Figure 4. ATPase activity of Pex6 D2 domains drive conformational changes.

(a) ATPase activities of wild-type Pex1/6 (Pex1/6^ATP), single Walker B (Pex1WB^ATP/6, Pex1/6WB^ATP) or double-Walker B mutants (Pex1/6 DWB^ATP) or Pex1 arginine finger (Pex1^R855K/6) or ISS motif (Pex1^D826V/6WB^ATP) mutants. Assays were performed in the presence of 1 mM ATP and 5 mM Mg²⁺ at 37 °C. Error bars represent s.d. of two independent experiments with five technical replicates each. *P<0.0001 in comparison with wild-type Pex1/6^ATP (one-way ANOVA). Michaelis–Menten plot of enzyme activity of Pex1/6^ATP, Pex1WB^ATP/6 or Pex1/6WB^ATP at various ATP concentrations (inset). Error bars represent s.d. of two independent experiments including three technical replicates. (b) Size-exclusion chromatography A_290 nm profiles of purified Pex1/6 complexes used for ATPase activity assays. (c) EM reconstructions of double- (Pex1/6 DWB^ATP) or single- (Pex1/6WB^ATP, Pex1WB^ATP/6) Walker B complexes in the presence of ATP as side views (upper row), followed by top views and cross-sections of the D1 and D2 rings (lower rows). (d) Side-view surface representation as in Fig. 3a. Underneath, a cartoon representation based on rigid body fits of homology models into negative stain EM maps is shown. Pex1/6^ATPγS and Pex1/6^ADP-AlFx are depicted in grey for comparison. Domains are coloured according to Fig. 1d. Green spheres depict residues Pex1^F771 and Pex6^Y805.