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- EMDB-40040: The structure of h12-LOX in dimeric form -

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Basic information

Entry
Database: EMDB / ID: EMD-40040
TitleThe structure of h12-LOX in dimeric form
Map dataThis is a composite map from the individual local refinements of 12-LOX "open" and "closed" subunits
Sample
  • Complex: Dimeric human 12-Lipoxygenase
    • Protein or peptide: Polyunsaturated fatty acid lipoxygenase ALOX12
  • Ligand: FE (II) ION
KeywordsLipoxygenase / platelets / lipid-modifying enzyme / lipid oxidation / OXIDOREDUCTASE
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 / Biosynthesis of Lipoxins (LX) / lipoxygenase pathway / arachidonate metabolic process / Oxidoreductases; Acting on single donors with incorporation of molecular oxygen (oxygenases); With incorporation of two atoms of oxygen / hepoxilin biosynthetic process / negative regulation of muscle cell apoptotic process / lipid oxidation / linoleic acid metabolic process / negative regulation of platelet aggregation / superoxide anion generation / fatty acid oxidation / establishment of skin barrier / lipid metabolic process / sarcolemma / 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
Polyunsaturated fatty acid lipoxygenase ALOX12
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsBlack KA / Mobbs JI / Venugopal H / Thal DM / Glukhova A
Funding support United States, 2 items
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, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40040.png

Downloads & links

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Map

FileDownload / File: emd_40040.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a composite map from the individual local refinements of 12-LOX "open" and "closed" subunits
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.82 Å/pix.
x 320 pix.
= 262.4 Å		0.82 Å/pix.
x 320 pix.
= 262.4 Å		0.82 Å/pix.
x 320 pix.
= 262.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 11.0
Minimum - Maximum-37.210006999999997 - 68.241425000000007
Average (Standard dev.)0.0035315345 (±1.3849361)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 262.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Dimeric human 12-Lipoxygenase

EntireName: Dimeric human 12-Lipoxygenase
Components
  • Complex: Dimeric human 12-Lipoxygenase
    • Protein or peptide: Polyunsaturated fatty acid lipoxygenase ALOX12
  • Ligand: FE (II) ION

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Supramolecule #1: Dimeric human 12-Lipoxygenase

SupramoleculeName: Dimeric human 12-Lipoxygenase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: Polyunsaturated fatty acid lipoxygenase ALOX12

MacromoleculeName: Polyunsaturated fatty acid lipoxygenase ALOX12 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Oxidoreductases; Acting on single donors with incorporation of molecular oxygen (oxygenases); With incorporation of two atoms of oxygen
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 76.582703 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MHHHHHHGRY RIRVATGAWL FSGSYNRVQL WLVGTRGEAE LELQLRPARG EEEEFDHDVA EDLGLLQFVR LRKHHWLVDD AWFCDRITV QGPGACAEVA FPCYRWVQGE DILSLPEGTA RLPGDNALDM FQKHREKELK DRQQIYCWAT WKEGLPLTIA A DRKDDLPP ...String:
MHHHHHHGRY RIRVATGAWL FSGSYNRVQL WLVGTRGEAE LELQLRPARG EEEEFDHDVA EDLGLLQFVR LRKHHWLVDD AWFCDRITV QGPGACAEVA FPCYRWVQGE DILSLPEGTA RLPGDNALDM FQKHREKELK DRQQIYCWAT WKEGLPLTIA A DRKDDLPP NMRFHEEKRL DFEWTLKAGA LEMALKRVYT LLSSWNCLED FDQIFWGQKS ALAEKVRQCW QDDELFSYQF LN GANPMLL RRSTSLPSRL VLPSGMEELQ AQLEKELQNG SLFEADFILL DGIPANVIRG EKQYLAAPLV MLKMEPNGKL QPM VIQIQP PSPSSPTPTL FLPSDPPLAW LLAKSWVRNS DFQLHEIQYH LLNTHLVAEV IAVATMRCLP GLHPIFKFLI PHIR YTMEI NTRARTQLIS DGGIFDKAVS TGGGGHVQLL RRAAAQLTYC SLCPPDDLAD RGLLGLPGAL YAHDALRLWE IIARY VEGI VHLFYQRDDI VKGDPELQAW CREITEVGLC QAQDRGFPVS FQSQSQLCHF LTMCVFTCTA QHAAINQGQL DWYAWV PNA PCTMRMPPPT TKEDVTMATV MGSLPDVRQA CLQMAISWHL SRRQPDMVPL GHHKEKYFSG PKPKAVLNQF RTDLEKL EK EITARNEQLD WPYEYLKPSC IENSVTI

UniProtKB: Polyunsaturated fatty acid lipoxygenase ALOX12

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Macromolecule #2: FE (II) ION

MacromoleculeName: FE (II) ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: FE2
Molecular weightTheoretical: 55.845 Da

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2736609
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: OTHER / Software - Name: cryoSPARC
Details: This is a composite map. The resolution (FSC 0.143) for the "open" subunit is 2.33, and for the "closed" subunit is 2.54
Number images used: 126914
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8ghc:
The structure of h12-LOX in dimeric form

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Atomic model buiding 2

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-8ghc:
The structure of h12-LOX in dimeric form

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