The molecular mechanism of Hsp100 chaperone inhibition by the prion curing agent guanidinium chloride

Abstract

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance. It is well established that low concentrations of guanidinium chloride (GdmCl) inhibit the ATPase activity of Hsp104, leading to so called "prion curing," the loss of prion-related phenotypes. Here, we present mechanistic details about the Hsp100 chaperone inhibition by GdmCl using the Hsp104 homolog ClpB from Thermus thermophilus. Initially, we demonstrate that NBD1 of ClpB, which was previously considered inactive as a separately expressed construct, is a fully active ATPase on its own. Next, we show that only NBD1, but not NBD2, is affected by GdmCl. We present a crystal structure of ClpB NBD1 in complex with GdmCl and ADP, showing that the Gdm(+) ion binds specifically to the active site of NBD1. A conserved essential glutamate residue is involved in this interaction. Additionally, Gdm(+) interacts directly with the nucleotide, thereby increasing the nucleotide binding affinity of NBD1. We propose that both the interference with the essential glutamate and the modulation of nucleotide binding properties in NBD1 is responsible for the GdmCl-specific inhibition of Hsp100 chaperones.

FIGURE 1.

The domain architecture of ClpB from T. thermophilus. The ClpB monomer (Protein Data Bank code 1QVR) comprises two nucleotide binding domains, NBD1 (green) and NBD2 (red). The darker colors indicate the helical bundles (also called small domains) of both NBDs. Bound nucleotides are shown as space-filling models. The long coiled coil M domain (yellow) is an insertion into NBD1. The ClpB constructs used in this study in addition to the full-length protein are NBD1-M(141–534) and NBD2(520–854), each containing only one NBD. The N-terminal domain (blue) is excluded for the separate constructs.

FIGURE 2.

Nucleotide binding parameters and ATPase activity of NBD1-M(141–534). A, kinetic fluorescence traces upon direct mixing of NBD1-M(141–534) and MANT-dADP. The final concentration of protein is 1 μm in all cases. The final MANT-dADP concentrations are 5 (blue), 8.3 (green), 12.5 (yellow), 18.75 (orange), and 25 μm (red). Single exponential fits are shown as colored lines (upper panel). The rate constants extracted from the kinetic traces are plotted against the MANT-dADP concentration to obtain the on-rate for MANT-dADP binding from the slope of the linear function (lower panel). The off-rate for MANT-dADP binding can be estimated from the y axis intercept but was determined separately by a dissociation experiment as described in the text. All nucleotide binding parameters obtained from these experiments are given in Table 2. B, fluorescence titrations to obtain K_D(ADP) and K_D(ATP). 2 μm NBD1-M(141–534) was incubated with 20 μm MANT-dADP and subsequently titrated with ADP (upper panel) or ATP (lower panel), respectively. For the ATP titration, phosphoenolpyruvate and pyruvate kinase were present as an ATP-regenerating system. The data were fitted with the cubic equation for competing ligands using K_D(MANT-dADP) obtained from the stopped flow experiments as an input value. All nucleotide binding parameters obtained from these experiments are given in Table 2. C, steady state ATPase turnover rates per molecule plotted against the ATP concentration (upper panel). The ATPase activity per molecule strongly depends on the protein concentration with [NBD1-M] = 5 (blue), 10 (green), 18 (yellow), 25 (orange), and 40 μm (red). The data were fitted with the Hill equation. The obtained Hill equation parameters k_cat, K_m, and the Hill coefficient are plotted against the protein concentration (lower panel). The low k_cat and high K_m values at low protein concentration and Hill coefficients significantly higher than 1.0 indicate that NBD1-M(141–534) oligomers represent the active form. a.u., arbitrary units.

FIGURE 3.

Nucleotide-dependent oligomerization of NBD1-M(141–534) in the presence and absence of GdmCl characterized by gel filtration and SLS. A, gel filtration profiles of NBD1-M(141–534) in nucleotide-free buffer (solid line) and with 2 mm ADP (dotted line) and 2 mm ATP (dashed line) present in the running buffer. The ATP-containing buffer was supplemented with phosphoenolpyruvate and pyruvate kinase as an ATP-regenerating system. In the presence of nucleotide, the peaks are broader and elute earlier, indicating a shift to higher molecular weight. Subsequent to the separation on the gel filtration column, a MALS detector was used to determine the average molecular mass of the eluted species, resulting in 44 (nucleotide-free), 58 (ADP), and 56 kDa (ATP). The actual molecular mass of the NBD1-M monomer is 45 kDa. B, the gel filtration runs were performed as described in A with 10 mm GdmCl present in the running buffer. The shift toward earlier elution in the presence of nucleotide is more pronounced than in A. The molecular masses determined by MALS data analysis are 44 (nucleotide-free) and 64 kDa (ADP). In the presence of ATP, two distinct masses were determined (86 and 48 kDa). The actual molecular mass of the NBD1-M monomer is 45 kDa. a.u., arbitrary units.

FIGURE 4.

Influence of GdmCl on the ATPase and disaggregation activity of ClpB. A, steady state (d)ATPase turnover rates of NBD1-M(141–534) wild type (green) and E209A (blue), NBD2(520–854) (red), and full-length ClpB (gray) in the presence of 0–15 mm GdmCl. Filled circles represent ATP hydrolysis data, and empty circles show dATP hydrolysis data (dATP lacks the 2′-OH group). ATP hydrolysis in NBD1, but not NBD2, is inhibited by low concentrations of GdmCl. NBD1-M(141–534) E209A is less active than the wild type; however, it is no longer affected by GdmCl. Hydrolysis of dATP is slower than for ATP; however, the inhibiting influence of GdmCl is decreased when the 2′-OH group is missing. From the hyperbolic fit of the (d)ATPase data, the K_D for GdmCl binding to NBD1-M(141–534) could be determined: K_D(GdmCl) = 2.3 ± 0.1 mm in the presence of ATP (green; filled) and K_D(GdmCl) = 8.3 ± 1.9 mm in the presence of dATP (green; empty). Fitting the ATPase data of full-length ClpB yielded K_D(GdmCl) = 2.0 ± 0.2 mm (gray). B, steady state ATPase turnover of NBD1-M(141–534) wild type for various concentrations of ATP in the presence of 15 mm GdmCl (green). Data fitting using the Hill equation yielded k_cat = 1.03 ± 0.03 min⁻¹, K_m = 0.30 ± 0.02 μm, and n = 1.9 ± 0.3. Steady state ATPase turnover of NBD1-M(141–534) E209A for various concentrations of ATP in the absence of GdmCl (blue) is also shown. Data fitting using the Hill equation yielded k_cat = 0.46 ± 0.01 min⁻¹, K_m = 0.94 ± 0.04 μm, and n = 2.9 ± 0.3. C, unfolding of NBD1-M(141–534) by GdmCl treatment. The change in molar ellipticity at 222 nm is plotted against the GdmCl concentration. The data were fitted assuming a two-state transition. The m value (m_d-n) is 3.63 ± 0.56 kJ mol⁻¹ m⁻¹. The obtained midpoint lies at [D]^50% = 3.57 ± 0.15 m, which is more than 200 times more GdmCl than used in the activity assays. D, chaperone-assisted reactivation of heat-aggregated α-glucosidase. The assay was performed as described under “Experimental Procedures.” The relative α-glucosidase activity (normalized against the positive control) is shown for different time points during the ClpB/DnaK/DnaJ/GrpE-assisted disaggregation reaction in the absence of GdmCl (red) and in the presence of 3 (orange), 6 (yellow), 9 (green), and 12 mm GdmCl (blue). Negative controls included only ClpB present (dark blue) and only DnaK/DnaJ/GrpE present (brown). E, the relative α-glucosidase activity (normalized against the positive control) at t = 120 min is plotted against the GdmCl concentration, showing that the disaggregation reaction is significantly impaired in the presence of GdmCl. The colors refer to the GdmCl concentrations in D. deg, degrees.

FIGURE 5.

The crystal structure of NBD1-M(141–534) in complex with ADP and GdmCl. A, the Gdm⁺ ion binds in the active site of ClpB NBD1, forming contacts with the side chain carboxyl group of glutamate 209 and the backbone carbonyl groups of aspartate 170 and proline 171. The Gdm⁺ ion interacts directly with the nucleotide via a hydrogen bond with the 2′-OH group of ADP. The F_o − F_c electron density map (contoured at 3σ) was obtained after initial phasing prior to modeling Gdm⁺ and ADP. The side chain of lysine 204, the catalytically essential Walker A residue, contacts the β-phosphate of ADP and is not involved in the Gdm⁺ interaction. Dotted lines refer to hydrogen bonds or ionic interactions. Distances are given in Å. B, structure alignment of ClpB NBD1 (green) and NBD2 (red) active sites. The P-loop regions, residues 195–215 of the NBD1-M structure (Protein Data Bank code 4HSE) and 592–612 of the published full-length structure (Protein Data Bank code 1QVR), respectively, were chosen for superposition. The equivalent residue to glutamate 209 in NBD1 is lysine 606, an amino acid of opposite charge, in NBD2. This together with steric restrictions explains the specificity of GdmCl binding to NBD1.

FIGURE 6.

Nucleotide binding behavior of NBD1-M(141–534) in the presence of GdmCl. A, MANT-ADP binding to NBD1. NBD1-M(141–534) and MANT-ADP were mixed directly in the presence (filled circles) or absence (empty circles) of 10 mm GdmCl. The fitted rate constants of the kinetic traces were plotted against the nucleotide concentration. Although the slope of the linear function (corresponding to the on-rate) is only slightly increased, the y axis intercept (corresponding to the off-rate) is significantly lower in the presence of GdmCl. The off-rates were determined separately by a dissociation experiment as described in the text, confirming a 4-fold increased binding affinity for MANT-ADP in the presence of GdmCl due to a slower off-rate. B, MANT-dADP binding to NBD1. The stopped flow experiments were performed as described in A using MANT-dADP, which lacks the 2′-OH group, instead of MANT-ADP. Nucleotide binding in the presence of 10 mm GdmCl (filled circles) is only slightly increased compared with the absence of GdmCl (empty circles), indicating that the 2′-OH group is essential for the GdmCl interaction. C, fluorescence titrations to obtain K_D(ADP) in the absence of GdmCl (empty circles; data from Fig. 2B) and in the presence of 10 mm GdmCl (filled circles). ADP binding is enhanced 6-fold in the presence of GdmCl. D, fluorescence titrations to obtain K_D(ATP) in the absence of GdmCl (empty circles; data from Fig. 2B) and in the presence of 10 mm GdmCl (filled circles). ATP binding is enhanced 12-fold in the presence of GdmCl. The measured fluorescence was normalized for direct comparison of different titrations in one graph. All nucleotide binding parameters obtained from the experiments shown in this figure are given in Table 2. a.u., arbitrary units.

FIGURE 7.

Energy diagram of a simplified ATPase reaction. The energy diagram schematically depicts an ATPase reaction consisting of three steps: substrate binding, hydrolysis, and product release. The energy barriers, ΔG^≠, for the individual steps (represented by vertical arrows between ground and transition states) are directly related to the reaction rates. The rate-limiting step of the overall reaction is characterized by the highest ΔG^≠. A possible modulation of the energy landscape due to the influence of GdmCl is shown in red. The higher nucleotide binding affinities in the presence of GdmCl stabilize both the ATP- and ADP-bound state (or substrate- and product-bound state, respectively), thereby increasing ΔG^≠ for both the hydrolysis and product release steps (assuming that the transition states are not changed to the same extent). Depending on which step is initially rate-limiting, the overall reaction can be significantly inhibited by modulated nucleotide binding affinities.