Characterization of the Interactions of some Bivalent Metal Ions with α-Crystallin and Identification of the Binding Regions by Biophysical, Mutational and Mass Spectrometric Approach

α-Crystallin is a major eye lens protein of vertebrates. It is a small heat shock protein and functions as a molecular chaperone by preventing aggregation of other proteins. Previous studies from our lab revealed that Zn2+ had a dramatic influence on the stability and chaperone function of α-crystallin, although the nature of the binding interaction and mechanism of structural stabilization were unknown. We have undertaken a detailed study to characterize the binding interactions, to explore the mechanism of stabilization of α-crystallin by some bivalent metal ions like Zn2+ and Cu2+ and to identify the binding sites. We found from the MALDI mass spectrometry of the zinc-alpha binding interaction, that the binding was heterogenous and of low affinity. We established inter-subunit bridge formation by Zn2+ from our dynamic light scattering and subunit exchange experiments. Cu2+ behaves somewhat similar to Zn2+ in its interaction with α-crystallin. Histidines are most preferred metal binding sites of proteins. We prepared some single point and one double histidine mutants of α-crystallin and found that point mutation of histidine residues of α-crystallin was unable to eliminate the Zn2+ binding and subsequent stabilization via intersubunit bridging. A more definitive approach was taken to locate histidine residues which bind Zn+2 in α-crystallin. This involved chemical modification of histidine residues by DEPC (carbethoxylation) in presence of the bound metal ions followed by trypsin digestion and MALDI MS. This method has detected clearly H79, H107 and H115 of αA-crystallin and H104, H111 and H119 of αB-crystallin to be involved in binding of Zn+2. All of these His residues are situated in the highly conserved α-crystallin domain of the protein. The involvement of H119 of αB-crystallin has been reconfirmed by use of another proteolytic enzyme chymotrypsin. Bivalent metal Cu2+ which also binds through H100, H107 and H115 of αA-crystallin  confirming the involvement of Beta5-Beta7 region of the α-crystallin domain in the intersubunit bridge formation. We believe these findings are crucial in the understanding of mechanisms of age related loss of chaperone function of α-crystallin leading to the formation of cataract.