A Cu(II) binding site involving Cys18 and His22 displays a buffering effect in copper-induced aggregation of cataract-related human γD crystallin.

Cataracts are the main cause of blindness in the world, and they are caused by aggregation of lens crystallin proteins. Metal ions have emerged as a potential factor in the development of cataract disease, as copper and zinc ions induce the non-amyloid aggregation of lens crystallins in vitro. Copper-induced aggregation of human γD crystallin (HγD) involves disulfide and metal bridging, partial unfolding of the protein and copper reductase activity. In this study, the effect of the double mutation C18S/H22Q in the copper-induced aggregation of HγD was evaluated by turbidity assays, electrophoresis and transmission electron microscopy, finding that the mutation renders HγD more susceptible to copper-induced aggregation. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) show that the C18S/H22Q mutation does not abolish copper reductase activity of HγD, and that Cys18 and His22 constitute a Cu(II) binding site with a 2N1S1O coordination sphere. This site displays a metal-buffering effect, and in its absence, copper-induced aggregation of HγD crystallin is exacerbated by a metal-bridging mechanism involving other Cu(II) binding sites. This study reveals the presence of a copper-buffering site in HγD crystallin that protects it from aggregation; this would be of functional relevance to prevent development of cataracts upon loss of cellular redox balance and metal homeostasis in the context of an aging lens.