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Molecular basis of vitamin-K-driven γ-carboxylation at the membrane interface

Nature volume 639, pages 816–824 (2025)Cite this article

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Abstract

The γ-carboxylation of glutamate residues enables Ca2+-mediated membrane assembly of protein complexes that support broad physiological functions, including haemostasis, calcium homeostasis, immune response and endocrine regulation1,2,3,4. Modulating γ-carboxylation levels provides prevalent treatments for haemorrhagic and thromboembolic diseases5. This unique post-translational modification requires vitamin K hydroquinone (KH2) to drive highly demanding reactions6 catalysed by the membrane-integrated γ-carboxylase (VKGC). Here, to decipher the underlying mechanisms, we determined cryo-electron microscopy structures of human VKGC in unbound form, with KH2 and four haemostatic and non-haemostatic proteins possessing propeptides and glutamate-rich domains in different carboxylation states. VKGC recognizes substrate proteins through knob-and-hole interactions with propeptides, thereby bringing tethered glutamate-containing segments for processive carboxylation within a large chamber that provides steric control. Propeptide binding also triggers a global conformational change to signal VKGC activation. Through sequential deprotonation and KH2 epoxidation, VKGC generates a free hydroxide ion as an exceptionally strong base that is required to deprotonate the γ-carbon of glutamate for CO2 addition. The diffusion of this superbase—protected and guided by a sealed hydrophobic tunnel—elegantly resolves the challenge of coupling KH2 epoxidation to γ-carboxylation across the membrane interface. These structural insights and extensive functional experiments advance membrane enzymology and propel the development of treatments for γ-carboxylation disorders.

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Fig. 1: Cryo-EM structure of VKGC with VKDPs.
Fig. 2: Structural components of VKGC.
Fig. 3: Propeptide recognition.
Fig. 4: The γ-carboxylation site.
Fig. 5: Coupling of KH2 epoxidation with γ-carboxylation and propeptide-induced VKGC activation.

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Data availability

The cryo-EM maps have been deposited into the Electron Microscopy Data Bank under accession numbers EMD-44939 (apo VKGC), EMD-44941 (VKGC–TMG2), EMD-44940 (VKGC–TMG2 with KH2), EMD-44935 (VKGC–FIX with KH2), EMD-44936 (VKGC–FX with KH2), EMD-44937 (VKGC–PC with KH2) and EMD-44942 (partially carboxylated VKGC–FIX with KH2). The coordinates have been deposited at the PDB under accession numbers 9BVO (apo VKGC), 9BVQ (VKGC–TMG2), 9BVP (VKGC–TMG2 with KH2), 9BVK (VKGC–FIX with KH2), 9BVL (VKGC–FX with KH2), 9BVM (VKGC–PC with KH2) and 9BVR (partially carboxylated VKGC–FIX with KH2). The MS data have been deposited in ProteomeXchange under identifier PXD052939. Source data are provided with this paper.

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Acknowledgements

We thank J.-K. Tie and M. Ferron for discussions and for providing the GGCX−/− reporter cell line and the anti-Gla antibody, respectively. W.L. is supported by the National Heart, Lung, and Blood Institute (R01 HL121718), the Vagelos Endowed Chair, the Established Investigator Award and Collaborative Sciences Award from American Heart Association, Children’s Discovery Institute (MCII 2020-854), the National Institute of Allergy and Infectious Diseases (R01 AI158500) and the Forefront of Science Award from W. M. Keck Foundation. W.L. and M.L.G. are supported by National Institute of General Medical Sciences (R01 GM131008). B.A.G. is supported by the National Institute of Neurological Disorders and Stroke (R01 NS111997), the Eunice Kennedy Shriver National Institute of Child Health & Human Development (R01 HD106051) and the 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.

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  1. Guomin Shen

    Present address: Department of Cell Biology, School of Basic Medical Sciences, Harbin Medical University, Harbin, People’s Republic of China

Authors and Affiliations

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

    Qing Cao, Aaron Ammerman, Mierxiati Saimi, Zongtao Lin, Guomin Shen, Shixuan Liu, Andrzej M. Krezel, Benjamin A. Garcia & Weikai Li

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

    Huaping Chen & Jinbin Xu

  3. Department of Chemistry, Washington University, St Louis, MO, USA

    Jie Sun, Mengqi Chai & Michael L. Gross

  4. Department of Internal Medicine, Washington University School of Medicine, St Louis, MO, USA

    Fong-Fu Hsu

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

    Bin Liu

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Contributions

Q.C. performed cryo-EM, functional and MS experiments. M.S. provided primary support to Q.C., and H.C., G.S., S.L. and J.X. assisted. B.L. and W.L. determined the structures. A.A., Z.L., F.-F.H., M.C. and J.S. performed MS analyses with the support of M.L.G. and B.A.G. W.L. conceived and directed the project, and wrote the manuscript with input from Q.C., A.M.K. and all of the other authors.

Corresponding authors

Correspondence to Bin Liu or Weikai Li.

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The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Darrel Stafford and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 The fusion strategy improves the structural resolution and protein expression while preserving VKGC catalytic activity.

a, 2D classification of wild-type VKGC and TMG2-BRIL-VKGC fusion protein. The particles of wild-type VKGC were extracted at box size of 320 pixels (1.1 Å/pixel), and TMG2-BRIL-VKGC at box size of 320 pixels (0.885 Å/pixel). The 2D classifications for both wild-type and fusion proteins show high-resolution features in many regions. b, Comparison of cryo-EM density maps of wild-type VKGC (left) and TMG2-BRIL-VKGC (right), coloured by domains (as in Fig. 1h). For wild-type VKGC, the 3D reconstruction yielded only a 4.4 Å resolution map with part of the VKGC extramembrane region disordered. c, Expression levels of TMG2-VKGC with and without BRIL insertion, assessed by Western blot using an anti-VKGC antibody. The original blots are shown in Supplementary Fig. 2. UN, untransfected. This experiment was performed once. d, FSEC profile comparison of TMG2-VKGC with and without BRIL insertion. These proteins are tagged with a C-terminal eGFP, expressed in HEK 293T cells, and extracted in DDM. e, The elution profile of size-exclusion chromatography during the protein purification of TMG2-BRIL-VKGC. f, SDS-PAGE of the peak fractions (17-22 corresponding to 16-18 mL) from size-exclusion chromatography. M, protein marker. The original gel is shown in Supplementary Fig. 3. This experiment was performed once. g, Immunoblots showing γ-carboxylation of TMG2-BRIL-VKGC in VKGC-knockout (k/o) cells treated with vitamin K (K, reduced to KH2 in cells). Carboxylation is not observed in untreated or warfarin (W) treated cells. The anti-Gla antibody recognizes wild-type (WT) VKGC due to self-carboxylation, and vitamin K treatment induces an electrophoresis-mobility shift85. This shift is not evident in the fusion protein, likely due to its larger size (with an eGFP tag) or hindrance of self-carboxylation by the fusion. For gel source data, see Supplementary Fig. 4. This experiment was performed once. h, Self-incorporation of 14CO2 into VKGC fusion proteins (20 pmol). The autoradiographs (left) were quantified to determine the 14CO2 incorporation levels (right). The radioactivity counting (mean ± s.d. from n = 3 biological replicates) did not include the upper bands, which represent slight aggregation of protein samples in the gel lanes. The TMG2 Prop-Glu fusion (i.e., TMG2-BRIL-VKGC) was used in the control without KH2. The original autoradiographs are shown in Supplementary Fig. 6. i, Competition of free FIX Prop against TMG2 Prop-Glu and VKGC fusion protein (1 µM). Fluorescein-labelled proFIX18 was used, and the protein-bound fraction was captured by crosslinking. Wild-type VKGC and BSA served as controls. The original gels are shown in Supplementary Fig. 7. This experiment was performed once. j, Epoxidase activities of wild-type VKGC protein (1 µM) is similar with 1:1 and 1:10 ratios of FIX Prop-Glu. k, Ammonium sulfate has little effect on the activity of the TMG2 Prop-Glu and VKGC fusion protein. l, Ammonium sulfate substantially increases the epoxidase activity with the FLEEL substrate. Reactions were conducted according to Sugiura, et al.21, with 0.01 µM wild-type VKGC, 64 µM proFIX18 (Prop) or proFIX28 (Prop-Glu), 3.6 mM FLEEL (sGlu), 222 µM KH2, 8 mM DTT, and a 30 min incubation. m, Higher DTT concentration slightly increases the epoxidase activity in presence of proFIX18 and FLEEL. In our conditions, ~2 mM DTT was introduced with KH2 addition, whereas Sugiura, et al.21 used an additional 8 mM DTT. Other conditions in this experiment are the same as in l. n, The relative activities of different substrates remain similar with different protein concentrations of wild-type VKGC. For comparison, the condition without ammonium sulfate and with 2 µM proFIX18 and 5 mM FLEEL was used. Data in j-n are mean ± s.d. from n = 3 biological replicates.

Source data

Extended Data Fig. 2 Quality of cryo-EM maps.

From Left to right, Local resolution illustrations of the cryo-EM maps, angular distribution plots, half-map FSC curves, and model-to-map FSCs. Respective representations are shown for fusion constructs of VKGC with TMG2 Prop-Glu (a), VKGC with TMG2 Prop-Glu and KH2 (b), VKGC with FIX Prop-Glu and KH2 (c), VKGC with FX Prop-Glu and KH2 (d), VKGC with PC Prop-Glu and KH2 (e), and VKGC with partially carboxylated FIX Prop-Glu and KH2 (f), and wild-type VKGC in unbound state (g).

Extended Data Fig. 3 Cryo-EM data processing.

The dataset and density maps of VKGC with Prop-Glu of TMG2 and KH2 are shown as a representative. Other datasets are similarly processed and quality of density maps are similar. a, A representative raw cryo-EM image. This experiment was performed once with thousands of raw images collected. b, Representative 2D classes. c, The data processing procedure. Two rounds of 2D classification and two rounds of heterogenous refinements generated a 3.3 Å final map. d, Density maps of VKGC individual regions. e, Density maps of KH2 and two bound phosphatidylcholine (PhosC) molecules. f, Density maps of TMG2 propeptide and Glu-rich regions.

Extended Data Fig. 4 The large VKGC chamber may permit the binding of the entire Glu-rich region and the folding of Gla-rich region into helices.

a, Model of the 64 AA Prop-Glu region of FIX in an extended conformation within the large chamber of VKGC. The modelling used HADDOCK76, with basic residues in VKGC and Glu residues in FIX Glu region selected for direct interactions. The modelled part of Glu-rich region is coloured in purple, and the propeptide and Glu-rich region observed in cryo-EM structure in orange and red, respectively. b, View of the FIX Prop-Glu model with VKGC shown in electrostatic surface. The lower part of the large chamber is positively charged, encircled by a belt of basic residues that potentially facilitate the binding of negatively charged Glu or Gla residues24,58 during processive γ-carboxylation. c, The cryo-EM model of partially carboxylated FIX in front (left) and side (right) views with the ER membrane modelled by PPM analysis25. The double arrow indicates proximity of the Gla-rich region to the membrane opening of VKGC. d, MS quantification of γ-carboxylation levels of individual Glu residues in partially carboxylated FIX fused with VKGC. Data are mean ± s.d. from n = 3 biological replicates. Glu/Gla residues detectable by ETD fragmentation were shown; ETD was used to optimally preserve γ-carboxylation86,87. The relative carboxylation levels are consistent with HCD fragmentation, but the absolute levels in HCD are relatively lower due to partial loss of this labile post-translational modification. e, MS-MS spectra of γ-carboxylated peptides (used in d) with the ETD fragmentation mode. f, Conformational change of the FIX Glu-rich region from the uncarboxylated state (propeptide-tethered, VKGC bound, and without Ca2+; Fig. 4a–e), the partially carboxylated state (propeptide-tethered, VKGC bound, and with Ca2+, as in c), to the carboxylated, functional state (in absence of propeptide and VKGC, but with Ca2+; PDB 1NL0)88. The uncarboxylated and partially carboxylated structures are superimposed, and the fully carboxylated structure is shown in a similar N to C direction. g, Density maps of the propeptide (left) and partially carboxylated Gla-rich region (right, unsharpened map) of FIX.

Source data

Extended Data Fig. 5 Structural location of VKCFD1 mutations.

a-b, Location of VKCFD1 mutations (red spheres) on the VKGC structure, in side (a) and top (b) views. c, Cluster of VKCFD1 mutations (in bold letters) near a propeptide-binding pocket (site III in Fig. 3). d, Cluster of VKCFD1 mutations near the c-Glu binding site. e, VKCFD1 mutations at the KH2 binding site. S300F and F299S are found in the dysfunctional allele of VKCFD1 patients (Supplementary Table 1) and exhibit no γ-carboxylation activities (Fig. 5f and Extended Data Fig. 8c)28. Another VKCFD1 mutation, S284P, may act as a helix breaker in RH7, altering several residues that interact with KH2 (Fig. 5d). f, VKCFD1 mutations located at the α/β domain interface interacting with other structural components. These mutations potentially interfere with the allosteric motion or propeptide binding of VKGC.

Extended Data Fig. 6 Binding interactions between VKGC and propeptides of VKDPs.

a, Binding of the VKDP propeptides (Prop) to the α/β and jellyroll domains. b, Polar interactions at the interface between the α/β and jellyroll domains. c, Sequence alignment of propeptides from human VKDPs and VKGC-binding affinities of these propeptides19. PS, protein S. PZ, protein Z. OCN, osteocalcin. MGP, matrix Gla protein. GRP, Gla-rich protein or unique cartilage matrix-associated protein. The MGP propeptide does not contain the recognition motif for furin cleavage and is not removed in mature protein. Uncommon residues in VKDPs at the three key sites are indicated by red letter, with those known to affect propeptide binding in bold. The sequence logo89 above depicts the residue conservation level at each position. n.g., negligible. d, Cellular γ-carboxylation activities of VKGC mutants at the propeptide binding sites I, II and III. FIX-Gla-PC is used as the γ-carboxylation reporter. Data are mean ± s.d. from n = 3 biological replicates.

Source data

Extended Data Fig. 7 Glu-rich regions of VKDPs.

a, VKGC binding interactions with the 5-AA Glu-containing segments from FIX, FX, TMG2, and PC. b, Electrostatic surface showing the binding grooves for individual side chains in the 5-AA segments. These interactions are highly adaptive to different Glu-containing sequences. c, Sequence alignment of the Glu-rich regions from human VKDPs. OCN, GRP, and MGP are shown separately due to their low sequence homology to other VKDPs in the Gla domain. The dots in MGP indicate position of the propeptide that is flanked by Glu-rich sequences. For clarity, the non-consensus Glu residues in OCN, MGP, and GRP, and those at the C-terminal (after +33) of other VKDPs are shown in red letters, and those known to be carboxylated in bold. The sequence alignments were generated by Clustal W v2.1. The grey and light grey shading denote highly and moderately conserved residues, respectively. The sequence logos89 above alignments depict the residue conservation level at each position. Secondary structures (ω-loop and helices h1, h2, and h3) and disulfide bond (S-S) in the Gla domain are indicated below. Inset, Structural location of the Gla residues, Ca2+ ions, and hydrophobic residues (in ω-loop). The crystal structure of human FIX Gla domain (PDB 1NL0)88 is shown with membrane (grey shading) insertion90.

Extended Data Fig. 8 Comparisons of VKGC-TMG2 fusion in bound and unbound states with KH2 or with propeptide.

a, Overall structures with and without bound KH2. b, The c-Glu and KH2 binding sites in the two states. The KH2-bound structure (coloured) and unbound structure (grey) share essentially the same conformation. c, Cellular γ-carboxylation activities of VKGC mutants at the KH2 binding pocket. FIX-Gla-PC is used as the γ-carboxylation reporter. Data are mean ± s.d. from n = 3 or 6 biological replicates. d, Structure of wild-type VKGC in unbound state (coloured as in Fig. 1h). The jellyroll and α/β domains are partially dissociated, creating a gap in between. e, Propeptide binding induces rotation of the jellyroll domain and bent helix (arrows), and an adaptive change in the α/β domain. The unbound state (coloured) and propeptide-bound state (grey) are superimposed by the membrane domain. f, Key residues in the bent helix and L11 that impact propeptide binding. Left, Front view of the propeptide binding site. V502, S510, and W512 are located at the back of α/β and jellyroll domains. Mutations of these residues have been shown to affect propeptide binding91. Right, A 180° rotated view of the back, showing the three residues (in grey) participating in key interactions with surrounding residues (coloured by domains) that stabilize the two domains for propeptide binding. Green lines indicate hydrogen bonds. g-i. Backbone conformational changes between the propeptide bound and unbound structures. The structures of unbound, wild-type VKGC (coloured by domains) and VKGC fused with TMG2 Prop-Glu (red) are superimposed by membrane domain. Changes in the jellyroll domain (g), HL9 and L11 (h), and HL5-6 (i) are discernible from 4.4 Å density map of the unbound VKGC. The red arrows indicate representative backbone changes in the superimposed Prop-Glu bound structure that are out of the map densities of the unbound structure.

Source data

Extended Data Fig. 9 Sequence alignment of VKGC homologues.

The alignment is between VKGC homologues from distinct eukaryotic species (Homo, Homo sapiens. Mus, Mus musculus. Xenopus, Xenopus laevis. Takifugu, Takifugu rubripes. Drosophila, Drosophila melanogaster. Conus, Conus textile. Ciona, Ciona intestinalis) and bacterial species (Lepto, Leptospira interrogans. Strep, Streptomyces paromomycinus). The sequence alignments were generated by Clustal W v2.1, and extra sequences in certain homologues are omitted (represented by dots) for simplicity. Key catalytic residues are coloured in red. Below the aligned sequences, the asterisks indicate conserved residues, and single and double dots represent varying levels of sequence similarity. Brown spheres above the alignment indicate VKCFD1 mutations. Residues participating in the bindings of propeptide (P), 5-AA Glu-containing segment (G), and KH2 (K), or forming the hydrophobic tunnel (T) are indicated in coloured letters above the alignment. The protein folding topology (Cyto: cytoplasmic side, Lum: ER lumenal side) and secondary structures (as in Fig. 2b. TM, transmembrane helices. RH, reentrant helices) are denoted further above.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Information (download PDF )

Supplementary Tables 1–2 and Supplementary Figs. 1–7.

Reporting Summary (download PDF )

Peer Review File (download PDF )

Supplementary Data 1 (download PDF )

Validation reports of deposited coordinates and maps.

Supplementary Video 1 (download MP4 )

Propeptide binding induces conformational changes that trigger KH2 epoxidation and facilitate binding of the Glu-rich region. The video shows structural changes of VKGC alternating between the unbound state and FIX Prop–Glu-bound state, superimposed by the membrane domain. Propeptide binding between the jellyroll and α/β domains induces the rotation of the jellyroll domain. This rotation causes the associated bent helix to move, in turn altering the conformations of HL5–6 and HL9. These HL regions form part of the KH2-binding pocket, with HL9 also serving as the binding site for the five-amino-acid Glu-containing segment. Thus, propeptide binding reshapes these binding sites to facilitate KH2 epoxidation and the binding of Glu-containing segments.

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Cao, Q., Ammerman, A., Saimi, M. et al. Molecular basis of vitamin-K-driven γ-carboxylation at the membrane interface. Nature 639, 816–824 (2025). https://doi.org/10.1038/s41586-025-08648-1

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