Calcium-dependent .alpha.-helical structure in osteocalcin

Abstract

Osteocalcin is an abundant Ca2+-binding protein of bone containing three residues of vitamin K dependent gamma-carboxyglutamic acid (Gla) among its 49 (human, monkey, cow) or 50 (chicken) amino acids. Gla side chains participate directly in the binding of Ca2+ ions and the adsorption of osteocalcin to hydroxylapatite (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 (1) requires an intact disulfide bridge, (2) is proportionally diminished when Gla residues are decarboxylated to Glu, (3) is insensitive to 1.5 m NaCl, and (4) 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 two opposing alpha-helical domains of 9-12 residues each, connected by a bea turn and stabilized by the Cys23-Cys29 disulfide bond. Cation binding permits realization of the full alph a-helical potential by partial neutralization of high anionic charge in the helical domains. Periodic Gla occurrence at positions 17, 21, and 24 has been strongly conserved throughout evolution and places all Gla side chains on the same face of one alpha helix spaced at intervals of approximately 5.4 A, 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.

Structural Evidence of a Fourth Gla Residue in Fish Osteocalcin: Biological Implications,

“…The spatial distribution of these residues has been recognized to be consistent with the interatomic distances of calcium sites in hydroxyapatite crystals (15,26,30), confirming the proposed biological role for osteocalcin as a controlling agent in bone.…”

“…Subsequent studies on the osteocalcin knockout mice using Fourier transform infrared microspectroscopy (FT-IRM) indicated an apparent impaired bone maturation in these animals, implying a role for osteocalcin in stimulating bone mineral maturation (23). Despite this growing block of information, much remained to be understood concerning the molecular mechanism of action of this protein and the way it could interact in ViVo with mineral ions and in particular with calcium.Circular dichroism (CD) (15,24,25) and 1 H NMR (24, 26) studies were performed to elucidate the structure of avian † During part of …”

“…These Gla residues are thought to facilitate adsorption of osteocalcin to hydroxyapatite (10,14,15) and are located in the central, conserved portion of the molecule. In contrast, its N-terminal part exhibits considerable sequence variation (4,8,9,13).…”

“…Circular dichroism (CD) (15,24,25) and 1 H NMR (24, 26) studies were performed to elucidate the structure of avian † During part of…”

“…Circular dichroism (CD) (15,24,25) and 1 H NMR (24,26) studies were performed to elucidate the structure of avian † During part of this work, D.C.S. was the recipient of a PRODEP fellowship awarded by the Portuguese Ministry of Education.…”