Extended Data Fig. 5: Inhibition of VPS4B-D135C by ASPIR-2.
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) VPS4B (tagless), and VTA1 constructs (Coomassie blue staining). b, ATP-concentration dependence of the steady-state activity of VPS4B-WT and VPS4B-D135C in the presence of 2-fold excess VTA1, analyzed using an NADH-coupled assay. Rates were fit to the Michaelis–Menten equation for cooperative enzymes (mean±range, n = 2 independent experiments). c, Chemical structure of ASPIR-2, the analog used for x-ray crystallography studies. d, Time-dependent inhibition of the ATPase activity of VPS4B-D135C by ASPIR-2. Graph shows percentage residual ATPase activity (mean ± range, n = 2 independent experiments). e, Concentration-dependent inhibition of the VTA1-stimulated, steady-state ATPase activity of WT and D135C VPS4B after 30 min incubation with ASPIR-2 (1 mM ATP; data represent mean ± range, n = 2 independent experiments). f, 2Fo-Fc electron density map of the crystal structure of VPS4B-D135C bound to ASPIR-2, contoured at 2.0 σ. g, Overlay of the structure of VPS4B-D135C in complex with ASPIR-2 with the RADD model for compound 1 bound to katanin, at the nucleotide-binding site (ASPIR-2: purple and blue, compound 1: pink and blue, stick representation; VPS4B-D135C: gray, katanin: white, ribbon representation; VPS4B residue Cys-135 is also shown). The unmodified gel image for (a) and data for the graphs in (b) and (d-e) are available as source data.
