Fig. 3: Benzbromarone interferes with the interaction between Hsp90 and Aha1 which consequentially modulating the ATPase activity of Hsp90-Aha1 system.
A The results for [1H, 15N] HSQC-based competitive binding experiments are shown. Zoomed view of the superimposed [1H, 15N] HSQC spectra for 15N-Aha1-CTD alone (red), 15N-Aha1-CTD with the presence of Hsp90-NTD (with AMPPNP, blue, molar ratio of 1:5 for 15N-Aha1-CTD to Hsp90-NTD), 15N-Aha1-CTD with the presence of Hsp90-NTD and Benzbromarone (with AMPPNP, green, molar ratio of 1:5:8 for 15N-Aha1-CTD to Hsp90-NTD to Benzbromarone), and 15N-Aha1-CTD with the presence of Benzbromarone (purple, molar ratio of 1:8 for 15N-Aha1-CTD to Benzbromarone). The directional changes in chemical shift perturbations induced by Hsp90-NTD or Benzbromarone binding are indicated by black arrow and red arrow, respectively. B The determined ATPase activity data of Hsp90β (solid circle) and Hsp90β:Aha1(solid triangle) upon the treatment of Benzbromarone are shown. The ATPase rates obtained from the NADH-coupled ATPase experiments were plotted, along with the mean ± SD derived from three times of independent repeats (n = 3; each repeat contains one experimental data extracted from technical triplicates). Statistical significance was determined using unpaired t-test (***p < 0.001, ****p < 0.0001). C The ATP hydrolysis reaction catalyzed by Hsp90β was monitored by collecting 1D 31P NMR spectra. Overlay of 1D 31P spectra of the Hsp90β:ATP (4 μM:1 mM) reaction system without (red) and with the addition of Benzbromarone (400 µM, green) acquired at the time points of 2 h and 4 h after the initiation of reaction, are shown. D The ATP hydrolysis reaction catalyzed by Hsp90β and Aha1 was monitored by collecting 1D 31P spectra. Overlay of 1D 31P spectra of the Hsp90β:Aha1:ATP (4 μM:20 μM:1 mM) reaction system without (red) and with the addition of Benzbromarone (400 µM, green) are presented. The spectra were acquired at the time points of 20 min and 1 h after the initiation of reaction.
