title: An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-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 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 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 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.
authors.affiliation: From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and.
authors.forename: Eugene
authors.initials: E
authors.lastname: Serebryany
authors.affiliation: the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138.
authors.forename: Jaie C
authors.initials: JC
authors.lastname: Woodard
authors.affiliation: the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138.
authors.forename: Bharat V
authors.initials: BV
authors.lastname: Adkar
authors.affiliation: From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shabab
authors.affiliation: From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and jaking@mit.edu.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: King
authors.affiliation: the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 shakhnovich@chemistry.harvard.edu.
authors.forename: Eugene I
authors.initials: EI
authors.lastname: Shakhnovich
mesh_terms: D019943:Amino Acid Substitution; D002386:Cataract; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D066329:Protein Aggregates; D000072417:Protein Domains; D017510:Protein Folding; D017433:Protein Structure, Secondary; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C504903:CRYGD protein, human; D004220:Disulfides; D066329:Protein Aggregates; D038222:gamma-Crystallins; D003545:Cysteine
keywords: all-atom Monte Carlo simulation; cataract; crystallin; disulfide; domain swapping; oxidative stress; protein aggregation; protein misfolding; unfolding intermediate