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- PDB-9w4k: Cryo-EM structure of inhibitor E822-1968 bound human urea transpo... -

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Basic information

Entry
Database: PDB / ID: 9w4k
TitleCryo-EM structure of inhibitor E822-1968 bound human urea transporter A2.
ComponentsUrea transporter 2
KeywordsMEMBRANE PROTEIN / Urea transporter
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
: / Urea transporter 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHuang, S. / Sun, J.
Funding support China, 1items
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, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Aug 5, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 5, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 5, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 5, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 5, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 5, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Urea transporter 2
B: Urea transporter 2
C: Urea transporter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,2716
Polymers130,2593
Non-polymers1,0123
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Urea transporter 2 / Solute carrier family 14 member 2 / Urea transporter / kidney


Mass: 43419.789 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC14A2, HUT2, UT2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q15849
#2: Chemical ChemComp-A1EU8 / 3-(3-methoxyphenyl)-~{N}-[(3-methoxyphenyl)methyl]-1~{H}-pyrazole-5-carboxamide


Mass: 337.372 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C19H19N3O3
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Homotrimer complex of human urea transporter / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: FREON 12

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Electron microscopy imaging

MicroscopyModel: FEI MORGAGNI
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2131097 / Symmetry type: POINT

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