Stability, Homodimerization, and Calcium-Binding Properties of a Single, Variant βγ-Crystallin Domain of the Protein Absent in Melanoma 1 (AIM1)

Abstract

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.

Calcium-binding Crystallins from Yersinia pestis

“…Near-UV CD of the D2 domain showed a broad band between 280 and 290 nm for Trp and Tyr with weak ellipticity (data not shown). The addition of calcium had no significant effect on the near-UV CD of D2, suggesting that this protein did not undergo conformational changes upon cation binding (data not shown), similar to the ␥-crystallin (17) and AIM1-g1 domain (18). Because we purified D2 from an inclusion body, it is likely that this domain failed to refold.…”

“…Other than Protein S and Spherulin 3a, ␥-crystallin and AIM1 are known to bind calcium ions (17,18), whereas others, such as SKLP (5), WmKT (10), and SMPI (11) have not been shown to bind calcium. The calcium-binding property of members of this protein family is, thus, debated.…”

Crystallization and preliminary X-ray crystallographic investigations on a βγ-crystallin domain of absent in melanoma 1 (AIM1), a protein fromHomo sapiens

“…The AIM1g1 DNA sequence corresponding to residues 1021-1117 of the protein sequence was cloned into pET-17b expression vector and transformed into Escherichia coli BL21 (DE3) plysS cells (Ray et al, 1997;Rajini et al, 2003). Overexpression and purification of the protein was carried out as described previously (Rajini et al, 2003) by growing a litre of culture to an OD of 0.6 in Luria-Bertani (LB) broth and inducing it with 1 mM IPTG for 5-6 h. The cells were harvested by centrifugation and lysed using lysis buffer containing 50 mM TrisHCl pH 7.8, 100 mM NaCl, 1 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride (PMSF).…”

“…Overexpression and purification of the protein was carried out as described previously (Rajini et al, 2003) by growing a litre of culture to an OD of 0.6 in Luria-Bertani (LB) broth and inducing it with 1 mM IPTG for 5-6 h. The cells were harvested by centrifugation and lysed using lysis buffer containing 50 mM TrisHCl pH 7.8, 100 mM NaCl, 1 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). The lysate was centrifuged at 15 000 rev min À1 in a Sorvall high-speed centrifuge to separate the supernatant from the pellet.…”

“…Although its functions are not clearly known, its expression appears to be altered in melanoma (Ray et al, 1997). Interestingly, the first -crystallin domain of AIM1, called AIM1g1, has been shown to bind calcium with an affinity comparable to its microbial homologues (Rajini et al, 2003). In the sequence of AIM1g1 there are substitutions of key residues in the N-terminal Greek-key motif and an 11-residue insertion in the C-terminal Greek-key motif, which makes it an interesting variant domain.…”