Calcium ion binding to δ- and to β-crystallins. The presence of the "EF-hand" motif in δ-crystallin that aids in calcium ion binding

Abstract

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca²⁺ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca²⁺ or 5 mM Tb³⁺ opacified. Fluorescence titration of crystallins with TbCl₃ revealed that this ion binds to δ- and β-crystallins in solution. Equilibrium dialysis showed that four Ca²⁺ ions bind to one δ-crystallin tetramer with an affinity of 4.3 × 10³ M^-1. Analysis of the amino acid sequence of δ-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. β-Crystallin also binds four Ca²⁺ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 × 10³ M^-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.