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- EMDB-44940: Vitamin K-dependent gamma-carboxylase with TMG2 propeptide and gl... -

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Basic information

Entry
Database: EMDB / ID: EMD-44940
TitleVitamin K-dependent gamma-carboxylase with TMG2 propeptide and glutamate-rich region and with vitamin K hydroquinone
Map data
Sample
  • Complex: Vitamin K-dependent gamma-carboxylase with Transmembrane gamma-carboxyglutamic acid protein 2 propeptide and Glu-rech region and with vitamin K hydroquinone
    • Protein or peptide: Vitamin K-dependent gamma-carboxylase
    • Protein or peptide: Transmembrane gamma-carboxyglutamic acid protein 2
  • Ligand: vitamin K1 hydroquinone
  • Ligand: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsGGCX / VKGC / Vitamin K / VKCFD / Hemophilia B / Warfarin / Carboxylation / Blood Coagulaton / Calcium homeostasis / TMG / MEMBRANE PROTEIN / LYASE-SUBSTRATE complex
Function / homology
Function and homology information


peptidyl-glutamate 4-carboxylase / gamma-glutamyl carboxylase activity / negative regulation of testosterone biosynthetic process / negative regulation of bone development / Defective gamma-carboxylation of F9 / vitamin binding / vitamin K metabolic process / negative regulation of neurotransmitter secretion / Gamma-carboxylation of protein precursors / protein maturation ...peptidyl-glutamate 4-carboxylase / gamma-glutamyl carboxylase activity / negative regulation of testosterone biosynthetic process / negative regulation of bone development / Defective gamma-carboxylation of F9 / vitamin binding / vitamin K metabolic process / negative regulation of neurotransmitter secretion / Gamma-carboxylation of protein precursors / protein maturation / protein modification process / blood coagulation / endoplasmic reticulum lumen / serine-type endopeptidase activity / calcium ion binding / endoplasmic reticulum membrane / extracellular space / membrane / plasma membrane
Similarity search - Function
Vitamin K-dependent gamma-carboxylase / HTTM / : / : / HTTM domain / Vitamin K-dependent gamma-carboxylase, lumenal domain / Horizontally Transferred TransMembrane Domain / : / Coagulation factor-like, Gla domain superfamily / RmlC-like cupin domain superfamily ...Vitamin K-dependent gamma-carboxylase / HTTM / : / : / HTTM domain / Vitamin K-dependent gamma-carboxylase, lumenal domain / Horizontally Transferred TransMembrane Domain / : / Coagulation factor-like, Gla domain superfamily / RmlC-like cupin domain superfamily / Vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain superfamily / Vitamin K-dependent carboxylation domain. / Gla domain profile. / Domain containing Gla (gamma-carboxyglutamate) residues. / RmlC-like jelly roll fold
Similarity search - Domain/homology
Transmembrane gamma-carboxyglutamic acid protein 2 / Vitamin K-dependent gamma-carboxylase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLi W / Liu B / Cao Q
Funding support United States, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL121718 United States
American Heart AssociationEstablished Investigator Award United States
American Heart AssociationCollaborative Sciences Award United States
W. M. Keck FoundationForefront of Science Award United States
Childrens Discovery Institute of Washington University and St. Louis Childrens HospitalMCII 2020-854 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI158500 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131008 United States
CitationJournal: Nature / Year: 2025
Title: Molecular basis of vitamin-K-driven γ-carboxylation at the membrane interface.
Authors: Qing Cao / Aaron Ammerman / Mierxiati Saimi / Zongtao Lin / Guomin Shen / Huaping Chen / Jie Sun / Mengqi Chai / Shixuan Liu / Fong-Fu Hsu / Andrzej M Krezel / Michael L Gross / Jinbin Xu / ...Authors: Qing Cao / Aaron Ammerman / Mierxiati Saimi / Zongtao Lin / Guomin Shen / Huaping Chen / Jie Sun / Mengqi Chai / Shixuan Liu / Fong-Fu Hsu / Andrzej M Krezel / Michael L Gross / Jinbin Xu / Benjamin A Garcia / Bin Liu / Weikai Li /
Abstract: The γ-carboxylation of glutamate residues enables Ca-mediated membrane assembly of protein complexes that support broad physiological functions, including haemostasis, calcium homeostasis, immune ...The γ-carboxylation of glutamate residues enables Ca-mediated membrane assembly of protein complexes that support broad physiological functions, including haemostasis, calcium homeostasis, immune response and endocrine regulation. Modulating γ-carboxylation levels provides prevalent treatments for haemorrhagic and thromboembolic diseases. This unique post-translational modification requires vitamin K hydroquinone (KH) to drive highly demanding reactions 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 KH 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 KH epoxidation, VKGC generates a free hydroxide ion as an exceptionally strong base that is required to deprotonate the γ-carbon of glutamate for CO addition. The diffusion of this superbase-protected and guided by a sealed hydrophobic tunnel-elegantly resolves the challenge of coupling KH 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.
History
DepositionMay 20, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44940.png

Downloads & links

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Map

FileDownload / File: emd_44940.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.89 Å/pix.
x 320 pix.
= 283.307 Å		0.89 Å/pix.
x 320 pix.
= 283.307 Å		0.89 Å/pix.
x 320 pix.
= 283.307 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88533 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.53469914 - 0.94089776
Average (Standard dev.)0.00053605053 (±0.018644752)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 283.30655 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44940_half_map_1.map
Projections & Slices
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Slices (1/2)
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Histogram (log scale)

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Half map: #1

Fileemd_44940_half_map_2.map
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Sample components

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Entire : Vitamin K-dependent gamma-carboxylase with Transmembrane gamma-ca...

EntireName: Vitamin K-dependent gamma-carboxylase with Transmembrane gamma-carboxyglutamic acid protein 2 propeptide and Glu-rech region and with vitamin K hydroquinone
Components
  • Complex: Vitamin K-dependent gamma-carboxylase with Transmembrane gamma-carboxyglutamic acid protein 2 propeptide and Glu-rech region and with vitamin K hydroquinone
    • Protein or peptide: Vitamin K-dependent gamma-carboxylase
    • Protein or peptide: Transmembrane gamma-carboxyglutamic acid protein 2
  • Ligand: vitamin K1 hydroquinone
  • Ligand: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Vitamin K-dependent gamma-carboxylase with Transmembrane gamma-ca...

SupramoleculeName: Vitamin K-dependent gamma-carboxylase with Transmembrane gamma-carboxyglutamic acid protein 2 propeptide and Glu-rech region and with vitamin K hydroquinone
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 112.5 KDa

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Macromolecule #1: Vitamin K-dependent gamma-carboxylase

MacromoleculeName: Vitamin K-dependent gamma-carboxylase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: peptidyl-glutamate 4-carboxylase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 85.092758 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SGPRQDSRIG KLLGFEWTDL SSWRRLVTLL NRPTDPASLA VFRFLFGFLM VLDIPQERGL SSLDRKYLDG LDVCRFPLLD ALRPLPLDW MYLVYTIMFL GALGMMLGLC YRISCVLFLL PYWYVFLLDK TSWNNHSYLY GLLAFQLTFM DANHYWSVDG L LNAHRRNA ...String:
SGPRQDSRIG KLLGFEWTDL SSWRRLVTLL NRPTDPASLA VFRFLFGFLM VLDIPQERGL SSLDRKYLDG LDVCRFPLLD ALRPLPLDW MYLVYTIMFL GALGMMLGLC YRISCVLFLL PYWYVFLLDK TSWNNHSYLY GLLAFQLTFM DANHYWSVDG L LNAHRRNA HVPLWNYAVL RGQIFIVYFI AGVKKLDADW VEGYSMEYLS RHWLFSPFKL LLSEELTSLL VVHWGGLLLD LS AGFLLFF DVSRSIGLFF VSYFHCMNSQ LFSIGMFSYV MLASSPLFCS PEWPRKLVSY CPRRLQQLLP LKAAPQPSVS CVY KRSRGK SGQKPGLRHQ LGAAFTLLYL LEQLFLPYSH FLTQGYNNWT NGLYGYSWDM MVHSRSHQHV KITYRDGRTG ELGY LNPGV FTQSRRWKDH ADMLKQYATC LSRLLPKYNV TEPQIYFDIW VSINDRFQQR IFDPRVDIVQ AAWSPFQRTS WVQPL LMDL SPWRAKLQEI KSSLDNHTEV VFIADFPGLH LENFVSEDLG NTSIQLLQGE VTVELVAEQK NQTLREGEKM QLPAGE YHK VYTTSPSPSC YMYVYVNTTE LALEQDLAYL QELKEKVENG SETGPLPPEL QPLLEGEVKG GPEPTPLVQT FLRRQQR LQ EIERRRNTPF HERFFRFLLR KLYVFRRSFL MTCISLRNLI LGRPSLEQLA QEVTYANLRP FEAVGELNPS NTDSSHSN P PESNPDPVHS EF

UniProtKB: Vitamin K-dependent gamma-carboxylase

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Macromolecule #2: Transmembrane gamma-carboxyglutamic acid protein 2

MacromoleculeName: Transmembrane gamma-carboxyglutamic acid protein 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.399892 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EETDQEVFLG PPEAQSFLSS HTRIPRANHW DLELLTPGNL ERECLEERCS WEEAREYFED NTLTERFWES YIYNGKGGRG RVDVASLAV GLTGGILLIV LAGLGAFWYL RWRQHRG

UniProtKB: Transmembrane gamma-carboxyglutamic acid protein 2

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Macromolecule #4: vitamin K1 hydroquinone

MacromoleculeName: vitamin K1 hydroquinone / type: ligand / ID: 4 / Number of copies: 1 / Formula: A1AVC
Molecular weightTheoretical: 452.712 Da

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Macromolecule #5: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-...

MacromoleculeName: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
type: ligand / ID: 5 / Number of copies: 2 / Formula: 6PL
Molecular weightTheoretical: 763.1 Da
Chemical component information

ChemComp-6PL:
(4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / phospholipid*YM

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
20.0 mMC8H18N2O4SHEPES
0.005 %C56H92O25Glyco-diosgenin
0.055 mMC31H48O2Vitamin K hydroquinone
1.84 mMNaHCO3Sodium bicarbonate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283.15 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 63.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Number grids imaged: 1 / Number real images: 6226 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 171392
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9bvp:
Vitamin K-dependent gamma-carboxylase with TMG2 propeptide and glutamate-rich region and with vitamin K hydroquinone

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