Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23

Abstract

The molecular chaperone Hsp90 binds and hydrolyses ATP, but how this ATPase activity regulates the interaction of Hsp90 with a polypeptide substrate is not yet understood. Using the glucocorticoid receptor ligand binding domain as a substrate, we show that dissociation of Hsp90 from bound polypeptide depends on the Hsp90 ATPase and is blocked by geldanamycin, a specific ATPase inhibitor. The co-chaperone p23 greatly stimulates Hsp90 substrate release with ATP, but not with the non-hydrolysable nucleotides ATPgammaS or AMP-PNP. Point mutants of Hsp90 with progressively lower ATPase rates are progressively slower in ATP-dependent substrate release but are still regulated by p23. In contrast, ATPase-inactive Hsp90 mutants release substrate poorly and show no p23 effect. These results outline an ATP-driven cycle of substrate binding and release for Hsp90 which differs from that of other ATP-driven chaperones. Conversion of the ATP state of Hsp90 to the ADP state through hydrolysis is required for efficient release of substrate polypeptide. p23 couples the ATPase activity to polypeptide dissociation and thus can function as a substrate release factor for Hsp90.

Fig. 1. ATP-dependent release of Hsp90 bound to substrate. (A) Coomassie stained SDS–PAGE of LBD–chaperone complexes. Lane 1, total RL; lanes 2–3, total immunoprecipitation (T) from RL without (lane 2) and with (lane 3) added LBD; lanes 4–7, isolated LBD complexes were eluted for 10 min with buffer alone (lanes 4–5) or containing 1 mM ATP (lanes 6–7) and supernatants (S) separated from the pellets (P). Ovalbumin (Ova) was added to the supernatant as a recovery control. LC, IgG light chain. Molecular weight standards are marked on the left. Right, lanes 2 and 3 immunoblotted for Hsp90, Hsc70 and Hop. (B) LBD was incubated with RL, ATP and [³H]triamcinolone acetonide with or without 90 µM GA, followed by gel filtration. Radioactivity in each fraction is plotted and the elution volumes of the void (V₀) and LBD monomer are marked. (C) Radiolabelled Hsp90 was eluted from LBD complexes for 10 min with buffer alone or containing 1 mM ATP or 500 mM NaCl (HS). A representative phosphoimager scan (top) and Coomassie staining of LBD (middle) are shown. Bottom, the amount of Hsp90 released as a percentage of total Hsp90 in the LBD complex. Error bars in all figures show standard deviations from the mean of at least three experiments.

Fig. 2. Hsp90 remains bound to substrate after disruption of the Hop–Hsc70 linkage. (A) Lanes 1–2, Coomassie staining of TPR2A (lane 1) and TPR1 (lane 2). Lanes 3–6, binding reactions containing 2 µM Hop and 2 µM GST–C90 (lanes 3–4) or GST–C70 (lanes 5–6) were recovered with glutathione–agarose after incubation without (lanes 3 and 5) or with a 25-fold excess of TPR2A (lane 4) or TPR1 (lane 6) and detected by Coomassie staining. (B) Radiolabelled Hsc70 from RL was co-precipitated with Hsp90 and eluted from immune pellets with buffer alone or containing 20 µM TPR1, 20 µM TPR2A or 500 mM NaCl (HS). Top, phosphoimager scan. Inset, schematic diagram of the experiment showing the interactions disrupted by TPR1 (open arrowhead) and TPR2A (closed arrowhead). (C) Radiolabelled Hsp90 was eluted from LBD complexes with buffer alone or containing 20 µM TPR1, 20 µM TPR2A or 500 mM NaCl (HS). Top, phosphoimager scan. Inset, schematic diagram as in (B).

Fig. 3. The nucleotide state of Hsp90 regulates substrate binding. (A) Radiolabelled Hsp90 was eluted from LBD complexes with buffer alone or buffer containing the indicated nucleotide (1 mM), either without or with 90 µM GA. Top, phosphoimager scan. (B) The experiment in (A) was repeated but with 20 µM TPR2A added to the elution buffers. (C) The elution of radiolabelled Hsc70 was tested similarly to (A). (D) The elution of Hsp90 from LBD complexes with 1 mM ATP, ATPγS, AMP-PNP or with buffer alone was monitored over time.

Fig. 4. p23 is an ATP-dependent substrate release factor for Hsp90. (A) Radiolabelled Hsp90 was eluted from LBD complexes with buffer alone or buffer containing the indicated nucleotide (1 mM) with 1 mM phosphate (P_i) where marked. Elutions were either without or with 5 µM p23. (B) The elution of Hsp90 with RL and 1 mM ATP, or with the indicated nucleotide (1 mM) with or without 5 µM p23 was monitored over time. Curves shown in dashed lines are taken from Figure 3. (C) Hsp90 was eluted with 1 mM ATP and varying concentrations of p23. (D) Hsp90 was eluted with or without 1 mM ATP in the presence of full-length (FL) p23 or the p23 mutants 1–128 and 1–120 having the C-terminal 32 and 40 amino acids, respectively, deleted.

Fig. 5. Effect of p23 and point mutations on the Hsp90 ATPase. (A) Purified Hsp90 (5 µM) was incubated at 30°C in 2 mM ATP containing [α-³²P]ATP either without or with 10 µM p23. ADP production over time was measured and plotted after subtraction of the low GA-inhibited background. (B) ATPase rates of Hsp90 were determined as in (A) at different concentrations of ATP, either without or with 10 µM p23. The rates are plotted against ATP concentration and fitted to the Michaelis–Menten equation v = V_max/(1 + K_M/[ATP]). (C) ATPase rates in 2 mM ATP of wild-type Hsp90 (WT) or the indicated point mutants were measured at 30 or 37°C.

Fig. 6. Substrate release of Hsp90 is linked to the ATP hydrolysis rate. (A) LBD complexes containing radiolabelled WT or mutant Hsp90 were eluted with buffer alone or buffer containing 1 mM ATP, 1 mM ATP and 5 µM p23, or 500 mM NaCl (HS). (B) Radiolabelled WT or mutant Hsp90 translated in RL was co-precipitated with antibodies against p23 either with or without 5 mM ATPγS. Top, input Hsp90. (C) The elution from LBD complexes of WT Hsp90 and the indicated point mutants with 5 mM ATP and 5 µM p23 was monitored over time.

Fig. 7. The ATP-driven substrate binding cycle of Hsp90. (1) Substrate protein (S) is stably bound by the nucleotide-free state of Hsp90. (2) Binding of ATP onto Hsp90 results in slow release of substrate. (3) Conversion of Hsp90 to the ADP state through ATP hydrolysis produces fast and complete release of substrate, enhanced by p23. (4) Substrate may be loaded onto Hsp90 at the nucleotide-free state via Hsc70 and Hop (Prodromou et al., 1999).