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- EMDB-35500: Cryo-EM structure of the gastric proton pump with bound DQ-02 -

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Basic information

Entry
Database: EMDB / ID: EMD-35500
TitleCryo-EM structure of the gastric proton pump with bound DQ-02
Map data
Sample
  • Complex: hetero dimer of alpha and beta subunit
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit alpha
    • Protein or peptide: Potassium-transporting ATPase subunit beta
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: MAGNESIUM ION
  • Ligand: 4-[[5-chloranyl-2-(4-chlorophenyl)phenyl]methoxy]-N-methyl-but-2-yn-1-amine
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL
  • Ligand: water
KeywordsP-type ATPase / P2-type ATPase / MEMBRANE PROTEIN
Function / homology
Function and homology information


regulation of proton transport / pH reduction / potassium:proton exchanging ATPase complex / P-type potassium:proton transporter activity / Ion transport by P-type ATPases / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / potassium ion import across plasma membrane ...regulation of proton transport / pH reduction / potassium:proton exchanging ATPase complex / P-type potassium:proton transporter activity / Ion transport by P-type ATPases / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / ATPase activator activity / potassium ion transmembrane transport / proton transmembrane transport / cell adhesion / response to xenobiotic stimulus / apical plasma membrane / magnesium ion binding / ATP hydrolysis activity / ATP binding
Similarity search - Function
Gastric H+/K+-transporter P-type ATPase, N-terminal / Gastric H+/K+-ATPase, N terminal domain / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit beta superfamily / : / Sodium / potassium ATPase beta chain / Sodium and potassium ATPases beta subunits signature 1. / Sodium and potassium ATPases beta subunits signature 2. / P-type ATPase subfamily IIC, subunit alpha / Cation-transporting P-type ATPase, C-terminal ...Gastric H+/K+-transporter P-type ATPase, N-terminal / Gastric H+/K+-ATPase, N terminal domain / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit beta superfamily / : / Sodium / potassium ATPase beta chain / Sodium and potassium ATPases beta subunits signature 1. / Sodium and potassium ATPases beta subunits signature 2. / P-type ATPase subfamily IIC, subunit alpha / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / Cation transport ATPase (P-type) / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / haloacid dehalogenase-like hydrolase / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
Sodium/potassium-transporting ATPase subunit alpha / Potassium-transporting ATPase subunit beta
Similarity search - Component
Biological speciesSus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsAbe K / Yokoshima S / Yoshimori A
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H02426 Japan
CitationJournal: Commun Biol / Year: 2023
Title: Deep learning driven de novo drug design based on gastric proton pump structures.
Authors: Kazuhiro Abe / Mami Ozako / Miki Inukai / Yoe Matsuyuki / Shinnosuke Kitayama / Chisato Kanai / Chiaki Nagai / Chai C Gopalasingam / Christoph Gerle / Hideki Shigematsu / Nariyoshi Umekubo / ...Authors: Kazuhiro Abe / Mami Ozako / Miki Inukai / Yoe Matsuyuki / Shinnosuke Kitayama / Chisato Kanai / Chiaki Nagai / Chai C Gopalasingam / Christoph Gerle / Hideki Shigematsu / Nariyoshi Umekubo / Satoshi Yokoshima / Atsushi Yoshimori /
Abstract: Existing drugs often suffer in their effectiveness due to detrimental side effects, low binding affinity or pharmacokinetic problems. This may be overcome by the development of distinct compounds. ...Existing drugs often suffer in their effectiveness due to detrimental side effects, low binding affinity or pharmacokinetic problems. This may be overcome by the development of distinct compounds. Here, we exploit the rich structural basis of drug-bound gastric proton pump to develop compounds with strong inhibitory potency, employing a combinatorial approach utilizing deep generative models for de novo drug design with organic synthesis and cryo-EM structural analysis. Candidate compounds that satisfy pharmacophores defined in the drug-bound proton pump structures, were designed in silico utilizing our deep generative models, a workflow termed Deep Quartet. Several candidates were synthesized and screened according to their inhibition potencies in vitro, and their binding poses were in turn identified by cryo-EM. Structures reaching up to 2.10 Å resolution allowed us to evaluate and re-design compound structures, heralding the most potent compound in this study, DQ-18 (N-methyl-4-((2-(benzyloxy)-5-chlorobenzyl)oxy)benzylamine), which shows a K value of 47.6 nM. Further high-resolution cryo-EM analysis at 2.08 Å resolution unambiguously determined the DQ-18 binding pose. Our integrated approach offers a framework for structure-based de novo drug development based on the desired pharmacophores within the protein structure.
History
DepositionFeb 28, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35500.png

Downloads & links

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Map

FileDownload / File: emd_35500.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.75 Å/pix.
x 450 pix.
= 338.4 Å		0.75 Å/pix.
x 450 pix.
= 338.4 Å		0.75 Å/pix.
x 450 pix.
= 338.4 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.752 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.557
Minimum - Maximum-3.6598399 - 6.19196
Average (Standard dev.)-0.0016792481 (±0.09381471)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 338.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35500_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_35500_half_map_1.map
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Half map: #1

Fileemd_35500_half_map_2.map
Projections & Slices
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Sample components

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Entire : hetero dimer of alpha and beta subunit

EntireName: hetero dimer of alpha and beta subunit
Components
  • Complex: hetero dimer of alpha and beta subunit
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit alpha
    • Protein or peptide: Potassium-transporting ATPase subunit beta
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: MAGNESIUM ION
  • Ligand: 4-[[5-chloranyl-2-(4-chlorophenyl)phenyl]methoxy]-N-methyl-but-2-yn-1-amine
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL
  • Ligand: water

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Supramolecule #1: hetero dimer of alpha and beta subunit

SupramoleculeName: hetero dimer of alpha and beta subunit / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 135 KDa

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Macromolecule #1: Sodium/potassium-transporting ATPase subunit alpha

MacromoleculeName: Sodium/potassium-transporting ATPase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 114.325547 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GKAENYELYQ VELGPGPSGD MAAKMSKKKA GRGGGKRKEK LENMKKEMEI NDHQLSVAEL EQKYQTSATK GLSASLAAEL LLRDGPNAL RPPRGTPEYV KFARQLAGGL QCLMWVAAAI CLIAFAIQAS EGDLTTDDNL YLALALIAVV VVTGCFGYYQ E FKSTNIIA ...String:
GKAENYELYQ VELGPGPSGD MAAKMSKKKA GRGGGKRKEK LENMKKEMEI NDHQLSVAEL EQKYQTSATK GLSASLAAEL LLRDGPNAL RPPRGTPEYV KFARQLAGGL QCLMWVAAAI CLIAFAIQAS EGDLTTDDNL YLALALIAVV VVTGCFGYYQ E FKSTNIIA SFKNLVPQQA TVIRDGDKFQ INADQLVVGD LVEMKGGDRV PADIRILQAQ GCKVDNSSLT GESEPQTRSP EC THESPLE TRNIAFFSTM CLEGTAQGLV VNTGDRTIIG RIASLASGVE NEKTPIAIEI EHFVDIIAGL AILFGATFFI VAM CIGYTF LRAMVFFMAI VVAYVPEGLL ATVTVCLSLT AKRLASKNCV VKNLEAVETL GSTSVICS(BFD)K TGTLTQNRMT VSHLWFDNH IHSADTTEDQ SGQTFDQSSE TWRALCRVLT LCNRAAFKSG QDAVPVPKRI VIGDASETAL LKFSELTLGN A MGYRERFP KVCEIPFNST NKFQLSIHTL EDPRDPRHVL VMKGAPERVL ERCSSILIKG QELPLDEQWR EAFQTAYLSL GG LGERVLG FCQLYLSEKD YPPGYAFDVE AMNFPTSGLC FAGLVSMIDP PRATVPDAVL KCRTAGIRVI MVTGDHPITA KAI AASVGI ISEGSETVED IAARLRVPVD QVNRKDARAC VINGMQLKDM DPSELVEALR THPEMVFART SPQQKLVIVE SCQR LGAIV AVTGDGVNDS PALKKADIGV AMGIAGSDAA KNAADMILLD DNFASIVTGV EQGRLIFDNL KKSIAYTLTK NIPEL TPYL IYITVSVPLP LGCITILFIE LCTDIFPSVS LAYEKAESDI MHLRPRNPKR DRLVNEPLAA YSYFQIGAIQ SFAGFT DYF TAMAQEGWFP LLCVGLRPQW ENHHLQDLQD SYGQEWTFGQ RLYQQYTCYT VFFISIEMCQ IADVLIRKTR RLSAFQQ GF FRNRILVIAI VFQVCIGCFL CYCPGMPNIF NFMPIRFQWW LVPMPFSLLI FVYDEIRKLG VRCCPGSWWD QELYY

UniProtKB: Sodium/potassium-transporting ATPase subunit alpha

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Macromolecule #2: Potassium-transporting ATPase subunit beta

MacromoleculeName: Potassium-transporting ATPase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 33.113844 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAALQEKKSC SQRMEEFQRY CWNPDTGQML GRTLSRWVWI SLYYVAFYVV MSGIFALCIY VLMRTIDPYT PDYQDQLKSP GVTLRPDVY GEKGLDISYN VSDSTTWAGL AHTLHRFLAG YSPAAQEGSI NCTSEKYFFQ ESFLAPNHTK FSCKFTADML Q NCSGRPDP ...String:
MAALQEKKSC SQRMEEFQRY CWNPDTGQML GRTLSRWVWI SLYYVAFYVV MSGIFALCIY VLMRTIDPYT PDYQDQLKSP GVTLRPDVY GEKGLDISYN VSDSTTWAGL AHTLHRFLAG YSPAAQEGSI NCTSEKYFFQ ESFLAPNHTK FSCKFTADML Q NCSGRPDP TFGFAEGKPC FIIKMNRIVK FLPGNSTAPR VDCAFLDQPR DGPPLQVEYF PANGTYSLHY FPYYGKKAQP HY SNPLVAA KLLNVPRNRD VVIVCKILAE HVSFDNPHDP YEGKVEFKLK IQK

UniProtKB: Potassium-transporting ATPase subunit beta

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Macromolecule #3: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 3 / Number of copies: 5 / Formula: PCW
Molecular weightTheoretical: 787.121 Da
Chemical component information

ChemComp-PCW:
1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DOPC, phospholipid*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: 4-[[5-chloranyl-2-(4-chlorophenyl)phenyl]methoxy]-N-methyl-but-2-...

MacromoleculeName: 4-[[5-chloranyl-2-(4-chlorophenyl)phenyl]methoxy]-N-methyl-but-2-yn-1-amine
type: ligand / ID: 5 / Number of copies: 1 / Formula: PWI
Molecular weightTheoretical: 334.24 Da
Chemical component information

ChemComp-PWI:
4-[[5-chloranyl-2-(4-chlorophenyl)phenyl]methoxy]-N-methyl-but-2-yn-1-amine

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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: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #7: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 7 / Number of copies: 2 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 369 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

Concentration8 mg/mL
BufferpH: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm

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Image processing

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

5ylv

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 354147
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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