S100C is a member of the S100 family of EF-hand-type Ca2+-binding proteins which are thought to bind to and thereby regulate the activity of cellular target proteins in a Ca2+-dependent manner. An intracellular ligand for S100C is the Ca2+/phospholipid-binding protein annexin I and we show here that complex-formation is mediated through unique domains within S100C and annexin I. Using a proteolytically truncated annexin I derivative as well as a number of N-terminal annexin I peptides in liposome co-pelleting and ligand-blotting assays we map the S100C-binding site to the N-terminal 13 residues of annexin I. Similar analyses employing recombinantly expressed S100C mutants reveal that residues D91 to I94 in the unique C-terminal extension of this S100 protein are indispensable for annexin I binding. Interaction between S100C and an N-terminal annexin I peptide containing a tryptophan at position 11 can also be monitored by fluorescence emission spectroscopy after tryptophan excitation. This analysis indicates that the local environment of the tryptophan in annexin I becomes less aqueous on S100C binding, suggesting a hydrophobic nature of the protein-protein interaction. Thus the structural basis of the annexin I-S100C complex-formation probably resembles to a large extent that of the well-characterized annexin II-p11 interaction.

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