Local unfolding of the HSP27 monomer regulates chaperone activity

Abstract

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of β-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.

Fig. 1

Reduction of HSP27 releases highly active monomers. a Domain architecture of the human molecular chaperone HSP27, which forms polydisperse oligomers that reach >500 kDa. Native mass spectra collected at 25 μM total monomer concentration for both oxidised (red) and reduced (blue, +250 μM dithiothreitol, DTT) HSP27 reveal the formation of polydisperse oligomers. More monomers are present in the reduced sample, although the oligomeric distributions are highly similar (Supplementary Fig. 1). b The chaperone activity of HSP27 was assayed by monitoring the increase in light scattering at 340 nm of 10 μM of CS in the presence or absence of 0.5 μM reduced (blue, 5 mM 2-mercaptoethanol, BME) or oxidised (red) HSP27. At this concentration, dimeric HSP27 comprises more than half of all populated stoichiometries. The average of two replicates is shown with error bars corresponding to ±1 standard deviation (SD)

Fig. 2

HSP27 monomers are potent chaperones in vitro. The aggregation of MDH (a, b) and GAPDH (d, e) was monitored by light scattering at 340 nm and seeded amyloid fibril formation by αS (g, h) was monitored by ThT fluorescence. Reduced (blue) or oxidised (red) HSP27 was added at either high (≥10 μM) concentrations (a, d, g) where it is predominantly oligomeric or low (≤1 μM) concentrations (b, e, h) where a large fraction of dimers (oxidised) or monomers (reduced) are populated. The y axes were normalised for comparison, and the x axes were scaled by a factor T_f to normalise time. The T_f values used for each substrate were 5 (a, b), 5 (d, e), and 170 (g, h). The chaperone activity of HSP27 against each substrate (c, f, i) is quantified as one minus the ratio of average signal over the time course with and without chaperone (Methods). Values of one and zero would respectively represent the complete inhibition of aggregation and no protection against aggregation. The average of three replicates is shown with error bars corresponding to ±1 SD

Fig. 3

cHSP27 monomers are potent chaperones in vitro. a The central ACD exists as a stable dimer in which residue C137 at the dimer interface can access both reduced (blue, PDB 4mjh) and oxidised (red, PDB 2n3j) states. b The cHSP27 variants used in this study together with their native mass spectra at 5 μM. For reduced cHSP27, 250 μM DTT was added. c Aggregation of 300 μM α-lactalbumin (αLac; black) at 37 °C and d 80 μM insulin at 40 °C following the addition of 2 mM DTT, both monitored by light scattering at 340 nm. The values of T_f are 10 (c) and 0.3 (d). Aggregation of both proteins is inhibited by C137S (green), reduced cHSP27 (blue), H124K/C137S (purple), and full length HSP27 (empty). For αLac and insulin, the sHSP concentrations were 70 and 40 μM, respectively. The traces represent the average of three experiments with errors bars indicating ± 1 SD. Under these conditions, C137S and reduced cHSP27 are predominantly dimeric whereas H124K/C137S is monomeric e Relative chaperone activity of the cHSP27 variants from panels c and d, with activity defined as one minus the ratio of the average signal with and without chaperone. f The self-aggregation of 800 μM C137S and H124K/C137S monitored by light scattering at 340 nm at 37 °C. The traces represent the average of six (C137S) or three (H124K/C137S) experiments with error bars indicating ± 1 SD. Inset: A representative transmission electron microscopy (TEM) micrograph at 40 h reveals large, non-fibrillar aggregates of H124K/C137S

Fig. 4

Redox-induced perturbations to the structure and dynamics of cHSP27. a Overlaid 2D ¹H-¹⁵N HSQC spectra of oxidised (red), reduced (blue, +5 mM BME), and C137S (green) cHSP27 reveal their structural similarities. Significant CSPs are indicated with arrows. b CSPs for individual residues between reduced (top) or C137S (bottom) and the oxidised state reveal differences that localise to the β5 and β6 + 7 strands. The resonance from C137 was broadened beyond detection in reduced cHSP27 (cyan). Proline residues that do not contribute to this spectrum and unassigned residues are indicated. CSPs were calculated as √(ΔH² + (0.2ΔN²)). c R_ex versus residue number, which qualitatively probes μs–ms motions. Errors bars are 1 SD, as derived from propagation of the fitted errors in measured R_2,eff values. Residues near L_5,6+7 show large R_ex values in oxidised, reduced, and C137S cHSP27. The motion extends into the β5 and β6 + 7 strands for reduced cHSP27 and C137S. d R_ex values > 2 s⁻¹ are mapped onto the structure of cHSP27 (PDB 4mjh), indicating that residues with slow dynamics cluster near the dimer interface

Fig. 5

Relaxation dispersion reveals partial unfolding of the cHSP27 monomer. ¹⁵N CPMG RD experiments quantify μs–ms motions in oxidised (red), reduced (blue), and C137S (green) cHSP27. Fitted curves from a global analysis are shown as solid lines. Significant CPMG RD curves were observed in the β5 strand (a), L_5,6+7 loop (b), and the β6 + 7 strand (c). Redox-independent motions were observed in L_5,6+7, which arise from unfolding of the loop, whereas only the non-covalent dimers in C137S and reduced cHSP27 show motions throughout β5 and β6 + 7. d (top) CPMG RD-derived ¹⁵N chemical shift changes in C137S (|Δω|) plotted onto the structure (PDB: 4mjh) reveal structural changes in L_5,6+7 and the β5 and β6 + 7 strands. (middle) The ¹⁵N |Δω| values in L_5,6+7 are similar in both C137S and oxidised cHSP27, and correlate with those expected for a transition to a random coil, indicating that unfolding of L_5,6+7 is independent of oxidation state. In L_5,6+7, D129 forms an intermolecular salt bridge with R140 from an adjacent subunit, and the amide nitrogen from E130 forms a hydrogen bond with the carbonyl of D129 within the same subunit. All error bars are derived from fitting and represent SD values. (bottom) ¹⁵N |Δω| values in L_5,6+7 and β6 + 7 upon monomerisation are compared to the changes expected for random coil formation. The agreement is reasonable, indicating the monomer is substantially disordered in these regions. Error bars represent SD values

Fig. 6

High pressure and low pH stabilise the cHSP27 monomer. a NMR spectra of C137S, focusing on residue G116 as a function hydrostatic pressure, pH, and concentration. Shown here are four rows of spectra: increasing pressures at pH 7 in a baro resistant buffer, increasing pressures at pH 6.8 at 1 bar in phosphate buffer (pH decreases with pressure), decreasing pH at 1 bar, and decreasing protein concentration at pH 7 at 1 bar. Resonances from the dimer (green), monomer (purple), and unfolded (black) state are readily distinguishable. Decreasing concentration or pH and increasing pressure favour the monomerisation. At pressures greater than ~1.5 kbar, the unfolded form becomes the principally populated state. b Variations in NMR signal intensities with pressure from four residues that were unambiguously assigned in all three conformations were well explained (solid lines) by the quantitative 3-state equilibrium model shown. c The K_d for dimerisation is shown as a function of pressure (black), pH (purple), or their combination (grey). d The three-state mechanism of C137S dimer dissociation, where low pH and high pressure favour the partially disordered monomer, before ultimately the monomers unfold

Fig. 7

Structural and dynamical characterisation of the cHSP27 monomer. a Overlaid NMR spectra of C137S at 20 μM and pH 7 (green) where it is predominantly dimeric or pH 4.5 (purple) where it is monomeric. The 1D projection onto the ¹H dimension reveals significant disorder in the monomer. Insets: minor resonances observable in the pH 7 sample correspond to the directly observed monomer species at pH 4.5. D/M corresponds to dimer/monomer, respectively. b (top) Combined and weighted ¹H, ¹⁵N chemical shift perturbations (CSPs) between the C137S dimer at pH 7 and monomer at pH 4.5, with CSPs computed as described above. (middle) β-strands in the C137S monomer (purple) and dimer (green) as identified by RCI. (bottom) {¹H}-¹⁵N NOEs (hetNOEs) for the C137S dimer and monomer (Supplementary Fig. 9). Error bars are derived from signal-to-noise. c The ACD monomeric fold from PDB 4mjh is shown with the difference (dimer-monomer) in {¹H}-¹⁵N NOE values (tube thickness) and magnitude of CSPs (colour). d Inherited mutations that are implicated in the onset of CMT or dHMN disease are indicated in the ACD of HSP27. The mutations cluster to the regions that become solvent exposed upon monomer formation and tend to lower the charge density in the region. e Overlaid dimer structures of human HSP27 (PDB 4mjh), human αB-crystallin (PDB 4m5s), bovine αA-crystallin (PDB 3l1f), and rat HSP20 (PDB 2wj5) are shown in ribbon format for one subunit of each dimer. Their similarity indicates the highly-conserved fold of vertebrate ACDs. The second subunit of HSP27 is shown in cartoon format. Highly conserved residues among human sHSPs (HSPB1-HSPB6) are shown as spheres with the same colour format as d. f Possible hierarchical mechanism of monomer formation. The oxidised, reduced, and C137S forms of cHSP27 exhibit similar dynamics in L_5,6+7 and form a disordered loop. In the absence of a disulphide bond, this motion in L_5,6+7 propagates, resulting in the eventual unfolding of the β5 and β6 + 7 strands in the free monomer. The disulphide bond (C137), L_5,6+7 (D129, E130), and H124 are indicated