Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence

Abstract

The abundant molecular chaperone Hsp90 is a key regulator of protein structure in the cytosol of eukaryotic cells. Although under physiological conditions a specific subset of proteins is substrate for Hsp90, under stress conditions Hsp90 seems to perform more general functions. However, the underlying mechanism of Hsp90 remained enigmatic. Here, we analyzed the function of conserved Hsp90 domains. We show that Hsp90 possesses two chaperone sites located in the N- and C-terminal fragments, respectively. The C-terminal fragment binds to partially folded proteins in an ATP-independent way potentially regulated by cochaperones. The N-terminal domain contains a peptide binding site that seems to bind preferentially peptides longer than 10 amino acids. Peptide dissociation is induced by ATP binding. Furthermore, the antitumor drug geldanamycin both inhibits the weak ATPase of Hsp90 and stimulates peptide release. We propose that the existence of two functionally different chaperone sites together with a substrate-selecting set of cochaperones allows Hsp90 to guide the folding of a subset of target proteins and, at the same time, to exhibit general chaperone functions.

Figure 1

Influence of Hsp90 on the aggregation of insulin B-chain. Insulin aggregation (45 μM) was monitored in the absence (•) or presence of Hsp90 fragments or the wild-type proteins from yeast, humans, and E. coli. Hsp90 or domains of Hsp90 exhibited no detectable influence on the turbidity of the solution. (A) Influence of increasing concentrations of N210 on insulin aggregation: 4.5 μM N210 (▴), 9 μM N210 (□), 22.5 μM N210 (○), 45 μM N210 (▵), and 225 μM N210 (▾). (B) Influence of increasing concentrations of 262C on insulin aggregation: 4.5 μM 262C (▪), 9 μM 262C (□), 22.5 μM 262C (▴), and 45 μM 262C (▾). (C) Influence of increasing concentrations of yeast Hsp90, human Hsp90, or E. coli Hsp90 on insulin aggregation; yeast Hsp90: 2 μM (▾), 5 μM (▿), 18 μM (□), 45 μM (⧫), E. coli Hsp90 18 μM (▴), and human Hsp90 18 μM (▵).

Figure 2

ATP dependence of the interaction between Hsp90 and nonnative protein. The effects of Hsp90 and fragments thereof (45 μM each) on insulin B-chain aggregation (45 μM) were monitored in the absence (•) and presence of 1 mM of nucleotides or 100 μM GA (see below). (A) Influence of ATP (○) or GA (▿) on the chaperone function of 262C (▾). (B) Influence of ATP (▿), ADP (◊), or GA (▪) on the chaperone function of N210 (▵). (C) Influence of N210 (▵) on insulin aggregation and the effect of ATP (▴) or GA (□) on preformed N210⋅insulin complexes. The arrow indicates the time of addition of ATP or GA. (D) Influence of N210 (▵) on insulin aggregation and the effect of ATP (▴) on preformed N210⋅insulin complexes in a long time kinetic. The arrow indicates the time of addition of ATP. (E) Influence of ATP (▿), ADP (▵), or GA (□) on the chaperone function of yeast Hsp90 (18 μM) (▴). Addition of ATP to preformed Hsp90⋅insulin⋅complexes (⧫) is indicated by an arrow. (F) Influence of 262C (▾), N210 (▵), and 262C+N210 without ATP (◊) and with ATP (□) on insulin aggregation. ATP addition to a preformed N210⋅insulin complex is indicated by an arrow.

Figure 3

Substrate specificity of the Hsp90 chaperone sites. The effects of Hsp90 and fragments thereof (45 μM each) on insulin aggregation (45 μM) were monitored in the presence of various peptides. (A) Influence of GR1 peptide (45 μM) on insulin binding to N210. Aggregation of insulin in the absence (•) and in the presence of N210 (▵), N210 + GR1 (▴), and addition of GR1 (indicated by the arrow) to a preformed N210 insulin complex (○). (B) Influence of GR1 peptide on insulin binding to 262C. Aggregation of insulin in the absence of Hsp90 (•), in the presence of 262C (▾), 262C + 45 μM GR1 (□), and 262C + 225 μM GR1 (▪) and addition of GR1 to a preformed 262C⋅insulin complex (▵). (C) Influence of various peptides on insulin binding to N210. Aggregation of insulin in the absence of Hsp90 (•) and in the presence of N210 (▵), N210 + GR1 (▿), N210 + VSV8 (▪), N210 + HD 131 (○), and N210 + HD25 (⧫). (D) Comparison of the influence of peptide on the chaperone activity of wild-type Hsp90 and fragments thereof. Aggregation of insulin in the absence of Hsp90 (•) and in the presence of Hsp90 + GR1 (▪), 262C + GR1 (○), and N210 + GR1 (□). Concentrations of the fragments were 45 μM, and concentration of wild-type Hsp90 was 18 μM (based on the respective monomers). GR1 was added at a 1:1 ratio to insulin.

Figure 4

Influence of Hsp90 fragments on the aggregation of CS. (A) Aggregation of chemically denatured CS (150 nM) in the absence (•) or in the presence of 3 μM N210 (▵) and 3 μM N210 + 100 μM GA (▿). (B) Aggregation of chemically denatured CS (150 nM) in the absence (•) or in the presence of BSA (1.5 μM) (○), 1.5 μM 262C (▪), and 1.5 μM 262C + 100 μM GA (□). (C) Aggregation of thermally denatured CS (150 nM) in the absence (•) or in the presence of 150 nM N210 (▵) or 3 μM N210 (▪). (D) Aggregation of thermally denatured CS (150 nM) in the absence (•) or in the presence of increasing amounts of 262C: 150 nM (▿), 750 nM (○), 1.5 μM (⧫), and 3 μM (□).

Figure 5

Model for the ATP-regulated chaperone activity of Hsp90. The model describes the substrate binding cycle of the N-terminal domain of Hsp90. In the absence of ATP or in the presence of ADP, N210 has a high affinity binding site for substrate. Addition of nonnative proteins results in efficient complex formation. Functional differences between the ADP form and the nucleotide-free form could not be detected so far (1). Binding of ATP to the nucleotide-free or ADP-bound complexes between N210 and nonnative protein results in a conformational change that decreases the affinity of N210 for nonnative proteins (2). This complex seems to be short-lived (indicated by brackets), and, as a consequence, immediate dissociation of the substrate is observed (3). Alternatively, GA can bind to N210 and, similar to ATP, promote the release of bound substrates. To restore the high affinity binding site, ATP can either be released (4) or hydrolyzed (5). It remains to be seen which of the two mechanisms for reset to the high affinity state is used in vivo. S, substrate.