Effect of mutations on the structure and stability of lens βγ crystallins and their phenotypic relevance

Abstract

Purpose: The βγ-crystallins, which are packed at very high concentrations in the lens nuclear region, are folded using a series of interlocking antiparallel β-sheet chain conformation, called the Greek key motif, which offers the molecule a compact globular shape of high thermodynamic and kinetic stability. About 60 mutations in them are associated with various forms of congenital cataract. We study here the effect of several such mutations on the structure and stability of two human γ-crystallins as representative examples, in order to gain a molecular structural rationale of the phenotypes. Methods: We have worked with human γD- and γS-crystallin mutants since the structures of the wild type (WT) molecules are known in detail. Mutants P24T, A36P, L45PL54P, R77S, E107A, Y134A, R140X and G165fs of γD-, mutants D26G, S39C and V42M of γS- , and the two WT molecules were cloned, expressed, isolated and purified by standard methods. Solution state structures were determined using CD and fluorescence spectroscopy, surface exposure of nonpolar residues and aggregation tendencies monitored using extrinsic fluorescence probes (bis-ANS, Nile Red, Thioflavin T), structural stability studied using chemical and thermal denaturation methods, aggregation in cellulo visualized by transfection of cDNAs in HLE3 cells. Molecular modelling was done using standard procedures. Results: Mutations A36P, L45PL54P, R140X, G165fs of γ-D, and V42M of γ-S are seen to disturb one or more Greek key motifs and expose otherwise buried nonpolar residues to the surface chain, causing aggregation and light-scattering particles. All these are associated with nuclear cataracts. Those that do not disturb the motif (P24T, R77S of γ-D, and D26G of γ-S) behave very similar to the wild type molecule, but are weaker in stability and display ‘native state aggregation’; and are associated with peripheral cataracts. Conclusions: Given that the nuclear region of the mammalian lens is very rich in βγ-crystallins, it is pertinent to note that mutations in them that disrupt the Greek key motif are associated with nuclear cataracts.