Stability, Homodimerization, and Calcium-Binding Properties of a Single, Variant βγ-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 βγ-crystallin superfamily, which contains six predicted βγ domains. The first βγ-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 βγ 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 γΒ-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 βγ 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 μM), and 45Ca when blotted on a membrane. AIM1-g1 binds calcium (K D 30 μM) with a comparatively higher affinity than bovine lens γ-crystallin (90 μM). 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 βγ-crystallins (βB2- or γS-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 βγ domain, binds calcium and undergoes dimerization.