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. 2022 Jun 21;121(12):2233-2250.
doi: 10.1016/j.bpj.2022.05.032. Epub 2022 May 26.

The mechanism for thermal-enhanced chaperone-like activity of α-crystallin against UV irradiation-induced aggregation of γD-crystallin

Hao Li  1 , Yingying Yu  2 , Meixia Ruan  3 , Fang Jiao  3 , Hailong Chen  3 , Jiali Gao  4 , Yuxiang Weng  5 , Yongzhen Bao  6
Affiliations

The mechanism for thermal-enhanced chaperone-like activity of α-crystallin against UV irradiation-induced aggregation of γD-crystallin

Hao Li et al. Biophys J. .

Abstract

Exposure to solar UV irradiation damages γ-crystallin, leading to cataract formation via aggregation. α-Crystallin, as a small heat shock protein, efficiently suppresses this irreversible aggregation by selectively binding the denatured γ-crystallin monomer. In this study, liquid chromatography tandem mass spectrometry was used to evaluate UV-325 nm irradiation-induced photodamage of human γD-crystallin in the presence of bovine α-crystallin, atomic force microscope (AFM) and dynamic light scattering (DLS) techniques were used to detect the quaternary structure changes of the α-crystallin oligomer, and Fourier transform infrared spectroscopy and temperature-jump nanosecond time-resolved IR absorbance difference spectroscopy were used to probe the secondary structure changes of bovine α-crystallin. We find that the thermal-induced subunit dissociation of the α-crystallin oligomer involves the breaking of hydrogen bonds at the dimeric interface, leading to three different spectral components at varied temperature regions as resolved from temperature-dependent IR spectra. Under UV-325 nm irradiation, unfolded γD-crystallin binds to the dissociated α-crystallin subunit to form an αγ-complex, then follows the reassociation of the αγ-complex to the partially dissociated α-crystallin oligomer. This prevents the aggregation of denatured γD-crystallin. The formation of the γD-bound α-crystallin oligomer is further confirmed by AFM and DLS analysis, which reveals an obvious size expansion in the reassociated αγ-oligomers. In addition, UV-325 nm irradiation causes a peptide bond cleavage of γD-crystallin at Ala158 in the presence of α-crystallin. Our results suggest a very effective protection mechanism for subunits dissociated from α-crystallin oligomers against UV irradiation-induced aggregation of γD-crystallin, at the expense of a loss of a short C-terminal peptide in γD-crystallin.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1
Figure 1
Protein structures of dimeric α-crystallin and monomeric γD-crystallin with local structural arrangement around Trp156, and MS spectra of γD-crystallin with or without UV-325 nm irradiation. (A and B) Dimeric structures of (A) human αA-crystallin (PDB: 6T1R) (55) and (B) αB-crystallin (PDB: 2YGD) (57) under neutral condition, with β-strands indicated as β3-9. The intermolecular hydrogen bonds at the dimeric interface are labeled as dashes, including the interaction between the side chains of R(Arg)107 and D(Asp)80 in human αB-crystallin (B). (C) The monomeric structure of human γD-crystallin (1HK0) (29) with four conserved Trp(W) residues labeled and its local structures around W(Trp)156-A(Ala)158. The minimum distance between the indole ring of Trp156 and the nitrogen atom next to Cα of Ala158 is 4.14 Å, which makes the radical reaction possible. (D) The terminal amino acid sequence of recombinant γD-crystallin. The sites of 6His, thrombin site, and T7 tags at the N-terminus are underlined. The molecular weight (MW) of the short peptide chain containing 15–17 residues is labeled at the C-terminus. M(Met) at the N-terminal end, which tends to be removed by methionyl aminopeptidase during the protein expression, is labeled in red. (E and F) MW distribution of γD-crystallin without UV irradiation (E), and aggregated γD-crystallin upon UV-325 nm exposure (F). Photoaggregated γD-crystallin was dissolved in 1% formic acid/H2O before LC-MS measurement. The MS data were deconvoluted with a 1.0 (inset) and 0.5 Da/channel resolution at a mass range of 15–50 (inset) and 18–26 kDa, respectively. (G) MW distribution of photodamaged γD-crystallin in the presence of α-crystallin. α-Crystallin (50 μM) was mixed with γD-crystallin (41 μM), followed by UV-325 nm irradiation at 40°C, and no protein precipitation can be detected after 30 min UV-325 nm exposure with an average power density of 0.05 W/cm2. The MS data were deconvoluted from the LC peak of γD-crystallin in Fig. S3A. Inset: the photodamaged αγ-oligomer is dissolved in 1% formic acid/H2O before LC-MS measurement.
Figure 2
Figure 2
Temperature-dependent protein protection efficiency of bovine α-crystallin and its linear correlation with the surface hydrophobicity after preheat treatment. (A) Chaperone-like activity of bovine α-crystallin against heating temperature. The measurements were performed using 164 and 20.5 μM γD-crystallin as the substrate protein, and the chaperone-like activity was evaluated using the formula of protein protection efficiency (%) = ((A0 − A)/A0) × 100, where A and A0 represent the turbidity of the solution after a duration of exposure to UV light at the given temperatures with or without bovine α-crystallins. (B) Protein protection efficiency of preheated α-crystallins against aggregation of UV irradiation-denatured γD-crystallin and TCEP-induced aggregation of insulin at RT. α-Crystallin were first incubated at a given higher temperature for 10 min, then cooled to RT, and kept overnight for equilibration. Preheated α-crystallins were mixed with the substrates at the indicated protein concentrations, and incubated at least 30 min in the dark, followed by the measurements at RT. (C) Preheat temperature-dependent fluorescence spectra of protein-bound bis-ANS. The preheated α-crystallin was incubated with 25 μM bis-ANS at a protein concentration of 5 μM followed by excitation at 390 nm. The maximum fluorescence intensity at 492 nm against the temperature for preheat is present in the graphic inset. (D) Linear correlations between the fluorescence intensity of protein-bound bis-ANS at 492 nm and the protein protection efficiency against aggregation of denatured γD-crystallin and insulin.
Figure 3
Figure 3
IR absorption spectra of α-crystallin and the linear correlation between the temperature-dependent SVD-resolved population of intermediate component (component 2) and the proportion of disrupted intermolecular hydrogen bonds at the dimeric interface. (A) Second derivative FTIR spectra of bovine α-crystallin at the indicated temperatures. The magnitude of the negative peak in the second derivative spectrum corresponds to the absorption intensity. (B) Second derivative FTIR spectra of truncated (WTtruncated) and intact bovine αB-crystallin, together with the single-site mutated truncated (D107Gtruncated) bovine αB-crystallin, demonstrating that the 1604 cm−1 absorption band is from the intermolecular hydrogen bonds at the dimeric interface. (C) Temperature-dependent IR absorbance of intermolecular hydrogen bonds (1604 cm−1) at the dimeric interface in terms of amplitude of the second derivative spectra. The absorbance is derived from the relative intensity of the second derivative peak at 1604 cm−1, as shown in (A), which is defined as intensity difference between the absorbance at 1604 and 1595 cm−1 (I1604-I1595, where I1595 serves as the background intensity). (D) Relationship between equilibrium constant (K(T)) for breaking the intermolecular hydrogen bonds (1604 cm−1) at the dimeric interface and heating temperatures. ln KH-bond(T) versus 1/T can be well fitted by a straight line at temperatures of 37–67°C, suggesting a continuous disruption of hydrogen bonds at the dimeric interface. (E and F) IR absorption spectra (E) and the corresponding second derivative (F) of the three major SVD-resolved species for component 1–3 during the heating process at temperatures of 25–64°C. (G) SVD-resolved temperature-dependent population curves for the three components in (E) and (F). Mtotal represents the total amount of the monomers in solution. Subouter and Subinner represent the outermost and inner layer subunits, respectively. (H) Linear correlation between the SVD-resolved population of component 2 and the proportion of disrupted hydrogen bonds at the dimeric interface as derived from (C). (I) The sequential structural transition for the α-crystallin oligomer at elevated temperatures in parallel to the three species-associated components resolved by SVD. Three different colors also represent three resolved species-associated IR spectra, while the corresponding numbers of the colored spheres represent the relative populations.
Figure 4
Figure 4
Temperature-dependent population of the dissociated subunits, the relationship between the equilibrium constant and heating temperatures, and UV irradiation-induced quaternary structure changes of crystallin oligomer. (A) Population of the dissociated subunits from the outer and inner layers of the oligomer. The population of component 2 in Fig. 3G represents the total amount of the dissociated monomers (Mtotal), and the amount of the dissociated monomers (Mouter) is derived from the outermost layer subunits (Subouter) in Fig. 3G. The amount of the dissociated monomers from the inner layer of the oligomer (Minner) is evaluated as the difference between Mtotal and Mouter. (B and C) Correlations between ln Kd(T) and 1/T for the subunit dissociation from the outer (B) and inner (C) layers of the oligomer. Kd denotes the corresponding equilibrium constant. The straight lines are linear fitting lines. (D) Correlations between ln (p/[Mtotal]) and 1/T in the presence of photodamaged γD-crystallin at the indicated protein concentrations. The relationship can be fitted by two straight lines with a transition temperature of 55°C, similar to the dissociation of the subunits from the outer layer of the oligomer in (B). p represents the protein protection efficiency. (E and F) The average size distribution of α-crystallin and αγ-oligomer upon UV-325 nm exposure as revealed by DLS (E) and AFM (F) measurements. The photodamaged αγ-oligomer was produced at 40°C. Preheated oligomers were detected at RT with 45 measurements for calculations in DLS. (G and H) AFM images of α-crystallin without UV irradiation (G) and photodamaged αγ-oligomer upon UV-325 nm exposure (H). AFM images were constructed using Nikon NIS-Elements AR software.
Figure 5
Figure 5
T-jump time-resolved IR absorbance difference spectra of bovine α-crystallin and typical kinetics for structural changes of hydrophilic β-sheets, random coils, and intermolecular hydrogen bonds. (A) Time-resolved IR absorption difference (ΔOD) spectra of bovine α-crystallin at a T-jump value of 10°C from RT delayed by 6 μs with respect to the heating laser pulse. (B, C, and E) Kinetics for structural changes of (B) hydrophilic β-sheets (1620 cm−1), (C) random coils (1648 cm−1), and (E) the intermolecular hydrogen bonds at the dimeric interface (1602 cm−1) after a T-jump of ΔT = 10°C at two different pH values. The fitted kinetics are shown as dotted lines, and the damped oscillation in the fitting residual for random coils and the intermolecular hydrogen bonds might be attributed to a heat propagation at the interface to the sample (83,84). (D and F) Fitting of T-jump transient kinetics for IR absorbance of (D) random coils (1648 cm−1) and (F) the intermolecular hydrogen bonds at the dimeric interface (1602 cm−1). The fitting kinetics in (C and E) can be decomposed into two processes, corresponding to the thermal-induced dissociation and reassociation.
Figure 6
Figure 6
Pathway for the photolytic cleavage of peptide backbone and mechanism for the thermal-enhanced chaperone-like activity of bovine α-crystallin. (A) A possible photolytic pathway for UV absorption of tryptophan (Trp) inducing peptide bond cleavage in γD-crystallin (36). (B) Mechanism for the thermal-enhanced chaperone-like activity of bovine α-crystallin involving the formation of a stable complex between the chaperone and substrate γD-crystallin proteins. This mechanism occurs at temperatures of 25–43°C, and the substrate (unfolded γD-crystallin) binds to the dissociated subunits in solution from the outer layer of the α-crystallin oligomer. Kd represents the equilibrium constant for the subunit dissociation. Kb represents the equilibrium constant for the substrate binding. Kbγ represents the equilibrium constant for the reassociation of the αγ-complex.
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