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- PDB-8ubr: Complex of the phosphorylated human cystic fibrosis transmembrane... -

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Basic information

Entry
Database: PDB / ID: 8ubr
TitleComplex of the phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) with CFTRinh-172 and ATP/Mg
ComponentsCystic fibrosis transmembrane conductance regulator
KeywordsMEMBRANE PROTEIN / Inhibitor / CFTR
Function / homology
Function and homology information


positive regulation of voltage-gated chloride channel activity / : / Sec61 translocon complex binding / channel-conductance-controlling ATPase / intracellularly ATP-gated chloride channel activity / positive regulation of enamel mineralization / transepithelial water transport / RHO GTPases regulate CFTR trafficking / amelogenesis / intracellular pH elevation ...positive regulation of voltage-gated chloride channel activity / : / Sec61 translocon complex binding / channel-conductance-controlling ATPase / intracellularly ATP-gated chloride channel activity / positive regulation of enamel mineralization / transepithelial water transport / RHO GTPases regulate CFTR trafficking / amelogenesis / intracellular pH elevation / chloride channel inhibitor activity / : / Golgi-associated vesicle membrane / multicellular organismal-level water homeostasis / cholesterol transport / bicarbonate transport / bicarbonate transmembrane transporter activity / chloride channel regulator activity / vesicle docking involved in exocytosis / membrane hyperpolarization / chloride transmembrane transporter activity / sperm capacitation / cholesterol biosynthetic process / RHOQ GTPase cycle / chloride channel activity / positive regulation of exocytosis / ATPase-coupled transmembrane transporter activity / chloride channel complex / ABC-type transporter activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / 14-3-3 protein binding / cellular response to forskolin / chloride transmembrane transport / response to endoplasmic reticulum stress / cellular response to cAMP / PDZ domain binding / establishment of localization in cell / clathrin-coated endocytic vesicle membrane / Defective CFTR causes cystic fibrosis / Late endosomal microautophagy / recycling endosome / ABC-family proteins mediated transport / transmembrane transport / recycling endosome membrane / Chaperone Mediated Autophagy / Aggrephagy / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / protein-folding chaperone binding / early endosome membrane / early endosome / endosome membrane / Ub-specific processing proteases / apical plasma membrane / lysosomal membrane / endoplasmic reticulum membrane / enzyme binding / cell surface / protein-containing complex / ATP hydrolysis activity / ATP binding / nucleus / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
: / CFTR regulator domain / Cystic fibrosis TM conductance regulator (CFTR), regulator domain / Cystic fibrosis transmembrane conductance regulator / : / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site ...: / CFTR regulator domain / Cystic fibrosis TM conductance regulator (CFTR), regulator domain / Cystic fibrosis transmembrane conductance regulator / : / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / CHOLESTEROL / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / : / Cystic fibrosis transmembrane conductance regulator
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsYoung, P.G. / Fiedorczuk, K. / Chen, J.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM079238 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structural basis for CFTR inhibition by CFTR-172.
Authors: Paul G Young / Jesper Levring / Karol Fiedorczuk / Scott C Blanchard / Jue Chen /
Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating ...The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTR-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTR-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTR-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTR-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor.
History
DepositionSep 24, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 21, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Category: citation / citation_author
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 / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cystic fibrosis transmembrane conductance regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,10010
Polymers168,3341
Non-polymers3,7669
Water724
1


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

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Cystic fibrosis transmembrane conductance regulator / CFTR / ATP-binding cassette sub-family C member 7 / Channel conductance-controlling ATPase / cAMP- ...CFTR / ATP-binding cassette sub-family C member 7 / Channel conductance-controlling ATPase / cAMP-dependent chloride channel


Mass: 168334.469 Da / Num. of mol.: 1 / Mutation: E1371Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CFTR, ABCC7 / Cell line (production host): HEK 293S GnTl- / Production host: Homo sapiens (human)
References: UniProt: P13569, channel-conductance-controlling ATPase

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Non-polymers , 6 types, 13 molecules

#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-LBN / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / (2R)-2-[(9Z)-9-Octadecenoyloxy]-3-(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate


Mass: 760.076 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#6: Chemical ChemComp-WG5 / 4-[(Z)-{(3M)-4-oxo-2-sulfanylidene-3-[3-(trifluoromethyl)phenyl]-1,3-thiazolidin-5-ylidene}methyl]benzoic acid


Mass: 409.402 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H10F3NO3S2 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: Complex of CFTR with the small molecule inhibitor CFTRinh-172
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.168 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenConc.: 5.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R0.6/1
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 65.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66267 / Symmetry type: POINT
RefinementCross valid method: NONE

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