The Functional Significance of High Cysteine Content in Eye Lens γ-Crystallins.

Cataract disease is strongly associated with progressively accumulating oxidative damage to the extremely long-lived crystallin proteins of the lens. Cysteine oxidation affects crystallin folding, interactions, and light-scattering aggregation especially strongly due to the formation of disulfide bridges. Minimizing crystallin aggregation is crucial for lifelong lens transparency, so one might expect the ubiquitous lens crystallin superfamilies (α and ßγ) to contain little cysteine. Yet, the Cys content of γ-crystallins is well above the average for human proteins. We review literature relevant to this longstanding puzzle and take advantage of expanding genomic databases and improved machine learning tools for protein structure prediction to investigate it further. We observe remarkably low Cys conservation in the ßγ-crystallin superfamily; however, in γ-crystallin, the spatial positioning of Cys residues is clearly fine-tuned by evolution. We propose that the requirements of long-term lens transparency and high lens optical power impose competing evolutionary pressures on lens ßγ-crystallins, leading to distinct adaptations: high Cys content in γ-crystallins but low in ßB-crystallins. Aquatic species need more powerful lenses than terrestrial ones, which explains the high methionine content of many fish γ- (and even ß-) crystallins. Finally, we discuss synergies between sulfur-containing and aromatic residues in crystallins and suggest future experimental directions.

Congenital coralliform cataract is the predominant consequence of a recurrent mutation in the CRYGD gene.

BACKGROUND: Congenital cataract is a leading cause of treatable childhood blindness and both clinically and genetically heterogeneous. Among the already characterized phenotypes, coralliform cataract is a rare special form of congenital cataracts. Although previous studies had shown that mutations in the γD-crystallin (CRYGD) can result in congenital coralliform cataracts, no conclusive genotype-phenotype correlation might be drawn. Here we aimed to identify the spectrum and frequency of CRYGD gene mutations in congenital coralliform cataracts of Chinese origin. METHODS: The medical records of 392 Chinese families with congenital cataracts were reviewed between January 2011 and December 2021. The families, clinically documented to have congenital coralliform cataracts, were screened for mutations in candidate CRYGD gene. The genomic DNA of all subjects was extracted from peripheral blood leukocytes. PCR amplified and direct sequencing were performed to identify the disease-causing mutation. RESULTS: A total of 12 families with coralliform cataracts were recruited in this study in the past 10 years, accounting for 3.1% of the families with congenital cataracts. Of the 12 families, all affected individuals presented with bilateral non-progressive coralliform cataracts since birth, with the best-corrected Snellen visual acuities ranging from 20/200 to 20/25. A recurrent c.70 C > A (p. P24T) mutation in CRYGD was identified in 10 families (83.3%) with congenital cataract, which co-segregated with all affected individuals and was not observed in unaffected family members or ethnically matched normal controls. CONCLUSIONS: The coralliform cataract is characterized by being bilateral, non-progressive and present at birth. A recurrent p.P24T CRYGD mutation occurs independently in 83.3% of the Chinese families with congenital coralliform cataracts and most likely represents a mutational hot spot, which underscore the relations between coralliform cataract and p.P24T CRYGD.

Mercury ions impact the kinetic and thermal stabilities of human lens γ-crystallins via direct metal-protein interactions.

Loss of metal homeostasis may be involved in several age-related diseases, such as cataracts. Cataracts are caused by the aggregation of lens proteins into light-scattering high molecular weight complexes that impair vision. Environmental exposure to heavy metals, such as mercury, is a risk factor for cataract development. Indeed, mercury ions induce the non-amyloid aggregation of human γC- and γS crystallins, while human γD-crystallin is not sensitive to this metal. Using Differential Scanning Calorimetry (DSC), we evaluate the impact of mercury ions on the kinetic stability of the three most abundant human γ-crystallins. The metal/crystallin interactions were characterized using Isothermal Titration Calorimetry (ITC). Human γD-crystallins exhibited kinetic stabilization due to the presence of mercury ions, despite its thermal stability being decreased. In contrast, human γC- and γS-crystallins are both, thermally and kinetically destabilized by this metal, consistent with their sensitivity to mercury-induced aggregation. The interaction of human γ-crystallins with mercury ions is highly exothermic and complex, since the protein interacts with the metal at more than three sites. The isolated domains of human γ-D and its variant with the H22Q mutation were also studied, revealing the importance of these regions in the mercury-induced stabilization by a direct metal-protein interaction.

Whole genome assembly of the armored loricariid catfish Ancistrus triradiatus highlights herbivory signatures.

The catfish Ancistrus triradiatus belongs to the species-rich family Loricariidae. Loricariids display remarkable traits such as herbivory, a benthic lifestyle, the absence of scales but the presence of dermal bony plates. They are exported as ornamental fish worldwide, with escaped fishes becoming a threat locally. Although genetic and phylogenetic studies are continuously increasing and developmental genetic investigations are underway, no genome assembly has been formally proposed for Loricariidae yet. We report a high-quality genome assembly of Ancistrus triradiatus using long and short reads, and a newly assembled transcriptome. The genome assembly is composed of 9530 scaffolds, including 85.6% of ray-finned fish BUSCOs, and 26,885 predicted protein-coding genes. The genomic GC content is higher than in other catfishes, reflecting the higher metabolism associated with herbivory. The examination of the SCPP gene family indicates that the genes presumably triggering scale loss when absent, are present in the scaleless A. triradiatus, questioning their explanatory role. The analysis of the opsin gene repertoire revealed that gene losses associated to the nocturnal lifestyle of catfishes were not entirely found in A. triradiatus, as the UV-sensitive opsin 5 is present. Finally, most gene family expansions were related to immunity except the gamma crystallin gene family which controls pupil shape and sub-aquatic vision. Thus, the genome of A. triradiatus reveals that fish herbivory may be related to the photic zone habitat, conditions metabolism, photoreception and visual functions. This genome is the first for the catfish suborder Loricarioidei and will serve as backbone for future genetic, developmental and conservation studies.

Oxidation of active cysteines mediates protein aggregation of S10R, the cataract-associated mutant of mouse GammaB-crystallin.

The Ser10 to Arg mutation in mouse γB-crystallin (MGB) has been associated with protein aggregation, dense nuclear opacity, and the degeneration of fiber cells in the lens core. Overexpression of the gap junction protein, connexin 46 (Cx46), was found to suppress the nuclear opacity and restore normal cell-cell contact. However, the molecular basis for the protein aggregation and related downstream effects were not evident from these studies. Here, we provide a comparison of the structures and solution properties of wild type MGB and the S10R mutant in vitro and show that, even though the mutation does not directly involve cysteine residues, some cysteines in the mutant protein are activated, leading to the enhanced formation of intermolecular disulfide-crosslinked protein aggregates relative to the wild-type. This occurs even as the protein structure is essentially unaltered. Thus, the primary event is enhanced protein aggregation due to the disulfide crosslinking of the mutant protein. We suggest that these aggregates eventually get deposited on fiber cell membranes. Since the gap junction protein, Cx46 is involved in the transport of reduced glutathione, we posit that these deposits interfere in Cx46-mediated glutathione transport and facilitate the oxidative stress-mediated downstream changes. Overexpression of Cx46 suppresses such oxidative aggregation. These studies provide a plausible explanation for the protein aggregation and other changes that accompany this mutation. If indeed cysteine oxidation is the primary event for protein aggregation also in vivo, then the S10R mutant mouse, which is currently available, could serve as a viable animal model for human age-onset cataract.

Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging.

Cataract, a clouding of the eye lens from protein precipitation, affects millions of people every year. The lens proteins, the crystallins, show extensive post-translational modifications (PTMs) in cataractous lenses. The most common PTMs, deamidation and oxidation, promote crystallin aggregation; however, it is not clear precisely how these PTMs contribute to crystallin insolubilization. Here, we report six crystal structures of the lens protein γS-crystallin (γS): one of the wild-type and five of deamidated γS variants, from three to nine deamidation sites, after sample aging. The deamidation mutations do not change the overall fold of γS; however, increasing deamidation leads to accelerated disulfide-bond formation. Addition of deamidated sites progressively destabilized protein structure, and the deamidated variants display an increased propensity for aggregation. These results suggest that the deamidated variants are useful as models for accelerated aging; the structural changes observed provide support for redox activity of γS-crystallin in the lens.

Cataract-causing mutation R48C increases γA-crystallin susceptibility to oxidative stress and ultraviolet radiation.

Among all congenital cataracts caused by genetic mutations, approximately half are caused by a mutation in crystallin genes, and accounts the leading cause of blindness in children globally. In this study, we investigated the underlying molecular mechanism of R48C mutation (c.142C > T; p.[Arg48Cys]) of γA-crystallin in a Mexican-Mestizo descent family causing congenital cataracts. We purified γA-crystallin wild-type (WT) and R48C mutant and compared their structural characteristics and biophysical properties by Spectroscopic experiments and environmental stress (oxidative stress, ultraviolet irradiation, pH disorders, thermal shock, or chemical denaturation). The R48C mutant did not affect the secondary and tertiary structure of monomer γA-crystallin, nor did it affect its stability to heat shock and chemicals. However, the R48C mutant destroys the oxidative stability of γA-crystallin, which makes the protein more prone to aggregation and precipitation under oxidative conditions. These might be the pathogenesis of γA-crystallin R48C mutant related to congenital cataract and help to develop anti-cataract strategies from the perspective of γA-crystallin.

Surface Exposed Free Cysteine Suppresses Crystallization of Human γD-Crystallin.

Human γD-crystallin (HGD) has remarkable stability against condensation in the human lens, sometimes over a whole lifetime. The native protein has a surface exposed free cysteine that forms dimers (Benedek, 1997; Ramkumar et al., 1864)1,2 without specific biological function and leads to further protein association and/or aggregation, which creates a paradox for understanding its stability. Previous work has demonstrated that chemical modification of the protein at the free cysteine (C110), increases the temperature at which liquid-liquid phase separation occurs (LLPS), lowers protein solubility and suggests an important role for this amino acid in maintaining its long-term resistance to condensation. Here we demonstrate that mutation of the cysteine does not alter the structure or solubility (liquidus) line for the protein, but dramatically increases the protein crystal nucleation rate following LLPS, suggesting that the free cysteine has a vital role in suppressing crystallization in the human lens.

The aging mouse lens transcriptome.

Age is a major risk factor for cataract (ARC). However, the influence of aging on the lens transcriptome is under studied. Lens epithelial (LEC) and fiber cells (LFC) were isolated from young (3 month old) and aged (24 month old) C57BL/6J mice, and the transcriptome elucidated via RNAseq. EdgeR estimated differential gene expression in pairwise contrasts, and Advaita's Ipathway guide and custom R scripts were used to evaluate the potential biological significance of differentially expressed genes (DEGs). This analysis revealed age-dependent decreases in lens differentiation marker expression in both LECs and LFCs, with gamma crystallin transcripts downregulating nearly 50 fold in aged LFCs. The expression of the transcription factors Hsf4 and Maf, which are known to activate lens fiber cell preferred genes, are downregulated, while FoxE3, which represses gamma crystallin expression, is upregulated in aged fibers. Aged LECs upregulate genes controlling the immune response, complement pathways, and cellular stress responses, including glutathione peroxidase 3 (Gpx3). Aged LFCs exhibit broad changes in the expression of genes regulating cell communication, and upregulate genes involved in antigen processing/presentation and cholesterol metabolism, while changes in the expression of mitochondrial respiratory chain genes are consistent with mitochondrial stress, including upregulation of NDufa4l2, which encodes an alternate electron transport chain protein. However, age did not profoundly affect the response of LECs to injury as both young and aged LECs upregulate inflammatory gene signatures at 24 h post injury to similar extents. These RNAseq profiles provide a rich data set that can be mined to understand the genetic regulation of lens aging and how this impinges on the pathophysiology of age related cataract.

Single cell transcriptomics of the developing zebrafish lens and identification of putative controllers of lens development.

The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos and larvae at single cell resolution through five days of development, identifying a number of novel putative regulators of lens development. Our data address open questions about the temperospatial expression of α-crystallins during lens development that will support future studies of their function and provide the first detailed view of ß- and γ-crystallin expression in and outside the lens. We describe divergent expression in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, membrane associated, RNA-binding, and transcription factor genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.

The long noncoding RNA CRYBG3 induces aneuploidy by interfering with spindle assembly checkpoint via direct binding with Bub3.

Aneuploidy is a hallmark of genomic instability that leads to tumor initiation, progression, and metastasis. CDC20, Bub1, and Bub3 form the mitosis checkpoint complex (MCC) that binds the anaphase-promoting complex or cyclosome (APC/C), a crucial factor of the spindle assembly checkpoint (SAC), to ensure the bi-directional attachment and proper segregation of all sister chromosomes. However, just how MCC is regulated to ensure normal mitosis during cellular division remains unclear. In the present study, we demonstrated that LNC CRYBG3, an ionizing radiation-inducible long noncoding RNA, directly binds with Bub3 and interrupts its interaction with CDC20 to result in aneuploidy. The 261-317 (S3) residual of the LNC CRYBG3 sequence is critical for its interaction with Bub3 protein. Overexpression of LNC CRYBG3 leads to aneuploidy and promotes tumorigenesis and metastasis of lung cancer cells, implying that LNC CRYBG3 is a novel oncogene. These findings provide a novel mechanistic basis for the pathogenesis of NSCLC after exposure to ionizing radiation as well as a potential target for the diagnosis, treatment, and prognosis of an often fatal disease.

Double Domain Swapping in Human γC and γD Crystallin Drives Early Stages of Aggregation.

Human γD (HγD) and γC (HγC) are two-domain crystallin (Crys) proteins expressed in the nucleus of the eye lens. Structural perturbations in the protein often trigger aggregation, which eventually leads to cataract. To decipher the underlying molecular mechanism, it is important to characterize the partially unfolded conformations, which are aggregation-prone. Using a coarse grained protein model and molecular dynamics simulations, we studied the role of on-pathway folding intermediates in the early stages of aggregation. The multidimensional free energy surface revealed at least three different folding pathways with the population of partially structured intermediates. The two dominant pathways confirm sequential folding of the N-terminal [Ntd] and the C-terminal domains [Ctd], while the third, least favored, pathway involves intermediates where both the domains are partially folded. A native-like intermediate (I*), featuring the folded domains and disrupted interdomain contacts, gets populated in all three pathways. I* forms domain swapped dimers by swapping the entire Ntds and Ctds with other monomers. Population of such oligomers can explain the increased resistance to unfolding resulting in hysteresis observed in the folding experiments of HγD Crys. An ensemble of double domain swapped dimers are also formed during refolding, where intermediates consisting of partially folded Ntds and Ctds swap secondary structures with other monomers. The double domain swapping model presented in our study provides structural insights into the early events of aggregation in Crys proteins and identifies the key secondary structural swapping elements, where introducing mutations will aid in regulating the overall aggregation propensity.

Cataract-causing mutations L45P and Y46D impair the thermal stability of γC-crystallin.

Crystallin gene mutations are responsible for about half of the congenital cataract caused by genetic disorders. L45P and Y46D mutations of γC-crystallin have been reported in patients with nuclear congenital cataract. In this study, we explored the thermal stability of wild type (WT), L45P, and Y46D mutants of γC-crystallin at low and high concentrations, as well as the effect of αA-crystallin on the thermal stability of mutants. Spectroscopic experiments were used to monitor the structural changes on temperature-gradient and time-course heating process. Intermediate morphologies were determined through cryo-electron microscopy. The thermal stability of WT and mutants at concentrations ranging up to hundreds of milligrams were assessed via the UNcle multifunctional protein stability analysis system. The results showed that L45P and Y46D mutations impaired the thermal stability of γC-crystallin at low (0.2 mg/mL) and high concentrations (up to 200 mg/mL). Notably, with increase in protein concentration, the thermal stability of L45P and Y46D mutants of γC-crystallin simultaneously decreased. Thermal stability of L45P and Y46D mutants could be rescued by αA-crystallin in a concentration-dependent manner. The dramatic decrease in thermal stability of γC-crystallin caused by L45P and Y46D mutations contributed to congenital cataract in the mature human lens.

A novel F30S mutation in γS-crystallin causes autosomal dominant congenital nuclear cataract by increasing susceptibility to stresses.

Despite of increasingly accumulated genetic variations of autosomal dominant congenital cataracts (ADCC), the causative genes of many ADCC patients remains unknown. In this research, we identified a novel F30S mutation in γS-crystallin from a three-generation Chinese family with ADCC. The patients possessing the F30S mutation exhibited nuclear cataract phenotype. The potential molecular mechanism underlying ADCC by the F30S mutation was investigated by comparing the structural features, stability and aggregatory potency of the mutated protein with the wild type protein. Spectroscopic experiments indicated that the F30S mutation did not affect γS-crystallin secondary structure compositions, but modified the microenvironments around aromatic side-chains. Thermal and chemical denaturation studies indicated that the mutation destabilized the protein and increased its aggregatory potency. The mutation altered the two-state unfolding of γS-crystallin to a three-state unfolding with the accumulation of an unfolding intermediate. The almost identical values in the changes of Gibbs free energies for transitions from the native state to intermediate and from the intermediate to unfolded state suggested that the mutation probably disrupted the cooperativity between the two domains during unfolding. Our results expand the genetic variation map of ADCC and provide novel insights into the molecular mechanism underlying ADCC caused by mutations in ß/γ-crystallins.

Assessing the Structures and Interactions of γD-Crystallin Deamidation Variants.

Cataracts involve the deposition of the crystallin proteins in the vertebrate eye lens, causing opacification and blindness. They are associated with either genetic mutation or protein damage that accumulates over the lifetime of the organism. Deamidation of Asn residues in several different crystallins has been observed and is frequently invoked as a cause of cataract. Here, we investigated the properties of Asp variants, deamidation products of γD-crystallin, by solution NMR, X-ray crystallography, and other biophysical techniques. No substantive structural or stability changes were noted for all seven Asn to Asp γD-crystallins. Importantly, no changes in diffusion interaction behavior could be detected. Our combined experimental results demonstrate that introduction of single Asp residues on the surface of γD-crystallin by deamidation is unlikely to be the driver of cataract formation in the eye lens.

Cataract-causing mutations L45P and Y46D promote γC-crystallin aggregation by disturbing hydrogen bonds network in the second Greek key motif.

Congenital cataracts caused by genetic disorders are the primary cause of child blindness across the globe. In this work, we investigated the underlying molecular mechanism of two mutations, L45P and Y46D of γC-crystallin in two Chinese families causing nuclear congenital cataracts. Spectroscopic experiments were performed to determine structural differences between the wild-type (WT) and the L45P or Y46D mutant of γC-crystallin, and the structural stabilities of the WT and mutant proteins were measured under environmental stress (ultraviolet irradiation, pH disorders, oxidative stress, or chemical denaturation). The L45P and Y46D mutants had lower protein solubility and more hydrophobic residues exposed, making them prone to aggregation under environmental stress. The dynamic molecular simulation revealed that the L45P and Y46D mutations destabilized γC-crystallin by altering the hydrogen bonds network around the Trp residues in the second Greek key motif. In summary, L45P and Y46D mutants of γC-crystallin caused more hydrophobic residues to be solvent-exposed, lowered the solubility of γC-crystallin, and increased aggregation propensity under environmental stress. These might be the pathogenesis of γC-crystallin L45P and Y46D mutants related to congenital cataract.

ATP differentially antagonizes the crowding-induced destabilization of human γS-crystallin and its four cataract-causing mutants.

αßγ-crystallins account for ∼90% of ocular proteins in lens with concentrations ≥400 mg/ml, which has to be soluble for the whole life-span and their aggregation results in cataract. So far, four cataract-causing mutants G18V, D26G, S39C and V42 M have been identified for human γS-crystallin. Mysteriously, lens maintains ATP concentrations of 3-7 mM despite being a metabolically-quiescent organ. Here by DSF and NMR, we characterized the binding of ATP to three cataract-causing mutants of human γS-crystallin as well as its effect on the solution conformations and thermal stability. The results together decode several novel findings: 1) ATP shows no detectable binding to WT and mutants, as well as no significant alternation of their conformations even at molar ratio of 1:200.2) Cataract-causing mutants show distinctive patterns of the crowding-induced destabilization. 3) ATP differentially antagonizes their crowding-induced destabilization. Our studies suggest that the crowding-induced destabilization of human γS-crystallin is also critically dependent of the hydration shell which could be differentially altered by four mutations. Most unexpectedly, ATP acts as an effective mediator for the protein hydration shell to antagonize the crowding-induced destabilization.

Cataract-Associated New Mutants S175G/H181Q of ßΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes.

ß/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of ß/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of ßΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant ß-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of ß/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

A novel CRYGC E128* mutation underlying an autosomal dominant nuclear cataract in a south Indian kindred.

PURPOSE: To identify the mutation causing an autosomal dominant congenital nuclear cataract in a south Indian family by whole exome sequencing and to characterize further phenotypically the same in a zebra fish model. METHODS: A six-generation family (DKEC1) with several affected members registered at the Regional Institute of Ophthalmology (RIO), Chennai was documented to have congenital nuclear cataract. Detailed clinical history and blood samples were collected from all available family members. Genomic DNA of the proband was subjected to whole exome sequencing. Sequence variations suggestive of putative mutations were further confirmed by bidirectional sequencing and restriction site analysis. Functional analysis of the mutant CRYGC E128* in zebrafish embryos was done to dissect out the pathogenicity. RESULTS: A unique variation viz., c.382 G > T in the coding region of the CRYGC gene, resulting in a premature stop codon at position 128 (E128*) was documented in the affected family members. The same was absent in unaffected family members and in 120 unrelated population controls checked. Bioinformatic tools predicted that the mutation might cause a deleterious effect on protein structure and function. Molecular function analysis of this novel mutation (p. E128*, CRYGC) in the zebrafish indicated this mutation to impair lens transparency. CONCLUSION: This study identified a novel CRYGC mutation, E128* to cause autosomal dominant congenital nuclear cataract in a large south Indian family. Our study provides a new insight onto how the mutation might affect the γC-crystallin structure and function besides emphasizing the need for genetic diagnosis toward vision restoration.

Cataract-causing G18V eliminates the antagonization by ATP against the crowding-induced destabilization of human γS-crystallin.

In lens, ∼90% of ocular proteins are αßγ-crystallins with concentrations ≥400 mg/ml, which need to remain soluble for the whole life-span and their aggregation leads to cataract. The G18V mutation of human γS-crystallin causes hereditary childhood-onset cortical cataract. Mysteriously, despite being a metabolically-quiescent organ, lens maintains ATP concentrations of 3-7 mM. Very recently, we found that ATP has no significant binding to γS-crystallin as well as no alternation of its conformation. Nevertheless, ATP antagonizes the crowding-induced destabilization of γS-crystallin even at 1:1, most likely by interacting with the hydration shell. Here by DSF and NMR, we characterized the effect of ATP on binding, conformation, stability of G18V γS-crystallin and its interactions with α-crystallin. The results reveal: 1) G18V significantly accelerates the crowding-induced destabilization with Tm of 67 °C reduced to 50.5 °C at 1 mM. 2) Most unexpectedly, G18V almost completely eliminates the antagonizing effect of ATP against the crowding-induced destabilization. 3) ATP shows no significant effect on the interactions of α-crystallin with both WT and G18V γS-crystallin. Results together decode for the first time that G18V causes cataract not only by accelerating the crowding-induced destabilization, but also by eliminating the antagonizing effect of ATP against the crowding-induced destabilization.