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Increased hydrophobic surface exposure in the cataract-related G18V variant of human ?S-crystallin.

ABSTRACT: The objective of this study was to determine whether the cataract-related G18V variant of human ?S-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by ?B-crystallin.We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecules to the proteins, starting with the solution-state NMR structures of ?S-WT and ?S-G18V.?S-G18V exhibits increased ANS fluorescence, suggesting increased exposed hydrophobic surface area. The specific residues involved in ANS binding were mapped by NMR chemical shift perturbation assays, revealing ANS binding sites in ?S-G18V that are not present in ?S-WT. Molecular docking predicts three binding sites that are specific to ?S-G18V corresponding to the exposure of a hydrophobic cavity located at the interdomain interface, as well as two hydrophobic patches near a disordered loop containing solvent-exposed cysteines, all but one of which is buried in ?S-WT.Although both proteins display non-specific binding, more residues are involved in ANS binding to ?S-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site.Characterization of changes in exposed hydrophobic surface area between wild-type and variant proteins can help elucidate the mechanisms of aggregation propensity and chaperone recognition, presented here in the context of cataract formation. Experimental data and simulations provide complementary views of the interactions between proteins and the small molecule probes commonly used to study aggregation. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

SUBMITTER: Khago D

PROVIDER: S-EPMC5942885 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin.

Khago Domarin D Wong Eric K EK Kingsley Carolyn N CN Freites J Alfredo JA Tobias Douglas J DJ Martin Rachel W RW

Biochimica et biophysica acta 20151014 1 Pt B

<h4>Background</h4>The objective of this study was to determine whether the cataract-related G18V variant of human γS-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by αB-crystallin.<h4>Methods</h4>We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecules to the proteins, starting ...[more]

PMID: 26459004

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Project description:Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (μs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

Project description:Deamidation of proteins is one of the most prevalent post-translational modifications found upon aging, and in age-onset diseases. Specific asparagine and glutamine residues are often selectively deamidated during this process. In the human lens, deamidation has been shown to occur in many crystallins, but it is not clear how these deamidated proteins lead to lens opacity or cataract. Here we have modeled in vitro the effect of deamidation of specific asparagine and glutamine residues in human recombinant ?S-crystallin (HGS) on the solution properties of the protein. The residues selected for deamidation in vitro are those that are found to be deamidated in aged and cataractous lenses in vivo. Two derivatives were prepared, one with Asn76 and Asn143 deamidated (2N-HGS) and the other with two additional Gln residues (92 and 120) deamidated (2N2Q-HGS). Isoelectric focusing measurements showed the expected lowering of the pI from 6.9 in HGS to ?6.5 in 2N-HGS and to ?6.1 in 2N2Q-HGS. However, spectroscopic studies showed no significant change in the secondary and tertiary structures of the deamidated proteins relative to the wild type. The stability of 2N-HGS and 2N2Q-HGS, as measured by guanidinium hydrochloride unfolding, also remained comparable to that of HGS. The main difference was the altered protein-protein interaction among the three proteins. The net repulsive interactions that are characteristic of HGS are diminished in the deamidated derivatives as evidenced by static light scattering measurements of the second virial coefficient, B2 (B2 values for HGS, 2N-HGS, and 2N2Q-HGS of 8.90 × 10(-4), 7.10 × 10(-4), and 6.65 × 10(-4) mL mol g(-2), respectively). Further substantiation is provided by estimates of the excess binding energy of protein-protein interactions in the condensed phase, obtained from measurements of the PEG-induced liquid-liquid phase separation profiles for the three proteins. The data suggest that enhanced attractive protein-protein interactions, arising from the deamidation of HGS, promote protein aggregation, thereby leading to increased light scattering and opacity over time.

Project description:Creatine kinase (CK) is an energy storage enzyme that plays an important role in energy metabolism. CK/phosphocreatine functions as an energy buffer and links ATP production sites with ATP utilization sites. Several key mutations in the ?A-crystallin (cryaa) and ?B-crystallin (cryab) genes have been linked with autosomal-dominant, hereditary human cataracts. The cryaa-R49C mutation was identified in a four-generation Caucasian family. We previously identified an increase in the quantity of CK complexed with ?-crystallin in the lenses of knock-in mice expressing the cryaa-R49C mutation using proteomic analyses. Increased levels of CK in postnatal cataractous lenses may indicate increased ATP requirements during early cataract development. To gain a further understanding of the relationship between CK and ?-crystallin, we investigated whether ?-crystallin interacts with and forms complexes with CK, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 ?-crystallin subunits, with a Kd of 3.3 × 10-7 M, and that the interaction between ?-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and ?-crystallin, suggesting that the interaction between CK and ?-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble ?-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and ?-crystallin formed partially-water-insoluble, high-molecular-mass complexes. Enzyme-linked immunosorbent assay (ELISA)-based enzymatic activity analyses of lens homogenates showed a 17-fold increase in CK activity in the postnatal lenses of cryaa-R49C knock-in mice. These studies indicate that the interaction between ?-crystallin and CK is functionally important and that increased CK levels may be necessary to meet the increased ATP demands of ATP-dependent functions in cataractous lenses.

Dataset Information

Increased hydrophobic surface exposure in the cataract-related G18V variant of human ?S-crystallin.

Publications

Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin.

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