Payel Dasa, Jonathan A. Kingb,1, and Ruhong Zhoua,c,1

Abstract

The prevalent eye disease age-onset cataract is associated with aggregation of humanγD-crystallins, one of the longest-lived proteins. Identification of theγ-crystallin precursors to aggregates is crucial for developing strategies to prevent and reverse cataract. Our microseconds of atomistic molecular dynamics simulations uncover the molecular structure of the experimentally detected aggregation-prone folding intermediate species of monomeric native γD-crystallin with a largely folded C-terminal domain and a mostly unfolded N-terminal domain. About 30 residues including a, b, and c strands from the Greek Key motif 4 of the C-terminal domain experience strong solvent exposure of hydrophobic residues as well as partial unstructuring upon N-terminal domain unfolding. Those strands comprise the domain–domain interface crucial for unusually high stability ofγD-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors, which reveals domain-swapped dimeric structures. In the simulated dimeric structures, the N-terminal domain of one monomer is frequently found in contact with residues 135–164 encompassing the a, b, and c strands of the Greek Key motif 4 of the second molecule. The present results suggest thatγD-crystallin may
polymerize through successive domain swapping of those three C-terminalβ-strands leading to age-onset cataract, as an evolutionary cost of its very high stability. Alanine substitutions of the hydrophobic residues in those aggregation-proneβ-strands, such as L145 and M147, hinder domain swapping as a pathway toward dimerization. These findings thus provide critical molecular insights onto the initial stages of age-onset cataract, which is important for understanding protein aggregation diseases