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- EMDB-40590: hPAD4 bound to inhibitory Fab hI365 -

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Basic information

Entry
Database: EMDB / ID: EMD-40590
TitlehPAD4 bound to inhibitory Fab hI365
Map dataRelion sharpened map of PAD4 bound to inhibitory fab 365
Sample
  • Complex: hPAD4 bound to inhibitory Fab hI365
    • Protein or peptide: Protein-arginine deiminase type-4
    • Protein or peptide: Fab hI365 light chain
    • Protein or peptide: Fab hI365 heavy chain
  • Ligand: CALCIUM IONCalcium
Keywordscomplex / 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 / cytosol / cytoplasm
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMaker A / Verba KA
Funding support United States, 6 items
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, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40590.png

Downloads & links

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Map

FileDownload / File: emd_40590.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRelion sharpened map of PAD4 bound to inhibitory fab 365
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 7.0
Minimum - Maximum-28.285236000000001 - 40.667994999999998
Average (Standard dev.)0.0068629757 (±0.44100812)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 427.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40590_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of PAD4 bound to inhibitory fab 365

Fileemd_40590_half_map_1.map
AnnotationHalf map 1 of PAD4 bound to inhibitory fab 365
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of PAD4 bound to inhibitory fab 365

Fileemd_40590_half_map_2.map
AnnotationHalf map 2 of PAD4 bound to inhibitory fab 365
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : hPAD4 bound to inhibitory Fab hI365

EntireName: hPAD4 bound to inhibitory Fab hI365
Components
  • Complex: hPAD4 bound to inhibitory Fab hI365
    • Protein or peptide: Protein-arginine deiminase type-4
    • Protein or peptide: Fab hI365 light chain
    • Protein or peptide: Fab hI365 heavy chain
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: hPAD4 bound to inhibitory Fab hI365

SupramoleculeName: hPAD4 bound to inhibitory Fab hI365 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 248 KDa

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Macromolecule #1: Protein-arginine deiminase type-4

MacromoleculeName: Protein-arginine deiminase type-4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: protein-arginine deiminase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 77.902953 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHASGG LNDIFEAQKI EWHEENLYFQ GTSAQGTLIR VTPEQPTHAV CVLGTLTQLD ICSSAPEDCT SFSINASPGV VVDIAHGPP AKKKSTGSST WPLDPGVEVT LTMKAASGST GDQKVQISYY GPKTPPVKAL LYLTGVEISL CADITRTGKV K PTRAVKDQ ...String:
HHHHHHASGG LNDIFEAQKI EWHEENLYFQ GTSAQGTLIR VTPEQPTHAV CVLGTLTQLD ICSSAPEDCT SFSINASPGV VVDIAHGPP AKKKSTGSST WPLDPGVEVT LTMKAASGST GDQKVQISYY GPKTPPVKAL LYLTGVEISL CADITRTGKV K PTRAVKDQ RTWTWGPCGQ GAILLVNCDR DNLESSAMDC EDDEVLDSED LQDMSLMTLS TKTPKDFFTN HTLVLHVARS EM DKVRVFQ ATRGKLSSKC SVVLGPKWPS HYLMVPGGKH NMDFYVEALA FPDTDFPGLI TLTISLLDTS NLELPEAVVF QDS VVFRVA PWIMTPNTQP PQEVYACSIF ENEDFLKSVT TLAMKAKCKL TICPEEENMD DQWMQDEMEI GYIQAPHKTL PVVF DSPRN RGLKEFPIKR VMGPDFGYVT RGPQTGGISG LDSFGNLEVS PPVTVRGKEY PLGRILFGDS CYPSNDSRQM HQALQ DFLS AQQVQAPVKL YSDWLSVGHV DEFLSFVPAP DRKGFRLLLA SPRSCYKLFQ EQQNEGHGEA LLFEGIKKKK QQKIKN ILS NKTLREHNSF VERCIDWNRE LLKRELGLAE SDIIDIPQLF KLKEFSKAEA FFPNMVNMLV LGKHLGIPKP FGPVING RC CLEEKVCSLL EPLGLQCTFI NDFFTYHIRH GEVHCGTNVR RKPFSFKWWN MVP

UniProtKB: Protein-arginine deiminase type-4

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Macromolecule #2: Fab hI365 light chain

MacromoleculeName: Fab hI365 light chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.157678 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QSSSSLVTFG QGTKVEIKRT VAAPSVFIFP PSDSQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QSSSSLVTFG QGTKVEIKRT VAAPSVFIFP PSDSQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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Macromolecule #3: Fab hI365 heavy chain

MacromoleculeName: Fab hI365 heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.615482 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFNFY YSIHWVRQAP GKGLEWVASI SPYSGYTSYA DSVKGRFTIS ADTSKNTAYL QMNSLRAED TAVYYCARKH PGSYPFWGWA LDYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP V TVSWNSGA ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFNFY YSIHWVRQAP GKGLEWVASI SPYSGYTSYA DSVKGRFTIS ADTSKNTAYL QMNSLRAED TAVYYCARKH PGSYPFWGWA LDYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP V TVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THT

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 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
Component:
ConcentrationFormulaName
50.0 mMTris-HClTrisTris hydrochloride
150.0 mMNaClSodium chloridesodium chloride
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: 1.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 72.0 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:

1wd9


Details: Fab: 6OTC, 1N8Z
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0) / Number images used: 45834
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8sml:
hPAD4 bound to inhibitory Fab hI365

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