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- EMDB-40047: Structure of human ENPP1 in complex with variable heavy domain VH27.2 -

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Basic information

Entry
Database: EMDB / ID: EMD-40047
TitleStructure of human ENPP1 in complex with variable heavy domain VH27.2
Map dataFrom cryosparc non-uniform refinement and local resolution filter
Sample
  • Complex: ENPP1-VH27 complex
    • Protein or peptide: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1, secreted form, Immunoglobulin gamma-1 heavy chain fusion
    • Protein or peptide: VH27
  • Ligand: ZINC ION
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM IONCalcium
  • Ligand: water
Keywordsphosphodiesterase / inhibitor / IMMUNE SYSTEM
Function / homology
Function and homology information


GTP diphosphatase activity / cyclic-GMP-AMP hydrolase activity / negative regulation of hh target transcription factor activity / inorganic diphosphate transport / Vitamin B2 (riboflavin) metabolism / UTP diphosphatase activity / phosphodiesterase I / 3'-phosphoadenosine 5'-phosphosulfate binding / dinucleotide phosphatase activity / Vitamin B5 (pantothenate) metabolism ...GTP diphosphatase activity / cyclic-GMP-AMP hydrolase activity / negative regulation of hh target transcription factor activity / inorganic diphosphate transport / Vitamin B2 (riboflavin) metabolism / UTP diphosphatase activity / phosphodiesterase I / 3'-phosphoadenosine 5'-phosphosulfate binding / dinucleotide phosphatase activity / Vitamin B5 (pantothenate) metabolism / nucleoside triphosphate catabolic process / nucleotide diphosphatase / 3'-phosphoadenosine 5'-phosphosulfate metabolic process / intracellular phosphate ion homeostasis / negative regulation of protein autophosphorylation / nucleoside triphosphate diphosphatase activity / nucleic acid metabolic process / sequestering of triglyceride / negative regulation of bone mineralization / ATP diphosphatase activity / negative regulation of glycogen biosynthetic process / phosphate ion homeostasis / melanocyte differentiation / phosphodiesterase I activity / scavenger receptor activity / negative regulation of glucose import / regulation of bone mineralization / phosphate-containing compound metabolic process / exonuclease activity / negative regulation of fat cell differentiation / polysaccharide binding / response to ATP / bone mineralization / immunoglobulin complex / phosphatase activity / 3',5'-cyclic-AMP phosphodiesterase activity / response to inorganic substance / ATP metabolic process / negative regulation of insulin receptor signaling pathway / generation of precursor metabolites and energy / insulin receptor binding / negative regulation of cell growth / cellular response to insulin stimulus / gene expression / basolateral plasma membrane / adaptive immune response / nucleic acid binding / immune response / lysosomal membrane / calcium ion binding / cell surface / protein homodimerization activity / extracellular space / zinc ion binding / extracellular region / ATP binding / membrane / plasma membrane
Similarity search - Function
Somatomedin B domain, chordata / Somatomedin B -like domains / Somatomedin B domain / Somatomedin B-like domain superfamily / Somatomedin B domain / Somatomedin B domain (SMB) signature. / Somatomedin B (SMB) domain profile. / Type I phosphodiesterase/nucleotide pyrophosphatase/phosphate transferase / Type I phosphodiesterase / nucleotide pyrophosphatase / Extracellular Endonuclease, subunit A ...Somatomedin B domain, chordata / Somatomedin B -like domains / Somatomedin B domain / Somatomedin B-like domain superfamily / Somatomedin B domain / Somatomedin B domain (SMB) signature. / Somatomedin B (SMB) domain profile. / Type I phosphodiesterase/nucleotide pyrophosphatase/phosphate transferase / Type I phosphodiesterase / nucleotide pyrophosphatase / Extracellular Endonuclease, subunit A / DNA/RNA non-specific endonuclease / DNA/RNA non-specific endonuclease / DNA/RNA non-specific endonuclease / DNA/RNA non-specific endonuclease / DNA/RNA non-specific endonuclease superfamily / His-Me finger superfamily / Alkaline-phosphatase-like, core domain superfamily / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin subtype / Immunoglobulin / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Immunoglobulin gamma-1 heavy chain / Ectonucleotide pyrophosphatase/phosphodiesterase family member 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsCarozza JA / Wang H / Solomon PE / Wells JA / Li L
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P41 CA196276 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA258427 United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Discovery of VH domains that allosterically inhibit ENPP1.
Authors: Paige E Solomon / Colton J Bracken / Jacqueline A Carozza / Haoqing Wang / Elizabeth P Young / Alon Wellner / Chang C Liu / E Alejandro Sweet-Cordero / Lingyin Li / James A Wells /
Abstract: Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine ...Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.
History
DepositionMar 10, 2023-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateJan 3, 2024-
Current statusJan 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40047.png

Downloads & links

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Map

FileDownload / File: emd_40047.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFrom cryosparc non-uniform refinement and local resolution filter
Voxel sizeX=Y=Z: 0.8677 Å
Density
Contour LevelBy AUTHOR: 0.216
Minimum - Maximum-0.95711213 - 1.7641419
Average (Standard dev.)0.0010517604 (±0.02670029)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 312.37198 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A from non-uniform refinement

Fileemd_40047_half_map_1.map
Annotationhalf map A from non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B from non-uniform refinement

Fileemd_40047_half_map_2.map
Annotationhalf map B from non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ENPP1-VH27 complex

EntireName: ENPP1-VH27 complex
Components
  • Complex: ENPP1-VH27 complex
    • Protein or peptide: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1, secreted form, Immunoglobulin gamma-1 heavy chain fusion
    • Protein or peptide: VH27
  • Ligand: ZINC ION
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM IONCalcium
  • Ligand: water

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Supramolecule #1: ENPP1-VH27 complex

SupramoleculeName: ENPP1-VH27 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1,...

MacromoleculeName: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1, secreted form, Immunoglobulin gamma-1 heavy chain fusion
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 115.488617 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GEKSWVEEPC ESINEPQCPA GFETPPTLLF SLDGFRAEYL HTWGGLLPVI SKLKKCGTYT KNMRPVYPTK AFPNHYSIVT GLYPESHGI IDNKMYDPKM NASFSLKSKE KFNPEWYKGE PIWVTAKYQG LKSGTFFWPG SDVEINGIFP DIYKMYNGSV P FEERILAV ...String:
GEKSWVEEPC ESINEPQCPA GFETPPTLLF SLDGFRAEYL HTWGGLLPVI SKLKKCGTYT KNMRPVYPTK AFPNHYSIVT GLYPESHGI IDNKMYDPKM NASFSLKSKE KFNPEWYKGE PIWVTAKYQG LKSGTFFWPG SDVEINGIFP DIYKMYNGSV P FEERILAV LQWLQLPKDE RPHFYTLYLE EPDSSGHSYG PVSSEVIKAL QRVDGMVGML MDGLKELNLH RCLNLILISD HG MEQGSCK KYIYLNKYLG DVKNIKVIYG PAARLRPSDV PDKYYSFNYE GIARNLSCRE PNQHFKPYLK HFLPKRLHFA KSD RIEPLT FYLDPQWQLA LNPSERKYCG SGFHGSDNVF SNMQALFVGY GPGFKHGIEA DTFENIEVYN LMCDLLNLTP APNN GTHGS LNHLLKNPVY TPKHPKEVHP LVQCPFTRNP RDNLGCSCNP SILPIEDFQT QFNLTVAEEK IIKHETLPYG RPRVL QKEN TICLLSQHQF MSGYSQDILM PLWTSYTVDR NDSFSTEDFS NCLYQDFRIP LSPVHKCSFY KNNTKVSYGF LSPPQL NKN SSGIYSEALL TTNIVPMYQS FQVIWRYFHD TLLRKYAEER NGVNVVSGPV FDFDYDGRCD SLENLRQKRR VIRNQEI LI PTHFFIVLTS CKDTSQTPLH CENLDTLAFI LPHRTDNSES CVHGKHDSSW VEELLMLHRA RITDVEHITG LSFYQQRK E PVSDILKLKT HLPTFSQEDT SSGGGGENLY FQSSGGGSGG GEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV D KSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKGGGG SGLNDIFEAQ KIEWHEG

UniProtKB: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1, Immunoglobulin gamma-1 heavy chain

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Macromolecule #2: VH27

MacromoleculeName: VH27 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.564035 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVQLVESGGG LVQPGGSLRL SCAASGFDIY YSYYIGWVRR APGKGEELVA RISPSYGSTS YADSVKGRFT ISADISKNTA YLQMNSLRV EDTAVYYCAR FAYPWYVADD ALDYWGQGTL VTVSS

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM / Adenosine monophosphate

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #7: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration2 mg/mL
BufferpH: 7.4 / Details: phosphate buffered saline
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 57.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6wfj

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77023
FSC plot (resolution estimation)

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