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Structure and mechanism of vitamin-K-dependent γ-glutamyl carboxylase

Nature volume 639, pages 808–815 (2025)Cite this article

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Abstract

γ-Glutamyl carboxylase (GGCX) is the sole identified enzyme that uses vitamin K (VK) as a cofactor in humans. This protein catalyses the oxidation of VK hydroquinone to convert specific glutamate residues to γ-carboxyglutamate residues in VK-dependent proteins (VDPs), which are involved in various essential biological processes and diseases1,2,3. However, the working mechanism of GGCX remains unclear. Here we report three cryogenic electron microscopy structures of human GGCX: in the apo state, bound to osteocalcin (a VDP) and bound to VK. The propeptide of the VDP binds to the lumenal domain of GGCX, which stabilizes transmembrane helices 6 and 7 of GGCX to create the VK-binding pocket. After binding of VK, residue Lys218 in GGCX mediates the oxidation of VK hydroxyquinone, which leads to the deprotonation of glutamate residues and the construction of γ-carboxyglutamate residues. Our structural observations and results from binding and cell biological assays and molecular dynamics simulations show that a cholesterol molecule interacts with the transmembrane helices of GGCX to regulate its protein levels in cells. Together, these results establish a link between cholesterol metabolism and VK-dependent pathways.

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Fig. 1: Overall structures of GGCX and the GGCX–BGP complex.
Fig. 2: The propeptide of VDPs stabilizes the TM6 and TM7 of GGCX.
Fig. 3: VK binding and the catalytic mechanism of GGCX.
Fig. 4: Cholesterol regulates GGCX protein levels in cells.

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Data availability

The 3D cryo-EM maps for GGCX, GGCX–BGP and GGCX–BGP–MK-4 have been deposited into the Electron Microscopy Data Bank with accession numbers EMD-44912, EMD-44917 and EMD-44924, respectively. The atomic coordinates for the atomic models have been deposited into the Protein Data Bank under accession codes 9BUM, 9BUR and 9BUX. The raw cryo-EM data for GGCX, GGCX–BGP and GGCX–BGP–MK-4 have been deposited into the Electron Microscopy Public Image Archive under accession numbers EMPIAR-12453, EMPIAR-12451 and EMPIAR-12452, respectively. The MD simulation data have been deposited into Zenodo (https://doi.org/10.5281/zenodo.14150943)69. Source data are provided with this paper.

References

  1. Wu, S. M., Cheung, W. F., Frazier, D. & Stafford, D. W. Cloning and expression of the cDNA for human γ-glutamyl carboxylase. Science 254, 1634–1636 (1991).

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Furie, B., Bouchard, B. A. & Furie, B. C. Vitamin K-dependent biosynthesis of γ-carboxyglutamic acid. Blood 93, 1798–1808 (1999).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Mladenka, P. et al. Vitamin K—sources, physiological role, kinetics, deficiency, detection, therapeutic use, and toxicity. Nutr. Rev. 80, 677–698 (2022).

    Article  PubMed  Google Scholar 

  5. Stafford, D. W. The vitamin K cycle. J. Thromb. Haemost. 3, 1873–1878 (2005).

    Article  CAS  PubMed  MATH  Google Scholar 

  6. Shearer, M. J. & Okano, T. Key pathways and regulators of vitamin K function and intermediary metabolism. Annu. Rev. Nutr. 38, 127–151 (2018).

    Article  CAS  PubMed  MATH  Google Scholar 

  7. Rishavy, M. A. & Berkner, K. L. Vitamin K oxygenation, glutamate carboxylation, and processivity: defining the three critical facets of catalysis by the vitamin K-dependent carboxylase. Adv. Nutr. 3, 135–148 (2012).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  8. Li, T. et al. Identification of the gene for vitamin K epoxide reductase. Nature 427, 541–544 (2004).

    Article  ADS  CAS  PubMed  MATH  Google Scholar 

  9. Rost, S. et al. Mutations in cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 427, 537–541 (2004).

    Article  ADS  CAS  PubMed  MATH  Google Scholar 

  10. Furie, B. & Furie, B. C. The molecular basis of blood coagulation. Cell 53, 505–518 (1988).

    Article  CAS  PubMed  Google Scholar 

  11. 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 

  12. Karsenty, G. & Olson, E. N. Bone and muscle endocrine functions: unexpected paradigms of inter-organ communication. Cell 164, 1248–1256 (2016).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  13. Shearer, M. J. Vitamin K deficiency bleeding (VKDB) in early infancy. Blood Rev. 23, 49–59 (2009).

    Article  CAS  PubMed  MATH  Google Scholar 

  14. Stock, M. & Schett, G. Vitamin K-dependent proteins in skeletal development and disease. Int. J. Mol. Sci. 22, 9328 (2021).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  15. Wen, L. P., Chen, J. P., Duan, L. L. & Li, S. Z. Vitamin K-dependent proteins involved in bone and cardiovascular health. Mol. Med. Rep. 18, 3–15 (2018).

    CAS  PubMed  PubMed Central  MATH  Google Scholar 

  16. Furie, B. C. et al. The γ-carboxylation recognition site is sufficient to direct vitamin K-dependent carboxylation on all adjacent glutamate-rich region of thrombin in a propeptide–thrombin chimera. J. Biol. Chem. 272, 28258–28262 (1997).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  MATH  Google Scholar 

  18. Freedman, S. J., Furie, B. C., Furie, B. & Baleja, J. D. Structure of the calcium ion-bound γ-carboxyglutamic acid-rich domain of factor-IX. Biochemistry 34, 12126–12137 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Spyropoulos, A. C., Hayth, K. A. & Jenkins, P. Anticoagulation with anisindione in a patient with a warfarin-induced skin eruption. Pharmacotherapy 23, 533–536 (2003).

    Article  PubMed  Google Scholar 

  20. Watzka, M. et al. Bleeding and non-bleeding phenotypes in patients with gene mutations. Thromb. Res. 134, 856–865 (2014).

    Article  CAS  PubMed  MATH  Google Scholar 

  21. Tie, J., Wu, S. M., Jin, D. Y., Nicchitta, C. V. & Stafford, D. W. A topological study of the human γ-glutamyl carboxylase. Blood 96, 973–978 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Tie, J. K. et al. Characterization of vitamin K-dependent carboxylase mutations that cause bleeding and nonbleeding disorders. Blood 127, 1847–1855 (2016).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  23. Tie, J. K. et al. Determination of disulfide bond assignment of human vitamin K-dependent γ-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J. Biol. Chem. 278, 45468–45475 (2003).

    Article  CAS  PubMed  MATH  Google Scholar 

  24. Holm, L., Laiho, A., Törönen, P. & Salgado, M. DALI shines a light on remote homologs: one hundred discoveries. Protein Sci. 32, e4519 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wu, S. M., Mutucumarana, V. P., Geromanos, S. & Stafford, D. W. The propeptide binding site of the bovine γ-glutamyl carboxylase. J. Biol. Chem. 272, 11718–11722 (1997).

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  27. Lin, P. J. et al. The putative vitamin K-dependent γ-glutamyl carboxylase internal propeptide appears to be the propeptide binding site. J. Biol. Chem. 277, 28584–28591 (2002).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  29. Parker, C. H. et al. A conformational investigation of propeptide binding to the integral membrane protein γ-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry. Biochemistry 53, 1511–1520 (2014).

    Article  CAS  PubMed  MATH  Google Scholar 

  30. 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 

  31. 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 

  32. Rishavy, M. A. et al. Bronsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation. Biochemistry 45, 13239–13248 (2006).

    Article  CAS  PubMed  MATH  Google Scholar 

  33. Rishavy, M. A. et al. A new model for vitamin K-dependent carboxylation: the catalytic base that deprotonates vitamin K hydroquinone is not Cys but an activated amine. Proc. Natl Acad. Sci. USA 101, 13732–13737 (2004).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mosley, S. T., Brown, M. S., Anderson, R. G. W. & Goldstein, J. L. Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3-methylglutaryl coenzyme A reductase. J. Biol. Chem. 258, 3875–3881 (1983).

    Article  Google Scholar 

  35. Goldstein, J. L. & Brown, M. S. Regulation of the mevalonate pathway. Nature 343, 425–430 (1990).

    Article  ADS  CAS  PubMed  MATH  Google Scholar 

  36. Metherall, J. E., Goldstein, J. L., Luskey, K. L. & Brown, M. S. Loss of transcriptional repression of three sterol-regulated genes in mutant hamster cells. J. Biol. Chem. 264, 15634–15641 (1989).

    Article  CAS  PubMed  Google Scholar 

  37. Yang, T. et al. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110, 489–500 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Qi, X., Friedberg, L., De Bose-Boyd, R., Long, T. & Li, X. Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling. Nat. Chem. Biol. 16, 1368–1375 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. Vitamin K and energy transduction—a base strength amplification mechanism. Science 269, 1684–1691 (1995).

    Article  ADS  CAS  PubMed  Google Scholar 

  40. Berkner, K. L. & Pudota, B. N. Vitamin K-dependent carboxylation of the carboxylase. Proc. Natl Acad. Sci. USA 95, 466–471 (1998).

    Article  ADS  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  41. Hallgren, K. W., Zhang, D., Kinter, M., Willard, B. & Berkner, K. L. Methylation of γ-carboxylated Glu (Gla) allows detection by liquid chromatography–mass spectrometry and the identification of Gla residues in the γ-glutamyl carboxylase. J. Proteome Res. 12, 2365–2374 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. de Boer-van den Berg, M. A., Thijssen, H. H. & Vermeer, C. The in vivo effects of acenocoumarol, phenprocoumon and warfarin on vitamin K epoxide reductase and vitamin K-dependent carboxylase in various tissues of the rat. Biochim. Biophys. Acta 884, 150–157 (1986).

    Article  PubMed  Google Scholar 

  43. Tie, J. K., Jin, D. Y., Straight, D. L. & Stafford, D. W. Functional study of the vitamin K cycle in mammalian cells. Blood 117, 2967–2974 (2011).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  44. Di Minno, A. et al. Old and new oral anticoagulants: food, herbal medicines and drug interactions. Blood Rev. 31, 193–203 (2017).

    Article  PubMed  Google Scholar 

  45. Goldstein, J. L. & Brown, M. S. A century of cholesterol and coronaries: from plaques to genes to statins. Cell 161, 161–172 (2015).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  46. Undas, A., Brummel-Ziedins, K. E. & Mann, K. G. Anticoagulant effects of statins and their clinical implications. Thromb. Haemost. 111, 392–400 (2014).

    CAS  PubMed  MATH  Google Scholar 

  47. Jiang, S. Y. et al. Schnyder corneal dystrophy-associated UBIAD1 mutations cause corneal cholesterol accumulation by stabilizing HMG-CoA reductase. PLoS Genet. 15, e1008289 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Schumacher, M. M., Elsabrouty, R., Seemann, J., Jo, Y. & DeBose-Boyd, R. A. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase. eLife 4, e05560 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  50. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  MATH  Google Scholar 

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

    Article  PubMed  PubMed Central  MATH  Google Scholar 

  52. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019).

    Article  PubMed  PubMed Central  MATH  Google Scholar 

  53. 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 

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

    Article  ADS  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  55. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  ADS  PubMed  MATH  Google Scholar 

  56. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  57. Pettersen, E. F. et al. UCSF chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  MATH  Google Scholar 

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

    Article  CAS  PubMed  MATH  Google Scholar 

  59. McFarlane, M. R. et al. Scap is required for sterol synthesis and crypt growth in intestinal mucosa. J. Lipid Res. 56, 1560–1571 (2015).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  60. Li, H., Robertson, A. D. & Jensen, J. H. Very fast empirical prediction and rationalization of protein pK values. Proteins 61, 704–721 (2005).

    Article  CAS  PubMed  MATH  Google Scholar 

  61. Brooks, B. R. et al. Charmm—a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217 (1983).

    Article  CAS  MATH  Google Scholar 

  62. Vanommeslaeghe, K. et al. CHARMM general force field: a force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J. Comput. Chem. 31, 671–690 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 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 

  64. Lomize, M. A., Lomize, A. L., Pogozheva, I. D. & Mosberg, H. I. OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006).

    Article  CAS  PubMed  Google Scholar 

  65. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  ADS  CAS  Google Scholar 

  66. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    Article  CAS  PubMed  PubMed Central  MATH  Google Scholar 

  67. MacKerell, A. D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998).

    Article  CAS  PubMed  MATH  Google Scholar 

  68. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593 (1995).

    Article  ADS  CAS  MATH  Google Scholar 

  69. Elghobashi-Meinhardt, N. Structure and mechanism of vitamin K-dependent gamma-glutamyl carboxylase (GGCX) MD simulation data. Zenodo https://doi.org/10.5281/zenodo.14150943 (2024).

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Acknowledgements

We thank M. Brown, J. Goldstein, E. Olson, B. Wang, R. DeBose-Boyd and X. Li for discussions; D. Stoddard, J. Martinez-Diaz, C. Baker, R. Welch, C. Brautigam, S. Tso, L. Esparza and Y. Qin for technical support; A. Lemoff at the UTSW Proteomics Core for mass spectrometry analysis; and P. Schmiege for editing the manuscript. Cryo-EM data were collected at the UT Southwestern Medical Center Cryo-EM Facility (funded in part by CPRIT Award RP220582). This work was supported by NIH P01HL160487 (to R.W.) and the Endowed Scholars Program in Medical Science of UT Southwestern Medical Center and CPRIT (RR230054 to X.Q.). N.E.-M. acknowledges the Irish Centre for High-End Computing (ICHEC) for the provision of computational facilities and support. X.Q. is a CPRIT Scholar and a Michael L. Rosenberg Scholar in Medical Research of UT Southwestern Medical Center.

Author information

Authors and Affiliations

  1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Rong Wang

  2. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Baozhi Chen, Alyssa Ayala, Ning Zhou & Xiaofeng Qi

  3. School of Chemistry, University College Dublin, Dublin, Ireland

    Nadia Elghobashi-Meinhardt

  4. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Jian-Ke Tie

  5. Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, TX, USA

    Xiaofeng Qi

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  1. Rong Wang
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Contributions

X.Q., together with R.W., conceived the project and designed the research. R.W., B.C. and X.Q. purified the proteins. R.W. and X.Q. carried out cryo-EM work and refined the structures. R.W., B.C., A.A., N.Z. and X.Q. performed the functional assays. N.E.-M. conducted the MD simulations. J.-K.T. provided the GGCX-knockout HEK293 cell line. R.W., N.E.-M. and X.Q. analysed the data. X.Q. wrote the manuscript.

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Correspondence to Xiaofeng Qi.

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Extended data figures and tables

Extended Data Fig. 1 Biochemical and cryo-EM analyses of GGCX and the GGCX–BGP complex.

a, Representative gel-filtration chromatograms of GGCX purified without any compound (blue), with menaquinone-4 (MK-4) (orange), with Anisindione (teal) and with vitamin K1 epoxide (VKO1) and Warfarin (magenta). The SDS-PAGE gels of the fractions used for cryo-EM study are shown. b, Preliminary cryo-EM maps of GGCX purified without any compound, with MK-4, and with Anisindione. The cleft of the detergent micelle in each map is indicated by a dashed line. c, Summary of cryo-EM data processing procedures of GGCX purified with VKO1 and Warfarin. d, Fourier shell correlation (FSC) curves between two half maps of GGCX purified with VKO1 and Warfarin. e, Local resolution of the cryo-EM map of GGCX purified with VKO1 and Warfarin. Maps are colored according to local resolution, estimated using cryoSPARC. f, Cryo-EM maps of the major structural elements of GGCX purified with VKO1 and Warfarin, as well as the cryo-EM map of cholesterol (CLR). TM, transmembrane helix. g, GGCX structure docked into the low-resolution cryo-EM map of GGCX purified with MK-4. The cholesterol density and the surrounding residues are shown in the lower panel. h, Comparison of the gel-filtration chromatograms of GGCX–BGP complex (green) and GGCX (blue) purified without any compound. The ratio of protein used for cryo-EM study in total protein is around 3.7% in GGCX and 23.4% in GGCX–BGP samples. The SDS-PAGE gel of the fractions of the GGCX–BGP complex used for cryo-EM study is shown. i, The SDS-PAGE gel and western blot result of the GGCX–BGP complexes expressed and purified with either MK-4 (GGCX–BGP–MK-4) or Anisindione (GGCX–BGP–ANI). The BGP bands in the SDS-PAGE gel were subjected for Mass spectrometry (MS) analysis. The western blot was performed with the same samples using the antibody specifically recognizing the Gla68 of BGP. BGPGla, carboxylated BGP protein. j, Mass spectrometry results for detecting the carboxylation of BGP in GGCX–BGP–MK-4 and GGCX–BGP–ANI samples.

Source Data

Extended Data Fig. 2 Cryo-EM analyses of the GGCX–BGP complex.

a, Summary of cryo-EM data processing procedures of the GGCX–BGP complex. b, Fourier shell correlation (FSC) curves between two half maps of the GGCX–BGP complex. c, Local resolution of cryo-EM map of the GGCX–BGP complex. Maps are colored according to local resolution, estimated using cryoSPARC. d, Cryo-EM map of the GGCX–BGP (unsharpened) at different contour levels. The strong cholesterol (yellow) is visible at a high contour level, while the unassigned lipid/detergent densities (cyan) are invisible. e, Cryo-EM maps of the major structural elements of GGCX and BGP, as well as the cholesterol and Phosphatidylcholine (PC) in GGCX–BGP complex.

Extended Data Fig. 3 Sequence alignment of GGCX.

Sequence alignment of GGCX proteins from Homo sapiens (NP_000812), Mus musculus (NP_062776), Parus major (XP_015504226), Xenopus tropicalis (NP_001116905), and Danio rerio (XP_003199339). The residue numbers and major structural elements are shown above the sequence. The conserved residues are colored in red.

Extended Data Fig. 4 Sequence alignment of human Gla proteins and the structures of Gla domains.

a, Sequence alignment of the propeptide and Gla domain of human Prothrombin (Factor II, F2), Factor VII (F7), Factor IX (F9), Factor X (F10), Protein C, Protein S, Protein Z, Transmembrane Gla proteins (TMGs) 1-4, Growth arrest-specific gene 6 (Gas6), Matrix Gla protein (MGP) and BGP. The propeptide and Gla domain are indicated by brown and orange lines, respectively. The conserved -16, -10 and -6 positions of the propeptide are labeled above the sequence. The disulfide bonds in Gla domains are indicated by black lines. The residues of BGP observed in the GGCX–BGP structure are indicated by a red rectangle. b and c, Crystal structures of the mature Gla domain of human F9 (PDB: 1NL0, light orange) (b) and mature porcine BGP (PDB: 1Q8H, grey) (c). The side chains of Gla residues are shown as sticks and Ca2+ ions are shown as green balls. d, Cryo-EM structure of unmature human BGP in GGCX–BGP complex. The propeptide and Gla domain are shown in brown and orange, respectively. The invisible residues are indicated by dashed lines.

Extended Data Fig. 5 Models of the propeptides from other VDPs binding to GGCX.

a, Propeptide of Prothrombin (residue 26-38) modeled to GGCX. The potential interactions are indicated by dashed lines. The residues of Prothrombin are denoted by underlining. b, Propeptide of F9 (residue 29-41) modeled to GGCX. The potential interactions are indicated by dashed lines. The residues of F9 are denoted by underlining. c, Propeptide of Protein C (residue 25-37) modeled to GGCX. The potential interactions are indicated by dashed lines. The residues of Protein C are denoted by underlining. d, Propeptide of MGP (residue 32-44) modeled to GGCX. The potential interactions are indicated by dashed lines. The residues of MGP are denoted by underlining.

Extended Data Fig. 6 Cryo-EM analyses of GGCX–BGP–MK-4.

a, Summary of cryo-EM data processing procedures of the GGCX–BGP–MK-4 complex. b, Fourier shell correlation (FSC) curves between two half maps of the GGCX–BGP–MK-4 complex. c, Local resolution of cryo-EM map of the GGCX–BGP–MK-4 complex. Maps are colored according to local resolution, estimated using cryoSPARC. d, Cryo-EM map of GGCX–BGP–MK-4 (unsharpened) at different contour levels. The strong cholesterol (yellow) is visible at a high contour level, while the unassigned lipid/detergent densities (cyan) are invisible. e, Comparison of the Gla-binding site in GGCX–BGP and GGCX–BGP–MK-4 cryo-EM maps. GGCX, the propeptide and Gla domain of BGP were colored in grey, brown, and orange, respectively. The Gla peptide observed in GGCX–BGP structure but not in GGCX–BGP–MK-4 structure was indicated by a red arrow. f, Cryo-EM maps of the major structural elements of GGCX and BGP, as well as the cholesterol and PC in GGCX–BGP–MK-4 complex.

Extended Data Fig. 7 Chemical structures of VKs and Warfarin modeled to the VK-binding pocket in GGCX.

a, Structure of VK1, VK2 family and VK3. b, Warfarin modeled into the VK-binding pocket. The head group of Warfarin was aligned to that of MKO-4. The potential steric clash between GGCX and Warfarin is indicated by a dashed oval. The chemical structure of Warfarin is shown.

Extended Data Fig. 8 Model of GGCX C-terminus self-carboxylation.

a, The flexible C-terminus of GGCX (residues 726-758) is modeled into the center cavity of GGCX. Glu733, Glu757 and Glu748 are shown and Glu748 is modeled into the reaction center. MKH2-4, MK-4 hydroquinone. b, the top view of a.

Extended Data Table 1 Cryo-EM data collection refinement, and validation statistics
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Supplementary information

Supplementary Information

This file contains Supplementary Fig. 1 (uncropped gels) and Supplementary Table 1.

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Supplementary Video 1

MD simulations of GGCX without cholesterol. MD simulations were performed for 200 ns. POPC is shown as sticks.

Supplementary Video 2

MD simulations of GGCX with cholesterol. MD simulations were performed for 200 ns. POPC and cholesterol are shown as sticks.

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Wang, R., Chen, B., Elghobashi-Meinhardt, N. et al. Structure and mechanism of vitamin-K-dependent γ-glutamyl carboxylase. Nature 639, 808–815 (2025). https://doi.org/10.1038/s41586-024-08484-9

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