Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism

Abstract

The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

Figure 1. Observation of conformational motions in Hsp90 by PET fluorescence quenching.

(a) PET reporter design. Left: Structural model of apo Hsp90 based on crystallographic data of the NTD (pdb id 1AM1) and the MC-domain (pdb id 2CGE). NTD (N), charged linker (CL), M-domain (M), and C-domain (C) are indicated. The nucleotide-binding pocket is indicated by an orange arrow. Right: Crystal structure of full-length Hsp90 in closed-clamp conformation with bound AMP-PNP (pdb id 2CG9). N-terminal β-strand and lid are colored magenta and green, respectively. Engineered Oxa and Trp are shown as red spheres and blue sticks, respectively. Amino acid side chains that were mutated to alter function are highlighted in cyan. (b) Fluorescence intensity time traces of reporter A2C-E162W for β-strand swap (magenta) and the corresponding control A2C (gray). AMP-PNP was added at t = 0 min. The black line is a three-exponential fit to the data. (c) Fluorescence intensity time traces of reporter S51C-A110W for lid closure (green) fitted using a bi-exponential function, and of variant S51W-A110C (inset, green). Controls S51C and A110C are shown in gray. (d) Fluorescence intensity time traces of reporter E192C-N298W for N/M-association (cyan) fitted using a bi-exponential function. Control E192C is shown in gray. (e) ATPase activity of wild-type Hsp90 and mean rate constants of β-strand swap, lid closure, and N/M-domain association obtained from PET fluorescence experiments. Data represent mean values ± s.d. of three measurements.

Figure 2. Modulation of conformational motions by mutagenesis.

(a) Crystal structure of the NTD (pdb id 1AM1) showing the engineered TrpZip motif introduced through double mutation A2W-L160W (blue sticks). N-terminal β-strand and lid segment are highlighted magenta and green. (b) Time-dependent fluorescence intensities of reporter A2C-E162W (β-strand swap) and mutants thereof. Fluorescence time traces of wild-type (gray), TrpZip (red), mutants T101I (orange), R380A (blue), and A107N (green) are shown. Black lines are exponential fits to the data. AMP-PNP was added at t = 0 min. (c) Time-dependent fluorescence intensities of reporter S51C-A110W (lid) and mutants thereof. The color code of panel (b) applies. (d) Time-dependent fluorescence intensities of reporter E192C-N298W (N/M-domain association) and mutants thereof. The color code of panel (b) applies. (e) Effects of mutation on the rate constant of ATP hydrolysis by Hsp90 (gray) and on the mean rate constants of β-strand swap (magenta), lid closure (green), and N/M-domain association (cyan). ATPase activities of de-activating mutants (TrpZip, T101I, and R380A) were measured at 37° C and plotted as relative rate constants. Data represent mean values ± s.d. of three measurements. X = no kinetics detectable.

Figure 3. Influence of Aha1 on kinetics of local motions.

(a) AMP-PNP-triggered fluorescence intensity time traces of β-strand swap (A2C+E162W, magenta), lid closure (S51C-A110W, green), and N/M-domain association (E192C-N298W, cyan). Samples were incubated with Aha1 before measurement and time traces were recorded using stopped-flow spectroscopy. Data in shaded color are control samples that lacked the engineered Trp. Fluorescence transients were fitted using a bi-exponential model including a linear baseline drift of minor amplitude (black line). (b) Rate constants of ATP hydrolysis (gray) by wild-type Hsp90 (wt) and mutant F349A together with the corresponding mean rate constants of β-strand swap (magenta), lid closure (green), and N/M-domain association (cyan) measured in absence and presence of Aha1 (X = no kinetics detectable). Data represent mean values ± s.d. of three measurements. (c) Equilibrium fluorescence intensities measured from reporters of N/M-domain association and of the lid on wild-type Hsp90 and mutant F349A after incubation with Aha1.

Figure 4. Equilibrium dynamics of lid and β-strand probed by PET-FCS.

(a) Reporter design. N-terminal β-strand and lid of the NTD (pdb id 1AM1) are highlighted magenta and green. Engineered pairs of Oxa (red sphere) and Trp (blue sticks) probing β-strand (Q14C-A2W) and lid (A112C-S25W) are indicated. (b) and (c), ACFs (G(τ)) recorded from Q14C-A2W (magenta) and A112C-S25W (green), respectively, on the isolated NTD. Data recorded after binding of ATP are shown in orange. Control samples lacking the engineered Trp are shown in gray. Black lines are fits to the data using a model for molecular diffusion containing two single-exponential relaxations. The ACF of the TrpZip construct (Q14C-A2W-L60W) is shown in blue in panel (b), and was described by a molecular diffusion model without additional relaxations. All ACFs were normalized to the average number of molecules in the detection focus for clarity. Broken lines indicate the amplitudes of the diffusion decays. (d) and (e) ACFs of same reporters engineered on the NM-domain. Same color code as in panels (b) and (c) applies. Black lines are fits to the data using a model for molecular diffusion containing three single-exponential relaxations. ACFs recorded in the presence of Aha1 are shown in cyan. (f) Rate constants of β-strand release (magenta) and lid release (green) in NTD and NM-domain. Effects of binding of ATP and Aha1 are shown. Data represent mean values ± s.d. of three measurements.

Figure 5. Possible origins of multi-exponential kinetics in protein dynamics.

Two-dimensional projection of a conformational free energy surfaces along an arbitrary reaction coordinate. (a) Ground-state heterogeneity. Multiple open-clamp conformations (o₁-o₃) of different free energy give rise to energy barriers of different height (broken arrows) along parallel pathways to the closed-clamp conformation (c). (b) On-pathway intermediates. Conformational change along a pathway containing a series of discrete intermediates (i₁-i₃) of different free energy. (c) Pathway heterogeneity. Open-clamp conformers of same free energy traverse to the closed-clamp conformation along different pathways that are characterized by different energy barrier heights.

Figure 6. Integration of results into the chaperone catalytic cycle.

(1) At the beginning of the catalytic cycle, apo Hsp90 populates a heterogeneous ensemble of open-clamp conformers. Lid (green) and N-terminal β-strand (magenta) are highly mobile structural elements with sub-millisecond reconfiguration times. (2) Binding of ATP to the NTD leads to rapid release of the lid to an intermediate conformational state. The co-chaperone Aha1 pre-associates N- and M-domains but also remodels the lid segment for accelerated closure. (3) Closure of the molecular clamp involves cooperative action of conformational switches. Closure of the lid over the ATP-binding pocket, cross-subunit swap of β-strands, and association of the N- and M-domains are slow and interdependent. Swap of the terminal β-strands is weakly coupled with the other motions. (4) Hydrolysis of ATP leads to a compact, ADP-bound conformation, which relaxes to an open state with concomitant release of ADP and inorganic phosphate. Opening of the molecular clamp reconstitutes Hsp90 for the next catalytic cycle.