1 mM ATP, 30 min incubation; data represent mean ± s.d., n = 3 independent experiments). (c-d) ATP concentration dependence of the steady-state activity of WT and D135C HS-VPS4B (c), and WT and D402C FIGL1 (d) analyzed using an NADH-coupled assay. Rates were fit to the Michaelis–Menten equation for cooperative enzymes (mean ± range, n = 2 independent experiments for HS-VPS4B-WT and HS-VPS4B-D135C; mean ± s.d., n = 5 independent experiments for FIGL1-WT, n = 7 independent experiments for FIGL1-D402C). Kinetic parameters were determined: kcat = 1.7 ± 0.1 s-1, K1/2 = 0.12 ± 0.07 mM for HS-VPS4B-WT; kcat = 0.5 ± 0.1 s-1, K1/2 = 0.15 ± 0.01 mM for HS-VPS4B-D135C; kcat = 3.4 ± 0.4 s-1, K1/2 = 0.2 ± 0.1 mM for FIGL1-WT; kcat = 2.3 ± 0.5 s-1, K1/2 = 0.3 ± 0.1 mM for FIGL1-D402C. (e-f) Differential scanning fluorimetry of WT and D135C HS-VPS4B (e) and WT and D402C FIGL1 (f) in the absence and presence of ADP (1 mM) (5% DMSO for both conditions). One representative experiment is shown (n = 2 independent experiments). The unmodified gel images for (a) and data for the graphs in (b-f) are available as source data.
