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- PDB-8ghr: Structure of human ENPP1 in complex with variable heavy domain VH27.2 -

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Basic information

Entry
Database: PDB / ID: 8ghr
TitleStructure of human ENPP1 in complex with variable heavy domain VH27.2
Components
  • Ectonucleotide pyrophosphatase/phosphodiesterase family member 1, secreted form, Immunoglobulin gamma-1 heavy chain fusion
  • VH27
KeywordsIMMUNE SYSTEM / phosphodiesterase / inhibitor
Function / homology
Function and homology information


cyclic-GMP-AMP hydrolase activity / negative regulation of hh target transcription factor activity / inorganic diphosphate transport / Vitamin B2 (riboflavin) metabolism / GTP diphosphatase activity / UTP diphosphatase activity / 3'-phosphoadenosine 5'-phosphosulfate binding / dinucleotide phosphatase activity / phosphodiesterase I / nucleoside triphosphate catabolic process ...cyclic-GMP-AMP hydrolase activity / negative regulation of hh target transcription factor activity / inorganic diphosphate transport / Vitamin B2 (riboflavin) metabolism / GTP diphosphatase activity / UTP diphosphatase activity / 3'-phosphoadenosine 5'-phosphosulfate binding / dinucleotide phosphatase activity / phosphodiesterase I / nucleoside triphosphate catabolic process / 8-oxo-(d)RTP hydrolase activity / nucleotide diphosphatase / 3'-phosphoadenosine 5'-phosphosulfate metabolic process / nucleic acid metabolic process / Vitamin B5 (pantothenate) metabolism / nucleoside triphosphate diphosphatase activity / ATP diphosphatase activity / negative regulation of glycogen biosynthetic process / negative regulation of bone mineralization / phosphate ion homeostasis / melanocyte differentiation / intracellular phosphate ion homeostasis / regulation of bone mineralization / negative regulation of D-glucose import / phosphate-containing compound metabolic process / phosphodiesterase I activity / exonuclease activity / scavenger receptor activity / immunoglobulin complex / response to ATP / negative regulation of fat cell differentiation / bone mineralization / phosphatase activity / polysaccharide binding / 3',5'-cyclic-AMP phosphodiesterase activity / 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 / basolateral plasma membrane / gene expression / adaptive immune response / nucleic acid binding / immune response / lysosomal membrane / calcium ion binding / cell surface / protein homodimerization activity / extracellular space / extracellular region / zinc ion binding / ATP binding / membrane / plasma membrane
Similarity search - Function
Extracellular Endonuclease; Chain A / 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 ...Extracellular Endonuclease; Chain A / 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 subtype / Immunoglobulin / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold / Immunoglobulins / Immunoglobulin-like / Sandwich / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / Immunoglobulin gamma-1 heavy chain / Ectonucleotide pyrophosphatase/phosphodiesterase family member 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsCarozza, J.A. / Wang, H. / Solomon, P.E. / Wells, J.A. / Li, L.
Funding support United States, 2items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 2, 2023Provider: repository / Type: Initial release
Revision 1.0Aug 2, 2023Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Aug 2, 2023Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.1Jan 3, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1, secreted form, Immunoglobulin gamma-1 heavy chain fusion
B: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1, secreted form, Immunoglobulin gamma-1 heavy chain fusion
C: VH27
D: VH27
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,72420
Polymers258,1054
Non-polymers3,61916
Water1086
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, size exclusion chromatography performed to establish dimer complex formation.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 2 types, 4 molecules ABCD

#1: Antibody Ectonucleotide pyrophosphatase/phosphodiesterase family member 1, secreted form, Immunoglobulin gamma-1 heavy chain fusion / Immunoglobulin gamma-1 heavy chain NIE


Mass: 115488.617 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ENPP1, M6S1, NPPS, PC1, PDNP1 / Production host: Homo sapiens (human) / References: UniProt: P22413, UniProt: P0DOX5
#2: Antibody VH27


Mass: 13564.035 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Sugars , 2 types, 8 molecules

#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 4 types, 14 molecules

#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#7: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: ENPP1-VH27 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4 / Details: phosphate buffered saline
SpecimenConc.: 2 mg/ml / Embedding 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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 57 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77023 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00214262
ELECTRON MICROSCOPYf_angle_d0.55819440
ELECTRON MICROSCOPYf_dihedral_angle_d6.0361980
ELECTRON MICROSCOPYf_chiral_restr0.0422118
ELECTRON MICROSCOPYf_plane_restr0.0052498

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