An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin
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CitationSerebryany, Eugene, Jaie C. Woodard, Bharat V. Adkar, Mohammed Shabab, Jonathan A. King, and Eugene I. Shakhnovich. 2016. “An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human ΓD-Crystallin.” Journal of Biological Chemistry 291 (36): 19172–83. https://doi.org/10.1074/jbc.M116.735977.
AbstractConsiderable 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 gamma-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 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 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 beta-hairpin and rearrangement of the native beta-sheet topology. Specific binding between the extruded hairpin and a distal beta-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation.
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