Unfolding of metastable linker region is at the core of Hsp33 activation as a redox-regulated chaperone

Abstract

Hsp33, a molecular chaperone specifically activated by oxidative stress conditions that lead to protein unfolding, protects cells against oxidative protein aggregation. Stress sensing in Hsp33 occurs via its C-terminal redox switch domain, which consists of a zinc center that responds to the presence of oxidants and an adjacent metastable linker region, which responds to unfolding conditions. Here we show that single mutations in the N terminus of Hsp33 are sufficient to either partially (Hsp33-M172S) or completely (Hsp33-Y12E) abolish this post-translational regulation of Hsp33 chaperone function. Both mutations appear to work predominantly via the destabilization of the Hsp33 linker region without affecting zinc coordination, redox sensitivity, or substrate binding of Hsp33. We found that the M172S substitution causes moderate destabilization of the Hsp33 linker region, which seems sufficient to convert the redox-regulated Hsp33 into a temperature-controlled chaperone. The Y12E mutation leads to the constitutive unfolding of the Hsp33 linker region thereby turning Hsp33 into a constitutively active chaperone. These results demonstrate that the redox-controlled unfolding of the Hsp33 linker region plays the central role in the activation process of Hsp33. The zinc center of Hsp33 appears to act as the redox-sensitive toggle that adjusts the thermostability of the linker region to the cell redox status. In vivo studies confirmed that even mild overexpression of the Hsp33-Y12E mutant protein inhibits bacterial growth, providing important evidence that the tight functional regulation of Hsp33 chaperone activity plays a vital role in bacterial survival.

FIGURE 1.

Location of the Hsp33 single site mutations. A, domain structure of full-length reduced Hsp33 from B. subtilis Hsp33 (PDB code: 1VZY) (7). Hsp33 is a two-domain protein with an N-terminal domain (blue), a highly flexible linker region (green), and a zinc binding domain (yellow) that harbors the four absolutely conserved cysteines, which coordinate one zinc ion (red sphere). The linker region appears to be stably folded on top of a large hydrophobic four-stranded β-sheet, which has been postulated to serve as a substrate binding site upon linker unfolding and activation of Hsp33 (see supplemental Fig. S1). Although reduced, inactive Hsp33 is monomeric in solution, it forms dimers in the crystal structure. B, to alter the hydrophobic character of the linker binding interface, either Tyr¹² in β-sheet 1 was replaced with glutamate (Hsp33-Y12E) or Met¹⁷² in β-sheet 9 was substituted with serine (Hsp33-M172S). To illustrate the location of the targeted residues in E. coli Hsp33, the x-ray structure of the truncated E. coli Hsp33_1–255 (PDB code:1HW7) was used.

FIGURE 2.

In vitro chaperone activity of Hsp33 and variants. A, activation kinetics of wild-type Hsp33 and its mutant proteins. Reduced wild-type Hsp33 (open circles), Hsp33-M172S (closed squares), or Hsp33-Y12E (open squares) were incubated in 2 mm H₂O₂ at either 43 or at 30 °C (inset). At the time points indicated, aliquots of Hsp33 (final concentration 300 nm) were tested for their ability to suppress the aggregation of chemically denatured CS (final concentration 75 nm) at 30 °C. B, chaperone activity of reduced, zinc-reconstituted wild-type Hsp33 and its variants. The influence of a 4-fold molar excess of the reduced Hsp33 variants on the aggregation of chemically unfolded CS (75 nm) at 30 °C or thermally unfolded CS (150 nm) at 43 °C was determined. 0% activity is defined as the light scattering signal 4 min after addition of CS in the absence of chaperones, and 100% activity corresponds to the light scattering signal of CS in the presence of a 4-fold molar excess of wild-type Hsp33 that had been activated for 180 min in 2 mm H₂O₂ at 43 °C (Hsp33_{ox43 °C}). To detect any potential changes in the thiol oxidation status of wild-type Hsp33 and the variants during the activity assay at 30 °C or 43 °C, aliquots were taken immediately after the end of the activity measurement (i.e. 4 min) and labeled with the thiol alkylating reagent AMS. The 490-Da AMS interacts irreversibly with any free thiol group in the protein and leads to a mass increase corresponding to the number of reduced cysteines. The change in mass can be visualized using non-reducing SDS-PAGE (top of panel). The double line (‖) in the top panel indicates the position at which a second Hsp33_ox control was removed from the image.

FIGURE 3.

Hydrophobicity of Hsp33 and variants. 3 μm freshly reduced wild-type Hsp33_red (trace a), Hsp33-M172S _red (trace b), Hsp33-Y12E_red (trace c), or active wild-type Hsp33_{ox43 °C} (trace a′), Hsp33-M172S _{ox43 °C} (trace b′), Hsp33-Y12E _{ox43 °C} (trace c′) were incubated with 10 μm of the hydrophobic probe bis-ANS in 40 mm Hepes buffer, pH 7.5. Fluorescence spectra were recorded and buffer corrected.

FIGURE 4.

Thermal stability of wild-type Hsp33 and variants. Preparations of freshly reduced wild-type Hsp33, Hsp33-Y12E, Hsp33-M172S, or fully active Hsp33_{ox43 °C} were heated to 50 °C (1 °C per minute) and changes in the molar ellipticity were recorded at 195 nm using a circular dichroism spectropolarimeter.

FIGURE 5.

Tyrosine 12: a central interaction hub in Hsp33. A, homology modeling of reduced E. coli Hsp33 was conducted with MODELLER using the crystal structure of reduced B. subtilis Hsp33 (PDB code: 1VZY) as a template. The N-terminal core domain is shown in the yellow surface representation, and the C-terminal redox switch domain is shown in cyan ribbon presentation. The zinc ion is depicted as red sphere. The N-terminal Tyr¹² (magenta spheres) appears to interact tightly with several linker residues (cyan spheres). A high GA341 score of 1 provided by the MODWEB modeling server indicates that the model is accurate. B, connectivity map of Tyr¹². Interactions between Tyr¹² and residues in the Hsp33 C terminus (amino acids 179–288) are depicted as magenta and cyan nodes, respectively. The interaction types shown are van der Waals interactions. Arrows point toward the residues that contribute a backbone atom to the interaction.

FIGURE 6.

Expression of constitutively active Hsp33-Y12E mutant causes growth defects in vivo. A, E. coli strains JH21 (BL21 ΔhslO) expressing either (closed circles) wild-type Hsp33, (closed squares) Hsp33-M172S, (open circles) Hsp33-Y12E or (open squares) no protein from a pET11a plasmid were cultivated in the presence of 50 μm IPTG in MOPS minimal medium at 37 °C. Growth was monitored at A₆₀₀. Aliquots of the four cell cultures were harvested by centrifugation as indicated by arrows. B, total cell lysates were prepared and separated into soluble proteins and insoluble aggregates. The amount of total cell lysate and supernatant loaded was adjusted to the number of cells harvested. A 10-fold higher amount of aggregates was loaded onto the SDS-PAGE to visualize cellular proteins that precipitate.