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. 2025 Nov;34(11):e70359.
doi: 10.1002/pro.70359.

Collaboration between two conserved sequence motifs drives ATPase stimulation of Hsp90 by Aha1

Desmond Prah Amoah  1 , Rebecca Mercier  1 , Gnin Alyousef  1 , Jill L Johnson  2 , Paul LaPointe  1
Affiliations

Collaboration between two conserved sequence motifs drives ATPase stimulation of Hsp90 by Aha1

Desmond Prah Amoah et al. Protein Sci. 2025 Nov.

Abstract

Hsp90 is a dimeric molecular chaperone essential for the folding, stabilization, activation, and maturation of hundreds of client proteins, which are critical for cellular function. Co-chaperones, such as Aha1, play a key role in regulating the ATP-dependent Hsp90 client activation cycle by modulating Hsp90's ATPase activity and controlling progression through the cycle. Two highly conserved motifs in Aha1-the NxNNWHW and RKxK motifs-are known to regulate specific aspects of the Hsp90 ATPase cycle. In this study, we demonstrate that the K60 residue within the RKxK motif facilitates the structural organization of the NxNNWHW motif prior to ATP hydrolysis. Mutation of the K60 residue partially impairs the in vivo functionality of yeast Aha1. Additionally, we reveal that each individual residue within the NxNNWHW motif modulates the ATPase rate and apparent affinity for ATP of Hsp90. These findings provide new insights into how conserved regions of Aha-type co-chaperones influence Hsp90 kinetics and its regulation of client protein folding.

Keywords: ATPase; Aha1; Hsp90; chaperone; co‐chaperone; protein folding; yeast.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Interaction between the yeast Aha1 co‐chaperone and Hsp90; K60 is essential for strong ATPase stimulation by Aha1. (a) Aha1 is a co‐chaperone of Hsp90 that consists of two distinct domains: an N domain and a C domain, connected by a flexible, unstructured linker. The N‐terminal domain features two conserved motifs: the NxNNWHW motif and the RKxK motif. (b) The structure of the Aha1‐Hsp90 complex in the nucleotide‐free (Apo) state (PDB: 6XLB) reveals that both the N‐terminal and C‐terminal domains of Aha1 are bound to the middle domain of Hsp90. The RKxK motif is structured in this complex but the NxNNWHW motif (except for W11) is not. (c) In the nucleotide‐bound state (PDB: 6XLF), the K60 residue within the RKxK motif of Aha1 (depicted in magenta) is repositioned toward the now‐structured NxNNWHW motif (orange) in the N‐terminal domain of Aha1. The R59 (cyan) and K62 (red) residues of the RKxK motif remain oriented toward the Hsp90 middle domain and away from the NxNNWHW motif (orange). (d) The stimulation of Hsc82 was measured using increasing concentrations of Aha1 (black circles), Aha1R59A (green squares), Aha1K60A (magenta triangles), and Aha1K62A (inverted blue triangles). Each reaction contained 1 μM Hsc82 and varying concentrations of the co‐chaperone (n = 3). Error bars represent the standard error of the mean. (e) The B max values derived from the experiments in panel (D) are plotted. The B max for Aha1K60A was significantly lower than that for wildtype Aha1. In contrast, the B max values for Aha1R59A and Aha1K62A were similar to that of wildtype Aha1. (f) Chart showing B max values from (e). (g) K app values derived from the experiments in panel (d) are plotted. The K app for Aha1K60A was significantly lower than that for wildtype Aha1. In contrast, the K app values for Aha1R59A and Aha1K62A were similar to that of wildtype Aha1. (h) Chart showing K app values from (g). Data information: in d, data points are the mean of three independent triplicate experiments and error bars represent the standard error of the mean. Reactions contained 1 μM Hsc82 and indicated concentration of co‐chaperone (N = 3). Error bars in e and g represent the standard deviation. Statistical significance in e and g was determined using Tukey's multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
FIGURE 2
FIGURE 2
The K60A mutation mimics the loss of the NxNNWHW motif. (a) Stimulation of Hsc82 by increasing concentrations of Aha1 (black circles), Aha1Δ11 (open black circles), Aha1R59A (green circles) or Aha1R59A/Δ11 (open green circles). (b) The B max values derived from the experiments in panel (a) are plotted. (c) Stimulation of Hsc82 by increasing concentrations of Aha1 (black circles), Aha1Δ11 (open black circles), Aha1K60A (purple triangles) or Aha1K60A/Δ11 (open purple triangles). (d) The B max values derived from the experiments in panel (c) are plotted. (e) Stimulation of Hsc82 by increasing concentrations of Aha1 (black circles), Aha1Δ11 (open black circles), Aha1K62A (blue inverted triangles) or Aha1K62A/Δ11 (open blue inverted triangles). (f) The B max values derived from the experiments in panel (e) are plotted. Data Information: In a, c, and e, data points are the mean of three independent triplicate experiments and error bars represent the standard error of the mean. Reactions contained 1 μM Hsc82 and indicated concentration of co‐chaperone (N = 3). Error bars in b, d, and f represent the standard deviation. Statistical significance in b, d, and f was determined using Tukey's multiple comparisons test (**p < 0.01; ****p < 0.0001).
FIGURE 3
FIGURE 3
The K60A mutation has the same effect on the apparent affinity for ATP as deletion of the NxNNWHW motif. (a) The apparent Km for ATP of Hsc82 was measured in the presence of Aha1 (black circles), Aha1R59A (green squares), Aha1K60A (purple triangles) Aha1K62A (blue inverted triangles), and Aha1Δ11 (open black circles). ATPase activity was measured using increasing ATP concentrations (12.5, 25, 50, 100, 200, 400, 800, 1600 μM), and the resulting ATPase rates were analyzed using the Michaelis–Menten non‐linear regression function in GraphPad Prism. The curve fits all had R 2 values >0.9. The apparent K m values for three independent experiments are plotted and the error bars represent the standard error (N = 3). Statistical significance was calculated using a Tukey's multiple comparisons test (*p < 0.05; ****p < 0.0001). (b) Table showing K m values plotted in (a).
FIGURE 4
FIGURE 4
K60A mutation impairs the action of the NxNNWHW of Aha1 in yeast. (a) Yeast strains expressing Hsc82S25P as the sole source of Hsp90 exhibit temperature‐sensitive growth. Yeast cells containing plasmids encoding the specified C‐terminally myc‐tagged Aha1 co‐chaperones were cultured overnight at 30°C in YPD medium supplemented with 200 mg/L Geneticin (for Aha1 plasmid selection). The cultures were then diluted to a concentration of 1 × 108 cells per milliliter. Ten‐fold serial dilutions were prepared, and 10 μL aliquots were spotted onto YPD agar plates containing 200 mg/L Geneticin. The plates were incubated for 2 days at temperatures of 30, 34, or 37°C. Rescue of growth of Hsc82S25P yeast by Aha1K60A was impaired compared to wild‐type Aha1, Aha1R59A, and Aha1K62A. (b) Western blot of total lysates extracted from the yeast strains shown in (a) was probed with anti‐Hsp90, anti‐actin, and anti‐myc (for Aha1) antibodies.
FIGURE 5
FIGURE 5
The stimulation of Hsc82S25P ATPase activity by Aha1 depends on the K60 residue, which supports the function of the NxNNWHW motif. (a) The ATPase activity of both wildtype Hsc82 (black circles) and Hsc82S25P (black diamonds) was enhanced by increasing concentrations of Aha1. Hsc82S25P ATPase activity was stimulated by Aha1R59A (green squares) and Aha1K62A (blue inverted triangles), but not by Aha1K60A (purple triangles) or Aha1Δ11 (open black diamonds). (b) The B max values derived from the experiments in panel (a) are plotted. Data Information: In a, data points are the mean of three independent triplicate experiments and error bars represent the standard error of the mean. Reactions contained 1 μM Hsc82 and indicated concentration of co‐chaperone (N = 3). Error bars in b represent the standard deviation. Statistical significance in b was determined using Tukey's multiple comparisons test (***p < 0.001; ****p < 0.0001).
FIGURE 6
FIGURE 6
Mutation of each residue of the NxNNWHW motif to alanine mimics deletion of NxNNWHW motif. (a) Schematic of Hsp90‐Aha1 complex in the nucleotide‐bound state (PDBID: 6XLF) depicting the N5 (cyan), N7 (yellow), N8 (magenta), W9 (red), H10 (orange) and W11 (gray) residues of the NxNNWHW motif located in the N‐domain of Aha1. RKxK motif is shown in tan. (b) Stimulation of Hsc82 ATPase activity by increasing concentrations of Aha1 (black circles), N5A, N7A, N8A (each in a different shaded of orange diamond), N5A/N7A/N8A (open orange diamonds), W9A (dark turquoise squares), H10A (pink squares), W11A (light turquoise squares), W9A/H10A/W11A (open purple squares) and Aha1Δ11 (open black circles). (c) The B max values derived from the experiments in panel (b) are plotted. Data Information: In b, data points are the mean of three independent triplicate experiments and error bars represent the standard error of the mean. Reactions contained 1 μM Hsc82 and indicated concentration of co‐chaperone (N = 3). Error bars in c represent the standard deviation. Statistical significance in c was determined using Tukey's multiple comparisons test (**p < 0.001; ***p < 0.001; ****p < 0.0001).
FIGURE 7
FIGURE 7
Mutations in the NxNNWHW motif increases the apparent for ATP of Hsc82. (a) The apparent K m for ATP of Hsc82 was measured in the presence of Aha1 (black circles), N5A, N7A, N8A (each in a different shaded of orange diamond), N5A/N7A/N8A (open orange diamonds), and Aha1Δ11 (open black circles). (b) The apparent K m for ATP of Hsc82 was measured in the presence of Aha1 (black circles), W9A (dark turquoise squares), H10A (pink squares), W11A (light turquoise squares), W9A/H10A/W11A (open purple squares), and Aha1Δ11 (open black circles). ATPase activity was measured using increasing ATP concentrations (12.5, 25, 50, 100, 200, 400, 800, 1600 μM), and the resulting ATPase rates were analyzed using the Michaelis–Menten non‐linear regression function in GraphPad Prism. The curve fits all had R 2 values >0.9. The apparent K m values for three independent experiments are plotted and the error bars represent the standard error (N = 3). Statistical significance was calculated using a Tukey's multiple comparisons test (*p < 0.05; ****p < 0.0001). (c) Table showing K m values plotted in (a). (d) Table showing K m values plotted in (B).
FIGURE 8
FIGURE 8
Schematic showing a model for the role of the RKxK motif in Hsp90 ATPase stimulation. Top panel: Aha1 binds to Hsp90 and stimulates ATPase activity. Middle panel: mutation of either R59 or K62 impairs binding affinity to Hsp90 but not ATPase stimulation. Bottom panel: mutation of K60 impairs binding affinity to Hsp90 and ATPase stimulation by preventing the action of the NxNNWHW motif.
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