The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function

Abstract

The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90's chaperone cycle. A unique linker, called charged linker (CL), connects the N- and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 kBT (2.7 kJ/mol) through binding of the CL to the N-domain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90's chaperone cycle governing client activation, cell viability, and stress tolerance.

Keywords: FRET; Hsp90 conformation; asymmetric state; optical tweezers; single molecule.

Fig. 1.

Unfolding of Hsp90 by optical tweezers shows a structured CL. (A) Schematics of the experimental setup (not to scale). Monomeric Hsp90 was clamped between two glass beads (spheres), which were trapped by focused laser beams to apply and measure forces. DNA (chain) and ubiquitins (circles) serve as handles to manipulate Hsp90 in the center of the optical tweezers setup. (B) A typical force–extension trace at constant velocity (here, 10 nm/s) shows the successive unfolding of Hsp90’s three domains as large peaks. The contour length increase obtained from WLC (worm-like chain) fits (dashed lines) for each domain matches the number of amino acids within the domain (average values are given between WLC fits). The first peak (violet to green) accounts for the unfolding of the C-domain, the second peak (green to blue) for the N-domain, and the last (blue to orange) for the M-domain. The colored trace shows filtered data; gray is the full-resolution data at 20 kHz. (Inset, violet) Magnified view of the region before any of the domains unfold (arrow). Rapid fluctuations between two states were observed, which are caused by docking and undocking of the CL.

Fig. 2.

The CL interacts with the N-domain in a sequence-dependent manner. (A) Close-up view of the force–extension trace of the WT protein showing the two-state behavior of the CL (flipping between the two dashed WLC fits). (B) Substitution of amino acids 211–263 (Upper) and amino acids 211–272 (Lower) by GGS repeats. No docked state is observed, indicating that the N–M interface is not stabilized anymore. (C) N-domain deletion prevents the docked state (Upper), whereas M- and C-domain deletion shows a two-state behavior similar to the WT protein. All Hsp90 mutants show similar length increases and domain stabilities like WT (SI Appendix, Tables S1 and S2).

Fig. 3.

Undocking–docking kinetics and energetics of the CL. (A) Equilibrium transitions between the docked (high force, blue) and the undocked (low force, red) state of the CL at two different average forces (pretensions). As expected the pretension changes the kinetics and the population of the two states. In optical tweezers experiments the CL was elongated (stretched) in the undocked state by up to 4 nm (deflection). (B) The force-dependent probability to be in the docked state (blue) or undocked state (red). A global fit (SI Appendix, Methods) allows extrapolation to zero force. Averaging all measured molecules (n = 34) resulted in a 75% probability of being in the docked and a 25% probability of being in the undocked state at zero force. (C) The force-dependent rate constants were fitted (SI Appendix, Methods) and yielded an undocking rate constant of 75 s⁻¹ and a docking rate constant of 173 s⁻¹. All data shown in this panel represent an example experiment from the exact same molecule.

Fig. 4.

N-terminal dimerization is modulated by the CL. (A) Histograms of FRET efficiencies in the absence of nucleotide (Left, apo) and in the presence of ATP (Right) for WT (black), GGS substitutions from amino acids 211–263 (blue), and amino acids 211–272 (orange). Each histogram contains a minimum of 923 (up to 5,565) FRET events. (B) Cross-linking experiments using the cysteine-specific cross-linker BM(PEG)₃ with homodimers of WT, Sub211-272, and Sub211-263. First lane is without cross-linking agent yielding monomer bands. Other lanes are with cross-linking agent (X-Linker) in apo (second lane), AMP-PNP (third lane), and ATPγS (forth lane) condition, respectively, yielding monomer and dimer bands. (C) ATPase activity of the mutants at 30 °C, error bars represent SD. (D) Different concentrations of the cochaperone Aha1 were added to the mutants, and the ATPase activity was determined. The figure shows the stimulation of the Hsp90 ATPase activity compared with the value obtained in the absence of Aha1. (E) The binding of the different Hsp90 constructs to Aha1 was analyzed by analytical ultracentrifugation. dc/dt plots are shown for Aha1-FAM (magenta) and the mixtures of Aha1-FAM with the indicated Hsp90 constructs in the absence of nucleotide. (F) Analytical ultracentrifugation experiments for the interaction between Atto488-labeled p23/Sba1 and the Hsp90 mutants in the presence of AMP-PNP.

Fig. 5.

CL conformations in Hsp90’s chaperone cycle. The Hsp90 cycle is driven by large and slow conformational changes, leading from the open to the closed state. In addition, the CL can rapidly adopt a docked or an undocked state. The docked conformation fixes the N-domain to the M-domain (e.g., Open 1a and Closed 1a), whereas the undocked conformation provides some flexibility, but still close proximity of the domains (e.g., Open 1b and Closed 1b). In principle, both CLs can undock at the same time, but we have omitted this unlikely case (6%) for clarity. Undocking of the CL is not restricted to the open conformation (Open 1b), thus leading to a previously unknown state of Hsp90, namely Closed 1b. In the Closed 2 state (Protein Data Bank ID 2CG9), the CL may adopt an additional conformation, which has not been measured directly (dashed box). Here a segment of the CL (amino acids 263–272) might be important for the integrity of the Closed 2 state, which is essential for proper biological function.