Hsp90 is regulated by a switch point in the C-terminal domain

Abstract

Heat shock protein 90 (Hsp90) is an abundant, dimeric ATP-dependent molecular chaperone, and ATPase activity is essential for its in vivo functions. S-nitrosylation of a residue located in the carboxy-terminal domain has been shown to affect Hsp90 activity in vivo. To understand how variation of a specific amino acid far away from the amino-terminal ATP-binding site regulates Hsp90 functions, we mutated the corresponding residue and analysed yeast and human Hsp90 variants both in vivo and in vitro. Here, we show that this residue is a conserved, strong regulator of Hsp90 functions, including ATP hydrolysis and chaperone activity. Unexpectedly, the variants alter both the C-terminal and N-terminal association properties of Hsp90, and shift its conformational equilibrium within the ATPase cycle. Thus, S-nitrosylation of this residue allows the fast and efficient fine regulation of Hsp90.

Figure 1

Sequence alignment of Heat shock protein 90 homologues and structure of the Hsp90 carboxy-terminal domain. (A) Schematic domain organization of yeast Heat shock protein 90 (yHsp90). Ala 577 is shown in red. (B–D) Alignment of Hsp90 family members. (B) For these Hsp90 proteins, a proteomic-based analysis revealed cysteine modification of Hsp90 by nitric oxide (Martinez-Ruiz et al, 2005; Jorge et al, 2007, Rhee et al, 2005; Lindermayr et al, 2005). In human Hsp90α (hHsp90α), Cys 597 was found to be S-nitrosylated. (C) Alignment of representatives of Hsp90 showing a conserved cysteine residue with the respective position highlighted in red. (D) Hsp90 family members lacking the conserved cysteine residue. (E) Surface presentation of yHsp90 in the closed conformation (PDB code: 2CG9). The position marked using a square indicates Ala 577, which corresponds to Cys 597 in hHsp90α. The enlargement (right) shows Ala 577 in red and hydrogen bonds as black dotted lines.

Figure 2

Carboxy-terminal modifications alter Heat shock protein 90 ATPase activity. (A) Steady state k_cat values of wild-type yeast Heat shock protein 90 (yHSP90) and yHsp90A577X mutants. (B) yHsp90 was treated with NO in vitro as described. The efficiency for S-nitrosylation of the yHsp90A577C mutant was 73%, as determined using an Ellman assay. (C–F) Stimulating effect of the Hsp90 co-chaperone Aha1 on the ATPase activity of Hsp90 variants. (C) Steady state k_cat values of wild-type yHsp90 (open circles), yHsp90A577C (open squares), yHsp90A577C modified with NO (filled squares) and yHsp90A577N (open triangles) using 1 μM of yHsp90 and increasing amounts of Aha1. Data were fitted according to equation (1); see supplementary information online. (D–F) Steady state k_catmax values at Aha1 saturation for variants of yHsp90 (D), human HSP90β (hHsp90β) (E) and hHsp90α (F). The inset in (E) represents the stimulation of wild-type hHsp90β (open circles), wild-type hHsp90β modified with NO (filled circles) and hHsp90C590A (open triangles). The inset in (F) represents stimulation of wild-type hHsp90α (open circles), wild-type hHsp90α modified with NO (filled circles) and hHsp90αC598A (open triangles). Experiments in (C–F) were measured at 37°C in 40 mM HEPES, pH 7.5, and 20 mM KCl. NO, nitric oxide; wt, wild type.

Figure 3

Effects of Heat shock protein 90 mutations on carboxy- and amino-terminal dimerization determined using FRET. (A) Subunit exchange of different yeast Heat shock protein 90 (yHsp90) dimers. The decrease in the fluorescein signal was monitored (λ_ex=496 nm; λ_em=520 nm) after mixing 100 nM FITC-labelled yHsp90 with 100 nM TAMRA-labelled yHsp90. The data were corrected for linear bleaching. Wild-type yHsp90, blue; yHsp90A577I, orange; and yHsp90A577N, green. ATPase activities for mutants are given in supplementary Table S2 online. (B) Amino-terminal dimerization of the preformed FRET pairs of yHsp90 variants after the addition of 2 mM ATPγS, monitored by following the decrease in the fluorescein signal as in (A). The colour code is the same as in (A). FITC, fluorescein isothiocyanate; FRET, fluorescence resonance energy transfer; TAMRA, tetramethyl-6-carboxyrhodamine; wt, wild-type.

Figure 4

Effect of Heat shock protein 90 nitric oxide modification and Hsp90 mutations on reverse transcriptase activation. (A–D) Priming competent RT–D-ɛ-RNA complexes were reconstituted as described in the supplementary information online. The ³²P-labelled RT was visualized by autoradiography, as shown in the Fig 4A inset. (A) Densitometric analysis of the effects of yeast Heat shock protein 90 (yHsp90) wild-type and mutants on the formation of the priming-active RT–D-ɛ-RNA complex. (B) Influence of NO on RT–D-ɛ-RNA complex formation. (C–D) Effects of human HSP90β (hHSP90β) variants (C) and hHsp90α variants (D) on RT–D-ɛ-RNA complex formation and the influence of NO on hHsp90 activity. Experiments were conducted as in (A) and (B). All experiments were carried out in triplicates; error bars indicate s.d. NO, nitric oxide; RT, reverse transcriptase; wt, wild-type.

Figure 5

In vivo chaperone effect of Heat shock protein 90 mutants. (A) A yeast HSC/HSP82^−/− deletion strain was transfected with HSP82 wild-type (wt) or single point mutants and a plasmid containing V-SRC. The phosphorylation activity of v-Src was analysed by Western blotting using a phosphotyrosine antibody (4G10), as shown in Fig 5A inset (see also supplementary Fig S6B online). The average of three independent experiments was analysed using densitometry. (B) The yeast deletion strain was transfected with human Heat shock protein 90β (hHSP90β) and hHSP90α wild-type or the alanine mutations, co-transfected with V-SRC and analysed as in (A). The error bars indicate s.d.