ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca(2+)-dependent manner

• Published in: Biochemical and biophysical research communications Vol. 291; no. 5; pp. 1166 - 1172
• Main Authors: Satoh, Hirokazu, Shibata, Hideki, Nakano, Yoshimi, Kitaura, Yasuyuki, Maki, Masatoshi
• Format: Journal Article
• Language: English
• Published: United States 15.03.2002
• Subjects: Amino Acid Sequence; Annexins - chemistry; Annexins - metabolism; Apoptosis Regulatory Proteins; Binding Sites; Biosensing Techniques; Calcium - metabolism; Calcium-Binding Proteins - metabolism; Fluorescent Dyes - metabolism; Humans; Molecular Sequence Data; Naphthalenesulfonates - metabolism; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Surface Plasmon Resonance; Two-Hybrid System Techniques
• ISSN: 0006-291X
• DOI: 10.1006/bbrc.2002.6600

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family. ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix. By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI. Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix. Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. The dissociation constant (K(d)) was estimated to be approximately 70 nM. The Ca(2+)-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca(2+)-dependently exposed hydrophobic surface of ALG-2.