Cataract-linked γD-crystallin mutants have weak affinity to lens chaperones α-crystallins

Abstract

To test the hypothesis that α-crystallin chaperone activity plays a central role in maintenance of lens transparency, we investigated its interactions with γ-crystallin mutants that cause congenital cataract in mouse models. Although the two substitutions, I4F and V76D, stabilize a partially unfolded γD-crystallin intermediate, their affinities to α-crystallin are marginal even at relatively high concentrations. Detectable binding required further reduction of γD-crystallin stability which was achieved by combining the two mutations. Our results demonstrate that mutants and possibly age-damaged γ-crystallin can escape quality control by lens chaperones rationalizing the observation that they nucleate protein aggregation and lead to cataract.

Figure 1

Ribbon representation of γD-crystallin structure (pdbID 1HK0). The N-terminal domain is colored in blue; sites of cataract-causing mutations are highlighted in red and cysteine 111 in green.

Figure 2

Room temperature CD analysis of γD-crystallin mutants. (A) Far-UV spectra showing a pronounced minimum at 218 nm. (B) Near-UV spectra reporting changes in the tertiary fold as a consequence of the mutations.

Figure 3

Denaturant unfolding curves of γD-crystallin mutants at (A) 25 °C and (B) 37 °C. The substitutions lead to the appearance of two explicit transitions compared with the WT monophasic curve. The first transition corresponds to the population of an intermediate with an unfolded N-terminal domain. The second transition corresponds to unfolding of the C-terminal domain. The solid lines represent non-linear least-squares fit yielding the parameters reported in Table 1.

Figure 4

Binding isotherms of γD-crystallin mutant to (A) αA-crystallin and (B) αB-crystallin and its phosphorylation mimic αB-D3. The concentration of the single mutants was 50 μM while the double mutant was 25 μM. Samples were incubated at 37 °C for two hours at the appropriate ratio as described in the methods section. The solid lines for the double mutant I4F/V76D in (A) and (B) are non-linear least-squares fit assuming high affinity binding where 4 α-crystallin subunits bind one γ-crystallin. The dissociation constants are 43±7 and 27±6 μM for αA-crystallin and αB-D3, respectively. For αB-crystallin, the dissociation constant was larger than 300 μM.

Figure 5

Analysis of γD-crystallin mutant binding to α-crystallin by size exclusion chromatography (SEC) detected by in-line absorbance at 280 nm and fluorescence at 475 nm. (A) Samples of αA-crystallin and γD-crystallin were incubated at the indicated molar ratios and injected on a Superose 6 column. The binding of γD-crystallin I4F/V76D is manifested by a fluorescence peak with retention time similar to that of αA-crystallin. (B) Comparative SEC analysis of binding to αB-crystallin and αB-D3. A 50-fold molar excess of the former is required for detectable binding of the double mutant while αB-D3 binds the single mutants. The y axes were scaled to show details of the complex peak. (C) The complex between αB-D3 and γD-crystallin is detected as a distinct peak, indicated by the arrow, migrating at a larger mass than αB-D3 alone.

Figure 6

Fraction of N, I and U as a function of changes in ΔG⁰_N→I for (A) ΔG⁰_I→U = 10 kcal/mol and (B) ΔG⁰_I→U= −2 kcal/mol. For (B) shifts in the ΔG⁰_N→I lead to a large increase in the fraction of U. These simulations apply to three-state unfolding equilibria.

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