An internal disulfide locks a misfolded aggregation-prone intermediate in cataract-linked mutants of human {\gamma}D-crystallin

TL;DR

This study reveals that an internal disulfide bond stabilizes a misfolded intermediate in human gamma-D crystallin mutants, promoting aggregation linked to cataracts, through combined simulations and experimental validation.

Contribution

It identifies a specific disulfide bond crucial for aggregation in cataract-linked mutants, providing mechanistic insight into amorphous protein aggregation in the eye lens.

Findings

Disulfide bond between Cys32 and Cys41 is necessary for aggregation.

Simulation predicts stabilization of an unfolding intermediate by this disulfide.

Mutagenesis confirms the bond's role in oxidative aggregation.

Abstract

Considerable mechanistic insight has been gained into amyloid aggregation; however, a large class of non-amyloid protein aggregates are considered 'amorphous,' and in most cases little is known about their mechanisms. Amorphous aggregation of {\gamma}-crystallins in the eye lens causes a widespread disease of aging, cataract. We combined simulations and experiments to study the mechanism of aggregation of two {\gamma}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 Cys32 and Cys41, 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 \b{eta}-hairpin and rearrangement of the native \b{eta}-sheet topology. Specific binding between the extruded hairpin and a distal \b{eta}-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation.