ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca(2+)-dependent manner.
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.
Satoh
Hirokazu
H
Laboratory of Molecular and Cellular Regulation, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Shibata
Hideki
H
Nakano
Yoshimi
Y
Kitaura
Yasuyuki
Y
Maki
Masatoshi
M
D000595:Amino Acid Sequence; D017302:Annexins / Q000737:chemistry / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
D017302:Annexins; D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C097753:PDCD6 protein, human; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D002118:Calcium