The charged region of Hsp90 modulates the function of the N-terminal domain

Abstract

Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90.

Figure 1

Influence of Hsp90 fragments on the aggregation of insulin B-chain. Insulin aggregation (50 μM) was monitored in the absence (●) or presence of Hsp90. Hsp90 fragments or wild-type Hsp90 exhibited no detectable influence on the turbidity of the solution. All measurements with N210, 262C, and yeast Hsp90 reflect experiments repeated as in ref. . (A) Influence of increasing concentrations of N210 on insulin aggregation: 5 μM N210 (▴), 10 μM N210 (□), 25 μM N210 (○), 50 μM N210 (▵), and 250 μM N210 (▾). (B) Influence of increasing concentrations of N272 on insulin aggregation: 2 μM N272 (□), 3 μM N272 (▴), 6 μM N272 (⋄), 12.5 μM N272 (■), 25 μM N272 (▿), 50 μM N272 (♦), and 100 μM N272 (▵). (C) Comparison of different Hsp90 fragments and wild-type Hsp90 (6 μM each). Influence of yeast Hsp90 (⋄), N210 (▴), 262C (□), N272 (■), and a coincubation of N272 and 262C (▵). (D) The effects of N272 (5 μM) on insulin aggregation were monitored in the absence (▿) or presence (□) of the peptide GR1 (50 μM). Addition of GR1 (50 μM) to a preformed N272⋅insulin complex showed no detectable influence on the aggregation kinetics (■). All concentrations are calculated for the respective monomers.

Figure 2

Titration of N210 with ADP as detected by calorimetry. (Upper) Raw data obtained for 32 automatic injections (4 μl each) of an ADP solution (16.9 mM) into the sample cell containing N210 at 180 μM in 10 mM potassium phosphate, pH 6.9/100 mM sodium chloride/30 mM MgCl₂. (Lower) Plot of processed data. The solid line corresponds to the best-fit curve obtained by least-squares deconvolution. The best values for fitting parameters are 1.03 for n (number of sites), 25,000 M⁻¹ for K (binding constant), and −17,100 cal/mol for ΔH. The standard deviation of points from the calculated line is 0.069% of the total integral heat for saturating all sites in the N210 sample. ATP hydrolysis did not interfere with the calorimetric experiments, since the turnover number of both N210 and N272 is very low under the conditions used (cf. ref. ; additional data not shown).

Figure 3

ATP dependence of the interaction between N272 and insulin. The effects of N272 (10 μM) on the aggregation of insulin B-chain (50 μM) (●) were monitored in the absence (○) and presence of 10 mM ATP and 20 mM MgCl₂ (■) or 150 μM GA (▵). Addition of MgATP/GA to a preformed N272⋅insulin complex showed no detectable effect in this assay (▴).

Figure 4

Model for the chaperone activity of N272. The model describes different functional states of the N-terminal peptide-binding site of Hsp90. In the absence of substrate, ATP, or GA, the fragment has a high affinity for these ligands [1]. The presence of ADP does not influence the affinity for the ligand (not shown). Binding of ATP or GA to N272 results in a conformational change [2] that decreases the affinity of N272 for substrates [3]. This is in accordance with the previously described substrate binding cycle of N210 (5). Binding of substrate is followed by a conformational switch [4], as the N272⋅substrate complex showed a decreased binding affinity for ATP/GA [5]. Furthermore, after the change in conformation, the bound substrate does not exchange with peptides free in solution [5]. The existence of the complex [5] is hypothetical. S and S′, substrates.