Biochemical characterization of the penta-EF-hand protein grancalcin and identification of L-plastin as a binding partner

Grancalcin is a recently described Ca2+-binding protein especially abundant in human neutrophils, Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca2+ although it has an unusual Ca2+ coordination loop and one that does not bind Ca2+ but is directly involved in homodimerization, We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated, This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca2+ load. Flow dialysis showed that recombinant grancalcin binds two Ca2+ per subunit with positive cooperativity and moderate affinity ([Ca2+](0.5) of 25 and 83 muM in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca2+-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca2+ and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca2+-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice verse.