beta-Strand interactions at the domain interface critical for the stability of human lens gammaD-crystallin

Abstract

Human age-onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens gammaD-crystallin protein and its isolated domains. A partially unfolded folding intermediate of gammaD-crystallin is detected in simulations with its C-terminal domain (C-td) folded and N-terminal domain (N-td) unstructured, in excellent agreement with biochemical experiments. 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. A hydrophobic folding core was identified within the C-td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non-native surface salt-bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non-native Glu135-Arg142 salt-bridge causes significant destabilization to the folding core of the isolated C-td, which, in turn, induces unfolding of the N-td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of gammaD-crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the gammaD-crystallin surface might lead to unfolding and subsequent aggregation.

Figure 1

(a) Cartoon representation of γD-crys monomer is shown by specifying the four Greek key motifs (N-td₁, N-td₂, C-td₃, and C-td₄) in different colors. The heavy sidechain of the residues at the interdomain surface are shown in ball-stick representation. The predicted substitution site E135 is also shown that forms a salt-bridge interaction with R142 in the wild-type protein during unfolding simulations. Yellow color is used for nonpolar residues, while polar residues are shown in orange. Acidic residues are colored in red and basic residues are colored in blue. (b) Time evolution of the fraction of native contacts, Q, for the single domains in isolation at two different temperatures, 380 K (dashed) and 425 K (solid). (c) Time evolution of the fraction of native contacts, Q, for the single domains within the full monomer at two different temperatures, 380 K (dashed) and 425 K (solid). (d) Time evolution of the fraction of native contacts, Q, of the N-td (solid line) in isolation and in the full monomer and the fraction of native contacts, Q, at the domain interface (dashed line).

Figure 2

(a) Probabilities of native contact formation for the unfolded conformational ensemble (RMSD_Cα >10 Å and Q <0.3) of the isolated C-td. Darker shades of blue indicate stronger native contacts. Secondary elements are shown for each residue, β-sheets in green and helices in red. (b) The probabilities of native contact formation per residue, Q_res, of C-td₄ as a function of simulation time during isolated C-td unfolding. The quantity, Q_res is colored according to the colorbar.

Figure 3

(a) Time evolution of the fraction of native contacts, Q, for the isolated C-td (solid line) and for the N-td in full protein (dashed line) of the wild-type protein (in black) and the E135A mutant (in red) at 425 K. (b) C_α-C_α Distance between E135 and residue 142 as a function of simulation time in the isolated C-td of the wild-type protein (in black) and the E135A mutant (in red). The time dependence of the distance between the E135-R142 ion-pair in the wild-type isolated C-td is also shown in dashed line. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Representative conformations of the isolated C-tds of the wild-type and the E135A mutant during 160 ns unfolding simulations. The cartoon representation of the protein is colored in gray to black from N-terminal to C-terminal. Residues 135 and 142 are shown in ball-stick representation. The colors used to represent different types of residues are similar to that used in Figure 1(a). For the wild-type, the non-native salt-bridge between Glu135 and Arg142 was formed within the first 5 ns of the unfolding simulation and maintained as a salt-bridge for the rest of the simulation. On the other hand, for the E135A mutant, there is no such salt-bridge, and the two residues are separated apart in far distance after about 100 ns simulation. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]