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- PDB-9bvr: Vitamin K-dependent gamma-carboxylase with factor IX propeptide a... -

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Basic information

Entry
Database: PDB / ID: 9bvr
TitleVitamin K-dependent gamma-carboxylase with factor IX propeptide and partially carboxylated glutamate-rich region and with vitamin K hydroquinone and calcium
Components
  • Coagulation factor IX
  • Vitamin K-dependent gamma-carboxylase
KeywordsMEMBRANE PROTEIN / LYASE/SUBSTRATE / GGCX / VKGC / Vitamin K / VKCFD / Hemophilia B / Warfarin / Carboxylation / Blood Coagulaton / Calcium homeostasis / 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
Chem-6PL / : / Coagulation factor IX / Vitamin K-dependent gamma-carboxylase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLi, W. / Liu, B. / Cao, Q.
Funding support United States, 7items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Mar 19, 2025Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.3Mar 26, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Vitamin K-dependent gamma-carboxylase
P: Coagulation factor IX
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,2829
Polymers92,8082
Non-polymers3,4737
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AP

#1: Protein Vitamin K-dependent gamma-carboxylase / Gamma-glutamyl carboxylase / Peptidyl-glutamate 4-carboxylase / Vitamin K gamma glutamyl carboxylase


Mass: 85005.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GGCX, GC / Cell line (production host): HEK293S / Production host: Homo sapiens (human)
References: UniProt: P38435, peptidyl-glutamate 4-carboxylase
#2: Protein Coagulation factor IX / Christmas factor / Plasma thromboplastin component / PTC


Mass: 7802.636 Da / Num. of mol.: 1 / Fragment: residues 29-92
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F9 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: P00740, coagulation factor IXa

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Sugars , 2 types, 4 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 3 molecules

#4: Chemical ChemComp-A1AVC / vitamin K1 hydroquinone / 2-methyl-3-[(2E,7R,11R)-3,7,11,15-tetramethylhexadec-2-en-1-yl]naphthalene-1,4-diol


Mass: 452.712 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H48O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical 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 / 1-PALMITOYL-2-STEAROYL-SN-GLYCERO-3-PHOSPHOCHOLINE


Mass: 763.100 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H85NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Vitamin K-dependent gamma-carboxylase with factor IX propeptide and partially carboxylated glutamate-rich region and with vitamin K hydroquinone and calcium
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / Plasmid: pEG BacMam
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
220 mMHEPESC8H18N2O4S1
30.005 %Glyco-diosgeninC56H92O251
40.055 mMVitamin K hydroquinoneC31H48O21
51.84 mMSodium bicarbonateNaHCO31
65 mMCalcium chlorideCaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 63 K
Image recordingElectron dose: 54.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4992
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 4092 / Height: 5760

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173076 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036422
ELECTRON MICROSCOPYf_angle_d0.5938687
ELECTRON MICROSCOPYf_dihedral_angle_d10.32492
ELECTRON MICROSCOPYf_chiral_restr0.038914
ELECTRON MICROSCOPYf_plane_restr0.0051093

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