Cooperative Nucleotide Binding in Hsp90 and Its Regulation by Aha1

Abstract

The function of the molecular chaperone Hsp90 depends on large conformational changes, the rearrangement of local motifs, and the binding and hydrolysis of ATP. The size and complexity of the Hsp90 system impedes the detailed investigation of their interplay using standard methods. To overcome this limitation, we developed a three-color single-molecule FRET assay to study the interaction of Hsp90 with a fluorescently labeled reporter nucleotide in detail. It allows us to directly observe the cooperativity between the two nucleotide binding pockets in the protein dimer. Furthermore, our approach disentangles the protein conformation and the nucleotide binding state of Hsp90 and extracts the kinetics of the state transitions. Thereby, we can identify the kinetic causes mediating the cooperativity. We find that the presence of the first nucleotide prolongs the binding of the second nucleotide to Hsp90. In addition, we observe changes in the kinetics for both the open and the closed conformation of Hsp90 in dependence on the number of occupied nucleotide binding sites. Our analysis also reveals how the co-chaperone Aha1, known to accelerate Hsp90's ATPase activity, affects those transitions in a nucleotide-dependent and independent manner, thereby adding another layer of regulation to Hsp90.

Figure 1

Data acquisition, data analysis, and state allocation. (a) Pictogram of the studied system consisting of an Hsp90 dimer with the labels Atto488 (blue) and Atto550 (green) attached and the reporter nucleotide AMP-PNP^∗ in solution, labeled with Atto647N (red). The protein is immobilized by NeutrAvidin/biotin interaction on the top of the flow chamber and excited by an evanescent field using alternating laser excitation with a blue and a green laser in a prism-type total internal reflection fluorescence geometry. The fluorescence light is collected by an objective, separated by dichroic mirrors, and detected with electron-multiplying charge-coupled device cameras. (b) Fluorescence intensity traces of a single particle after the excitation of the blue (top) and the green (center) dye measured in the absence of additional unlabeled nucleotide or co-chaperone. The partial fluorescence traces ($P{F}_{\text{em}}^{\text{ex}}$, with excitation of the ex dye and emission of the em dye) calculated from the intensity traces are shown below. (c) Pictograms of the distinguishable states and the respective identifiers used in this work. The first two populations represent the same functional state, namely open Hsp90 with nucleotide bound. (d) 3D representation of the Gaussians (isosurface at FWHM) fitted to the partial fluorescence data, which represent the five different populations. The same color code as in (c) is used. (e) The resulting state allocation for the fluorescence intensity traces is shown in (b).

Figure 2

The average dwell time of the reporter nucleotide AMP-PNP^∗ bound to Hsp90 is prolonged by additional nucleotide. (a) Pictogram of the observed dissociation of labeled AMP-PNP^∗ (PNP) from the Hsp90 dimer. (b) Average dwell time of AMP-PNP^∗ bound to Hsp90 in the absence of additional nucleotide (PNP^∗) and in the presence of 250 μM unlabeled ATP or unlabeled AMP-PNP. The underlying dwell time distributions are shown in Fig. S9. Error bars represent the SD estimated from jackknife resampling. Differences between the dwell time distributions are significant with ^∗∗∗p < 0.001. To see this figure in color, go online.

Figure 3

The effects of nucleotide on Hsp90’s conformation and its state transitions. (a) Populations of open and closed conformation for Hsp90 bound to AMP-PNP^∗ (normalized to unity) in the absence of additional nucleotide (PNP^∗) and in the presence of 250 μM ATP or AMP-PNP. Error bars represent the SD within 10 subsets, each comprising 75% of the full dataset. The addition of nucleotide results in a significant population shift with ^∗∗∗p < 0.001. (b) Transition rates for Hsp90 bound to labeled AMP-PNP^∗ in dependence on additional nucleotide. Error bars represent the 99% CI; differences with ^∗∗p < 0.01 (CIs do not overlap) are highlighted. To see this figure in color, go online.

Figure 4

The effects of Aha1 (10 μM), ATP (250 μM), and Aha1 combined with ATP on Hsp90 and AMP-PNP^∗. (a) The mean dwell time of AMP-PNP^∗ on Hsp90 is significantly (^∗∗∗p < 0.001) increased by Aha1. The effect observed for Aha1+ATP is smaller than for Aha1 or ATP alone. Error bars are calculated as in Fig. 2. (b) Effects on the normalized population of open and closed conformation for Hsp90 bound to AMP-PNP^∗. Error bars are calculated as in Fig. 3a. (c) Transition rates and the effects of nucleotide, co-chaperone, and their combination. Errors bars represent the 99% CI; differences with ^∗∗p < 0.01 are highlighted. To see this figure in color, go online.

Figure 5

The effects of ATP and Aha1 on the state transitions of Hsp90 and their possible structural origins. (a) A minimal model for the state transitions of Hsp90 in the presence of AMP-PNP^∗ and the effects of ATP and Aha1 on these transitions. Only the most frequent transitions are shown. ATP increases the closing rate of Hsp90 with AMP-PNP^∗ bound, and decelerates the reverse reaction, as well as the dissociation of AMP-PNP^∗. Aha1 also accelerates the closing of AMP-PNP^∗-bound Hsp90. In a combination of Aha1 and ATP, their effects on the closing of AMP-PNP^∗-bound Hsp90 add up, whereas Aha1 prevents the decelerating effects of ATP. (b) Our observations could be caused by the depicted local rearrangements, which have been proposed to be affected by Aha1 and nucleotide binding. (Left) Reported rearrangement of the catalytic loop upon binding of Aha1 (green and dark green, superposition of the crystal structures PDB: 2CG9 (35) and 1USV (10)). (Right) Reported conformation of the nucleotide lid in the AMP-PNP or the ADP-bound crystal structures (dark and light blue, superposition of the crystal structures PDB: 2CG9 and 2WEP (36)).