An ATCUN-like copper site in B2-crystallin plays a protective role in cataract-associated aggregation

03 March 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Cataracts is the leading cause of blindness worldwide and it is caused by crystallin damage and aggregation. Senile cataractous lenses have relatively high levels of metals, while some metal ions can directly induce aggregation of human -crystallins. Here we evaluated the impact of divalent metal ions in the aggregation of human B2-crystallin, one of the most abundant crystallins in the lens. Turbidity assays showed that Pb2+, Hg2+, Cu2+, and Zn2+ ions induce the aggregation of B2-crystallin. Metal-induced aggregation is partially reverted by a chelating agent, indicating formation of metal-bridged species. Our study focused on the mechanism of copper-induced aggregation of B2-crystallin, finding that it involves metal-bridging, disulfide-bridging, and loss of protein stability. Circular dichroism (CD) and electron paramagnetic resonance (EPR) revealed the presence of at least three Cu2+ binding sites in B2-crystallin; one of them with spectroscopic features typical of Cu2+ bound to an amino-terminal copper and nickel binding motif (ATCUN), a motif found in Cu transport proteins. The ATCUN-like Cu binding site is located at the unstructured N-terminus of B2-crystallin, and it could be modeled by a peptide with the first six residues in the protein sequence (NH2-ASDHQF-). Removal of the N-terminus yields an N-truncated form of B2-crystallin that is more susceptible to Cu-induced aggregation and loss of thermal stability, indicating a protective role for the ATCUN-like site. EPR and X-ray absorption spectroscopy (XAS) studies reveal the presence of a copper redox active site in B2-crystallin that is associated to metal-induced aggregation and formation of disulfide-bridged oligomers. Our study demonstrates metal-induced aggregation of cataract-related B2-crystallin and the presence of putative copper binding sites in the protein. Whether the copper-transport ATCUN-like site in B2-crystallin plays a functional/protective role or constitute a vestige from its evolution as a lens structural protein, remains to be elucidated.


metal ions
ATCUN site
metal-induced aggregation
cataract disease

Supplementary materials

Supporting Information
Sequence alignment of human βB2-crystallin with the more abundant γ- and β-crystallins. Comparison of EPR and CD spectra of copper-bound βB2-crystallin and HSA. SDS-PAGE analysis of Cu-induced aggregates of N-truncated βB2-crystallin. Copper EPR simulations parameters for the ATCUN-like site in βB2- crystallin. Thermodynamic parameters for the Gibbs-Helmholtz fit of the thermal denaturation experiments with βB2-crystallin WT and the N-truncated protein.


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