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- PDB-8ghe: The structure of h12-LOX in tetrameric form bound to endogenous i... -

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Basic information

Entry
Database: PDB / ID: 8ghe
TitleThe structure of h12-LOX in tetrameric form bound to endogenous inhibitor oleoyl-CoA
ComponentsPolyunsaturated fatty acid lipoxygenase ALOX12
KeywordsOXIDOREDUCTASE/INHIBITOR / Lipoxygenase / platelets / lipid-modifying enzyme / lipid oxidation / OXIDOREDUCTASE / OXIDOREDUCTASE-INHIBITOR complex
Function / homology
Function and homology information


unsaturated fatty acid metabolic process / hepoxilin-epoxide hydrolase activity / leukotriene A4 metabolic process / lipoxin B4 biosynthetic process / Synthesis of Hepoxilins (HX) and Trioxilins (TrX) / Biosynthesis of DPAn-6 SPMs / Hydrolases; Acting on ether bonds; Ether hydrolases / arachidonate 12-lipoxygenase / lipoxin A4 biosynthetic process / Biosynthesis of DHA-derived SPMs ...unsaturated fatty acid metabolic process / hepoxilin-epoxide hydrolase activity / leukotriene A4 metabolic process / lipoxin B4 biosynthetic process / Synthesis of Hepoxilins (HX) and Trioxilins (TrX) / Biosynthesis of DPAn-6 SPMs / Hydrolases; Acting on ether bonds; Ether hydrolases / arachidonate 12-lipoxygenase / lipoxin A4 biosynthetic process / Biosynthesis of DHA-derived SPMs / Biosynthesis of DPAn-3-derived maresins / arachidonate 15-lipoxygenase / arachidonate 15-lipoxygenase activity / arachidonate 12(S)-lipoxygenase activity / Synthesis of 12-eicosatetraenoic acid derivatives / linoleate 13S-lipoxygenase activity / Synthesis of Lipoxins (LX) / lipoxygenase pathway / Oxidoreductases; Acting on single donors with incorporation of molecular oxygen (oxygenases); With incorporation of two atoms of oxygen / arachidonic acid metabolic process / lipid oxidation / negative regulation of muscle cell apoptotic process / hepoxilin biosynthetic process / linoleic acid metabolic process / negative regulation of platelet aggregation / superoxide anion generation / fatty acid oxidation / establishment of skin barrier / sarcolemma / lipid metabolic process / iron ion binding / extracellular exosome / membrane / cytosol / cytoplasm
Similarity search - Function
Lipoxygenase, vertebrates, PLAT domain / Lipoxygenase, mammalian / Lipoxygenase, conserved site / Lipoxygenases iron-binding region signature 2. / Lipoxygenase, iron binding site / Lipoxygenases iron-binding region signature 1. / Lipoxygenase / Lipoxygenase, C-terminal / Lipoxigenase, C-terminal domain superfamily / Lipoxygenase ...Lipoxygenase, vertebrates, PLAT domain / Lipoxygenase, mammalian / Lipoxygenase, conserved site / Lipoxygenases iron-binding region signature 2. / Lipoxygenase, iron binding site / Lipoxygenases iron-binding region signature 1. / Lipoxygenase / Lipoxygenase, C-terminal / Lipoxigenase, C-terminal domain superfamily / Lipoxygenase / Lipoxygenase iron-binding catalytic domain profile. / Lipoxygenase homology 2 (beta barrel) domain / PLAT/LH2 domain / PLAT/LH2 domain superfamily / PLAT/LH2 domain / PLAT domain profile.
Similarity search - Domain/homology
Chem-3VV / : / Polyunsaturated fatty acid lipoxygenase ALOX12
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.05 Å
AuthorsBlack, K.A. / Mobbs, J.I. / Venugopal, H. / Thal, D.M. / Glukhova, A.
Funding support United States, 2items
OrganizationGrant numberCountry
Other private
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM131835 United States
CitationJournal: Blood / Year: 2023
Title: Cryo-EM structures of human arachidonate 12S-lipoxygenase bound to endogenous and exogenous inhibitors.
Authors: Jesse I Mobbs / Katrina A Black / Michelle Tran / Wessel A C Burger / Hariprasad Venugopal / Theodore R Holman / Michael Holinstat / David M Thal / Alisa Glukhova /
Abstract: Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the ...Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.
History
DepositionMar 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 11, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Polyunsaturated fatty acid lipoxygenase ALOX12
C: Polyunsaturated fatty acid lipoxygenase ALOX12
D: Polyunsaturated fatty acid lipoxygenase ALOX12
A: Polyunsaturated fatty acid lipoxygenase ALOX12
hetero molecules


Theoretical massNumber of molelcules
Total (without water)307,5869
Polymers306,3314
Non-polymers1,2555
Water14,952830
1


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

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Components

#1: Protein
Polyunsaturated fatty acid lipoxygenase ALOX12 / Arachidonate (12S)-lipoxygenase / 12S-LOX / 12S-lipoxygenase / Arachidonate (15S)-lipoxygenase / ...Arachidonate (12S)-lipoxygenase / 12S-LOX / 12S-lipoxygenase / Arachidonate (15S)-lipoxygenase / Linoleate (13S)-lipoxygenase / Lipoxin synthase 12-LO / Platelet-type lipoxygenase 12


Mass: 76582.703 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ALOX12, 12LO, LOG12 / Production host: Homo sapiens (human)
References: UniProt: P18054, Oxidoreductases; Acting on single donors with incorporation of molecular oxygen (oxygenases); With incorporation of two atoms of oxygen, arachidonate 12-lipoxygenase, ...References: UniProt: P18054, Oxidoreductases; Acting on single donors with incorporation of molecular oxygen (oxygenases); With incorporation of two atoms of oxygen, arachidonate 12-lipoxygenase, arachidonate 15-lipoxygenase, Hydrolases; Acting on ether bonds; Ether hydrolases
#2: Chemical
ChemComp-FE2 / FE (II) ION


Mass: 55.845 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe
#3: Chemical ChemComp-3VV / S-{(3R,5R,9R)-1-[(2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxy-3-(phosphonooxy)tetrahydrofuran-2-yl]-3,5,9-trihydroxy-8,8-dimethyl-3,5-dioxido-10,14-dioxo-2,4,6-trioxa-11,15-diaza-3lambda~5~,5lambda~5~-diphosphaheptadecan-17-yl} (9Z)-octadec-9-enethioate (non-preferred name) / oleoyl-CoA


Mass: 1031.980 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C39H68N7O17P3S / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 830 / Source method: isolated from a natural source / Formula: H2O
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: Tetrameric human 12-Lipoxygenase bound to endogenous inhibitor oleoyl-CoA
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 / Calibrated magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
4Gctf1.18CTF correction
7UCSF Chimeramodel fitting
12cryoSPARC3D reconstruction
19PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2736609
3D reconstructionResolution: 2.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 311579 / Symmetry type: POINT
Atomic model building
IDProtocol
1FLEXIBLE FIT
2
Atomic model building
ID 3D fitting-IDSource nameType
11AlphaFoldin silico model
22AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00421931
ELECTRON MICROSCOPYf_angle_d0.65729821
ELECTRON MICROSCOPYf_dihedral_angle_d11.7018211
ELECTRON MICROSCOPYf_chiral_restr0.0473207
ELECTRON MICROSCOPYf_plane_restr0.0093884

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