Characterization of the calcyclin (S100A6) binding site of annexin XI-A by site-directed mutagenesis.
Residues in annexin XI-A essential for binding of calcyclin (S100A6) were examined by site-directed mutagenesis. GST fusion proteins with the calcyclin binding site of annexin XI-A, GST-AXI 34-62 and GST-AXI 49-77 bound to calcyclin-Sepharose Ca2+-dependently. The mutants GST-AXI L52E, M55E, A56E and M59E lost the binding ability, whereas GST-AXI A57E retained the ability. These results demonstrate that the hydrophobic residues L52, M55, A56 and M59 on one side surface of the alpha-helix are critical for the binding. Assays with GST fusion proteins and synthesized peptides corresponding to the calcyclin binding site indicated that other regions around the calcyclin binding site are important to stabilize the conformation.
Sudo
T
T
Department of Pharmacology, Nagoya University School of Medicine, Japan. tsudo@tsuru.med.nagoya-u.ac.jp
Hidaka
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017302:Annexins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009418:S100 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D017302:Annexins; D010455:Peptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D009418:S100 Proteins; D002118:Calcium