Apoptosis-linked gene product ALG-2 is a new member of the calpain small subunit subfamily of Ca2+-binding proteins

Biochemistry. 1999 Jun 8;38(23):7498-508. doi: 10.1021/bi990034n.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins
  • Apoptosis* / genetics
  • 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

Substances

  • Apoptosis Regulatory Proteins
  • Calcium-Binding Proteins
  • Pdcd6 protein, mouse
  • Peptide Fragments
  • Recombinant Proteins
  • Solutions
  • Calpain
  • Calcium