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- PDB-6rtw: Crystal structure of the Patched-1 (PTCH1) ectodomain in complex ... -

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Basic information

Entry
Database: PDB / ID: 6rtw
TitleCrystal structure of the Patched-1 (PTCH1) ectodomain in complex with nanobody NB64 and cholesterol-hemisuccinate
Components
  • Llama-derived nanobody NB64
  • Protein patched homolog 1
KeywordsMEMBRANE PROTEIN / ---- Hedgehog morphogen receptor / receptor-nanobody complex / cholesterol / palmitate / lipid-protein-modification
Function / homology
Function and homology information


neural plate axis specification / cell differentiation involved in kidney development / hedgehog receptor activity / response to chlorate / neural tube patterning / cell proliferation involved in metanephros development / smoothened binding / hedgehog family protein binding / Ligand-receptor interactions / hindlimb morphogenesis ...neural plate axis specification / cell differentiation involved in kidney development / hedgehog receptor activity / response to chlorate / neural tube patterning / cell proliferation involved in metanephros development / smoothened binding / hedgehog family protein binding / Ligand-receptor interactions / hindlimb morphogenesis / epidermal cell fate specification / spinal cord motor neuron differentiation / prostate gland development / Activation of SMO / limb morphogenesis / somite development / patched binding / negative regulation of cell division / cellular response to cholesterol / dorsal/ventral neural tube patterning / smooth muscle tissue development / pharyngeal system development / mammary gland epithelial cell differentiation / mammary gland duct morphogenesis / cell fate determination / commissural neuron axon guidance / metanephric collecting duct development / regulation of smoothened signaling pathway / dorsal/ventral pattern formation / Class B/2 (Secretin family receptors) / embryonic limb morphogenesis / ciliary membrane / negative regulation of multicellular organism growth / smoothened signaling pathway / branching involved in ureteric bud morphogenesis / cholesterol binding / positive regulation of epidermal cell differentiation / dendritic growth cone / keratinocyte proliferation / spermatid development / positive regulation of cholesterol efflux / negative regulation of keratinocyte proliferation / negative regulation of osteoblast differentiation / embryonic organ development / Hedgehog 'off' state / axonal growth cone / negative regulation of smoothened signaling pathway / heart morphogenesis / response to mechanical stimulus / response to retinoic acid / negative regulation of stem cell proliferation / regulation of mitotic cell cycle / cyclin binding / stem cell proliferation / caveola / neural tube closure / protein localization to plasma membrane / liver regeneration / animal organ morphogenesis / Hedgehog 'on' state / negative regulation of DNA-binding transcription factor activity / brain development / protein processing / regulation of protein localization / endocytic vesicle membrane / apical part of cell / response to estradiol / glucose homeostasis / heparin binding / midbody / in utero embryonic development / response to xenobiotic stimulus / intracellular membrane-bounded organelle / protein-containing complex binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / plasma membrane
Similarity search - Function
Transmembrane receptor, patched / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
CHOLESTEROL HEMISUCCINATE / Protein patched homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsRudolf, A.F. / Kowatsch, C. / El Omari, K. / Malinauskas, T. / Kinnebrew, M. / Ansell, T.B. / Bishop, B. / Pardon, E. / Schwab, R.A. / Qian, M. ...Rudolf, A.F. / Kowatsch, C. / El Omari, K. / Malinauskas, T. / Kinnebrew, M. / Ansell, T.B. / Bishop, B. / Pardon, E. / Schwab, R.A. / Qian, M. / Duman, R. / Covey, D.F. / Steyaert, J. / Wagner, A. / Sansom, M.S.P. / Rohatgi, R. / Siebold, C.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Cancer Research UKC20724/A14414 United Kingdom
Cancer Research UKC20724/A26752 United Kingdom
European Research Council647278 United Kingdom
CitationJournal: Nat Chem Biol / Year: 2019
Title: The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism.
Authors: Amalie F Rudolf / Maia Kinnebrew / Christiane Kowatsch / T Bertie Ansell / Kamel El Omari / Benjamin Bishop / Els Pardon / Rebekka A Schwab / Tomas Malinauskas / Mingxing Qian / Ramona Duman ...Authors: Amalie F Rudolf / Maia Kinnebrew / Christiane Kowatsch / T Bertie Ansell / Kamel El Omari / Benjamin Bishop / Els Pardon / Rebekka A Schwab / Tomas Malinauskas / Mingxing Qian / Ramona Duman / Douglas F Covey / Jan Steyaert / Armin Wagner / Mark S P Sansom / Rajat Rohatgi / Christian Siebold /
Abstract: Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C ...Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.
History
DepositionMay 27, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.3Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein patched homolog 1
B: Llama-derived nanobody NB64
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,3336
Polymers48,1822
Non-polymers1,1504
Water3,783210
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: surface plasmon resonance
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2650 Å2
ΔGint5 kcal/mol
Surface area20320 Å2
MethodPISA
Unit cell
Length a, b, c (Å)47.260, 65.720, 151.360
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Protein patched homolog 1 / PTC1


Mass: 34138.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human PTC-H1ECD1 residues 139-423, containing three N-terminal extra residues derived from the expression vector pHLsec, and a C-terminal His6-Tag for IMAC purification.
Source: (gene. exp.) Homo sapiens (human) / Gene: PTCH1, PTCH / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q13635
#2: Antibody Llama-derived nanobody NB64


Mass: 14043.568 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: pMES4y / Production host: Escherichia coli (E. coli) / Variant (production host): WK6su-
#3: 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.: 3
Source method: isolated from a genetically manipulated source
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
#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 210 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.44 Å3/Da / Density % sol: 49.57 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.1 M HEPES/MOPS pH 7.5, 0.03 M of each halide (sodium fluoride, sodium bromide, sodium iodide), 12.5% (v/v) MPD, 12.5% (w/v) PEG 1000, 12.5% (w/v) PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.9686 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 30, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9686 Å / Relative weight: 1
ReflectionResolution: 1.9→37.19 Å / Num. obs: 37866 / % possible obs: 99.4 % / Redundancy: 5.2 % / CC1/2: 0.998 / Rmerge(I) obs: 0.111 / Rpim(I) all: 0.053 / Net I/σ(I): 9.1
Reflection shellResolution: 1.9→1.95 Å / Redundancy: 4.2 % / Mean I/σ(I) obs: 0.8 / Num. unique obs: 2618 / CC1/2: 0.36 / Rpim(I) all: 0.867 / % possible all: 94.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
xia2data reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6RTY

6rty


Resolution: 1.9→37.19 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.949 / SU B: 4.429 / SU ML: 0.119 / Cross valid method: THROUGHOUT / ESU R: 0.142 / ESU R Free: 0.138 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22985 1851 4.9 %RANDOM
Rwork0.18529 ---
obs0.18746 35951 99.32 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 39.641 Å2
Baniso -1Baniso -2Baniso -3
1--1.69 Å2-0 Å2-0 Å2
2---0.07 Å20 Å2
3---1.77 Å2
Refinement stepCycle: 1 / Resolution: 1.9→37.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3159 0 77 210 3446
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0133331
X-RAY DIFFRACTIONr_bond_other_d0.0010.0172990
X-RAY DIFFRACTIONr_angle_refined_deg1.5341.6594535
X-RAY DIFFRACTIONr_angle_other_deg1.3531.5846981
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.6995401
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.13623.354164
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.91515547
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.4711514
X-RAY DIFFRACTIONr_chiral_restr0.0660.2430
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.023686
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02662
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it3.0783.9621607
X-RAY DIFFRACTIONr_mcbond_other3.0673.9591606
X-RAY DIFFRACTIONr_mcangle_it4.3055.9242007
X-RAY DIFFRACTIONr_mcangle_other4.3055.9272008
X-RAY DIFFRACTIONr_scbond_it4.3954.5121724
X-RAY DIFFRACTIONr_scbond_other4.3774.5091723
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.5416.5872529
X-RAY DIFFRACTIONr_long_range_B_refined8.08975.14813320
X-RAY DIFFRACTIONr_long_range_B_other8.0575.08313173
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.9→1.949 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.351 128 -
Rwork0.336 2489 -
obs--94.72 %

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