The Three-Dimensional Structure of Bovine Calcium Ion-Bound Osteocalcin Using 1H NMR Spectroscopy†

The family of vitamin K-dependent γ-carboxylated calcium binding proteins is important in a variety of tissues and cellular functions. The formation of γ-carboxyglutamic acid (Gla)1 in the liver derived blood clotting factors, for example, results in calcium-dependent conformational changes in the proteins and functionally important interactions with acidic phospholipid surfaces (1–3). The predominant Gla protein found in bone matrix is osteocalcin (4, 5), a small Ca2+ binding protein containing three Gla residues which are thought to facilitate the protein’s adsorption to hydroxy-apatite (6). Because of its unique calcium binding properties, early functional studies suggested that osteocalcin controlled the nucleation or deposition of mineral in bone (7–10). However, more recent evidence indicates that osteocalcin is not related to events which allow mineral deposition to occur but, rather, that it participates in regulation of mineralization or bone turnover. For example, several studies have suggested that osteocalcin functions as a matrix signal for resorption through the recruitment and differentiation of bone resorbing cells (11–15). On the other hand, the osteocalcin knock-out mouse has been shown to have an increased bone mass and increased bone formation rates, implying that osteocalcin may function to limit bone formation (16). Further studies on the knock-out mouse also suggest a role for osteocalcin in mineral maturation and bone remodeling (17). Despite the increase in functional knowledge, structural information on the protein is limited. In all species examined, the central portion of the molecule is strongly conserved (18). This region includes a disulfide loop (Cys23–Cys29) and three γ-carboxyglutamic acid residues (Gla) at positions 17, 21, and 24. Circular dichroism (CD) and 1H NMR studies show that the apo-protein is in a random coil conformation while the addition of millimolar levels of Ca2+ induces an R-helical conformation in the molecule (19–23). Empirical modeling predicts two antiparallel α-helical domains, several β-turns, and a β-sheet structure resulting in an accessible COOH terminus (19). In this model, spatial projections of the side chains of the three Gla residues coincide with the spacing between the Ca2+ atoms in the hydroxyapatite crystal (24) allowing for binding to the bone via uncoordinated Gla carboxyl groups. At present there is no high-resolution structure of osteocalcin nor of any noncollagenous bone protein. This is in sharp contrast to other calcium binding proteins such as calmodulin (25, 26), troponin C (27, 28), and calbindin (29) or other Gla-containing proteins (1–3). A high-resolution structure of Ca2+-osteocalcin will provide information on a new class of proteins. It may also shed light on the function of the protein and its interaction with bone mineral at the molecular level. The purpose of this study is to determine the high-resolution 3D structure of bovine Ca2+-osteocalcin using 2D 1H NMR techniques.