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- PDB-8e50: Cryo-EM structure of human glycerol-3-phosphate acyltransferase 1... -

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Basic information

Entry
Database: PDB / ID: 8.0E+50
TitleCryo-EM structure of human glycerol-3-phosphate acyltransferase 1 (GPAT1) in complex with CoA and palmitoyl-LPA
ComponentsGlycerol-3-phosphate acyltransferase 1, mitochondrial
KeywordsMEMBRANE PROTEIN / acyltransferase / LPA / monotopic / mitochondrial
Function / homology
Function and homology information


glycerol-3-phosphate 1-O-acyltransferase / glycerol-3-phosphate O-acyltransferase activity / sn-1-glycerol-3-phosphate C16:0-DCA-CoA acyl transferase activity / phosphatidylglycerol biosynthetic process / CDP-diacylglycerol biosynthetic process / Triglyceride biosynthesis / negative regulation of activation-induced cell death of T cells / triglyceride biosynthetic process / phosphatidic acid biosynthetic process / acyl-CoA metabolic process ...glycerol-3-phosphate 1-O-acyltransferase / glycerol-3-phosphate O-acyltransferase activity / sn-1-glycerol-3-phosphate C16:0-DCA-CoA acyl transferase activity / phosphatidylglycerol biosynthetic process / CDP-diacylglycerol biosynthetic process / Triglyceride biosynthesis / negative regulation of activation-induced cell death of T cells / triglyceride biosynthetic process / phosphatidic acid biosynthetic process / acyl-CoA metabolic process / glycerol-3-phosphate metabolic process / Synthesis of PA / activation-induced cell death of T cells / diacylglycerol biosynthetic process / phospholipid biosynthetic process / phospholipid homeostasis / positive regulation of multicellular organism growth / activated T cell proliferation / RUNX1 regulates estrogen receptor mediated transcription / positive regulation of activated T cell proliferation / fatty acid homeostasis / response to glucose / regulation of cytokine production / fatty acid metabolic process / Activation of gene expression by SREBF (SREBP) / mitochondrial membrane / defense response to virus / Estrogen-dependent gene expression / mitochondrial outer membrane / plasma membrane
Similarity search - Function
Glycerol-3-phosphate O-acyltransferase/Dihydroxyacetone phosphate acyltransferase / Glycerol-3-phosphate acyltransferase, PlsB / GPAT/DHAPAT, acyltransferase domain / GPAT/DHAPAT, C-terminal domain / Glycerol-3-phosphate acyltransferase C-terminal region / Phospholipid/glycerol acyltransferase / Acyltransferase / Phosphate acyltransferases
Similarity search - Domain/homology
COENZYME A / Chem-NKO / Glycerol-3-phosphate acyltransferase 1, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsWasilko, D.J. / Johnson, Z.L. / Ammirati, M. / Chang, J.S. / Han, S. / Wu, H.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis of the acyl-transfer mechanism of human GPAT1.
Authors: Zachary Lee Johnson / Mark Ammirati / David Jonathan Wasilko / Jeanne S Chang / Stephen Noell / Timothy L Foley / Hyejin Yoon / Kathleen Smith / Shoh Asano / Katherine Hales / Min Wan / ...Authors: Zachary Lee Johnson / Mark Ammirati / David Jonathan Wasilko / Jeanne S Chang / Stephen Noell / Timothy L Foley / Hyejin Yoon / Kathleen Smith / Shoh Asano / Katherine Hales / Min Wan / Qingyi Yang / Mary A Piotrowski / Kathleen A Farley / Tamara Gilbert / Lisa M Aschenbrenner / Kimberly F Fennell / Jason K Dutra / Mary Xu / Chunyang Guo / Alison E Varghese / Justin Bellenger / Alandra Quinn / Christopher W Am Ende / Graham M West / Matthew C Griffor / Donald Bennett / Matthew Calabrese / Claire M Steppan / Seungil Han / Huixian Wu /
Abstract: Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to ...Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.
History
DepositionAug 19, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 28, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Feb 1, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glycerol-3-phosphate acyltransferase 1, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,0973
Polymers86,9191
Non-polymers1,1782
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
MethodPISA

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Components

#1: Protein Glycerol-3-phosphate acyltransferase 1, mitochondrial / GPAT-1


Mass: 86919.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GPAM, GPAT1, KIAA1560 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q9HCL2, glycerol-3-phosphate 1-O-acyltransferase
#2: Chemical ChemComp-COA / COENZYME A / Coenzyme A


Mass: 767.534 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H36N7O16P3S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NKO / (2R)-2-hydroxy-3-(phosphonooxy)propyl hexadecanoate / 16:0 LPA / palmitoyl lysophosphatidic acid


Mass: 410.483 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H39O7P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: glycerol-3-phosphate acyltransferase 1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.086 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
2150 mMSodium Chloride1
30.06 PercentDigitonin1
41 mMDTT1
53 mMFluorinated Fos-Choline-81
6150 uMpalmitoyl-LPA1
71 mMCoA1
SpecimenConc.: 6.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot force -5, blot time 3 sec

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 78 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9153
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 50

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9RELION3.1.2initial Euler assignment
10RELION3.1.2final Euler assignment
12RELION3.1.23D reconstruction
13PHENIXmodel refinement
14REFMAC5model refinement
Image processingDetails: collected in super resolution mode; gain normalized and binned by 2 during motion correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1704123
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76033 / Symmetry type: POINT
Atomic model buildingPDB-ID: 8E4Y

8e4y


Pdb chain-ID: A
RefinementResolution: 3.67→98 Å / Cor.coef. Fo:Fc: 0.903 / SU B: 53.606 / SU ML: 0.691 / ESU R: 1.452
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.37256 --
obs0.37256 25788 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 169.051 Å2
Baniso -1Baniso -2Baniso -3
1--1.32 Å20.66 Å2-0.88 Å2
2--2.11 Å2-1.53 Å2
3----0.79 Å2
Refinement stepCycle: 1 / Total: 5277
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.0135379
ELECTRON MICROSCOPYr_bond_other_d0.0010.0155279
ELECTRON MICROSCOPYr_angle_refined_deg1.2751.6367282
ELECTRON MICROSCOPYr_angle_other_deg1.0141.5712106
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.7045645
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.48421.429273
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.40215949
ELECTRON MICROSCOPYr_dihedral_angle_4_deg11.1871537
ELECTRON MICROSCOPYr_chiral_restr0.0450.2711
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.025921
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021260
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.0817.8912598
ELECTRON MICROSCOPYr_mcbond_other6.08117.892597
ELECTRON MICROSCOPYr_mcangle_it10.04926.853237
ELECTRON MICROSCOPYr_mcangle_other10.04826.8523238
ELECTRON MICROSCOPYr_scbond_it5.65218.3882781
ELECTRON MICROSCOPYr_scbond_other5.65118.3882782
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.78727.4014046
ELECTRON MICROSCOPYr_long_range_B_refined16.3236086
ELECTRON MICROSCOPYr_long_range_B_other16.3226087
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.67→3.765 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.568 1911 -
obs--100 %

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