Dataset Information

An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human ?D-Crystallin.

ABSTRACT: Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered "amorphous," and in most cases, little is known about their mechanisms. Amorphous aggregation of ?-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two ?D-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys(32) and Cys(41), was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal ?-hairpin and rearrangement of the native ?-sheet topology. Specific binding between the extruded hairpin and a distal ?-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation.

SUBMITTER: Serebryany E

PROVIDER: S-EPMC5009285 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin.

Serebryany Eugene E Woodard Jaie C JC Adkar Bharat V BV Shabab Mohammed M King Jonathan A JA Shakhnovich Eugene I EI

The Journal of biological chemistry 20160714 36

Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered "amorphous," and in most cases, little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the lat ...[more]

PMID: 27417136

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Project description:BackgroundThe eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human ?S-crystallin (?S-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein ?B-crystallin does not interact with wild-type ?S-crystallin, but does bind its G18V variant. The specific molecular determinants of ?B-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of ?S, ?S-G18A, was created to test the limits of ?B-crystallin selectivity.MethodsMolecular dynamics simulations were used to investigate the structure and dynamics of ?S-G18A. The overall fold of ?S-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between ?B-crystallin and ?S-G18A.Results?S-G18A exhibits minimal structural changes, but has compromised thermal stability relative to ?S-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human ?B-crystallin does not bind the G18A variant, in contrast to previous observations for ?S-G18V, which aggregates at physiological temperature.Conclusions?B-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant.General significanceHuman ?B-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related ?S-G18V variant, but not the function-preserving ?S-G18A variant, which is monomeric at physiological temperature.

Dataset Information

An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human ?D-Crystallin.

Publications

An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin.

Similar Datasets

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