Systematic Mutant Analyses Elucidate General and Client-Specific Aspects of Hsp90 Function

Abstract

To probe the mechanism of the Hsp90 chaperone that is required for the maturation of many signaling proteins in eukaryotes, we analyzed the effects of all individual amino acid changes in the ATPase domain on yeast growth rate. The sensitivity of a position to mutation was strongly influenced by proximity to the phosphates of ATP, indicating that ATPase-driven conformational changes impose stringent physical constraints on Hsp90. To investigate how these constraints may vary for different clients, we performed biochemical analyses on a panel of Hsp90 mutants spanning the full range of observed fitness effects. We observed distinct effects of nine Hsp90 mutations on activation of v-src and glucocorticoid receptor (GR), indicating that different chaperone mechanisms can be utilized for these clients. These results provide a detailed guide for understanding Hsp90 mechanism and highlight the potential for inhibitors of Hsp90 that target a subset of clients.

Figure 1. Effects of individual amino acid changes in the N-domain of Hsp90 on yeast growth rate

(A) Outline of the bulk competition strategy utilized to analyze the effects of Hsp90 mutants on yeast growth rate. (B) Full biological replicates of Hsp90 mutations spanning amino acids 12-21 and 92-101 are strongly correlated. Growth effects are plotted as selection coefficients (0=no effect compared to wildtype, and −1=null). Silent mutations that do not change the protein sequence are plotted in green, stop codons are shown in red and amino acid changes in black. (C) Estimates of growth effects from bulk competitions correlate strongly with measurements of mono-culture growth rate for a panel of Hsp90 mutants. The following mutations were analyzed in monoculture: L18F, Y24L, R32A, N37S, D40E, K54E, E57K, R65L, Q72D, G83T, I96A, S99A, I117F, F120A, G121A, F124A, Y125W, L129M, E165K, I172D, and K191F. (D) Heatmap representation of the fitness landscape observed for individual amino acid changes across amino acids 2-231 of Hsp90. See also Figure S1 and tableS1.

Figure 2. Relationship between evolutionary conservation and experimental fitness effects

(A) Comparison of the experimental fitness sensitivity of each position in the N-domain to conservation observed in Hsp90 sequences from diverse eukaryotes. Positions exhibiting the strongest evolutionary conservation exhibit a broad range of experimental sensitivity to mutation while the most evolutionary variable positions are almost universally experimentally tolerant to mutations (B) Experimental fitness effects at amino acid positions that exhibited the greatest and least evolutionary variation. (C&D) Structural representations of the Hsp90 N-domain from 2CG9.PDB (Ali et al., 2006) where the width of the backbone trace and the color indicate the evolutionary variation (C) or experimental sensitivity to mutation (D) of each position.

Figure 3. Structural distribution of experimentally sensitive and tolerant positions

(A&B) Structural representations of the Hsp90 ATPase domain illustrating the location of the eight amino acids that are experimentally sensitive to any amino acid change (indicated with cyan spheres), and the 55 positions that experimentally tolerate any amino acid change (indicated with yellow spheres). Structural representations are shown for the ADP bound state from 1AM1.PDB (Prodromou et al., 1997) (A) and the ATP bound state from 2CG9.PDB (Ali et al., 2006) (B).(C) Sensitivity to mutations correlates with proximity to atoms in ATP. The atoms of ATP are colored based on the observed correlation between experimental fitness effects and distance to the Cα atoms of each amino acid residue. Distance to the γ-phosphate of ATP exhibits the strongest correlation and explains more than half of the observed variance in mutational sensitivity. See also Figure S2, S3, and S4.

Figure 4. Contacts between D79 and adenine can be altered without compromising Hsp90 function

(A) Structural representation of the N-domain of Hsp90 highlighting the near ideal geometry of the hydrogen bond formed between N6 of adenine and the side chain of D79. (B) Growth rate of budding yeast in monoculture for D79E, D79C, and D79N Hsp90 variants. (C) ATPase rates observed for purified Hsp90 proteins (2.5 μM) in the presence and absence of the co-chaperone Aha1 (10 μM). (D) Ability of D79E, D79C and D79N Hsp90 to mature the GR client in yeast. (E) Efficiency of v-src maturation supported by D79E, D79C, and D79N Hsp90 variants. The black vertical line indicates where lanes were removed for figure clarity. D79N along with appropriate controls were analyzed on a separate blot that is shown on the right side of this panel. See also Figure S5.

Figure 5. Identification of Hsp90 mutants that differentially impact the maturation of GR and v-src

(A) Structural representation of the N-domain of Hsp90 indicating mutations identified as having a strong potential for separation of function. (B) The effects of individually cloned Hsp90 variants on activation of GR and v-src compared to effects on yeast growth rate. (C) Comparison of the effects of Hsp90 mutants on activation efficiency for GR versus v-src identifies eight mutations with severe deficiency for v-src that are capable of mediating the efficient activation of GR. (D) Structural representation of the N-domain of Hsp90 and the Cdc37 co-chaperone based on 1US7.PDB (Roe et al., 2004) indicating the location of the sites of mutations that caused client-specific effects. See also Figure S6.

Figure 6. Distinct impact of Hsp90 mutations on maturation of v-src and Ste11 kinases

(A) Comparison of the effects of Hsp90 mutants on activation efficiency for Ste11 versus v-src. Hsp90 mutations with severe defects for v-src activation exhibited impacts on Ste11 activation that ranged from severe defects (colored pink) to intermediate defects (colored orange) to no observable defect (colored blue). (B) The locations of mutations with severe defects for v-src activation are indicated on the structure of Hsp90 bound to Cdc37. The impacts of these mutations on Ste11 activation are indicated by color as in panel A. The location where nucleotide binds is indicated in green.