Fig 2: The Ahsa1 ICD modulates ATPase stimulation of Hsp90.ATPase stimulation of Hsp90α and Hsp90β by Ahsa1 constructs (A). Schematic of Ahsa1 constructs used. (B, C) Stimulation of Hsp90α (B) and Hsp90β (C) ATPase activity by increasing concentrations of full-length Ahsa1 (black circles), Ahsa1Δ20 (blue squares), and Ahsa1Δ27 (purple triangles). (D) Bmax values of each of the three replicate reactions shown in (A) blue) and (B) (green). (E) Kapp values of the reactions shown in (A) (blue) and (B) (green). Reactions contained 2 μM Hsp90α or Hsp90β and indicated concentration of cochaperone. N = 3. Data Information: In (B, C), data for each concentration of cochaperone are presented as mean +/− SEM of three independent experiments (N = 3; each N is one experiment carried out with technical triplicates as described in “Methods”). (D, E) The individual Bmax or Kapp values, respectively, are plotted as well as the mean +/− SEM of the three independent experiments shown in (B, C) (N = 3; each N is one experiment carried out with technical triplicates as described in “Methods”). Statistical significance in (D, E) was calculated using a Tukey’s multiple comparisons test (*P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001) (P value symbols in black apply to Hsp90α and Hsp90β; P value symbols in blue apply to Hsp90α; P value symbols in green apply to Hsp90β). Source data are available online for this figure.

Fig 3: Model of ICD autoinhibition of Ahsa1-stimulated Hsp90 ATPase activity, and Ahsa1 mediated client folding.(A) ICD only partially inhibits the NxNNWHW motif, whether it inhibits Hsp90 directly or another region of Ahsa1 is unknown. (B) Three possible roles for the ICD in complex recovery. (1) Ahsa1 is recruited in an ICD-dependent manner to Hsp90/GR complexes (TOP), and the complex remains stable because of slow ATP hydrolysis. (2) Ahsa1Δ20 is recruited in an ICD-independent manner to Hsp90/GR complexes (MIDDLE) but rapid ATP hydrolysis results in destabilization of the complex. (3) Deletion of the ICD prevents Ahsa1 recruitment to Hsp90/GR complexes (BOTTOM). (C) Pymol structure (6XLF) showing the extensive contacts made by the yeast Aha1p (shown in pink) residues WHW (dark blue; residues 9, 10, and 11 in yeast Aha1p) with Hsp90 (cyan). NxNN residues are shown in orange.

Fig 4: Mutations in the WHW portion of the NxNNWHW motif reduce the apparent Km of Hsp90 for ATP.Mutations in the WHW portion of the NxNNWHW motif results in higher apparent affinity for ATP in both Hsp90α (A) and Hsp90β (B). The apparent Km values for three experiments are shown in the scatter plots. Data Information: In (A, B), the apparent Km values for three independent experiments are plotted as well as the mean +/− SEM (N = 3; each N is one experiment carried out with technical triplicates as described in “Methods”). Statistical significance was calculated using a Dunnett’s multiple comparisons test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). N = 3. Source data are available online for this figure.

Fig 5: Complex formation between Ahsa1 and GR requires Ahsa1 interaction with Hsp90.Band intensity of three replicate experiments was measured by densitometry. After background subtraction, intensities of Hsp90 (A) and GR (B) were normalized against the amount of Ahsa1 recovered and expressed as a percentage of each protein with wildtype Ahsa1. Data Information: In (A, B), data from three individual experiments are plotted as individual points as well as the mean +/− SD (N = 3). Statistical significance was calculated for (A, B) using a Dunnett’s multiple comparison’s test (***P ≤ 0.001; ****P ≤ 0.0001). Source data are available online for this figure.