Investigating the mechanism of suppression of human γD-crystallin aggregation by myo-inositol.

Abstract

Aggregation of γ-crystallins in the lens of the eye is implicated in cataract disease. Misfolding of this protein under oxidative conditions, such as those present in the ageing eye lens, and mutations in the CRYGD gene correlate with cataract formation. Previous studies from our group indicated that non-native disulfide bonds in early unfolding intermediates kinetically trap misfolded conformations of γD-crystallin, which then aggregate by a mechanism akin to domain-swapping. Recent biophysical experiments from our group have shown that myo-inositol, a small molecule that is abundant in the healthy lens, disrupts the aggregation of human γD-crystallin in vitro and inhibits formation of the aggregation precursors. In this project, theoretical techniques are used to complement these experiments by providing more detailed, atomistic insights into the mechanism of aggregation suppression. Monte Carlo (MC) simulations with a statistical potential, replica exchange and umbrella biasing are used to generate structures for folding intermediates for γD-crystallin and several constructs with experimentally relevant, aggregation-prone mutations, and folding pathways are elucidated from these simulations using our group's previously validated DBFOLD algorithm. Theoretical results are validated against experimental data by comparing calculated misfolding parameters with known experimental aggregation propensities. In addition, Molecular Dynamics (MD) simulations are used to probe the transient chemical interactions between myo-inositol and γD-crystallin folding intermediates, and results from these simulations are compared to insights from aggregation assays, NMR data, and other spectroscopic experiments. The aim is to predict and characterize key chemical interactions whereby myo-inositol shifts γD-crystallin away from aggregation-permissive conformations, which would enable development of improved drug candidates to treat or prevent cataract disease. Our approach to simulating structures of γD-crystallin aggregation precursors and modelling their interactions with small molecules may generalize to other protein misfolding diseases.