Domain exchange experiments in duck δ-crystallins: Functional and evolutionary implications

Abstract

δ-Crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two δ crystallins, denoted δ1 and δ2. Duck δ2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to δ2/ASL in the amino acid sequence, δ1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras—221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, δ1 or δ2, respectively—were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to δ1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in δ1. The thermodynamic characterization of human ASL (hASL) and duck δ1 and δ2 indicate that δ crystallins are slightly less stable than hASL, with the δ1 being the least stable. The ΔGs of unfolding are 57.25, 63.13, and 70.71 kcal mol⁻¹ for δ1, δ2, and hASL, respectively. This result was unexpected, and we speculate that δ crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein–protein and protein–solvent interactions.