Calcium-dependent .alpha.-helical structure in osteocalcin

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Abstract
Osteocalcin is an abundant Ca2+-binding protein of bone containing 3 residues of vitamin K dependent .gamma.-carboxyglutamic acid (Gla) among its 49 (human, monkey and cow) or 50 (chicken) amino acids. Gla side chains participate directly in the binding of Ca2+ ions and the adsorption of osteocalcin to hydroxyapatite (HA) surfaces in vivo and in vitro. Osteocalcin exhibits a major conformational change when Ca2+ is bound. Metal-free chicken osteocalcin is a random coil with only 8% of its residues in the .alpha. helix as revealed by circular dichroism. In the presence of physiological levels of Ca2+, 38% of the protein adopts the .alpha.-helical conformation with a transition midpoint at 0.75 mM Ca2+ in a rapid, reversible fashion which requires an intact disulfide bridge, is proportionally diminished when Gla residues are decarboxylated to Glu, is insensitive to 1.5 M NaCl and can be mimicked by other cations. Tyr fluorescence, UV difference spectra and Tyr reactivity to tetranitromethane corroborate the conformational change. Homologous monkey osteocalcin also exhibits Ca2+-dependent structure. Integration of predictive calculations from osteocalcin sequence has yielded a structural model for the protein, the dominant features of which include 2 opposing .alpha.-helical domains of 9-12 residues each, connected by a .beta. turn and stabilzed by the Cys23.sbd.Cys29 disulfide bond. Cation binding permits realization of the full .alpha.-helical potential by partial neutralization of high anionic charge in the helical domains. Periodic Gla occurrence at positions 17, 21 and 24 was strongly conserved throughout evolution and places all Gla side chains on the same face of one .alpha. helix spaced at intervals of .apprx. 5.4 .ANG., closely paralleling the interatomic separation of Ca2+ in the HA lattice. Helical osteocalcin has greatly increased affinity for HA; thus, the Ca2+-induced structural transition may perform an informational role related to bone metabolism.