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) mere 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. (C) 1999 Federation of European Biochemical Societies.