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- EMDB-40039: The structure of h12-LOX in monomeric form -

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Basic information

Entry
Database: EMDB / ID: EMD-40039
TitleThe structure of h12-LOX in monomeric form
Map dataThe unsharpened map from Non-Uniform refinement in CryoSPARC
Sample
  • Complex: Monomeric 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 / Synthesis of Lipoxins (LX) / Oxidoreductases; Acting on single donors with incorporation of molecular oxygen (oxygenases); With incorporation of two atoms of oxygen / lipoxygenase pathway / 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 / 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: 2.76 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-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40039.png

Downloads & links

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Map

FileDownload / File: emd_40039.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe unsharpened map from Non-Uniform refinement in CryoSPARC
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-0.7337574 - 1.6171471
Average (Standard dev.)0.0004512657 (±0.0339399)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_40039_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally filtered map after Non-Uniform refinement in CryoSPARC....

Fileemd_40039_additional_1.map
AnnotationLocally filtered map after Non-Uniform refinement in CryoSPARC. Sharpened with B -30.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 from Non-Uniform refinement in CryoSPARC

Fileemd_40039_half_map_1.map
AnnotationHalf map 1 from Non-Uniform refinement in CryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from Non-Uniform refinement in CryoSPARC

Fileemd_40039_half_map_2.map
AnnotationHalf map 2 from Non-Uniform refinement in CryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Monomeric human 12-Lipoxygenase

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

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Supramolecule #1: Monomeric human 12-Lipoxygenase

SupramoleculeName: Monomeric human 12-Lipoxygenase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 75 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: 1 / 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: 76.451508 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: HHHHHHGRYR IRVATGAWLF SGSYNRVQLW LVGTRGEAEL ELQLRPARGE EEEFDHDVAE DLGLLQFVRL RKHHWLVDDA WFCDRITVQ GPGACAEVAF PCYRWVQGED ILSLPEGTAR LPGDNALDMF QKHREKELKD RQQIYCWATW KEGLPLTIAA D RKDDLPPN ...String:
HHHHHHGRYR IRVATGAWLF SGSYNRVQLW LVGTRGEAEL ELQLRPARGE EEEFDHDVAE DLGLLQFVRL RKHHWLVDDA WFCDRITVQ GPGACAEVAF PCYRWVQGED ILSLPEGTAR LPGDNALDMF QKHREKELKD RQQIYCWATW KEGLPLTIAA D RKDDLPPN MRFHEEKRLD FEWTLKAGAL EMALKRVYTL LSSWNCLEDF DQIFWGQKSA LAEKVRQCWQ DDELFSYQFL NG ANPMLLR RSTSLPSRLV LPSGMEELQA QLEKELQNGS LFEADFILLD GIPANVIRGE KQYLAAPLVM LKMEPNGKLQ PMV IQIQPP SPSSPTPTLF LPSDPPLAWL LAKSWVRNSD FQLHEIQYHL LNTHLVAEVI AVATMRCLPG LHPIFKFLIP HIRY TMEIN TRARTQLISD GGIFDKAVST GGGGHVQLLR RAAAQLTYCS LCPPDDLADR GLLGLPGALY AHDALRLWEI IARYV EGIV HLFYQRDDIV KGDPELQAWC REITEVGLCQ AQDRGFPVSF QSQSQLCHFL TMCVFTCTAQ HAAINQGQLD WYAWVP NAP CTMRMPPPTT KEDVTMATVM GSLPDVRQAC LQMAISWHLS RRQPDMVPLG HHKEKYFSGP KPKAVLNQFR TDLEKLE KE ITARNEQLDW PYEYLKPSCI ENSVTI

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: 1 / 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
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: AIR / Details: 15mA
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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 FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2736609
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 35994
FSC plot (resolution estimation)

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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-8ghb:
The structure of h12-LOX in monomeric form

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