Crystal structure of human grancalcin, a member of the penta-EF-hand protein family

Grancalcin is a Ca2+-binding protein expressed at high level in neutrophils . It belongs to the PEF family, proteins containing five EF-hand motifs and which are known to associate with membranes in Ca2+-dependent manner. Prot otypic members of this family are Ca2+-binding domains of calpain. Our rece nt finding that grancalcin interacts with L-plastin, a protein known to hav e actin bundling activity, suggests that grancalcin may play a role in regu lation of adherence and migration of neutrophils. The structure of human gr ancalcin has been determined at 1.9 Angstrom resolution in the absence of c alcium (R-factor of 0.212 and R-free of 0.249) and at 2.5 Angstrom resoluti on in the presence of calcium (R-factor of 0.226 and R-free of 0.281). The molecule is predominantly alpha-helical: it contains eight alpha-helices an d only two short stretches of two-stranded beta-sheets between the loops of paired EF-hands. Grancalcin forms dimers through the association of the un paired EF5 hands in a manner similar to that observed in calpain, confirmin g this mode of association as a paradigm for the PEF family. Only one Ca2was found per dimer under crystallization conditions that included CaCl2. T his cation binds to EF3 in one molecule, while this site in the second mole cule of the dimer is unoccupied. This unoccupied site shows higher mobility . The structure determined in the presence of calcium, although does not re present a fully Ca2+-loaded form, suggests that calcium induces rather smal l conformational rearrangements. Comparison with calpain suggests further t hat the relatively small magnitude of conformational changes invoked by cal cium alone may be a characteristic feature of the PEF family. Moreover, the largest differences are localized to the EF1, thus supporting the notion t hat calcium signaling occurs through this portion of the molecule and that it may involve the N-terminal Gly/Pro rich segment. Electrostatic potential distribution shows significant differences between grancalcin and calpain domain VI demonstrating their distinct character. (C) 2000 Academic Press.