Why Do N-terminal and C-terminal Domain Mutants of Human Gamma Crystallins Lead to Different Congenital Cataract Phenotypes?

Abstract

Purpose: : Over 16 mutations seen in gamma crystallin genes are associated with congenital cataracts. Analysis shows that mutations in the N-terminal domain (N-td) of these two-domain proteins are associated with peripheral cataracts, while those in the C-terminal domain (C-td) lead to nuclear cataract. We investigated this dichotomy by comparing the structural properties of the various mutant proteins. Methods: : cDNAs of wild type (wt), P23T and R77S (N-td mutants), E107A, R140X, W156X and G165fs (C-td mutants) of human gamma-D crystallin were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. The conformational properties and structural stability of the proteins were compared using spectroscopic analysis. Their ability to bind Ca2+ was studied using the mimic dye Stains-all. Comparative modeling of wt and mutants was also done. Results: : We find that: (1) C-td mutants are far less soluble than the wt or N-td mutants; (2) N-td mutants, while structurally very similar to wt, display minor ‘sticky patches’ in their tertiary structure, promoting inter-protein aggregation, whereas C-td mutants expose a far larger number of apolar residues; (3) while wt and N-td mutants bind to the Ca2+-mimic dye Stains-all, C-td mutants do so far weaker, suggesting that the Greek Key folds (Ca2+ binding sites; Rajini et al., J Biol Chem., 2001) in the C-terminal domain are disturbed in them but maintained in the N-td mutants. . Conclusions: : This partial unfolding of the C-terminal structural motif in the C-td proteins (not seen in the N-td mutants) destabilizes the structural integrity and stability, causing rapid aggregation (Moreau KL, King J. J Biol Chem. 2009), weakened Ca2+ binding and possible activation of proteolysis (Sanderson J. et al., IOVS 2000; Biswas S et al., Mol Cell Biochem. 2003), while N-tds display ‘native state aggregation’ (Sandilands A et al., EMBO J. 2002). These structural differences between the N-td and C-td mutants appear to lead to the phenotype differences.