Apoptosis-Linked Gene Product ALG-2 Is a New Member of the Calpain Small Subunit Subfamily of Ca2+-Binding Proteins

• Published in: Biochemistry (Easton) 1999-06, Vol.38 (23), p.7498-7508
• Main Authors: Lo, Kevin W.-H, Zhang, Qiang, Li, Ming, Zhang, Mingjie
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis - genetics; Apoptosis Regulatory Proteins; Calcium - chemistry; Calcium - metabolism; Calcium-Binding Proteins - biosynthesis; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - isolation & purification; Calpain - chemistry; Calpain - genetics; Dimerization; Escherichia coli - genetics; Mice; Models, Molecular; Multigene Family; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Point Mutation; Protein Conformation; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Sequence Homology, Amino Acid; Solutions
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi990034n

ALG-2 is a newly discovered Ca2+-binding protein which has been demonstrated to be directly linked to apoptosis. Structurally, ALG-2 is expressed as a single polypeptide chain corresponding to a 22 kDa protein containing five putative EF-hand Ca2+-binding sites. In this work, we have developed an efficient expression and purification scheme for recombinant ALG-2. Utilizing this protocol, we can routinely obtain purified recombinant protein with a yield of approximately 100 mg per liter of bacterial cell cultures. Gel filtration and chemical cross-linking experiments have shown that Ca2+-free ALG-2 forms a weak homodimer in solution. Biochemical and spectroscopic studies of truncated and point mutants of ALG-2 demonstrated that the fifth EF-hand Ca2+-binding motif is likely to participate in the formation of the dimer complex. Experimentally, both the amino- and carboxyl-terminal truncated mutants of ALG-2 have shown their ability to retain the structural, as well as, Ca2+-binding integrity when individually expressed in bacteria. In this respect, the N-terminal domain encompasses the first two EF-hands, and the C-terminal domain contains the remaining three EF-hands. Combining mutagenesis and spectroscopic studies, we showed that ALG-2 possesses two strong Ca2+-binding sites. Employing fluorescence spectroscopy and circular dichroism, we showed that the binding of Ca2+ to ALG-2 induced significant conformational changes in both the N-terminal and C-terminal domains of the protein. Furthermore, our studies demonstrated that Ca2+ binding to both strong Ca2+-binding sites of ALG-2 is required for ion-induced aggregation of the protein. We also report here the expression, purification, and partial characterization of a Ca2+-binding-deficient ALG-2 mutant (Glu47Ala/Glu114Ala). In light of its much decreased affinity for Ca2+, this mutant could prove to be instrumental in elucidating the Ca2+-mediated function of ALG-2 within the context of its cellular environment.