Hydrophobic Core Mutations Associated with Cataract Development in Mice Destabilize Human γD-Crystallin

Abstract

The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human γD-crystallin (HγD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key β-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the γ-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract in mice, presumably an aggregated state of the mutant crystallins. It has not been possible to identify the aggregating precursor within lens tissues. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of γ-crystallins, mouse mutant substitutions were introduced into HγD-Crys. The mutant proteins L5S, V75D, and I90F were expressed and purified from Escherichia coli. WT HγD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded and the C-td native-like. L5S and V75D in the N-td also displayed three-state unfolding transitions, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. I90F destabilized the C-td, shifting the overall unfolding transition to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments showed that the N-tds of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded more rapidly. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.

The Human W42R γD-Crystallin Mutant Structure Provides a Link between Congenital and Age-related Cataracts*

“…After irradiation, the intensity of the Trp-42 H⑀1 resonance was reduced by 50%, that of Trp-68 was reduced by 37%, whereas that of Trp-156 was only decreased by 5% (Fig. 7A) 15 N HSQC spectrum after irradiation, indicating that new protein species had appeared. We estimated which parts (amino acids) of HGD experienced damage by measuring the intensities of resonances that were associated with undamaged protein over the time course of the irradiation.…”

“…To further confirm that the small resonances do not arise from an unrelated chemical species, but belong to the partially folded minor I-state that can exchange with the major N-state, we also performed H/D exchange experiments for 15 N HSQC spectra were recorded as a function of time. In the folded state, slow exchange of amide hydrogens with deuterons occurs (43), whereas in the unfolded state, exchange is fast.…”

“…Several small, well resolved resonances that were not observed at 600 MHz were detected. For assignment of these resonances, this spectrum was compared with that of the V75D mutant of HGD (V75D) that is known to possesses an unfolded N-td and folded C-td at 3.6 M urea and 37°C, as shown by fluorescence spectroscopy (15) and solution NMR spectroscopy. 3 Comparing the 1 H-15 N HSQC spectra of native W42R, trypsin-digested W42R, HGD C-td, and V75D in 3.6 M urea, many minor resonances were assigned to residues that belong to the C-td (Fig.…”

“…For the remainder of the sample, a two-dimensional 1 H-15 N HSQC spectrum was recorded and compared with that of nonirradiated 15 N-labeled HGD. NMR Spectroscopy-NMR spectra for resonance assignments were acquired at 25°C, using both 15 N-labeled and 13 C, 15 N-labeled samples on Bruker AVANCE 900, AVANCE 800, or AVANCE 600 spectrometers, equipped with 5-mm triple-resonance z-axis gradient cryoprobes. Temperature calibration was performed using the chemical shift differences between methyl and hydroxyl protons of 100% methanol.…”

Aggregation of γ-crystallins associated with human cataracts via domain swapping at the C-terminal β-strands

“…We also find that, upon N-td unfolding the C-td undergoes a net approximatley 23% increase in solvent-exposed surface area (probed using a sphere of 1.4 Å) from the crystal structure. Recent experiments also suggested that a partial unfolding of the C-td is required for aggregation, further demonstrating the structural similarity between the simulated folding intermediate and the experimentally detected aggregation-prone species (23). To map this structural change of the C-td at the residue level, the root-mean-square fluctuation (RMSF) and the %SASA increase in the I2 ensemble as well as the hydrophobicity of each residue within the C-td were calculated (Fig.…”