Journal of Molecular Biology
The Structure and Stability of the Disulfide-Linked γS-Crystallin Dimer Provide Insight into Oxidation Products Associated with Lens Cataract Formation
Graphical abstract
Introduction
The eye lens contains a high concentration of crystallin proteins arranged in a well-ordered, short-range array that allows for lens transparency and the refraction of light onto the retina, thus ensuring proper vision [1], [2], [3]. In mammals, the crystallins comprise three types (α, β, and γ), of which there are several isoforms. The α-crystallins are members of the small heat-shock protein family, whereas the β- and γ-crystallins are a structurally homologous superfamily of proteins that are not related to small heat-shock proteins [4], [5], [6]. β- and γ-crystallins have a monomeric mass of approximately 20 kDa and consist of two domains, each containing two Greek-key β-sheet motifs, which are adjoined by a short linking peptide [6], [7], [8], [9].
There is little protein turnover in the eye lens, and thus, the crystallins are long-lived proteins that must maintain their structural integrity throughout life to preserve lens transparency [10], [11], [12]. Cataract occurs due to a loss of crystallin protein stability and the subsequent propensity of crystallins to partially unfold, leading to aggregation and precipitation [6], [13]. While cataract acquired during early life commonly stems from destabilizing, inheritable mutations in crystallin proteins, age-related cataract is thought to originate from cumulative post-translational modifications (PTMs) [14], [15]. Oxidation is a prevalent crystallin PTM in both aged and cataractous lenses [16], [17] that increases the aggregation propensity of some crystallins in vitro [18], [19], [20]. Cysteine residues are the principal site of protein oxidation [16], [21], and disulfide-linked crystallins are a major component of the insoluble fraction of cataractous lenses [22], [23]. The key factor in preventing crystallin oxidation is the cellular reductant glutathione, the levels of which diminish with age, to the extent that it is severely depleted in cataractous lenses [24], [25], [26].
γS-crystallin (γS) is one of the major crystallins in the human lens [27], and its abundance increases with age due to postnatal expression [28]. Human γS in cataractous lenses is oxidized at specific cysteine residues [17], including S-methylation [29], S-glutathionylation [30], and intermolecular disulfide bond formation [23]. Indeed, γS forms disulfide-linked dimers in vitro [31]. Disulfide-linked dimerization similarly occurs for the R14C mutant of γD-crystallin, leading to increased aggregation propensity and hereditary juvenile-onset cataract [32]. In light of this and the enhanced oxidative conditions in the aging lens, a detailed understanding of the structural and physiological implications of disulfide-linked dimerization of wild-type γS is needed. Herein, we isolated disulfide-linked, dimeric human γS and determined its structure by X-ray crystallography and small-angle X-ray scattering (SAXS). The significance of the disulfide bonding arrangement of the three clustered cysteine residues at positions 22, 24, and 26 is discussed. Furthermore, we provide biophysical and biochemical evidence for the role of the γS dimer in age-related cataract and the potential molecular mechanisms underlying this role.
Section snippets
Structure of human γS disulfide-linked dimer
Human γS monomer was expressed heterologously in Escherichia coli and purified using anion exchange and size exclusion chromatography (SEC). Previously, γS was reported to undergo time-dependent dimer formation under ambient, oxidative conditions at slightly elevated pH, for example, pH 8 [31]. In the present study, γS monomer was readily converted to dimer at physiological pH (i.e., pH 7) by concentrating the monomer to a minimum of 20 mg mL−1 and leaving the protein at 4 °C for 1 week.
Discussion
The evidence for cysteine oxidation, including disulfide crosslinks, between crystallin proteins in aging and cataractous lenses is extensive [16], [17], [21], [22], [23], [47]. Previous studies have shown that disulfide bonds can form in vitro in γ-crystallins [18], [31], [32], [48], but most studies do not demonstrate the ability for these disulfides to be viable in a reducing environment commensurate with that of the lens. Human lens GSH concentrations decrease with age from approximately 6
Production of γS monomer and dimer
A pET43.1 plasmid encoding recombinant human γS (178 amino acids; UniProt P22914) was purchased from Genscript and expressed in BL21(DE3) E. coli cells. Cells were cultured initially at 37 °C for 4–5 h. Expression was then induced with 500 μM IPTG, and the cell culture was incubated overnight at 30 °C. Cells were pelleted, resuspended in DEAE column buffer (20 mM Tris–HCl, pH 8.0), and lysed using sonication. The cell lysate was loaded onto an anion-exchange column (HiPrep DEAE FF 16/10; GE
Acknowledgments
We thank Prof. Roger Truscott and Ms. Jen Xiang for insightful discussions. We acknowledge Dr. Paul Carr for helpful discussions related to protein crystallography. The Centre for Advanced Microscopy at ANU is acknowledged for their assistance with TEM. We are grateful to Dr. Adam Carroll and the team at the Joint Mass Spectrometry Facility, ANU, for help and guidance in performing and designing mass spectrometry experiments. We are grateful to Dr. Robert Knott at the Australian Nuclear Science
References (75)
- et al.
The molecular refractive function of lens γ-crystallins
J. Mol. Biol.
(2011) - et al.
Characterization of the human β-crystallin gene Hu β A3/A1 reveals ancestral relationships among the beta gamma-crystallin superfamily
J. Biol. Chem.
(1986) - et al.
Ageing and vision: structure, stability and function of lens crystallins
Prog. Biophys. Mol. Biol.
(2004) Lens organelle degradation
Exp. Eye Res.
(2002)- et al.
Identification of long-lived proteins reveals exceptional stability of essential cellular structures
Cell
(2013) - et al.
Protein misfolding and aggregation in cataract disease and prospects for prevention
Trends Mol. Med.
(2012) - et al.
The etiology of human age-related cataract. Proteins don't last forever
Biochim. Biophys. Acta Gen. Subj.
(2016) Age-related nuclear cataract—oxidation is the key
Exp. Eye Res.
(2005)- et al.
Proteomic analysis of the oxidation of cysteine residues in human age-related nuclear cataract lenses
Biochim. Biophys. Acta Proteins Proteom.
(2008) - et al.
An internal disulfide locks a misfolded aggregation-prone intermediate in cataract-linked mutants of human γD-crystallin
J. Biol. Chem.
(2016)
Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: implications for age-related cataract
Biochim. Biophys. Acta Mol. Basis Dis.
Oxidative changes in human lens proteins during senile nuclear cataract formation
Biochim. Biophys. Acta
The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage
Exp. Eye Res.
Sequence analysis of βA3, βB3, and βA4 crystallins completes the identification of the major proteins in young human lens
J. Biol. Chem.
Ontogeny of human lens crystallins
Exp. Eye Res.
Lens crystallins and oxidation: the special case of γS
Biophys. J.
Interior and surface of monomeric proteins
J. Mol. Biol.
Human βB2-crystallin forms a face-en-face dimer in solution: an integrated NMR and SAXS study
Structure
Inference of macromolecular assemblies from crystalline state
J. Mol. Biol.
A 1H NMR spectroscopic comparison of γS- and γB-crystallins
Exp. Eye Res.
Preferential and specific binding of human αB-crystallin to a cataract-related variant of γS-crystallin
Structure
Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens
Free Radic. Biol. Med.
The X-ray crystal structure of human γS-crystallin C-terminal domain
J. Biol. Chem.
Disulphide cross-linked protein of high molecular weight in human cataractous lens
Exp. Eye Res.
Spectroscopic studies on the mixed disulfide formation of lens crystallin with glutathione
Exp. Eye Res.
Protein-thiol mixed disulfides in human lens
Exp. Eye Res.
Separating instability from aggregation propensity in γS-crystallin variants
Biophys. J.
Equilibrium folding intermediates of a Greek key β-barrel protein
J. Mol. Biol.
A single destabilizing mutation (F9S) promotes concerted unfolding of an entire globular domain in γS-crystallin
J. Mol. Biol.
The βγ-crystallins: native state stability and pathways to aggregation
Prog. Biophys. Mol. Biol.
A combined NMR and SAXS analysis of the partially folded cataract-associated V75D γD-crystallin
Biophys. J.
Determination of domain structure of proteins from X-ray solution scattering
Biophys. J.
Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing
Biophys. J.
Small heat shock proteins prevent α-synuclein aggregation via transient interactions and their efficacy is affected by the rate of aggregation
J. Biol. Chem.
Short-range order of crystallin proteins accounts for eye lens transparency
Nature
Role of short-range protein interactions in lens opacifications
Mol. Vis.
Primary structure of the bovine β-crystallin Bp chain: internal duplication and homology with γ-crystallin
Eur. J. Biochem.
Cited by (32)
The 18th amino acid glycine plays an essential role in maintaining the structural stabilities of γS-crystallin linking with congenital cataract
2023, International Journal of Biological Macromoleculesα-Crystallin chaperone mimetic drugs inhibit lens γ-crystallin aggregation: Potential role for cataract prevention
2022, Journal of Biological ChemistryAcquired Disorder and Asymmetry in a Domain-Swapped Model for γ-Crystallin Aggregation: Acquired asymmetry and disorder in a crystallin
2022, Journal of Molecular BiologyDeamidation of the human eye lens protein γS-crystallin accelerates oxidative aging
2022, StructureCitation Excerpt :Solvent-exposed cysteines would appear to be disadvantageous for a long-lived protein. Indeed, the dimer of γS, formed by the intermolecular disulfide linkage of C25 and C25 of two γS monomers, is less stable and more aggregation-prone than the monomer (Thorn et al., 2019). The functional advantage of these solvent-accessible cysteines in γS is still under investigation, although they have been demonstrated to play a critical role in metal interactions.
Redox chemistry of lens crystallins: A system of cysteines
2021, Experimental Eye ResearchCitation Excerpt :The natural glutathione redox buffer in the lens becomes depleted with age, especially so in the course of cataract development, to the extent that γ-crystallin concentration exceeds glutathione concentration in the aged lens (Friedburg and Manthey, 1973; Giblin, 2000). We (Serebryany et al., 2018; Thorn et al., 2019) and others (Kaiser et al., 2019; Quinlan and Hogg, 2018; Roskamp et al., 2020; Skouri-Panet et al., 2001; Srikanthan et al., 2004) have suggested that these sequence-proximal protein thiols, which can form disulfides without changing the overall protein structure, may supplement and eventually inherit glutathione's redox buffer function. However, formation of disulfides between Cys residues that are distal in the sequence and structure has been noted in both γ- and β-crystallins (Hanson et al., 1998; Serebryany et al., 2016b; Takemoto, 1997a,b).
The cataract-related S39C variant increases γS-crystallin sensitivity to environmental stress by destroying the intermolecular disulfide cross-links
2020, Biochemical and Biophysical Research CommunicationsCitation Excerpt :PyMOL, a molecular modeling program (http://www.pymol.org/), was used for analyzing the MD simulations [18]. The structure of the γS-crystallin dimer was constructed based on the human γS-crystallin dimer template (6FD8) [19] by PyMOL. Then, the conditions, a water box with dissolved 150 mM NaCl, were simulated by GROMACS.
- †
D.C.T. and A.B.G. contributed equally to this work.
- 1
Current address: P.D. Mabbitt, MRC Protein Phosphorylation and Ubiquitination Unit, University of Dundee, Dundee, UK.