The three-dimensional structure of bovine calcium ion-bound osteocalcin using 1H NMR spectroscopy

Structural information on osteocalcin or other noncollagenous bone proteins is very limited. We have solved the three-dimensional structure of calcium bound osteocalcin using (1)H 2D NMR techniques and proposed a mechanism for mineral binding. The protons in the 49 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. Distance constraints, dihedral angle constraints, hydrogen bonds, and (1)H and (13)C chemical shifts were all used to calculate a family of 13 structures. The tertiary structure of the protein consisted of an unstructured N terminus and a C-terminal loop (residues 16-49) formed by long-range hydrophobic interactions. Elements of secondary structure within residues 16-49 include type III turns (residues 20-25) and two alpha-helical regions (residues 27-35 and 41-44). The three Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm-molecular dynamics simulation approach was used to place three calcium atoms on the NMR-derived structure. One calcium atom was coordinated by three side chain oxygen atoms, two from Asp30, and one from Gla24. The second calcium atom was coordinated to four oxygen atoms, two from the side chain in Gla 24, and two from the side chain of Gla 21. The third calcium atom was coordinated to two oxygen atoms of the side chain of Gla17. The best correlation of the distances between the uncoordinated Gla oxygen atoms is with the intercalcium distance of 9.43 A in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.     

The (1)H NMR structure of bovine Pb(2+)-osteocalcin and implications for lead toxicity

Structural information on the effect of Pb(2+) on proteins under physiologically relevant conditions is largely unknown. We have previously shown that low levels of lead increased the amount of osteocalcin bound to hydroxyapatite (BBA 1535:153). This suggested that lead induced a more compact structure in the protein. We have determined the 3D structure of Pb(2+)-osteocalcin (49 amino acids), a bone protein from a target tissue, using (1)H 2D NMR techniques. Lead, at a stoichiometry of only 1:1, induced a similar fold in the protein as that induced by Ca(2+) at a stoichiometry of 3:1. The structure consisted of an unstructured N-terminus and an ordered C-terminal consisting of a hydrophobic core (residues 16-49). The genetic algorithm-molecular dynamics simulation predicted the lead ion was coordinated by the Gla 24 and Gla 21 residues. It is proposed that mineral binding occurs via uncoordinated Gla oxygen ions binding to calcium in hydroxyapatite. A comparison of Pb(2+)- and Ca(2+)-osteocalcin suggests Pb(2+), at a lower stoichiometry, may induce similar conformational changes in proteins and subsequent molecular processes normally controlled by calcium alone. This may contribute to a molecular mechanism of lead toxicity for calcium binding proteins. Lead exposure may alter the amount of mineral bound osteocalcin and contribute to abnormal bone remodeling.     

Structure,activity, and distribution of fish osteocalcin

Osteocalcin (bone Gla protein) is an extracellular matrix protein synthesized by osteoblasts that is a marker of bone. Osteocalcin probably originated in the ancestors of Teleostei or bony fish and of the Tetrapoda or amphibians, reptiles, birds, and mammals. We have characterized the Cyprinus carpio (carp) osteocalcin for mineral binding to hydroxyapatite, amino acid sequence, and extent of secondary structure. Hydroxyapatite binding is enhanced in the presence of calcium. The alpha-helical content of teleost osteocalcin increases and beta-sheet structure decreases upon calcium binding, similar to findings in calf osteocalcin. The gene structure and primary sequence of prepro-osteocalcin from 2 pufferfish compared with carp shows that there are many conserved features in teleost osteocalcin genes. Using an immunoassay for carp osteocalcin, we determined that the relative content of osteocalcin is highest in dorsal fin spines and other bones and lowest in scales. The carp osteocalcin antibodies, cross-reactive to other species of fish, were used to study the role of osteocalcin in teleost model systems.     

The presence of γ-carboxyglutamic acid in the proteins associated with ectopic calcification

γ-Carboxyglutamic acid (Gla) is identified in the proteins associated with several types of ectopic calcifications in which hydroxyapatite is the major mineral component. These included the calcified skin and subcutaneous plaques from a patient with dermatomyositis, the calcium containing material extruded from the skin of a patient with scleroderma, and the calcified, atheromatous plaques from aorta. Alkaline hydrolysis of biopsy material from these and from normal tissue revealed the presence of Gla only in the plaque specimens. Since a γ-carboxyglutamic acid-containing protein is normally present in bone and absent in unmineralized tissues, the presence of Gla in soft tissue calcifications is a potentially significant finding, especially in view of its known calcium and phospholipid binding properties.     

Calcium-dependent alpha-helical structure in osteocalcin

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.     

Influence of specific gamma-carboxyglutamic acid residues on the integrity of the calcium-dependent conformation of human protein C.

The concentration of Ca2+ that produced 50% of the saturable intrinsic fluorescence change (C50) of wild-type (wt) recombinant (r) human protein C (PC) was 0.40 mM. The C50 for Ca2+ increased < 2.5-fold for the following r-PC variants (Gla is gamma-carboxyglutamic acid): [Gla6-->Asp]r-PC, [Gla7-->Asp]r-PC, [Gla14-->Asp]r-PC, [Gla19-->Asp]r-PC, or [Gla25-->Asp]r-PC, and approximately 4-6-fold for [Gla20-->Asp]r-PC and [Gla29-->Asp]r-PC. Much more dramatic increases in the C50 for Ca2+ were observed for [Gla16-->Asp]r-PC (> 75-fold) and [Gla26-->Asp]r-PC (ca. 30-fold). A substantially larger maximum fluorescence change (> 3-fold) as compared to that for wtr-PC, was also found in the case of the Ca2+/[Gla16-->Asp]r-PC complex, suggesting that the final Ca(2+)-induced conformation for this variant is dissimilar to that for wtr-PC and the above mutants. When a mutation was constructed at Arg15 ([Arg15-->Leu]r-PC), a residue conserved in all Gla-containing coagulation proteins, no fluorescence alteration occurred upon addition of Ca2+. The C50 for Ca2+ for promotion of the binding of the Ca(2+)-dependent, Gla-domain-directed, conformational monoclonal antibodies, JTC-1 and JTC-3, to wtr-PC was 3.0 and 4.0 mM, respectively. A similar C50 value was found for [Gla25-->Asp]r-PC. In the case of each antibody, approximately 4-6-fold higher C50 values for Ca2+ were found for the mutants; [Gla14-->Asp]r-PC, [Gla19-->Asp]r-PC, and [Gla29-->Asp]r-PC. Ca2+ did not promote binding of either of these antibodies to the following variants; [Gla6-->Asp]r-PC, [Gla7-->Asp] r-PC, [Arg15-->Leu]r-PC, [Gla16-->Asp]r-PC, [Gla20-->Asp]r-PC, and [Gla26-->Asp]r-PC. The results of this study suggest that adoption of the Ca(2+)-dependent conformation of PC is greatly dependent upon the presence of specific essential Gla residues, particularly those, namely Gla16 and Gla26, shown in the crystal structure of the prothrombin Gla domain/Ca2+ complex to be involved with coordination of Ca2+ ions not exposed to the surface. Of similar importance is Arg15. On the other hand, Gla residues at positions 14 and 19 are much less important in directing this same conformation. This finding is readily reconciled with the above crystal structure, which shows that these latter 2 residues are mainly responsible for coordination of a surface-exposed Ca2+ that is present at the end of the Ca(2+)-ion channel.     

Laser Raman spectroscopy of calf bone Gla protein

The Raman spectra of solid calf bone Gla protein in its native state, decarboxylated, with reduced disulfide bond, and as the calcium salt have been obtained. The amide I and III bands are consistent with the presence of alpha-helical, antiparallel beta-sheet, and random-coil regions in all four forms of bone Gla protein. Random coil appears to be the prevailing conformation. The protein conformation in the calcium salt exhibits an increased alpha-helix character compared to the native protein. No significant differences in the backbone conformation are observed among the native, decarboxylated, and reduced forms of bone Gla protein. The Raman band at 504 cm-1, due to the disulfide stretching vibration in native bone Gla protein, is unchanged upon decarboxylation and binding of Ca2+ to the protein, indicating the absence of any changes in the conformation around the disulfide bond in these protein species. The tryptophan and most of the tyrosine residues appear to be 'exposed' rather than 'buried' in the native protein. The environment of at least one of the phenylalanine residues changes when Ca2+ is bound to bone Gla protein. A small change also appears to take place in the environment of at least one of the tyrosine residues upon Ca2+-binding or reduction of the disulfide bond.     

Bone markers during a 6-month space flight: effects of vitamin K supplementation

Rapid bone loss is a serious health problem for astronauts during long lasting missions in space. We have recorded the changes of biochemical markers for bone metabolism in one of the astronauts during the 6-month space flight of the EUROMIR-95 mission. Immediately after launch both bone resorption markers and urinary calcium excretion increased about two fold, whereas bone formation markers remained unchanged. After 12 1/2 weeks the astronaut received vitamin K1 (10 mg/day for 6 weeks). Vitamin K is known to be involved in the formation of gamma-carboxyglutamate (Gla) in proteins, such as the calcium-binding bone Gla-proteins osteocalcin and matrix Gla-protein. Concomitant with the start of vitamin K treatment, the calcium-binding capacity of osteocalcin increased, and so did the urinary excretion of free Gla. This is suggestive for a subclinical vitamin K-deficiency in the astronaut before vitamin K-supplementation. During periods of high vitamin K status markers for bone formation (osteocalcin and bone alkaline phosphatase) had increased as compared to the first part of the flight. The mean increases were 14 and 23%, respectively. Our data suggest that increased intake of vitamin K may contribute to counteracting microgravity-induced loss of bone mass during long lasting space missions, but need confirmation in more astronauts.     

Correlation of the appearance of γ-carboxyglutamic acid with the onset of mineralization in developing endochondral bone

γ-Carboxyglutamic acid (Gla) is a constituent of the non-collagenous bone protein osteocalcin. The appearance of γ-carboxyglutamic acid during $\text{de}\text{novo}$ differentiation and development of endochondral bone has been correlated with the onset of mineralization. Discrete stages of endochondral bone development were studied by subcutaneous implantation of demineralized rat diaphyseal bone matrix. Residual Gla in acid-demineralized bone matrix was lost rapidly on implantation. Gla levels were basal during mesenchymal cell proliferation (day 3) and chondrogenesis (days 5–7). Gla and calcium levels began to increase during cartilage mineralization (day 9) and continuously increased after day 10 concomitant with bone differentiation.     

Modeling zymogen protein C

A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the gamma-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII(a)/tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX(a). The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158-169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII(a) and IX(a). The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II(a)/thrombomodulin interaction.     

Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.     

A calcium-driven conformational switch of the N-terminal and core domains of annexin A1

In 1993, Huber and co-workers published the structure of an N-terminally truncated version of human annexin A1 lacking the first 32 amino acid residues (PDB code: 1AIN). In 2001, we reported the structure of full-length porcine annexin A1 including the N-terminal domain in the absence of calcium ions (PDB code: 1HM6). The latter structure did not reflect a typical annexin core fold, but rather a surprising interaction of the N-terminal domain and the core domain. Comparing these two structures revealed that in the full-length structure the first 12 residues of the N-terminal domain insert into the core of the protein, thereby replacing and unwinding one of the alpha-helices (helix D in repeat 3) that is involved in calcium binding. We hypothesized that this structure in the absence of calcium ions represents the inactive form of the protein. Furthermore, we proposed that upon calcium binding, the N-terminal domain would be expelled from the core domain and that the core D-helix would reform in the proper conformation for calcium coordination. Herein, we report the X-ray structure of full-length porcine annexin A1 in the presence of calcium. This new structure shows a typical annexin core structure as we hypothesized, with the D-helix back in place for calcium coordination while parts of the now exposed N-terminal domain are disordered. We could locate eight calcium ions in this structure, two of which are octa-coordinated and two of which were not observed in the structure of the N-terminally truncated annexin A1. Possible implications of this calcium-induced conformational switch for the membrane aggregation properties of annexin A1 will be discussed.     

The binding of Gla-containing proteins to phospholipids

It is demonstrated here that osteocalcin, the Gla-containing protein from bone, is unable to interfere with the binding of the blood coagulation factors to phospholipid vesicles. Therefore, it seems that besides the Gla residues other structural features of the coagulation factors are required for their effective binding to phospholipid surfaces.     

Osteocalcin induces chemotaxis, secretion of matrix proteins, and calcium-mediated intracellular signaling in human osteoclast-like cells

Osteocalcin, also called Bone Gla Protein (BGP), is the most abundant of the non-collagenous proteins of bone produced by osteoblasts. It consists of a single chain of 46-50 amino acids, according to the species, and contains three vitamin K-dependent gamma-carboxyglutamic acid residues (GLA), involved in its binding to calcium and hydroxylapatite. Accumulating evidences suggest its involvement in bone remodeling, its physiological role, however, is still unclear. In this study the adhesion properties and the biological effects of osteocalcin on osteoclasts have been analyzed using as an experimental model, human osteoclast-like cells derived from giant cell tumors of bone (GCT). Osteocalcin promoted adhesion and spreading of these cells, triggering the release of bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN), that in turn induced the clustering in focal adhesions of beta 1 and beta 3 integrin chains. Spreading was dependent upon the synthesis of these proteins. In fact, when the cells were incubated in the presence of monensin during the adhesion assay, they still adhered but spreading did not occur, focal adhesions disappeared and BSP, OPN, and FN were accumulated in intracellular granules. Furthermore osteocalcin induced chemotaxis in a dose-dependent manner. The action of BGP on osteoclasts was mediated by an intracellular calcium increase due to release from thapsigargin-sensitive stores. These results provide evidences that BGP exerts a role in the resorption process, inducing intracellular signaling, migration and adhesion, followed by synthesis and secretion of endogenous proteins.     

The metal-free and calcium-bound structures of a gamma-carboxyglutamic acid-containing contryphan from Conus marmoreus, glacontryphan-M

Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca(2+) channels expressed in mouse pancreatic beta-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453-32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a D-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two gamma-carboxyglutamic acid (Gla) residues, formed by post-translational modification of glutamic acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional (1)H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys(11)-Cys(5)-Pro(6) region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla(2) and Ser(3), moves away from the His(8) and Trp(10) aromatic rings and the alignment of the D-Trp(7) and His(8) aromatic rings with respect to the Trp(10) rings is altered. The blockage of L-type voltage-gated Ca(2+) channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone C alpha conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 A, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation.     

Structure of Ca2+ prothrombin fragment 1 including the conformation of the Gla domain

The structure of Ca2+ prothrombin fragment 1 has been solved at 2.8-A resolution by X-ray crystallographic methods. Most of the Gla domain of fragment 1 (residues 1-48), which is high homologous with the N-terminal regions of six other blood proteins, cannot be identified in the electron density map of the apo structure. This is not the case when crystals are grown in the presence of Ca2+ ions where the Gla domain exhibits a well-defined folded structure. The folding of the Gla domain is dominated by secondary structure: (a) 3.0 turns of alpha-helix (25%) and (b) five short beta-strands arranged into two beta-structural units (40%). The Cys18-Cys23 disulfide of the small conserved loop of Gla domains is close to a cluster of conserved aromatic residues. The resulting interaction is probably responsible for the fluorescence quenching event accompanying Ca2+ ion binding. Since the Gla domain approximates a discoid, all the Gla residues are easily accessible to solvent. The arrangement of the paired Gla residues (7-8, 20-21, 26-27) is highly suggestive in that they essentially line one edge of the Gla domain creating a potentially intense electronegative environment. This region might well be that associated with phospholipid binding. The kringle structure of Ca2+ fragment 1 is essentially indistinguishable from that of the apoprotein at this stage.     

Alterations of the gamma-carboxyglutamic acid and osteocalcin concentrations in vitamin D-deficient chick bone

The content of osteocalcin and protein bound gamma-carboxyglutamic acid (Gla) was studied as a function of bone maturation and mineralization in normal and vitamin D-deficient, rachitic chickens. The Gla/Ca2+ ratio was elevated in rachitic bone, particularly in the most undermineralized regions. For example, there is a 10- to 20-fold elevation in Gla/Ca2+ in the newly synthesized, least mineralized rachitic bone fraction, which progressively decreases to a 1.5-fold elevation in the most highly mineralized areas of rachitic tissue. Osteocalcin, which is the principal Gla-containing protein of mature bone, was quantitated by radioimmunoassay using specific antiserum to the 5670-dalton chicken protein. Surprisingly, the osteocalcin concentration is decreased 50% in vitamin D-deficient bone. From this we infer that accumulated Gla-containing protein in vitamin D-deficient and poorly mineralized bone may possibly represent a precursor of osteocalcin.     

Structural and functional characteristics of activated human factor IX after chemical modification of gamma-carboxyglutamic acid residues

Activated human factor IX (factor IXa) was treated under mildly acidic conditions with a mixture of formaldehyde and morpholine. This reagent has been shown to react preferentially with gamma-carboxyglutamyl (Gla) residues and to convert these residues to gamma-methyleneglutamyl residues (Wright, S.F., Bourne, C.D., Hoke, R.A., Koehler, K.A., and Hiskey, R.G. (1984) Anal. Biochem. 139, 82-90). The modified enzyme was evaluated for coagulant activity and calcium-dependent fluorescence quenching. [14C]Formaldehyde was employed to allow quantitation of the modification and to facilitate localization of the modified residues in the primary structure of factor IXa. In the presence of the [14C]formaldehyde/morpholine reagent, factor IXa rapidly lost coagulant activity, which corresponded to incorporation of radiolabel. Examination of the relationship between protein modification (radiolabel incorporation) and the loss of coagulant activity suggested that modification of 1 mol of Gla/mol of factor IXa results in complete loss of factor IXa coagulant activity. Primary structure analysis of the radioactivity labeled factor IXa suggested that modification of any one of 11 Gla residues was responsible for the loss of coagulant activity. In the presence of calcium, modified factor IXa exhibited a smaller Gla-dependent decrease in protein fluorescence than native factor IXa, but the Gla-independent fluorescence change was the same for both proteins. It therefore appears that the Gla domain of factor IXa must be completely intact for the enzyme to undergo a functionally important calcium-dependent conformational change necessary for coagulant activity.