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- EMDB-44942: Vitamin K-dependent gamma-carboxylase with factor IX propeptide a... -

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Basic information

Entry
Database: EMDB / ID: EMD-44942
TitleVitamin K-dependent gamma-carboxylase with factor IX propeptide and partially carboxylated glutamate-rich region and with vitamin K hydroquinone and calcium
Map data
Sample
  • Complex: Vitamin K-dependent gamma-carboxylase with factor IX propeptide and partially carboxylated glutamate-rich region and with vitamin K hydroquinone and calcium
    • Protein or peptide: Vitamin K-dependent gamma-carboxylase
    • Protein or peptide: Coagulation factor IX
  • 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 / MEMBRANE PROTEIN / LYASE-SUBSTRATE complex
Function / homology
Function and homology information


peptidyl-glutamate 4-carboxylase / gamma-glutamyl carboxylase activity / Defective F9 secretion / coagulation factor IXa / Defective gamma-carboxylation of F9 / vitamin binding / vitamin K metabolic process / Defective F9 activation / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant ...peptidyl-glutamate 4-carboxylase / gamma-glutamyl carboxylase activity / Defective F9 secretion / coagulation factor IXa / Defective gamma-carboxylation of F9 / vitamin binding / vitamin K metabolic process / Defective F9 activation / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / zymogen activation / Extrinsic Pathway of Fibrin Clot Formation / Protein hydroxylation / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Gamma-carboxylation of protein precursors / Removal of aminoterminal propeptides from gamma-carboxylated proteins / Intrinsic Pathway of Fibrin Clot Formation / protein maturation / protein modification process / Golgi lumen / blood coagulation / : / endopeptidase activity / endoplasmic reticulum lumen / serine-type endopeptidase activity / calcium ion binding / endoplasmic reticulum membrane / proteolysis / extracellular space / extracellular exosome / extracellular region / metal ion binding / 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 / Peptidase S1A, coagulation factor VII/IX/X/C/Z / : / Coagulation factor-like, Gla domain superfamily ...Vitamin K-dependent gamma-carboxylase / HTTM / : / : / HTTM domain / Vitamin K-dependent gamma-carboxylase, lumenal domain / Horizontally Transferred TransMembrane Domain / Peptidase S1A, coagulation factor VII/IX/X/C/Z / : / Coagulation factor-like, Gla domain superfamily / Coagulation Factor Xa inhibitory site / RmlC-like cupin domain superfamily / EGF-like domain / EGF-type aspartate/asparagine hydroxylation site / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Aspartic acid and asparagine hydroxylation site. / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / 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. / Epidermal growth factor-like domain. / EGF-like domain profile. / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain / RmlC-like jelly roll fold / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Serine proteases, trypsin family, histidine active site. / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Coagulation factor IX / Vitamin K-dependent gamma-carboxylase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 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_44942.png

Downloads & links

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Map

FileDownload / File: emd_44942.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88533 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.12907445 - 0.311849
Average (Standard dev.)0.00025993134 (±0.010510907)
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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Additional map: #1

Fileemd_44942_additional_1.map
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Half map: #2

Fileemd_44942_half_map_1.map
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Half map: #1

Fileemd_44942_half_map_2.map
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Sample components

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Entire : Vitamin K-dependent gamma-carboxylase with factor IX propeptide a...

EntireName: Vitamin K-dependent gamma-carboxylase with factor IX propeptide and partially carboxylated glutamate-rich region and with vitamin K hydroquinone and calcium
Components
  • Complex: Vitamin K-dependent gamma-carboxylase with factor IX propeptide and partially carboxylated glutamate-rich region and with vitamin K hydroquinone and calcium
    • Protein or peptide: Vitamin K-dependent gamma-carboxylase
    • Protein or peptide: Coagulation factor IX
  • 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 factor IX propeptide a...

SupramoleculeName: Vitamin K-dependent gamma-carboxylase with factor IX propeptide and partially carboxylated glutamate-rich region and with vitamin K hydroquinone and calcium
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 110 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: 85.00568 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPRQDSRIGK LLGFEWTDLS SWRRLVTLLN RPTDPASLAV FRFLFGFLMV LDIPQERGLS SLDRKYLDGL DVCRFPLLDA LRPLPLDWM YLVYTIMFLG ALGMMLGLCY RISCVLFLLP YWYVFLLDKT SWNNHSYLYG LLAFQLTFMD ANHYWSVDGL L NAHRRNAH ...String:
GPRQDSRIGK LLGFEWTDLS SWRRLVTLLN RPTDPASLAV FRFLFGFLMV LDIPQERGLS SLDRKYLDGL DVCRFPLLDA LRPLPLDWM YLVYTIMFLG ALGMMLGLCY RISCVLFLLP YWYVFLLDKT SWNNHSYLYG LLAFQLTFMD ANHYWSVDGL L NAHRRNAH VPLWNYAVLR GQIFIVYFIA GVKKLDADWV EGYSMEYLSR HWLFSPFKLL LSEELTSLLV VHWGGLLLDL SA GFLLFFD VSRSIGLFFV SYFHCMNSQL FSIGMFSYVM LASSPLFCSP EWPRKLVSYC PRRLQQLLPL KAAPQPSVSC VYK RSRGKS GQKPGLRHQL GAAFTLLYLL EQLFLPYSHF LTQGYNNWTN GLYGYSWDMM VHSRSHQHVK ITYRDGRTGE LGYL NPGVF TQSRRWKDHA DMLKQYATCL SRLLPKYNVT EPQIYFDIWV SINDRFQQRI FDPRVDIVQA AWSPFQRTSW VQPLL MDLS PWRAKLQEIK SSLDNHTEVV FIADFPGLHL ENFVSEDLGN TSIQLLQGEV TVELVAEQKN QTLREGEKMQ LPAGEY HKV YTTSPSPSCY MYVYVNTTEL ALEQDLAYLQ ELKEKVENGS ETGPLPPELQ PLLEGEVKGG PEPTPLVQTF LRRQQRL QE IERRRNTPFH ERFFRFLLRK LYVFRRSFLM TCISLRNLIL GRPSLEQLAQ EVTYANLRPF EAVGELNPSN TDSSHSNP P ESNPDPVHSE F

UniProtKB: Vitamin K-dependent gamma-carboxylase

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Macromolecule #2: Coagulation factor IX

MacromoleculeName: Coagulation factor IX / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: coagulation factor IXa
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.802636 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
TVFLDHENAN KILNRPKRYN SGKLEEFVQG NLERECMEEK CSFEEAREVF ENTERTTEFW KQYV

UniProtKB: Coagulation factor IX

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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
5.0 mMCaCl2Calcium chloride
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: 4992 / Average electron dose: 54.8 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: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 173076
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-9bvr:
Vitamin K-dependent gamma-carboxylase with factor IX propeptide and partially carboxylated glutamate-rich region and with vitamin K hydroquinone and calcium

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