The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner.
The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.
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.
Nakano
Yoshimi
Y
Shibata
Hideki
H
Maki
Masatoshi
M
D017310:Annexin A7 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017302:Annexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020832:EF Hand Motifs; D006801:Humans; D007700:Kinetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance
D017310:Annexin A7; D017302:Annexins; D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; C097753:PDCD6 protein, human; D011994:Recombinant Proteins; D002118:Calcium