Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Molecular Simulations Unravel Key Amino Acid Interactions Regulating Stability and Aggregation of Human Lens γD-crystallin
The prevalent eye disease age-onset cataract is associated with aggregation of human γD-crystallins, one of the longest-lived proteins of the body. The molecular determinants of the unusually high stability and (un)folding/aggregation of γD-crystallin remain to be unidentified. Determination of complex dynamics during protein (un)folding and aggregation events require molecular simulations at long time scales (micro to milliseconds). Even though simulations that are tens of nanoseconds long for certain systems (1-4) are routine, it remains challenging to perform them at longer time scales for atomistic models of biologically relevant proteins. In this study, we perform extensive atomistic molecular dynamics simulations using massively parallel IBM Blue Gene/L supercomputer (5) to characterize unfolding (6) and oligomerization (7) of human gamma D crystallin to advance our current understanding of cataract.
Using large-scale atomistic simulations (6), we have shown that the isolated N-terminal domain (N-td) of γD-crystallin is less stable than its isolated C-terminal domain (C-td), in addition to being the less stable domain in the full-length protein (Figure 1A and 1B), in agreement with biochemical experiments. Sequential unfolding of individual Greek key motifs was revealed within each isolated domain. Our simulations strongly indicate that the stability and the folding mechanism of the N-td are regulated by the interdomain interactions, consistent with experimental observations. We also found that the a and b strands from the Greek Key motif 4 comprising the interdomain interface are the most stable structures within the full protein. Detailed analysis uncovers a surprising Glu-Arg salt-bridge at the topologically equivalent positions of residues E135 and R142 that plays a significant role in determining the stability of a Greek Key motif (see Figure 1A). Disrupting the E135-R142 salt-bridge in silico resulted in destabilizing the inter-domain interface and facilitated the N-td unfolding. These findings (6) indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of γD-crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the γD-crystallin surface might lead to unfolding.
1IBM Thomas J. Watson Research Center, Yorktown Heights, NY 10598
Figure 1: Schematic summary of human γD-crystallin polymerization.
Identification of the aggregation precursors of γ-crystallins is extremely crucial for developing strategies to prevent and reverse cataract. We have used large-scale simulations to determine the structural basis of the pathogenic monomeric state and the intermolecular association of γD-crystallin. Our microseconds of atomistic molecular dynamics simulations (7) 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 (see Figure 1B). 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 that is crucial for the unusually high stability of γD-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors (7), which reveals domain-swapped dimeric structures (Figure 1C & 1D). In the simulated dimeric structure, 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 polymerize through successive domain swapping of those three C-terminal Β-strands leading to age-onset cataract, as an evolutionary cost of its very high stability (Figure 1). These findings (7) thus provide critical molecular insights onto the initial stages of age-onset cataract formation, which is important toward understanding protein aggregation diseases.