Mechanistic insights into the switch of αB-crystallin chaperone activity and self-multimerization

Abstract

αB-Crystallin (αBc) is a small heat shock protein that protects cells against abnormal protein aggregation and disease-related degeneration. αBc is also a major structural protein that forms polydisperse multimers that maintain the liquid-like property of the eye lens. However, the relationship and regulation of the two functions have yet to be explored. Here, by combining NMR spectroscopy and multiple biophysical approaches, we found that αBc uses a conserved β4/β8 surface of the central α-crystallin domain to bind α-synuclein and Tau proteins and prevent them from aggregating into pathological amyloids. We noted that this amyloid-binding surface can also bind the C-terminal IPI motif of αBc, which mediates αBc multimerization and weakens its chaperone activity. We further show that disruption of the IPI binding impairs αBc self-multimerization but enhances its chaperone activity. Our work discloses the structural mechanism underlying the regulation of αBc chaperone activity and self-multimerization and sheds light on the different functions of αBc in antagonizing neurodegeneration and maintaining eye lens liquidity.

Keywords: Tau protein (Tau); amyloid; crystallin; molecular chaperone; protein aggregation; protein misfolding; protein quality control; protein structure; α-synuclein; αB-crystallin.

Figure 1.

αBc and CαBc inhibit aggregation of αSyn and K19. A, domain architecture of αBc. The CαBc is flanked by a flexible NR and a flexible CT containing a conserved IPI motif. B, ThT kinetics of αSyn aggregation inhibited by αBc (top) and CαBc (bottom), respectively. C, negative-stain EM images of αSyn (top) and K19 (bottom) fibrils with and without αBc/CαBc at different concentrations. D, comparison of chaperone activity of αBc and CαBc for preventing aggregation of αSyn (left) and K19 (right). The ThT value was taken at the 55-h time point from the ThT kinetics curves. Error bars correspond to mean ± S.E. with n = 3. * indicates p < 0.05, and *** indicates p < 0.005. a.u., absorbance units.

Figure 2.

N terminus of αSyn binds to both CαBc and αBc. A, an overlay of the 2D ¹H-¹⁵N HSQC spectra of 25 μm αSyn in the absence (black) and presence of CαBc at molar ratios (αSyn:CαBc) of 1:10 (blue) and 1:20 (red), respectively. Resonances with relatively large chemical shift perturbations are highlighted on the right. B, CSDs of 25 μm αSyn titrated by CαBc (top) and αBc (bottom), respectively. The CSD values were calculated using the empirical equation CSD = [ΔHN² + 0.0289(ΔN)²]^1/2 where ΔHN and ΔN represent the chemical shift differences of ¹H and ¹⁵N, respectively. The domain organization of αSyn is shown on the top of the graph. NAC stands for non amyloid-β component. C, the inhibitory effects of αBc and CαBc on the aggregation of αSyn and αSyn(21–140), respectively. The ThT value was taken at the 60-h time point from the ThT kinetics curves. Error bars correspond to mean ± S.E. with n = 3. *** indicates p < 0.005, and N.S. indicates not significant.

Figure 3.

Identification of the binding surface of CαBc and αBc to αSyn. A, an overlay of the 2D ¹H-¹⁵N HSQC spectra of 200 μm CαBc in the absence (black) and presence of 400 μm αSyn (red). Residues with significant resonances changing are labeled. Resonances of the four key interacting residues, Lys⁹⁰, Lys⁹², Thr¹³⁴, and Ser¹³⁵, are highlighted on the right. B, CSD profile of CαBc upon addition of αSyn. Deviations higher than 0.015 ppm are highlighted in red. Secondary structure assignment of CαBc is on the top of the graph. C, residues with large CSD (>0.015 ppm) upon αSyn titration are highlighted in red on the structure of CαBc (Protein Data Bank (PDB) code 2klr) with ribbon (top) and surface representation (bottom). Two key interacting residues, Lys⁹⁰ and Lys⁹², are shown in a zoomed-in view on the right. D, inhibitory effects of αBc and its variants on the amyloid aggregation of αSyn (100 μm). The ThT value was taken at the 58-h time point from the ThT kinetics curves. Error bars correspond to mean ± S.E. with n = 3. *** indicates p < 0.005, and ** indicates p < 0.01.

Figure 4.

Competitive binding of the CαBc β4/β8 surface by IPI peptide and αSyn. A, an overlay of the 2D ¹H-¹⁵N HSQC spectra of 200 μm CαBc alone (black) and after incubation with 40 μm IPI peptide (blue). Resonances of four residues, Val⁹¹, Lys⁹², Leu¹³¹, and Thr¹³⁴, that underwent significant changes are displayed in a zoomed-in view. The resonances of the same residues of CαBc (200 μm) in the presence of 100 μm IPI-AAA peptide (dark yellow) are shown on the right. B, residue-specific CSD (top) and intensity changes (I/I₀; bottom) of CαBc in the presence of the IPI peptide. Residues with CSD >0.015 ppm and I/I₀ <0.4 are highlighted in blue, respectively. C, resonance changes of Leu¹³¹, Ser¹³⁶, and Lys⁹⁰ of CαBc (black) in the presence of αSyn alone (left column; red), αSyn (middle column; red) followed by titration of the IPI peptide (middle column; blue), and the IPI peptide alone (right column; blue), respectively. The inset shows the direction of chemical shift changes upon titration. A cartoon of the sequential titrations of αSyn and the IPI peptide to CαBc is shown on top. D, addition of the IPI peptide weakens the chaperone activity of CαBc for inhibiting αSyn aggregation. The ThT value was taken at the 80-h time point from the ThT kinetics curves. Error bars correspond to mean ± S.E. with n = 3. *** indicates p < 0.005.

Figure 5.

Influence of the IPI-β4/β8 interaction in αBc multimerization and chaperone activity. A, sedimentation velocity analysis of αBc(69–175), αBc(69–175)-KA, and αBc(69–175)-AAA at 20 °C at a concentration of 5 mg/ml. B, comparison of the chaperone activity of CαBc, αBc(69–175), αBc(69–175)-KA, and αBc(69–175)-AAA for preventing αSyn aggregation. The ThT value was taken at the 58-h time point from the ThT kinetics curves. Error bars correspond to mean ± S.E. with n = 3. *** indicates p < 0.005, and ** indicates p < 0.01. C, sedimentation (Sed) velocity analysis of αBc (0.7 mg/ml) and αBc-AAA (0.7 mg/ml) at 20 °C. D, comparison of the chaperone activities of αBc and αBc-AAA for inhibiting αSyn aggregation. Error bars correspond to mean ± S.E. with n = 3. *** indicates p < 0.005.

Figure 6.

Schematic diagram of the regulation of αBc for chaperone activity and multimerization. Under normal conditions, αBc forms polydisperse multimers (left) with limited chaperone activity in which the β4/β8 surface is occupied by the neighboring IPI peptide. In lens, multimerization enables αBc to act as a structural protein that packs into higher-order structures to maintain the scattering and transparency of lens. Under stress or disease conditions, αBc disassembles to small multimers (e.g. dimers and hexamers) in response to different stimuli, e.g. stress or phosphorylation (PTM), and exhibits much enhanced chaperone activity. The activated αBc (right) may capture different pathological amyloid clients (e.g. αSyn, Aβ, and Tau) with a more exposed β4/β8 surface and prevent them from forming irreversible amyloid aggregations, which are closely associated with a variety of neurodegenerative diseases (ND). The regulation of αBc between these two functions is accomplished by the competitive binding of the IPI motif and amyloid clients to the key β4/β8 surface of αBc.