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- PDB-7k65: Hedgehog receptor Patched (PTCH1) in complex with conformation se... -

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Basic information

Entry
Database: PDB / ID: 7k65
TitleHedgehog receptor Patched (PTCH1) in complex with conformation selective nanobody TI23
Components
  • Protein patched homolog 1
  • nanobody TI23
KeywordsMEMBRANE PROTEIN
Function / homology
Function and homology information


Ligand-receptor interactions / Activation of SMO / neural plate axis specification / response to chlorate / cell differentiation involved in kidney development / hedgehog receptor activity / cell proliferation involved in metanephros development / neural tube formation / smoothened binding / hedgehog family protein binding ...Ligand-receptor interactions / Activation of SMO / neural plate axis specification / response to chlorate / cell differentiation involved in kidney development / hedgehog receptor activity / cell proliferation involved in metanephros development / neural tube formation / smoothened binding / hedgehog family protein binding / hindlimb morphogenesis / Hedgehog 'on' state / epidermal cell fate specification / spinal cord motor neuron differentiation / prostate gland development / Hedgehog 'off' state / patched binding / negative regulation of cell division / somite development / smooth muscle tissue development / dorsal/ventral neural tube patterning / pharyngeal system development / cellular response to cholesterol / mammary gland duct morphogenesis / pattern specification process / mammary gland epithelial cell differentiation / cell fate determination / commissural neuron axon guidance / metanephric collecting duct development / mammary gland development / dorsal/ventral pattern formation / regulation of growth / embryonic limb morphogenesis / negative regulation of multicellular organism growth / branching involved in ureteric bud morphogenesis / cholesterol binding / positive regulation of epidermal cell differentiation / dendritic growth cone / keratinocyte proliferation / spermatid development / epidermis development / positive regulation of cholesterol efflux / negative regulation of keratinocyte proliferation / embryonic organ development / response to retinoic acid / negative regulation of osteoblast differentiation / response to mechanical stimulus / axonal growth cone / heart morphogenesis / negative regulation of stem cell proliferation / liver regeneration / cyclin binding / regulation of mitotic cell cycle / animal organ morphogenesis / epithelial cell proliferation / stem cell proliferation / negative regulation of smoothened signaling pathway / protein localization to plasma membrane / neural tube closure / brain development / protein processing / caveola / apical part of cell / negative regulation of epithelial cell proliferation / response to estradiol / glucose homeostasis / heparin binding / regulation of cell population proliferation / regulation of protein localization / midbody / in utero embryonic development / postsynaptic membrane / cilium / response to xenobiotic stimulus / negative regulation of cell population proliferation / negative regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / protein-containing complex binding / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / Golgi apparatus / signal transduction / extracellular region / zinc ion binding / plasma membrane
Similarity search - Function
Transmembrane receptor, patched / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Chem-Q7G / Protein patched homolog 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZhang, Y. / Bulkley, D.P. / Liang, J. / Manglik, A. / Cheng, Y. / Beachy, P.A.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM102498 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM098672 United States
National Institutes of Health/Office of the DirectorDP5OD023048 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit.
Authors: Yunxiao Zhang / Wan-Jin Lu / David P Bulkley / Jiahao Liang / Arthur Ralko / Shuo Han / Kelsey J Roberts / Anping Li / Wonhwa Cho / Yifan Cheng / Aashish Manglik / Philip A Beachy /
Abstract: Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, ...Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.
History
DepositionSep 18, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Protein patched homolog 1
B: nanobody TI23
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,63312
Polymers156,4412
Non-polymers6,19210
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Protein patched homolog 1 / PTC1


Mass: 142619.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ptch1, Ptch / Cell line (production host): freestyle 293 / Production host: Homo sapiens (human) / References: UniProt: Q61115
#2: Antibody nanobody TI23


Mass: 13821.417 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Chemical
ChemComp-Q7G / 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl}-4-{[(3beta,9beta,14beta,17beta,25R)-spirost-5-en-3-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranoside


Mass: 1165.315 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C56H92O25
Has ligand of interestN
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

Component
IDNameTypeEntity IDParent-IDSource
1Complex between Hedgehog receptor PTCH1 and nanobody TI23COMPLEX#1-#20MULTIPLE SOURCES
2Hedgehog receptor Patched PTCH1COMPLEX#11RECOMBINANT
3nanobody TI23COMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Homo sapiens (human)9606HEK293
23Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
30.02 %glycol-diosgeninGDN1
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 85.6 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 7046
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10cryoSPARC2initial Euler assignment
11cryoSPARC2final Euler assignment
12cryoSPARC2classification
13cisTEM13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3621265
Details: Particles were picked first using a Gaussian blob as the template and then were re-picked using the 2D classes generated from the first round.
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 307652 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6MG8

6mg8


Pdb chain-ID: A / Accession code: 6MG8 / Source name: PDB / Type: experimental model
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 135.37 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00379024
ELECTRON MICROSCOPYf_angle_d0.697912365
ELECTRON MICROSCOPYf_chiral_restr0.04171481
ELECTRON MICROSCOPYf_plane_restr0.00391527
ELECTRON MICROSCOPYf_dihedral_angle_d13.48855622

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