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

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 γ-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 γD-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. Twochain all-atom simulations predicted that one nonnative 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 β-hairpin and rearrangement of the native β-sheet topology. Specific binding between the extruded hairpin and a distal β-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation. Partially unfolded or misfolded, aggregation-prone protein conformational states are linked to a wide array of age-related protein misfolding diseases. The best studied of these conditions include amyotrophic lateral sclerosis (superoxide dismutase), Parkinson’s disease (α-synuclein), serpinopathies (α1-antitrypsin), cancers with P53 and P21 tumor suppressor defects, and lens cataract (crystallins) (1-5). Some of these aggregates contain the well-known amyloid structure, and others do not; even in cases where amyloid is the final aggregated state, oligomers, prefibrillar species, and amorphous aggregates are often closely linked to pathology (6,7). Structures and interactions of specific locally unfolded or misfolded intermediate states are critically important in the mechanisms of non-amyloid aggregation (1). Here we investigate such an intermediate-based aggregation mechanism for a lens γ-crystallin.