Stabilization of oligomeric structure of α-crystallin by Zn+² through intersubunit bridging

α-Crystallin, the major protein of mammalian eye lens, is a member of the small heat shock protein family and is a molecular chaperone. We previously reported that its molecular chaperone function as well as stability increased in presence of Zn+². Despite the effect of Zn+² on the structure and function of α-crystallin, evidence for direct interaction between them remained elusive. We now present the MALDI mass spectrometric data that shows direct evidence of Zn+² binding to recombinant αA- and αB-crystallin. The binding stoichiometry was over three Zn+² per subunit of α-crystallin at zinc/protein molar ratio of 20. Observation of multiple Zn+² binding is consistent with the large increase in thermodynamic stability. Sequence-based analysis of αA- and αB-crystallin predicted both proteins to be nonzinc binding proteins. Our dynamic light scattering data shows that Zn+² stabilizes the oligomeric structure of α-crystallin by bridging neighboring subunits in multiple centers. Despite the low affinity binding, the intersubunit bridging by multiple Zn+² makes the oligomer so stable that oligomer breakdown does not occur even at 6M urea. The subunit bridging has been supported by our FRET data that showed absence of subunit exchange in presence of zinc. MALDI data also showed that the interaction of α-crystallin with Zn+² is quite different from other bivalent metal ions. Bound Zn+² could be easily removed by dialysis of the complex. The relevance of such weak interaction on the stability of the oligomeric structure of α-crystallin and its function in the eye lens has been discussed.