Increase in Surface Hydrophobicity of the Cataract-Associated P23T Mutant of Human γD-Crystallin Is Responsible for Its Dramatically Lower, Retrograde Solubility

Abstract

The cataract-associated Pro23 to Thr (P23T) mutation in human γD-crystallin (HGD) has a variety of phenotypes and is geographically widespread. Therefore there is considerable interest in understanding the molecular basis of cataract formation due to this mutation. We showed earlier [Pande, et al. (2005) Biochemistry 44, 2491-2500] that the probable basis of opacity in this case is the severely compromised, retrograde solubility and aggregation of P23T relative to HGD. The dramatic solubility change occurs even as the structure of the mutant protein remains essentially unchanged in vitro. We proposed that the retrograde solubility and aggregation of P23T were mediated by net hydrophobic, protein-protein interactions. Based on these initial findings for P23T and related mutants, and the subsequent finding that they show atypical phase behavior, we concluded that the protein clusters formed in solutions of the mutant proteins were held together by net hydrophobic, anisotropic interactions. Here we show, using chemical probes, that the surface hydrophobicities of these mutants are inversely related to their solubility. Furthermore, by probing the isolated N-terminal domains of HGD and P23T directly, we find that the increase in surface hydrophobicity of P23T is localized in the N-terminal domain. Modeling studies suggest the presence of sticky patches on the surface of the N-terminal domain that could be engaged in forming protein clusters via hydrophobic protein-protein interactions. This work thus provides direct evidence for the dominant role played by net hydrophobic and anisotropic protein-protein interactions in the aggregation of P23T.

Cataract-associated mutant E107A of human γD-crystallin shows increased attraction to α-crystallin and enhanced light scattering

“…S2B), the Bis-ANS data show a small increase in the net surface hydrophobicity for E107A compared to HGD, while Nile Red does not. Because Bis-ANS is known to be a more sensitive probe for surface hydrophobicity compared to Nile Red (28), this difference is not surprising. The slight increase in surface hydrophobicity is consistent with the substitution of Glu by Ala in the mutant.…”

Exome sequencing identifies novel and recurrent mutations in GJA8 and CRYGD associated with inherited cataract

“…Further, NMR-spectroscopy and X-ray crystallography have indicated that the Pro24 and Thr24 proteins are structurally similar overall [21,22]. However, the Thr24 mutant exhibits local conformational and dynamic differences that may initiate aggregation or polymerization and in vitro experiments have shown that the Thr24 protein exhibits reduced solubility-a property that is likely to trigger cataract formation [23][24][25].…”

Gamma crystallins of the human eye lens

“…For example, in γD-, replacing the residue P24 with T (or other residues such as S or V), as reported in detail by the Pandes [56][57][58][59][60], decreases the solubility of the protein by increasing hydrophobicity and restricting backbone flexibility, altering the temperaturedependent solubility and the phase diagram. The roles of other amino acids in the sequence, whose replacement is associated with lens malfunction, are yet to be understood.…”