Abstract
Purpose :
Vertebrate βγ-crystallins adopt two homologous domains connected via a linking peptide which is considered a key determinant of their tertiary and quaternary arrangements. In vitro, γ-crystallins are monomeric whereas β-crystallins form mainly dimers that are thought to associate further via domain-swapping to form larger oligomers. This study aims to better understand the oligomeric preferences of βγ-crystallins.
Methods :
The propensity to form domain-swapped dimers in mutant γ-crystallins was predicted with AlphaFold Multimer and initially assessed by Superdex-75 column size-exclusion chromatography. The dimeric structure was determined by X-ray crystallography. Aggregation propensity was assessed by turbidimetry. Thermal stability was measured by circular dichroism and differential scanning calorimetry.
Results :
Several γ-crystallin mutants adopted non-covalently associated dimers in solution under both reducing and non-reducing conditions. The crystal structure of one select mutant shows a domain-swapped dimer, distinct from the compact ‘face en face’ dimeric structure of β-crystallin in solution. The domain structures show no structural perturbation and the conserved interface between the N- and C-terminal domains is preserved. The domain-swapped dimers of γ-crystallin are not markedly aggregation-prone at physiological temperature relative to their wildtype monomeric counterparts; however, they exhibit reduced thermodynamic stability.
Conclusions :
Domain-swapped dimers of vertebrate βγ-crystallins are less stable and are unfavorable in an aging lens where the risk of protein misfolding and aggregation is pronounced due to cumulative post-translational modifications. The long-lived crystallin proteins have therefore been engineered to avoid domain-swapped dimerization in order to retain their soluble, folded conformations throughout adulthood and avoid diseases of the lens such as cataract.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.