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- PDB-9dwq: PKD2 ion channel, F629S variant -

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Basic information

Entry
Database: PDB / ID: 9dwq
TitlePKD2 ion channel, F629S variant
ComponentsPolycystin-2
KeywordsMEMBRANE PROTEIN / Ion channel / TRP channel / polycystin
Function / homology
Function and homology information


detection of nodal flow / metanephric smooth muscle tissue development / metanephric cortex development / metanephric cortical collecting duct development / metanephric distal tubule development / polycystin complex / mesonephric tubule development / mesonephric duct development / metanephric part of ureteric bud development / renal tubule morphogenesis ...detection of nodal flow / metanephric smooth muscle tissue development / metanephric cortex development / metanephric cortical collecting duct development / metanephric distal tubule development / polycystin complex / mesonephric tubule development / mesonephric duct development / metanephric part of ureteric bud development / renal tubule morphogenesis / determination of liver left/right asymmetry / metanephric ascending thin limb development / metanephric mesenchyme development / metanephric S-shaped body morphogenesis / basal cortex / renal artery morphogenesis / calcium-induced calcium release activity / HLH domain binding / migrasome / cilium organization / VxPx cargo-targeting to cilium / detection of mechanical stimulus / muscle alpha-actinin binding / regulation of calcium ion import / voltage-gated monoatomic ion channel activity / placenta blood vessel development / cellular response to hydrostatic pressure / cellular response to fluid shear stress / cation channel complex / outward rectifier potassium channel activity / non-motile cilium / actinin binding / cellular response to osmotic stress / determination of left/right symmetry / : / voltage-gated monoatomic cation channel activity / neural tube development / voltage-gated sodium channel activity / aorta development / motile cilium / branching involved in ureteric bud morphogenesis / ciliary membrane / protein heterotetramerization / negative regulation of G1/S transition of mitotic cell cycle / spinal cord development / heart looping / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytoplasmic side of endoplasmic reticulum membrane / centrosome duplication / voltage-gated potassium channel activity / potassium channel activity / cell surface receptor signaling pathway via JAK-STAT / embryonic placenta development / voltage-gated calcium channel activity / monoatomic cation channel activity / transcription regulator inhibitor activity / cytoskeletal protein binding / release of sequestered calcium ion into cytosol / potassium ion transmembrane transport / sodium ion transmembrane transport / cellular response to calcium ion / basal plasma membrane / cytoplasmic vesicle membrane / cellular response to cAMP / lumenal side of endoplasmic reticulum membrane / cellular response to reactive oxygen species / protein tetramerization / phosphoprotein binding / establishment of localization in cell / liver development / calcium ion transmembrane transport / Wnt signaling pathway / intracellular calcium ion homeostasis / positive regulation of nitric oxide biosynthetic process / mitotic spindle / cell-cell junction / calcium ion transport / lamellipodium / regulation of cell population proliferation / heart development / ATPase binding / protein homotetramerization / basolateral plasma membrane / transmembrane transporter binding / cell surface receptor signaling pathway / regulation of cell cycle / cilium / ciliary basal body / signaling receptor binding / negative regulation of cell population proliferation / calcium ion binding / endoplasmic reticulum membrane / positive regulation of gene expression / endoplasmic reticulum / Golgi apparatus / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular exosome / identical protein binding / membrane
Similarity search - Function
Ferredoxin I 4Fe-4S cluster domain / : / Polycystic kidney disease type 2 protein / Polycystin domain / Polycystin domain / Polycystin cation channel, PKD1/PKD2 / Polycystin cation channel / Voltage-dependent channel domain superfamily / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.76 Å
AuthorsEsarte Palomero, O. / DeCaen, P.G.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01 DK131118-01 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01 DK123463-01 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)(F32DK137477-01A1 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)TL1DK132769 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)U2CDK129917 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Pathogenic variants in the polycystin pore helix cause distinct forms of channel dysfunction.
Authors: Orhi Esarte Palomero / Eduardo Guadarrama / Paul G DeCaen /
Abstract: PKD2 is a member of the polycystin subfamily of transient receptor potential (TRP) ion channel subunits which traffic and function in primary cilia organelle membranes. Millions of individuals carry ...PKD2 is a member of the polycystin subfamily of transient receptor potential (TRP) ion channel subunits which traffic and function in primary cilia organelle membranes. Millions of individuals carry pathogenic genetic variants in PKD2 that cause a life-threatening condition called autosomal dominant polycystic kidney disease (ADPKD). Although ADPKD is a common monogenetic disorder, there is no drug cure or available therapeutics which address the underlying channel dysregulation. Furthermore, the structural and mechanistic impacts of most disease-causing variants are uncharacterized. Using direct cilia electrophysiology, cryogenic electron microscopy (cryo-EM), and superresolution imaging, we have found mechanistic differences in channel dysregulation caused by three germline missense variants located in PKD2's pore helix 1. Variant C632R reduces protein thermal stability, resulting in impaired channel assembly and abolishes primary cilia trafficking. In contrast, variants F629S and R638C retain native cilia trafficking but exhibit gating defects. Cryo-EM structures (2.7 to 2.8 Å resolution) indicate loss of critical pore helix interactions which precipitate allosteric collapse of the channels inner gate. Results demonstrate how ADPKD-causing mutations cause mechanistically divergent and ranging impacts on PKD2 function, despite their shared structural proximity. These unexpected findings highlight the need for structural and biophysical characterization of polycystin variants, which will guide rational drug development of ADPKD therapeutics.
History
DepositionOct 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Sep 3, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polycystin-2
B: Polycystin-2
C: Polycystin-2
D: Polycystin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)340,2135
Polymers340,1734
Non-polymers401
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, native gel electrophoresis, gel filtration, homology
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Polycystin-2 / PC2 / Autosomal dominant polycystic kidney disease type II protein / Polycystic kidney disease 2 ...PC2 / Autosomal dominant polycystic kidney disease type II protein / Polycystic kidney disease 2 protein / Polycystwin / R48321 / Transient receptor potential cation channel subfamily P member 2


Mass: 85043.344 Da / Num. of mol.: 4 / Mutation: F629S
Source method: isolated from a genetically manipulated source
Details: PKD2 variant F629S (residues 52-793) was expressed with an N-terminal StrepII-MBP-TEV cleaved prior to macromolecular analysis
Source: (gene. exp.) Homo sapiens (human) / Gene: PKD2, TRPP2 / Plasmid: pCMV / Cell line (production host): expi293 / Production host: Homo sapiens (human) / References: UniProt: Q13563
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: PKD2 ion channel F629S variant protomer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.3396 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 / Cell: expi293 / Plasmid: pCMV
Buffer solutionpH: 7.4
Details: 25 mM HEPES-NaOH, 150 mM NaCl, 1 mM CaCl2, 1 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
225 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
31 mMCalcium ChlorideCaCl21
41 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Stabilized in amphipol A8-35. Monodisperse sample after gel filtration in a Superdex 200 column.
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 278 K
Details: Vitrification carried in air. Ethane temperature -183 C

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9145 / Details: 50 frame movie stacks. Super-resolution mode
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5particle selection
2SerialEMv4.1.0betaimage acquisition
4cryoSPARC4.5CTF correction
7UCSF ChimeraX1.8model fitting
8ISOLDE1.8model fitting
10cryoSPARC4.5initial Euler assignment
11cryoSPARC4.5final Euler assignment
12cryoSPARCclassification
13cryoSPARC4.53D reconstruction
14ISOLDE1.8model refinement
15PHENIX1.21.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1046126
Details: Denoised micrographs were blob picked (130-170 angstrom)
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 215875 / Algorithm: FOURIER SPACE
Details: Final reconstruction performed with C4 symmetry imposed
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 107.9 / Space: REAL
Details: The initial Alphafold model was fitted to the map using ChimeraX. Refinement was performed using ISOLDE and PHENIX.
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215952
ELECTRON MICROSCOPYf_angle_d0.4121644
ELECTRON MICROSCOPYf_dihedral_angle_d4.0652065
ELECTRON MICROSCOPYf_chiral_restr0.0362420
ELECTRON MICROSCOPYf_plane_restr0.0032688

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