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. 2025 Oct 22;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,✉
PMCID: PMC12541893  PMID: 41123251

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: Aha1, ATPase, chaperone, co‐chaperone, Hsp90, protein folding, yeast

1. INTRODUCTION

The 90‐kiloDalton heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that facilitates the folding and maturation of a broad but specific group of substrates called client proteins (Echeverria et al., 2011; Eckl & Richter, 2013; Taipale et al., 2012; Van Oosten‐Hawle et al., 2017). These clients include membrane proteins, hormone receptors, transcription factors, and other key regulators of signal transduction, the cell cycle, and stress responses (Citri et al., 2004; Morishima et al., 2000; Whitesell et al., 1994). The maturation of client proteins by the Hsp90 dimer takes place within an ATP‐dependent functional cycle, during which Hsp90 undergoes extensive conformational changes involving both inter‐ and intra‐protomer interactions (Chadli et al., 2000; Cunningham et al., 2008). The ATP‐dependent functional cycle complex is regulated by an array of proteins called co‐chaperones (Armstrong et al., 2012; Chang et al., 1997; Fang et al., 1998; Knoblauch & Garabedian, 1999; Lee et al., 2012; Li et al., 2013; Prodromou et al., 1999; Siligardi et al., 2004). Co‐chaperones influence ATP binding and hydrolysis, and modulate its interactions with client proteins (Panaretou et al., 2002; Richter et al., 2004). Moreover, the Hsp90 functional cycle is controlled by post‐translational modifications (PTMs), which can affect the Hsp90 conformational dynamics by altering client proteins and co‐chaperone recruitment (Mollapour et al., 2010; Mollapour et al., 2014; Mollapour, Tsutsumi, Truman, et al., 2011; Preuss et al., 2015; Xu et al., 2012). Although the regulation of the Hsp90 functional cycle during client maturation is not well understood, it is evident that ATP hydrolysis is necessary for Hsp90 to efficiently mature clients (Obermann et al., 1998; Panaretou et al., 1998; Prodromou & Bjorklund, 2022).

Each Hsp90 monomer consists of three major domains: an N‐terminal domain with an ATP‐binding pocket, a middle domain (connected to the N domain by a long charged linker), and a C‐terminal dimerization domain, which facilitates Hsp90 dimer formation (Jahn et al., 2014; Meyer et al., 2004; Ratzke et al., 2010). The interplay between these domains is essential for the catalytic activity of Hsp90 (Cunningham et al., 2008). The Hsp90 functional cycle begins with ATP binding to the N‐terminal domain, which induces interactions of the γ‐phosphate of ATP with an Arg 380 (R380) residue in the middle domain (Cunningham et al., 2012; Meyer et al., 2004). This interaction leads to the docking of the N‐ and middle domains of Hsp90, initiating ATP hydrolysis (Richter et al., 2001). The ATP lid, a small region in the N‐terminal domain, undergoes a conformational shift, trapping the ATP molecule and triggering the rotation of helices in the N‐terminal domains of opposite monomers, which exposes hydrophobic residues that facilitate N‐terminal dimerization (Cunningham et al., 2008; Wandinger et al., 2008). Although Hsp90 can adopt multiple structural states even in the absence of nucleotides (Bron et al., 2008), ATP binding is thought to lower the energy barrier between the apo (open) state and the closed, N‐terminally dimerized state, enabling efficient client maturation (Hessling et al., 2009).

The regulation of Hsp90 ATPase activity has become a focal point for understanding its role in client protein folding (LaPointe et al., 2020; Panaretou et al., 2002). Aha1, Activator of Hsp90 ATPase 1, is the most robust known stimulator of the normally very low Hsp90 ATPase activity (Lotz et al., 2003; Meyer et al., 2004). Aha1 is recruited to Hsp90 through various PTMs, such as phosphorylation and SUMOylation (Mollapour et al., 2010; Mollapour et al., 2014; Mollapour, Tsutsumi, Kim, et al., 2011), and is crucial for the folding and activation of diverse proteins, including Glucocorticoid receptor, v‐src, and cystic fibrosis transmembrane conductance regulator (CFTR) (Holmes et al., 2008; Wang et al., 2006). Aha1 enhances the ATPase cycle of Hsp90, promoting its transition to a catalytically competent state, although the exact mechanism remains poorly understood (Armstrong et al., 2012; Li et al., 2013; Mercier et al., 2019; Wolmarans et al., 2016). Aha1 consists of two functional domains: an N‐terminal domain and a C‐terminal domain, connected by an unstructured linker (Figure 1a) (Koulov et al., 2010; Li et al., 2013; Mercier et al., 2019; Retzlaff et al., 2010; Wolmarans et al., 2016). The N‐terminal domain contains two highly conserved motifs—the NxNNWHW (residues 5–11 in yeast) and RKxK (residues 59–62 in yeast) motifs that are known to regulate Hsp90 ATPase activity (Figure 1a) (Amoah et al., 2025; Horvat et al., 2014; Hussein et al., 2024; Mercier et al., 2019; Meyer et al., 2004). The NxNNWHW motif, located within the first 11 amino acids of yeast Aha1 and the first 27 amino acids of mammalian Aha1 (Ahsa1), is essential for maximal stimulation of the Hsp90 ATPase activity (Amoah et al., 2025; Hussein et al., 2024; Mercier et al., 2019). Our previous work has shown that the NxNNWHW motif is required for the robust stimulation of the intrinsically low Hsp90 ATPase activity, and our recent findings indicate that this motif also enforces ordered ATP hydrolysis within the Hsp90 dimer (Amoah et al., 2025; Hussein et al., 2024; Mercier et al., 2019). The in vivo action of Aha1 is also dependent on the presence of the NxNNWHW motif (Amoah et al., 2025; Mercier et al., 2019). On the other hand, the role of the RKxK motif of Aha1 on the function of this co‐chaperone is not yet well understood.

FIGURE 1.

FIGURE 1

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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).

Recent cryo‐EM structures have provided insights into how Aha1 interacts with Hsp90 to stimulate ATPase activity (Liu et al., 2020). In the absence of nucleotides, Aha1 binds to the middle domain of Hsp90 on opposing subunits, preventing the N‐domains of Hsp90 from interacting with the middle domain (Figure 1b). Upon ATP binding, Aha1 undergoes a conformational change, allowing its N‐terminal domain to interact with the N‐domains of Hsp90. This leads to the structural ordering of the NxNNWHW motif, which then participates in interactions with various residues in both the N domains of Hsp90 and certain residues in the RKxK motif of the N domain of Aha1 (Figure 1c). Interestingly, the RKxK motif is situated beneath the NxNNWHW motif, suggesting that they may act together to promote ATP hydrolysis.

To investigate the relationship between the conserved motifs in Aha1, we carried out site‐directed mutagenesis of the NxNNWHW and RKxK motifs in yeast Aha1, substituting alanine for each residue. These alanine mutants were compared to wildtype Aha1 and Aha1Δ11 (Aha1 missing the first 11 amino acids containing the NxNNWHW motif) to assess their capacity to stimulate ATPase activity in vitro. We found that mutation of lysine 60 to alanine (K60A) in the RKxK motif significantly impaired ATPase stimulation by Aha1, mimicking the loss of the NxNNWHW motif. Furthermore, we showed that the K60A mutation partially blocks the action of the NxNNWHW motif in yeast. Our results suggest that these conserved motifs act together to regulate the Hsp90 ATPase cycle.

2. ONLINE METHODS

2.1. Protein expression and purification

Saccharomyces cerevisiae Hsc82, Hsc82S25P, Aha1, Aha1Δ11, Aha1R59A, Aha1K60A, Aha1K62A, Aha1R59A/Δ11, Aha1K60A/Δ11, Aha1K62A/Δ11, Aha1N5A, Aha1N7A, Aha1N8A, Aha1N5/7/8A, Aha1W9A, Aha1H10A, Aha1W11A, and Aha1WHW/AAA were expressed in Escherichia coli strain BL21 (DE3) (New England Biolabs) from pET11d (Stratagene, La Jolla, CA, USA). Two versions of pET11d were used to express these proteins. Hsc82 and Hsc82S25P were expressed with N terminal 6xHis tags, and Aha1, Aha1Δ11, Aha1R59A, Aha1K60A, Aha1K62A, Aha1R59A/Δ11, Aha1K60A/Δ11, Aha1K62A/Δ11, Aha1N5A, Aha1N7A, Aha1N8A, Aha1N5/7/8A, Aha1W9A, Aha1H10A, Aha1W11A, and Aha1WHW/AAA were expressed with C terminal 6xHis tags. Cells were grown at 37°C to an OD600 of 0.8–1.0 and induced with 1 mM isopropyl‐1‐thio‐D‐galactopyranoside (IPTG). Cells expressing Hsc82, Hsc82T22E, Hsc82V387E, Hsc82E33A, Hsc82D79N, Hsc82T22E/E33A and Hsc82T22E/V387E were harvested after overnight growth at 16°C. Cells expressing Aha1 and Aha1Δ11 were harvested after 4 h at 37°C. Cells were harvested by centrifugation and stored at −80°C. Cells were resuspended in lysis buffer (50 mM KH2PO4, pH 8.0, 500 mM KCl, 10% Glycerol, 10 mM Imidazole, 5 mM β‐mercaptoethanol) and lysed using Avestin Emulsiflex C3 (Avestin, Ottawa, Ontario, Canada). Lysates were clarified by ultracentrifugation and His‐tagged proteins were isolated on a HisTrap FF column using an AKTA Explorer FPLC (GE Healthcare). Isolated 6xHis‐tagged proteins were then concentrated and subjected to anion exchange chromatography on a Hitrap Q FF column using an AKTA Explorer FPLC (GE Healthcare). The isolated proteins were further purified by size exclusion chromatography on a Hiload Superdex 200 pg column (GE Healthcare). Purity of each protein preparation was >95% as verified by Coomassie‐stained SDS‐PAGE analysis.

2.2. ATPase assays

ATPase assays were carried out using the enzyme‐coupled assay as previously described (Armstrong et al., 2012; Horvat et al., 2014; Mercier et al., 2019; Wolmarans et al., 2016; Wolmarans et al., 2019). All reactions were carried out in at least three independent experiments, with every condition in triplicate 100 μL (for 96‐well plates) or 50 μL (for 384‐well plates) reactions. Absorbance at 340 nm was measured every 1 (for 96‐well plates) or 2 (for 384‐well plates) minutes for 90 min using a Bio Tek Synergy 4 and the path‐length correction function. Average values of the experiments are shown with error expressed as standard error of the mean. The decrease in NADH absorbance at 340 nm was converted to micromoles of ATP using Beer's Law and then expressed as a function of time. The final conditions of all the reactions are 25 mM HEPES (pH 7.2), 12.5 or 16 mM NaCl (in titration and cycling experiments, respectively), 5 mM MgCl2, 1 mM DTT, 0.6 mM NADH, 2 mM ATP (co‐chaperone titration and cycling experiments), 1 mM phosphoenol pyruvate (PEP), 2.5 μL of pyruvate kinase/lactate dehydrogenase (PK/LDH) (Sigma), and 5% DMSO. To correct for contaminating ATPase activity, identical reactions were quenched with 100 μM NVP‐AUY922 and subtracted from unquenched reactions (DMSO control). In the titration experiments, 1 μM of Hsc82 or Hsc82S25P was added to reactions containing either 1, 2, 4, 8, or 16 μM of Aha1 (or Aha1 mutants). The ATPase assay was started by the addition of the regenerating system consisting of MgCl2, DTT, NADH, ATP, PEP, and PK/LDH. Fit lines were calculated according to the following equation (Y = ((B max × X)/(K app + X)) + X 0). As mentioned above, B max and K app are calculated in each triplicate experiment and this is done at least three times.

2.3. Yeast growth assays

Yeast cells were transformed by lithium acetate methods and grown in either YPD or defined synthetic complete media supplemented with 2% dextrose. Growth was examined by spotting 10‐fold serial dilutions of yeast cultures on appropriate media, followed by incubation for 2 days at the indicated temperature. Hsc82S25P was expressed in strain JJ816 (aha1hsc82hsp82/Yep24‐HSP82) after plasmid shuffling using 5‐fluoroorotic acid (5‐FOA) (Toronto Research Chemicals). Plasmids encoding wildtype and mutant Aha1 (p41KanTEF‐Aha1‐myc) have been described previously (Mercier et al., 2019). G418 was obtained from Sigma. Quantification of yeast growth assays was done as previously described (Mercier et al., 2023; Petropavlovskiy et al., 2020).

2.4. Statistical analysis

Statistical analysis (for Figures 1e,g, 2b,d,f, 3a, 5b, 6c, and 7a,b) was carried out in GraphPad Prism using Tukey's multiple comparisons test. Statistical significance is indicated in each figure.

FIGURE 2.

FIGURE 2

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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

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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 5.

FIGURE 5

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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

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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

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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).

3. RESULTS

3.1. K60 is required for robust Hsp90 ATPase stimulation

The NxNNWHW and RKxK motifs in Aha1 are important for stimulating Hsp90 ATPase activity (Amoah et al., 2025; Horvat et al., 2014; Hussein et al., 2024; Mercier et al., 2019; Meyer et al., 2004). However, while the NxNNWHW motif is necessary for robust ATPase stimulation by Aha1, the role of the RKxK motif is less well defined. To better understand the significance of the residues in the highly conserved RKxK motif, we introduced alanine residues at each position. We expressed and purified each of these constructs (harboring a C‐terminal 6xHis‐tag) for testing in ATPase stimulation assays. The K60A mutation (Aha1K60A) caused a significant reduction in ATPase stimulation of Hsc82 (the constitutively expressed yeast Hsp90) when compared to wildtype Aha1, Aha1R59A, or Aha1K62A (Figure 1d–f). Titrating co‐chaperones into our ATPase assays allows us to calculate both the maximally stimulated rate for each construct (i.e., B max) but also an apparent affinity of each construct for Hsc82 (i.e., K app; the concentration at which half of the B max is reached). This represents an advantage over physical methods to measure affinity that cannot distinguish between Aha1‐Hsc82 interactions that contribute to ATPase stimulation and those that do not. Interestingly, all three mutations reduced the apparent affinity of Aha1 for Hsc82 compared to wildtype, with the greatest effect observed for Aha1K60A (Figure 1g,h). This suggests that the conserved residues of the RKxK motif play distinct roles in binding to Hsp90 and stimulation of ATPase activity.

3.2. Mutation of K60 to an alanine mimics the loss of the NxNNWHW motif

As mentioned earlier, cryo‐EM structures of the Hsp90‐Aha1 complex show that the NxNNWHW motif is positioned near the RKxK residues upon ATP binding by Hsp90 (Figure 1b,c). Comparing the nucleotide‐free and ATP‐bound states reveals that K60, but not R59 and K62, reorients toward the NxNNWHW motif after ATP binding (Figure 1b,c). We hypothesized that the RKxK motif, and K60 in particular, enabled the participation of the NxNNWHW motif in ATPase stimulation. To test this, we measured the Aha1‐mediated ATPase stimulation activity of single point mutations in the RKxK motif in combination with deletion of the NxNNWHW motif (Figure 2a–f). As expected, deletion of the NxNNWHW motif reduced ATPase stimulation by Aha1, Aha1R59A, and Aha1K62A to the same degree. However, deletion of the NxNNWHW motif from Aha1K60A did not result in a further decrease in ATPase stimulation (Figure 2c,d). In other words, the mutation of K60 to alanine had the same effect as deletion of the NxNNWHW motif and no significant additional reduction occurred when the two mutations are combined. That these two mutations mimic one another suggested to us that K60 is required for the action of the NxNNWHW motif during ATPase stimulation.

3.3. Mutation of K60 to an alanine has the same effects on the apparent affinity for ATP as deletion of the NxNNWHW motif

In previous studies, we demonstrated that the loss of the NxNNWHW in yeast Aha1 affects the apparent affinity of Hsp90 for ATP (Mercier et al., 2019). Since the K60A mutation blocks the action of the NxNNWHW motif, we hypothesized that K60A would increase the apparent affinity of Hsp90 for ATP similar to the effects observed for the deletion of the NxNNWHW motif. Consistent with our hypothesis, the apparent affinity of Hsc82 for ATP in the presence of Aha1K60A was increased to a similar degree as for Aha1Δ11 (Figure 3a,b). While the difference between the apparent K m for ATP with these two Aha1 variants was statistically significant, this difference was small. Importantly, there was no significant difference in apparent affinity of ATP for Hsc82 in the presence of wildtype Aha1, Aha1R59A, or Aha1K62A (Figure 3a,b). This further supported the idea that K60 is required for the action of the NxNNWHW motif.

3.4. K60A mutation blocks the action of the NxNNWHW of Aha1 in vivo

We previously showed that the NxNNWHW motif is required for the in vivo action of Aha1 in yeast (Mercier et al., 2019). Specifically, overexpression of Aha1, but not Aha1Δ11, rescued the temperature‐sensitive growth phenotype exhibited by yeast expressing Hsc82S25P. Since the K60A mutation mimics deletion of the NxNNWHW motif, we hypothesized that the K60A mutation would prevent the rescue of temperature‐sensitive growth of yeast expressing Hsc82S25P. To test this, we expressed our different Aha1 constructs in a yeast strain expressing Hsc82S25P in which the endogenous AHA1 gene had been deleted. This strain grew poorly at elevated temperatures but was restored with the overexpression of Aha1, Aha1R59A, and Aha1K62A, but not Aha1K60A (Figure 4a). Importantly, all of our Aha1 constructs were expressed to a comparable degree, suggesting that Aha1K60A is functionally impaired in a manner that Aha1R59A and Aha1K62A are not (Figure 4b).

FIGURE 4.

FIGURE 4

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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.

3.5. ATPase stimulation of Hsc82S25P by RKxK mutants

We previously showed that ATPase stimulation of Hsc82S25P by Aha1 requires the NxNNWHW motif (Mercier et al., 2019). Importantly, deletion of the NxNNWHW motif and mutation of K60 (but not of R59 and K62) both resulted in a near‐complete loss of ATPase stimulation of Hsc82S25P (Figure 5a,b), suggesting that these two alterations result in the same impairment.

3.6. All residues in the NxNNWHW motif contribute to its function

The cryo‐EM structure of the Hsp90‐Aha1 complex in the ATP‐bound state shows that the NxNN and the WHW portions of the motif are positioned on opposite sides of a turn in the polypeptide backbone (Figure 6a). More specifically, the WHW portion of the motif appears to make contact with the RKxK motif and parts of Hsp90, while most of the NxNN portion makes contact with other parts of Aha1. To determine the contribution of each residue to ATPase stimulation, we constructed Aha1 point mutants at each position, as well as two different combination mutants where all asparagine residues of the NxNN were mutated or all residues of the WHW were mutated. Surprisingly, all mutants of Aha1 were profoundly compromised in their ability to stimulate the ATPase activity of Hsc82 (Figure 6b,c). Only Aha1N7A could stimulate the ATPase activity of Hsc82 to a degree greater than Aha1 lacking the entire NxNNWHW motif (Aha1Δ11). This suggests that each residue in the NxNNWHW motif is required for the robust Aha1‐mediated ATPase stimulation of Hsp90.

3.7. Mutation of the NxNNWHW motif to alanine increases the apparent for ATP

We previously demonstrated that deletion of the NxNNWHW motif of Aha1 increases the apparent affinity of Hsp90 for ATP (Mercier et al., 2019). To determine if the point mutations we introduced into the NxNNWHW motif also corresponded to a decrease in the apparent K m for ATP, we tested our constructs in ATPase stimulation assays with increasing concentrations of ATP. Every mutation in the NxNNWHW (except N7) increased the apparent affinity of Hsc82 for ATP compared to wildtype Aha1 and to a similar extent as Aha1Δ11 (Figure 7a–d). That the N7A mutation resulted in the smallest change in maximal stimulation rate and apparent affinity for ATP suggests that this residue, despite being strongly conserved, is the least important for the function of the NxNNWHW motif.

4. DISCUSSION

Cryo‐EM structures of Aha1 in complex with Hsp90 have provided insight into the basis for ATPase regulation of Hsp90 (Liu et al., 2020). In the nucleotide‐free state, the N and C‐terminal domains of Aha1 are each bound to the middle domain of a different subunit of the Hsp90 dimer. The R59, K60, and K62 residues of the RKxK motif are positioned in the vicinity of the middle domain residues (310–390) of Hsp90 (Figure 1b). Upon ATP binding by Hsp90, the Aha1 N domain tilts toward the now‐resolved N domains of Hsp90, and the NxNNWHW motif acquires structure near residues in Hsp90 that are important for ATP hydrolysis. Interestingly, K60 reorients toward the now‐structured NxNNWHW motif within 4 angstroms of W9 and W11 (Figure 1c). That the K60A mutation mimics the deletion of the NxNNWHW in all the assays we tested suggests that this residue is required for the action of the NxNNWHW motif. Moreover, the function of the NxNNWHW motif is sensitive to mutations at any amino acid position, suggesting an intricate network of interactions that lead to the hydrolysis of ATP as well as the reverse process, where ADP is released (Mercier et al., 2019). It was not clear to us from the structure of the Hsp90‐Aha1 complex why mutations at N7 were less disruptive than mutations of N5 or N8. Further analysis will be required to determine this.

Interestingly, despite K60 being the only residue that impaired the function of the NxNNWHW motif (Figure 1d–f), all three residues of RKxK are important for binding to Hsp90 (Figure 8). Mutations of any of the three conserved residues resulted in a significant reduction in apparent binding affinity (Figure 1g,h). The dual role that the RKxK motif plays in the binding and positioning of the NxNNWHW motif leads us to refer to the RKxK motif as the Binding and Positioning Motif (BPM). Additionally, the critical role that the NxNNWHW motif plays in ATPase stimulation, but not binding, leads us to refer to it as the ATPase Stimulation Loop (ASL). It is important to note that our binding affinity calculations are indirect (calculated from our enzymatic assays). While direct biophysical methods would be useful to reinforce our model for binding, our indirect measurements allow us to focus on the binding events that impact ATPase stimulation.

FIGURE 8.

FIGURE 8

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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.

Consistent with our model for the role of K60 in promoting the action of the ASL, mutation of K60, but not R59 or K62, impacted the affinity of Hsp90 for ATP, consistent with the effects observed from the deletion of the ASL (Figure 3a,b). This suggests that the K60 residue of the BPM in Aha1 plays a crucial role in controlling whether Hsp90 binds ATP tightly or loosely, as well as in how ATP is retained in the nucleotide‐binding pocket through regulation of lid dynamics, which might be through supporting the ASL. This would be entirely consistent with work suggesting that nucleotide exchange (and not hydrolysis) is the only absolute requirement for Hsp90 function in cells (Reidy et al., 2023; Reidy & Masison, 2020; Zierer et al., 2016). We tested the biological activity of Aha1 in yeast expressing Hsc82S25P. Serine 25 has previously been shown to be a key phosphorylation site required for the induction of autophagy (Backe et al., 2023). That Aha1 overexpression rescues the growth of yeast expressing Hsc82S25P likely suggests that larger structural perturbations caused by the introduction of a proline at this site can be corrected by proper assembly of the ASL and BPM upon Aha1 binding.

Our data indicate that each residue in the ASL in yeast Aha1 (except N7) plays a similarly important role in regulating ATPase activity and the apparent affinity of Hsp90 for ATP (Figures 6 and 7). We previously showed that ATPase stimulation by mammalian Aha1 (Ahsa1) was similarly impacted by mutations in the NxNN and WHW sections of the ASL (Hussein et al., 2024). In contrast, only mutations in the WHW portion of the ASL of mammalian Ahsa1 affected the apparent affinity for ATP. This suggests that the yeast and mammalian Aha1 proteins regulate Hsp90 differently despite the complete conservation of these sequence motifs. A possible explanation for this discrepancy is that yeast Aha1 lacks the first 20 amino acids found in other Aha1 from other non‐yeast organisms (Hussein et al., 2024). This 20 amino acid N‐terminal extension may interact with clients directly (Liu & Wang, 2022; Tang et al., 2023; Tripathi et al., 2014), with Hsp90 directly, or with other parts of Ahsa1. Additionally, the recruitment and activity of mammalian Aha1 are dependent on phosphorylation events, such as that of tyrosine 223 raising the possibility that the action of the BPM can be modulated by such events (Dunn et al., 2015). Without structural information regarding the human Ahsa1‐Hsp90 complex, it is difficult to determine how subtle nucleotide‐dependent conformational changes occur.

The data presented here suggest that the complete RKxK motif (that we now call the BPM) of Aha1 plays a critical role in binding Aha1 to Hsp90, perhaps following recruitment through modifications on specific Hsp90 residues (Mollapour et al., 2014; Mollapour, Tsutsumi, Kim, et al., 2011; Mollapour, Tsutsumi, Truman, et al., 2011; Xu et al., 2012). Specifically, the K60 residue is likely essential for the structural alignment of the NxNNWHW motif of Aha1 (that we now call the ASL), positioning it near key catalytic residues that are necessary for ATP hydrolysis. Additionally, K60 may be crucial in regulating ATP binding within the ATP‐binding pocket by influencing lid dynamics, which in turn impacts ATP‐dependent client processing. While our findings provide valuable insights into how the different components of Aha1's conserved motifs influence the Hsp90 chaperone cycle, further research is needed to fully understand how the BPM and ASL specifically contribute to the regulation of client protein folding.

AUTHOR CONTRIBUTIONS

Desmond Prah Amoah: Conceptualization; investigation; writing – original draft; methodology; formal analysis; visualization. Rebecca Mercier: Conceptualization; investigation; writing – review and editing; methodology; visualization. Gnin Alyousef: Investigation; methodology; writing – review and editing. Jill L. Johnson: Conceptualization; resources; funding acquisition; writing – review and editing; methodology. Paul LaPointe: Conceptualization; funding acquisition; writing – original draft; writing – review and editing; supervision; resources; formal analysis; methodology; visualization.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This work was supported with funding from the Canadian Institutes of Health Research (178282) and the Natural Sciences and Engineering Research Council (RGPIN‐2019‐04967) (Paul LaPointe), as well as the National Institutes of Health (NIH R01 GM127675) (Jill L Johnson).

Amoah DP, Mercier R, Alyousef G, Johnson JL, LaPointe P. Collaboration between two conserved sequence motifs drives ATPase stimulation of Hsp90 by Aha1. Protein Science. 2025;34(11):e70359. 10.1002/pro.70359

Review Editor: Zengyi Chang

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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