Calcyclin-binding site located on the NH₂-terminal domain of rabbit CAP-50 (Annexin-XI): Functional expression of CAP-50 in Escherichia coli

CAP-50 (annexin XI) is a member of annexin family proteins originally identified and characterized as a target protein for calcyclin (H. Tokumitsu et al. (1992) J. Biol. Chem. 267, 8919-8924). In the present work, the calcyclin-binding site of CAP-50 was determined by proteolytic study and by using various deletion mutants expressed in Escherichia coli. The 43-kDa fragment of CAP-50 digested with Staphylococcus aureus V8 protease did not bind to calcyclin in the presence of Ca²⁺. CAP-50 fusion proteins, including various NH₂-terminal deletion mutants were expressed in E. coli using rabbit CAP-50 cDNA and the calcyclin-binding potential was examined using the ¹²⁵I-calcyclin gel overlay method and coprecipitation with phosphatidylserine-containing vesicles in the presence of Ca²⁺. All recombinant protein carried the potential for Ca²⁺-dependent phospholipid binding, due to the presence of the COOH-terminal domain (core domain). Calcyclin-binding experiments showed that CAP-50 molecules lacking the NH₂-terminal 26 residues retain binding potential for calcyclin; however, deletion of an additional 26 amino acids from the NH₂ terminus abolishes specific calcyclin binding. These observations suggest that the calcyclin-binding site is located on the NH₂-terminal region of CAP-50, probably adjacent to or within the region from Tyr²⁷ to Leu⁵².