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- PDB-8sml: hPAD4 bound to inhibitory Fab hI365 -

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Basic information

Entry
Database: PDB / ID: 8sml
TitlehPAD4 bound to inhibitory Fab hI365
Components
  • Fab hI365 heavy chain
  • Fab hI365 light chain
  • Protein-arginine deiminase type-4
KeywordsHYDROLASE/IMMUNE SYSTEM / complex / deiminase / enzyme / arginine / inflammation / calcium binding / Fab / IMMUNE SYSTEM / HYDROLASE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


histone H3R2 arginine deiminase activity / histone H3R8 arginine deiminase activity / histone H3R17 arginine deiminase activity / histone arginine deiminase activity / histone H3R26 arginine deiminase activity / protein-arginine deiminase / protein-arginine deiminase activity / stem cell population maintenance / Chromatin modifying enzymes / post-translational protein modification ...histone H3R2 arginine deiminase activity / histone H3R8 arginine deiminase activity / histone H3R17 arginine deiminase activity / histone arginine deiminase activity / histone H3R26 arginine deiminase activity / protein-arginine deiminase / protein-arginine deiminase activity / stem cell population maintenance / Chromatin modifying enzymes / post-translational protein modification / protein modification process / nucleosome assembly / chromatin organization / chromatin remodeling / innate immune response / calcium ion binding / protein-containing complex / nucleoplasm / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Protein-arginine deiminase / Protein-arginine deiminase, C-terminal / Protein-arginine deiminase (PAD), N-terminal / Protein-arginine deiminase (PAD), central domain / Protein-arginine deiminase, central domain superfamily / PAD, N-terminal domain superfamily / Protein-arginine deiminase (PAD) / Protein-arginine deiminase (PAD) N-terminal domain / Protein-arginine deiminase (PAD) middle domain / Cupredoxin
Similarity search - Domain/homology
Protein-arginine deiminase type-4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMaker, A. / Verba, K.A.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (NIH/NIBIB)K99EB030587 United States
National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (NIH/NIBIB)R00EB030587 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)1P41CA1962 76 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA191018 United States
Damon Runyon Cancer Research FoundationDRG-2297-17 United States
Damon Runyon Cancer Research FoundationDFS-52-22 United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4.
Authors: Xin Zhou / Sophie Kong / Allison Maker / Soumya G Remesh / Kevin K Leung / Kliment A Verba / James A Wells /
Abstract: Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody ...Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.
History
DepositionApr 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 12, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein-arginine deiminase type-4
B: Fab hI365 light chain
C: Fab hI365 heavy chain
D: Protein-arginine deiminase type-4
E: Fab hI365 light chain
F: Fab hI365 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)251,59312
Polymers251,3526
Non-polymers2406
Water00
1


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

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Components

#1: Protein Protein-arginine deiminase type-4 / HL-60 PAD / Peptidylarginine deiminase IV / Protein-arginine deiminase type IV


Mass: 77902.953 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PADI4, PAD4, PADI5, PDI5 / Variant: 9606 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UM07, protein-arginine deiminase
#2: Antibody Fab hI365 light chain


Mass: 23157.678 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab hI365 heavy chain


Mass: 24615.482 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
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: hPAD4 bound to inhibitory Fab hI365 / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.248 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris hydrochlorideTris-HCl1
2150 mMsodium chlorideNaCl1
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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 72 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2particle selection
2SerialEMimage acquisition
4cryoSPARC2CTF correction
9Rosettamodel refinement
10ISOLDEmodel refinement
11cryoSPARC2initial Euler assignment
12cisTEM1final Euler assignment
13cisTEM1classification
14cisTEM13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45834 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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