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The structure and oxidation of the eye lens chaperone αA-crystallin


The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.

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Fig. 1: Cryo-EM 3D reconstructions of human αA-crystallin (reduced) oligomers.
Fig. 2: Pseudoatomic models of the human αA-crystallin (reduced) 16-mer.
Fig. 3: CTR interactions in the reduced αA-crystallin oligomer ensemble.
Fig. 4: Intramolecular disulfide cross-linking in human αA-crystallin.
Fig. 5: Oligomer architecture of oxidized human αA-crystallin.
Fig. 6: Dynamics of oxidized human αA-crystallin.
Fig. 7: αA-crystallin is capable of transferring disulfide bonds to human p53.

Data availability

The cryo-EM density maps of αA-crystallin oligomers have been deposited in the EMBD under accession codes EMD-4895 (12-mer), EMD-4894 (16-mer) and EMD-4896 (20-mer). The coordinates for the 16-mer model were deposited in the wwPDB under accession number PDB 6T1R. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with dataset identifier PXD013587. The1H, 15N and 13C chemical shifts of reduced αA-crystallin are available at the BioMagResBank (BMRB) with accession number BMRB-27109. All other data are available from the corresponding authors upon reasonable request.


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We are grateful to J. Plitzko (Max Planck Institute for Biochemistry) for continuous support with EM and critical discussions. We thank D. Balchin for his comments on H/DX-MS data analysis, and M.-L. Jokisch, R. Ciccone and G. Feind for technical assistance during initial experiments. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 1035) and CIPSM to J.B., B. Reif, M.Z. and S.W. Cross-linking/mass spectrometry work was supported by the Wellcome Trust (103139). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149).

Author information




C.J.O.K., J.B. and S.W. designed and conceived the research plan. C.P., B. Rockel and C.J.O.K. performed EM experiments and processed the data. C.J.O.K. carried out, with contributions from P.W.N.S., E.V.M. and M.H., the experiments for the biochemical and biophysical characterization. V.D. provided full-length human recombinant p53. M.S. and S.A. performed NMR experiments. M.S. and B. Reif analyzed the NMR data. J.Z. conducted cross-linking/mass spectrometry experiments. J.Z. and J.R. analyzed the cross-linking data. M.Z. performed molecular dynamics simulations and model building. C.J.O.K., J.B. and S.W. wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to Johannes Buchner or Sevil Weinkauf.

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The authors declare no competing interests.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cryo-EM/3D reconstruction of human αA-crystallin oligomers.

a, Cryo-EM micrograph of human αA-crystallin (reduced, αAred). Top- and side-views are highlighted by white and black circles, respectively. Scale bar: 50 nm. b, Reference-free 2D class averages of top-views (top and middle rows) with corresponding eigenimages indicating size variations and 3-, 4- and 5-fold symmetries (bottom row). c, Reference-free 2D class averages of side-views (top and middle rows) with corresponding eigenimages indicating 2-fold symmetry (bottom row). df, Characteristic final class averages (top row) with the corresponding 2D reprojections of the 3D model (bottom row) of the αA-crystallin 12-mer (d), 16-mer (e) and 20-mer (f). Box size in bf, 17.3 nm. g, Angular distribution plots, that is the distributions of the Euler angles of the final class averages contributing to the 3D reconstructions of the αA-crystallin 12-, 16- and 20-mer. h, Fourier shell correlation (FSC) curves between maps from two independently refined half data sets of 12-, 16- and 20-mer populations. According to the 0.143 gold standard criterion, the resolutions for 12-, 16- and 20-mer 3D reconstructions are 9.2, 9.8 and 9.0 Å, respectively.

Extended Data Fig. 2 Structural variability of human αA-crystallin (reduced) oligomers.

a, c, e, Top and side views of the cryo-EM maps of αA-crystallin (reduced) 12-mer (a), 16-mer (c) and 20-mer (e) (mesh presentation) overlaid with the most important 3D eigenvector (red) indicating the positions of main variances (variance map). b, d, f, Representative 3D class averages of the 12-mer (b), 16-mer (d) and 20-mer (f). The map used for modeling of the 16-mer in 3D domain-swapped configuration is marked in (d) by an asterisk. Scale bar, 10 nm.

Extended Data Fig. 3 Cross-linking of human αA-crystallin.

a, Cross-linker titration of αA-crystallin, denaturing NuPAGE gel. Reduced (left) and oxidized (right) αA-crystallin were incubated for 1 h at room temperature with BS3 cross-linker at the indicated molar BS3:αA-crystallin ratios. Excised monomer (450:1, blue), dimer (450:1 and 900:1, red) and oligomer (450:1, green) gel bands for both αAred and αAox were digested with trypsin and further analyzed. Sequence coverages: αAred-monomer: 97.1 %, αAred-dimer: 99.4 %, αAred-oligomer: 100 %, αAox-monomer: 83.2 %, αAox-dimer: 94.8 %, αAox-oligomer: 100 %. b, Fragmentation spectrum of a cross-linked peptide with an intramolecular link between K70 and K99. c, Fragmentation spectrum of a cross-linked peptide with an intermolecular cross-link between M1 and M1.

Extended Data Fig. 4 Cross-links observed in reduced and oxidized human αA-crystallin.

a, Primary sequence of human αA-crystallin. BS3 reactive K, S, T, Y residues and the N-terminus are coloured red. b, Linkage maps comparing the cross-linked residue pairs observed in monomer, dimer and oligomer pools of αAred and αAox. In total, 113 auto-validation cross-links are shown. Colour code: blue, shared cross-links between αAred and αAox (44 shared cross-links, 39 %); black, unique cross-links in αAox (63 cross-links, 56 %); orange, unique cross-links in αAred (6 cross-links, 5 %). Colour code for the sequence regions of αA-crystallin: NTR (residues 1–60), sienna; ACD (residues 61-145), gray; CTR (residues 146-173), green. c, Histograms of Cα-Cα distances of cross-links observed in αAred. The distances were measured between corresponding residues resolved in the crystal structures of truncated versions of zebrafish (PDB 3N3E, left) and bovine (PDB 3L1E, right) αA-crystallin.

Extended Data Fig. 5 Secondary structure prediction and modeling of the N-terminal region of human αA-crystallin.

a, Summary of sequence-based secondary structure predictions of the NTR as obtained from 15 different web-based prediction programs. The predictions reproduce all β-strand segments (blue) present in metazoan sHsp structures. According to the predictions, the NTR most likely contains 3-4 α-helical segments (orange). b, A possible 3D structure model of the NTR of human αA-crystallin predicted using I-Tasser. c, Examples of possible conformations of the NTR of apical (Map) and d, equatorial protomers (Meq) obtained upon structure modeling by molecular dynamics flexible fitting. Although the positions of the three helices within the EM-density in both Map and Meq differ, their arrangement relative to each other is well preserved in comparison to the I-Tasser model mRMSD 2 Å).

Extended Data Fig. 6 Superposition of 1H,15N correlation spectra of 15N-αAred and 15N-αAred-IPSL.

The superposition of 1H,15N correlation spectra of 15N-αAred (black) and 15N-αAred-IPSL treated with ascorbic acid (reduced) shows chemical shift perturbations for residues, for which we have observed an attenuation of the signal intensity for the oxidized 15N-αAred -IPSL sample. In particular, residues T153, A155, E156, R157 display significant chemical shift changes, consistent with the PRE results. At the same time, the chemical shifts of the C-terminal residues (T168, S169, A170, S172, S173) are not affected by the presence of the nitroxyl moiety.

Extended Data Fig. 7 Impact of oxidation on αA-crystallin structure.

a,b, Far-UV (a) and near-UV (b) CD spectra of αAred (black line) and αAox (gray line). Note that the chemical microenvironment of tyrosins, phenylalanines and W9 are affected by oxidation. c, SEC elution profiles of αAred (black line) and αAox (gray line) on a Superose 6 10/300 GL column. Inset: a segment of the calibration curve using the filtration standard mixture from BioRad. The calculated average molecular masses are 380 kDa for αAred and 770 kDa for αAox, respectively (ThG: bovine thyroglobulin, 670 kDa; γG: bovine γ-globulin, 158 kDa). Note the peak broadening, that is increased polydispersity in αAox. d, Analysis of αAred (black line) and αAox (gray line) by sedimentation velocity aUC in a concentration range from 2µM to 150 µM using SEDFIT. The concentrations are 2 µM, 10 µM, 20 µM, 50 µM and 150 µM. The inset shows the concentration dependence of the sedimentation coefficient. e, A set of the class averages used for the 3D reconstruction of αAox 32-mer. f, 2D reprojections of the reconstructed 3D volume corresponding to the orientations of the class averages shown in (e). Box size in e and f: 26.7 nm.

Extended Data Fig. 8 Impact of oxidation on αA-crystallin stability.

a, Oligomeric states of αAred (black circles) and αAox (gray circles) in the presence of urea as determined by sedimentation velocity aUC at 20 °C. The oligomers of both proteins dissociate successively with increasing urea concentrations. Note that αAred and αAox form a 2S species at urea concentrations of 4.5 M and 3.5 M, respectively, suggesting destabilization of αAox oligomers. b, Intrinsic fluorescence urea unfolding transitions for αAred and αAox at 20 °C. The midpoints of the cooperative transition are at 2.7 M for αAox and at 3.8 M urea for αAred, indicating destabilization of the NTR in the case of αAox. The spectral settings of the fluorimeter were chosen to selectively assess the transition of W9 located within the NTR.

Extended Data Fig. 9 αA-crystallin is capable of transferring disulfide bonds to MDH.

a, Denaturing, non-reducing PAGE of samples withdrawn at the indicated timepoints (red arrows) from the aggregation assays in the presence of αAred and recombinant reduced E. coli DsbA as shown in Fig. 7a. Note that disulfide-bridged species of p53 are formed only in marginal amounts. b, Heat-induced aggregation of recombinant malate dehydrogenase (MDH, 4µM) in the presence of an equimolar amount of GSSG, αAred, αAox and reduced (DsbAred) or oxidized (DsbAox) E. coli DsbA. Note that the aggregation of MDH is fully suppressed in the presence of αAred and almost fully suppressed in the presence of αAox. c, Relative intensity of the MDH monomer band as a fraction of the initial intensity (amount of monomer) at the beginning of each aggregation kinetics experiment (t = 0 min). d,e, Denaturing, non-reducing PAGE of samples withdrawn at the indicated timepoints (red arrows) from the aggregation assays shown in (b). Experiments were performed in the presence of GSSG, αAox or DsbAox (d), in the absence of GSSG (MDH only) and in the presence of αAred or DsbAred (e). Note that disulfide-bridged species of MDH are formed in the presence of αAox.

Supplementary information

Supplementary Information

Supplementary Note, Supplementary Fig. 1 and Table 1.

Reporting Summary

Supplementary Table 1

Cross-links observed in monomers, dimers and oligomers of reduced αA-crystallin.

Supplementary Table 2

Cross-links observed in monomers, dimers and oligomers of oxidized αA-crystallin.

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Kaiser, C.J.O., Peters, C., Schmid, P.W.N. et al. The structure and oxidation of the eye lens chaperone αA-crystallin. Nat Struct Mol Biol 26, 1141–1150 (2019).

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