Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural insights into the vitamin K-dependent γ-carboxylation of osteocalcin

Cell Research volume 35, pages 735–749 (2025)Cite this article

Subjects

Abstract

The γ-carboxylation state of osteocalcin determines its essential functions in bone mineralization or systemic metabolism and serves as a prominent biomarker for bone health and vitamin K nutrition. This post-translational modification of glutamate residues is catalyzed by the membrane-embedded vitamin K-dependent γ-carboxylase (VKGC), which typically recognizes protein substrates through their tightly bound propeptide that triggers γ-carboxylation. However, the osteocalcin propeptide exhibits negligible affinity for VKGC. To understand the underlying molecular mechanism, we determined the cryo-electron microscopy structures of VKGC with osteocalcin carrying a native propeptide or a high-affinity variant at different carboxylation states. The structures reveal a large chamber in VKGC that maintains uncarboxylated and partially carboxylated osteocalcin in partially unfolded conformations, allowing their glutamate-rich region and C-terminal helices to engage with VKGC at multiple sites. Binding of this mature region together with the low-affinity propeptide effectively stimulates VKGC activity, similar to high-affinity propeptides that differ only in closely fitting interactions. However, the low-affinity propeptide renders osteocalcin prone to undercarboxylation at low vitamin K levels, thereby serving as a discernible biomarker. Overall, our studies reveal the unique interaction of osteocalcin with VKGC and provide a framework for designing therapeutic strategies targeting osteocalcin-related bone and metabolic disorders.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Cryo-EM structures of human VKGC with OCN.
Fig. 2: Structure domains of the VKGC–OCN complex.
Fig. 3: Binding interactions of OCN propeptides.
Fig. 4: VKGC binding of the OCN mature region.
Fig. 5: OCN propeptide-induced allosteric motions triggering VKGC epoxidase activity.
Fig. 6: Structure of VKGC with OCN in a partially carboxylated state.

Similar content being viewed by others

Data availability

Structure coordinates and cryo-EM maps have been deposited in the Protein Data Bank and the Electron Microscopy Data Bank under accession numbers 9MQE and EMD-48522 for VKGC–OCN (native propeptide) with KH2, 9MQC and EMD-48520 for VKGC–OCN (high-affinity propeptide) with KH2, 9MQB and EMD-48519 for partially carboxylated VKGC–OCN (high-affinity propeptide) with KH2. The MS data have been deposited in ProteomeXchange with the accession number PXD059315.

References

  1. Poser, J. W., Esch, F. S., Ling, N. C. & Price, P. A. Isolation and sequence of the vitamin K-dependent protein from human bone. Undercarboxylation of the first glutamic acid residue. J. Biol. Chem. 255, 8685–8691 (1980).

    Article  CAS  PubMed  Google Scholar 

  2. Al-Qtaitat, A. I. & Aldalaen, S. M. A review of non-collagenous proteins; their role in bone. Am. J. Life Sci. 2, 351–355 (2014).

    Google Scholar 

  3. Booth, S. L., Centi, A., Smith, S. R. & Gundberg, C. The role of osteocalcin in human glucose metabolism: marker or mediator?. Nat. Rev. Endocrinol. 9, 43–55 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Zoch, M. L., Clemens, T. L. & Riddle, R. C. New insights into the biology of osteocalcin. Bone 82, 42–49 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Liska, D. J. & Suttie, J. W. Location of gamma-carboxyglutamyl residues in partially carboxylated prothrombin preparations. Biochemistry 27, 8636–8641 (1988).

    Article  CAS  PubMed  Google Scholar 

  6. Hoang, Q. Q., Sicheri, F., Howard, A. J. & Yang, D. S. Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 425, 977–980 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Price, P. A., Otsuka, A. A., Poser, J. W., Kristaponis, J. & Raman, N. Characterization of a gamma-carboxyglutamic acid-containing protein from bone. Proc. Natl. Acad. Sci. USA 73, 1447–1451 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Boskey, A. L. et al. Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone 23, 187–196 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. Ducy, P. et al. Increased bone formation in osteocalcin-deficient mice. Nature 382, 448–452 (1996).

    Article  CAS  PubMed  Google Scholar 

  10. Poundarik, A. A. et al. Dilatational band formation in bone. Proc. Natl. Acad. Sci. USA 109, 19178–19183 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nikel, O., Laurencin, D., McCallum, S. A., Gundberg, C. M. & Vashishth, D. NMR investigation of the role of osteocalcin and osteopontin at the organic-inorganic interface in bone. Langmuir 29, 13873–13882 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hauschka, P. V., Lian, J. B., Cole, D. E. & Gundberg, C. M. Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiol. Rev. 69, 990–1047 (1989).

    Article  CAS  PubMed  Google Scholar 

  13. Lee, N. K. et al. Endocrine regulation of energy metabolism by the skeleton. Cell 130, 456–469 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fulzele, K. et al. Insulin receptor signaling in osteoblasts regulates postnatal bone acquisition and body composition. Cell 142, 309–319 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Oury, F. et al. Endocrine regulation of male fertility by the skeleton. Cell 144, 796–809 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Oury, F. et al. Osteocalcin regulates murine and human fertility through a pancreas-bone-testis axis. J. Clin. Invest. 123, 2421–2433 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Patterson-Buckendahl, P. et al. Decreased sensory responses in osteocalcin null mutant mice imply neuropeptide function. Cell Mol. Neurobiol. 32, 879–889 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Oury, F. et al. Maternal and offspring pools of osteocalcin influence brain development and functions. Cell 155, 228–241 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Brown, J. P. et al. Serum bone Gla-protein: a specific marker for bone formation in postmenopausal osteoporosis. Lancet 1, 1091–1093 (1984).

    Article  CAS  PubMed  Google Scholar 

  20. Booth, S. L. Roles for vitamin K beyond coagulation. Annu. Rev. Nutr. 29, 89–110 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Binkley, N. C., Krueger, D. C., Engelke, J. A., Foley, A. L. & Suttie, J. W. Vitamin K supplementation reduces serum concentrations of under-gamma-carboxylated osteocalcin in healthy young and elderly adults. Am. J. Clin. Nutr. 72, 1523–1528 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Hao, Z., Jin, D. Y., Stafford, D. W. & Tie, J. K. Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study. Haematologica 105, 2164–2173 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Murshed, M., Schinke, T., McKee, M. D. & Karsenty, G. Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins. J. Cell Biol. 165, 625–630 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mosnier, L. O., Zlokovic, B. V. & Griffin, J. H. The cytoprotective protein C pathway. Blood 109, 3161–3172 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Dahlback, B. Vitamin K-dependent protein S: beyond the protein C pathway. Semin. Thromb. Hemost. 44, 176–184 (2018).

    Article  PubMed  Google Scholar 

  26. Furie, B. & Furie, B. C. Molecular basis of vitamin K-dependent gamma-carboxylation. Blood 75, 1753–1762 (1990).

    Article  CAS  PubMed  Google Scholar 

  27. Sugiura, I., Furie, B., Walsh, C. T. & Furie, B. C. Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state. Proc. Natl. Acad. Sci. USA 94, 9069–9074 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Suttie, J. W. et al. Vitamin K-dependent carboxylase: possible role of the substrate “propeptide” as an intracellular recognition site. Proc. Natl. Acad. Sci. USA 84, 634–637 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Stanley, T. B., Jin, D.-Y., Lin, P.-J. & Stafford, D. W. The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase. J. Biol. Chem. 274, 16940–16944 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Houben, R. J. et al. Characteristics and composition of the vitamin K-dependent gamma-glutamyl carboxylase-binding domain on osteocalcin. Biochem. J. 364, 323–328 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Houben, R. J. et al. Osteocalcin binds tightly to the gamma-glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins. Biochem. J. 341, 265–269 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Morris, D. P., Stevens, R. D., Wright, D. J. & Stafford, D. W. Processive post-translational modification. Vitamin K-dependent carboxylation of a peptide substrate. J. Biol. Chem. 270, 30491–30498 (1995).

    Article  CAS  PubMed  Google Scholar 

  33. Stenina, O., Pudota, B. N., McNally, B. A., Hommema, E. L. & Berkner, K. L. Tethered processivity of the vitamin K-dependent carboxylase: factor IX is efficiently modified in a mechanism which distinguishes Gla’s from Glu’s and which accounts for comprehensive carboxylation in vivo. Biochemistry 40, 10301–10309 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Suttie, J. W. Vitamin K-dependent carboxylase. Annu. Rev. Biochem. 54, 459–477 (1985).

    Article  CAS  PubMed  Google Scholar 

  35. Berkner, K. L. Vitamin K-dependent carboxylation. Vitam. Horm. 78, 131–156 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Rost, S. et al. Compound heterozygous mutations in the γ-glutamyl carboxylase gene cause combined deficiency of all vitamin K-dependent blood coagulation factors. Br. J. Haematol. 126, 546–549 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. De Vilder, E. Y., Debacker, J. & Vanakker, O. M. GGCX-associated phenotypes: An overview in search of genotype-phenotype correlations. Int. J. Mol. Sci. 18, 240 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Cao, Q. et al. Molecular basis of vitamin-K-driven gamma-carboxylation at the membrane interface. Nature 639, 816–824 (2025).

    Article  CAS  PubMed  Google Scholar 

  39. Jorgensen, M. J. et al. Recognition site directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX. Cell 48, 185–191 (1987).

    Article  CAS  PubMed  Google Scholar 

  40. Pan, L. C. & Price, P. A. The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors. Proc. Natl. Acad. Sci. USA 82, 6109–6113 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Stanley, T. B., Humphries, J., High, K. A. & Stafford, D. W. Amino acids responsible for reduced affinities of vitamin K-dependent propeptides for the carboxylase. Biochemistry 38, 15681–15687 (1999).

    Article  CAS  PubMed  Google Scholar 

  42. Mutucumarana, V. P., Acher, F., Straight, D. L., Jin, D.-Y. & Stafford, D. W. A conserved region of human vitamin K-dependent carboxylase between residues 393 and 404 is important for its interaction with the glutamate substrate. J. Biol. Chem. 278, 46488–46493 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Rishavy, M. A. & Berkner, K. L. Insight into the coupling mechanism of the vitamin K-dependent carboxylase: mutation of histidine 160 disrupts glutamic acid carbanion formation and efficient coupling of vitamin K epoxidation to glutamic acid carboxylation. Biochemistry 47, 9836–9846 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Hao, Z. et al. gamma-Glutamyl carboxylase mutations differentially affect the biological function of vitamin K-dependent proteins. Blood 137, 533–543 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zacchi, L. F. et al. Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product. Commun. Biol. 4, 390 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kaufman, R. J., Wasley, L. C., Furie, B. C., Furie, B. & Shoemaker, C. B. Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells. J. Biol. Chem. 261, 9622–9628 (1986).

    Article  CAS  PubMed  Google Scholar 

  47. Cairns, J. R. & Price, P. A. Direct demonstration that the vitamin K-dependent bone Gla protein is incompletely gamma-carboxylated in humans. J. Bone Miner. Res. 9, 1989–1997 (1994).

    Article  CAS  PubMed  Google Scholar 

  48. Malashkevich, V. N., Almo, S. C. & Dowd, T. L. X-ray crystal structure of bovine 3 Glu-osteocalcin. Biochemistry 52, 8387–8392 (2013).

    Article  CAS  PubMed  Google Scholar 

  49. Huang, M., Furie, B. C. & Furie, B. Crystal structure of the calcium-stabilized human factor IX Gla domain bound to a conformation-specific anti-factor IX antibody. J. Biol. Chem. 279, 14338–14346 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Lin, P. J., Straight, D. L. & Stafford, D. W. Binding of the factor IX gamma-carboxyglutamic acid domain to the vitamin K-dependent gamma-glutamyl carboxylase active site induces an allosteric effect that may ensure processive carboxylation and regulate the release of carboxylated product. J. Biol. Chem. 279, 6560–6566 (2004).

    Article  CAS  PubMed  Google Scholar 

  51. Sekhri, A. et al. The conundrum of “warfarin hypersensitivity”: prolonged partial thromboplastin time from factor IX propeptide mutation. Am. J. Ther. 23, e911–e915 (2016).

    Article  PubMed  Google Scholar 

  52. Chu, K., Wu, S. M., Stanley, T., Stafford, D. W. & High, K. A. A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism. J. Clin. Invest. 98, 1619–1625 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Chu, R. et al. Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography. J. Mol. Biol. 323, 253–262 (2002).

    Article  CAS  PubMed  Google Scholar 

  54. Li, C. et al. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol. 11, 92 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  57. Rubinstein, J. L. & Brubaker, M. A. Alignment of cryo-EM movies of individual particles by optimization of image translations. J. Struct. Biol. 192, 188–195 (2015).

    Article  PubMed  Google Scholar 

  58. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. D. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  63. Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    Article  CAS  PubMed  Google Scholar 

  64. Ren, F. et al. X-ray structures of the high-affinity copper transporter Ctr1. Nat. Commun. 10, 1386 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Presnell, S. R., Tripathy, A., Lentz, B. R., Jin, D. Y. & Stafford, D. W. A novel fluorescence assay to study propeptide interaction with gamma-glutamyl carboxylase. Biochemistry 40, 11723–11733 (2001).

    Article  CAS  PubMed  Google Scholar 

  66. Lomize, A. L., Todd, S. C. & Pogozheva, I. D. Spatial arrangement of proteins in planar and curved membranes by PPM 3.0. Protein Sci. 31, 209–220 (2022).

    Article  CAS  PubMed  Google Scholar 

  67. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).

    Article  CAS  PubMed  Google Scholar 

  68. Higgins-Gruber, S. L. et al. Effect of vitamin K-dependent protein precursor propeptide, vitamin K hydroquinone and glutamate substrate binding on the γ-glutamyl carboxylase’s structure and function. J. Biol. Chem. 285, 31502–31508 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Reza Dastvan and Andrzej Krezel for providing access to HPLC systems. W.L. is supported by National Heart, Lung, and Blood Institute (R01 HL121718), the Vagelos Endowed Chair, the Established Investigator Award and Collaborative Sciences Award from American Heart Association, National Institute of General Medical Sciences (R01 GM131008), Children’s Discovery Institute (MCII 2020-854), National Institute of Allergy and Infectious Diseases (R01 AI158500), and the Forefront of Science Award from W. M. Keck Foundation. B.A.G. is supported by National Institute of Neurological Disorders and Stroke (R01 NS111997), the Eunice Kennedy Shriver National Institute of Child Health & Human Development (R01 HD106051), and National Science Foundation (CHE 2127882). Z.L. is supported by a BMB Research Seed Grant (PJ000027587) and National Institute on Aging (P30 AG066444). B.L. is supported by the Hormel Institute, University of Minnesota.

Author information

Author notes

  1. These authors contributed equally: Qing Cao, Jianjun Fan.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA

    Qing Cao, Jianjun Fan, Aaron Ammerman, Samjhana Awasthi, Zongtao Lin, Saimi Mierxiati, Benjamin A. Garcia & Weikai Li

  2. Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA

    Huaping Chen & Jinbin Xu

  3. The Hormel Institute, University of Minnesota, Austin, MN, USA

    Bin Liu

Authors
  1. Qing Cao
    View author publications

    Search author on:PubMed Google Scholar

  2. Jianjun Fan
    View author publications

    Search author on:PubMed Google Scholar

  3. Aaron Ammerman
    View author publications

    Search author on:PubMed Google Scholar

  4. Samjhana Awasthi
    View author publications

    Search author on:PubMed Google Scholar

  5. Zongtao Lin
    View author publications

    Search author on:PubMed Google Scholar

  6. Saimi Mierxiati
    View author publications

    Search author on:PubMed Google Scholar

  7. Huaping Chen
    View author publications

    Search author on:PubMed Google Scholar

  8. Jinbin Xu
    View author publications

    Search author on:PubMed Google Scholar

  9. Benjamin A. Garcia
    View author publications

    Search author on:PubMed Google Scholar

  10. Bin Liu
    View author publications

    Search author on:PubMed Google Scholar

  11. Weikai Li
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Q.C. carried out the cryo-EM, functional, and MS experiments. J.F. analyzed OCN and VKGC mutants with assistance from S.A. and S.M. A.A. and Z.L. performed the MS analyses with B.A.G.’s support. H.C. and J.X. assisted with the in vitro γ-carboxylation assay. B.L. determined the structures. W.L. conceived and directed the project, and wrote the manuscript with input from Q.C. and other authors.

Corresponding authors

Correspondence to Bin Liu or Weikai Li.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cao, Q., Fan, J., Ammerman, A. et al. Structural insights into the vitamin K-dependent γ-carboxylation of osteocalcin. Cell Res 35, 735–749 (2025). https://doi.org/10.1038/s41422-025-01161-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41422-025-01161-0

This article is cited by

Search

Advanced search

Quick links