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.
Mesh Terms:
Amino Acid Sequence, Annexins, Apoptosis Regulatory Proteins, Binding Sites, Biosensing Techniques, Calcium, Calcium-Binding Proteins, Fluorescent Dyes, Humans, Molecular Sequence Data, Naphthalenesulfonates, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Surface Plasmon Resonance, Two-Hybrid System Techniques
Biochem. Biophys. Res. Commun.
Date: Mar. 15, 2002
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