Stability, homodimerization, and calcium-binding properties of a single, variant betagamma-crystallin domain of the protein absent in melanoma 1 (AIM1).

AIM1 (absent in melanoma), a candidate suppressor of malignancy in melanoma, is a nonlens member of the betagamma-crystallin superfamily, which contains six predicted betagamma domains. The first betagamma-crystallin domain of AIM1 (AIM1-g1) diverges most in sequence from the superfamily consensus. To examine its ability to fold and behave like a normal betagamma domain, we cloned AIM1-g1 and overexpressed it in Escherichia coli as a recombinant protein. The recombinant domain was found to be a stable, soluble protein, similar to lens protein gammaBeta-crystallin in secondary structure. The tertiary structure of AIM1-g1 is dominated by the contribution of aromatic amino acids and cysteine. AIM1-g1 undergoes concentration-independent, noncovalent homodimerization with no trace of monomer, similar to a one-domain protein spherulin 3a. Since many betagamma domain proteins bind calcium, we have also investigated the calcium-binding properties of AIM1-g1 by various methods. AIM1-g1 binds the calcium-mimic dye Stains-all, the calcium probe terbium (with K(D) 170 microM), and (45)Ca when blotted on a membrane. AIM1-g1 binds calcium (K(D) 30 microM) with a comparatively higher affinity than bovine lens gamma-crystallin (90 microM). However, calcium binding does not induce significant change in the protein conformation in the near- and far-UV CD and in fluorescence. The AIM1-g1 domain is as stable as domains of betagamma-crystallins (betaB2- or gammaS-crystallins) as monitored by guanidinium chloride unfolding (midpoint of unfolding transition is 1.8 M GdmCl), and the stability of the protein is not altered upon binding calcium as evaluated by equilibrium unfolding. These results show that, despite the sequence variation, AIM1-g1 folds such as a betagamma domain, binds calcium and undergoes dimerization.