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- EMDB-65636: Cryo-EM structure of inhibitor E822-1968 bound human urea transpo... -

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Basic information

Entry
Database: EMDB / ID: EMD-65636
TitleCryo-EM structure of inhibitor E822-1968 bound human urea transporter A2.
Map data
Sample
  • Complex: Homotrimer complex of human urea transporter
    • Protein or peptide: Urea transporter 2
  • Ligand: 3-(3-methoxyphenyl)-~{N}-[(3-methoxyphenyl)methyl]-1~{H}-pyrazole-5-carboxamide
KeywordsUrea transporter / MEMBRANE PROTEIN
Function / homology
Function and homology information


urea transmembrane transport / urea transport / amine transport / SLC-mediated transport of organic cations / urea transmembrane transporter activity / cell adhesion molecule binding / apical plasma membrane / membrane / plasma membrane
Similarity search - Function
Urea transporter / Urea transporter / Ammonium/urea transporter
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHuang S / Sun J
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82304601 China
CitationJournal: Nat Commun / Year: 2026
Title: Hotspot pocket-based discovery of urea transporter selective inhibitors.
Authors: Lei Liu / Zhi Li / Chao Zhang / Yan Zhang / Zhizhen Huang / Daolai Zhang / Dongfang Li / Juanjuan Zhao / Yuhao Miao / Boyang Cai / Kongkai Zhu / Jin-Peng Sun / Guige Hou / Ying Sun / Baoxue ...Authors: Lei Liu / Zhi Li / Chao Zhang / Yan Zhang / Zhizhen Huang / Daolai Zhang / Dongfang Li / Juanjuan Zhao / Yuhao Miao / Boyang Cai / Kongkai Zhu / Jin-Peng Sun / Guige Hou / Ying Sun / Baoxue Yang / Xiao Yu / Shenming Huang /
Abstract: Urea transporter (UT) inhibitors are a promising class of diuretics, as selective inhibitors targeting UT-A subtypes have demonstrated considerable therapeutic potential. Herein, we employ a two- ...Urea transporter (UT) inhibitors are a promising class of diuretics, as selective inhibitors targeting UT-A subtypes have demonstrated considerable therapeutic potential. Herein, we employ a two-round progressive hotspot pocket-based virtual screening approach combined with biological validation to identify M353-0039 as a highly potent and selective inhibitor of UT-A2. We conduct cryo-electron microscopy to solve the structures of UT-A2 bound with the two inhibitors, M353-0039 and E822-1968, at the resolution of 2.7 Å and 2.9 Å respectively, and elucidate the structural mechanism underlying the superior efficacy and selectivity of M353-0039. Compared with the inhibitor HQA2 and E822-1968, M353-0039 occupies a deeper binding pocket and forms more interactions with UT-A2, thus leading to greater inhibitory potency. We demonstrate that the selectivity of M353-0039 is driven by the nonconserved residues C285 and G322 within the "T-T" subpocket of UT-A2. Finally, we validate the selective effects of M353-0039 in inhibiting UT-A2 function both in mouse models and hepatic cell. These findings not only identify a selective inhibitor as a tool that can be applied to elucidate the unique physiological roles of UT-A2 but also provide an available method for efficiently developing UT-A-selective inhibitors with potent activity as the next-generation diuretics.
History
DepositionJul 31, 2025-
Header (metadata) releaseAug 5, 2026-
Map releaseAug 5, 2026-
UpdateAug 5, 2026-
Current statusAug 5, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_65636.png

Downloads & links

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Map

FileDownload / File: emd_65636.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 256 pix.
= 235.52 Å		0.92 Å/pix.
x 256 pix.
= 235.52 Å		0.92 Å/pix.
x 256 pix.
= 235.52 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.92 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.9209265 - 1.4743474
Average (Standard dev.)0.0014894798 (±0.042694673)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 235.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_65636_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_65636_half_map_2.map
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Sample components

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Entire : Homotrimer complex of human urea transporter

EntireName: Homotrimer complex of human urea transporter
Components
  • Complex: Homotrimer complex of human urea transporter
    • Protein or peptide: Urea transporter 2
  • Ligand: 3-(3-methoxyphenyl)-~{N}-[(3-methoxyphenyl)methyl]-1~{H}-pyrazole-5-carboxamide

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Supramolecule #1: Homotrimer complex of human urea transporter

SupramoleculeName: Homotrimer complex of human urea transporter / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Urea transporter 2

MacromoleculeName: Urea transporter 2 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.419789 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEESSEIKVE TNISKTSWIR SSMAASGKRV SKALSYITGE MKECGEGLKD KSPVFQFFDW VLRGTSQVMF VNNPLSGILI ILGLFIQNP WWAISGCLGT IMSTLTALIL SQDKSAIAAG FHGYNGVLVG LLMAVFSDKG DYYWWLLLPV IIMSMSCPIL S SALGTIFS ...String:
MEESSEIKVE TNISKTSWIR SSMAASGKRV SKALSYITGE MKECGEGLKD KSPVFQFFDW VLRGTSQVMF VNNPLSGILI ILGLFIQNP WWAISGCLGT IMSTLTALIL SQDKSAIAAG FHGYNGVLVG LLMAVFSDKG DYYWWLLLPV IIMSMSCPIL S SALGTIFS KWDLPVFTLP FNITVTLYLA ATGHYNLFFP TTLLQPASAM PNITWSEVQV PLLLRAIPVG IGQVYGCDNP WT GGIFLIA LFISSPLICL HAAIGSTMGM LAALTIATPF DSIYFGLCGF NSTLACIAIG GMFYVITWQT HLLAIACALF AAY LGAALA NMLSVFGLPP CTWPFCLSAL TFLLLTTNNP AIYKLPLSKV TYPEANRIYY LSQERNRRAS IITKYQAYDV S

UniProtKB: Urea transporter 2

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Macromolecule #2: 3-(3-methoxyphenyl)-~{N}-[(3-methoxyphenyl)methyl]-1~{H}-pyrazole...

MacromoleculeName: 3-(3-methoxyphenyl)-~{N}-[(3-methoxyphenyl)methyl]-1~{H}-pyrazole-5-carboxamide
type: ligand / ID: 2 / Number of copies: 3 / Formula: A1EU8
Molecular weightTheoretical: 337.372 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: FREON 12

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Electron microscopy

MicroscopeFEI MORGAGNI
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: OTHER / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3k3f

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 2131097
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: OTHER

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