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- PDB-6rvc: Crystal structure of Patched-1 ectodomain 2 (PTCH1-ECD2) in compl... -

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Basic information

Entry
Database: PDB / ID: 6rvc
TitleCrystal structure of Patched-1 ectodomain 2 (PTCH1-ECD2) in complex with nanobody 75
Components
  • Nanobody NB75
  • 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 / positive regulation of cholesterol efflux / branching involved in ureteric bud morphogenesis / smoothened signaling pathway / cholesterol binding / negative regulation of osteoblast differentiation / positive regulation of epidermal cell differentiation / regulation of mitotic cell cycle / dendritic growth cone / keratinocyte proliferation / cyclin binding / spermatid development / negative regulation of keratinocyte proliferation / Hedgehog 'off' state / embryonic organ development / axonal growth cone / heart morphogenesis / response to retinoic acid / negative regulation of stem cell proliferation / response to mechanical stimulus / negative regulation of smoothened signaling pathway / liver regeneration / stem cell proliferation / caveola / neural tube closure / protein localization to plasma membrane / animal organ morphogenesis / Hedgehog 'on' state / brain development / negative regulation of DNA-binding transcription factor activity / protein processing / regulation of protein localization / endocytic vesicle membrane / glucose homeostasis / apical part of cell / response to estradiol / heparin binding / midbody / in utero embryonic development / response to xenobiotic stimulus / intracellular membrane-bounded organelle / positive regulation of DNA-templated transcription / protein-containing complex binding / 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
Protein patched homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 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 31, 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 / struct_ncs_dom_lim
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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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: Protein patched homolog 1
C: Protein patched homolog 1
D: Nanobody NB75
E: Nanobody NB75
F: Nanobody NB75
hetero molecules


Theoretical massNumber of molelcules
Total (without water)136,9779
Polymers136,4386
Non-polymers5383
Water3,639202
1
A: Protein patched homolog 1
D: Nanobody NB75
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,7013
Polymers45,4792
Non-polymers2211
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Protein patched homolog 1
E: Nanobody NB75


Theoretical massNumber of molelcules
Total (without water)45,4792
Polymers45,4792
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Protein patched homolog 1
F: Nanobody NB75
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,7974
Polymers45,4792
Non-polymers3172
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)76.180, 101.120, 106.180
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13B
23C
14D
24E
15D
25F
16E
26F

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11LYSLYSALAALAAA842 - 93580 - 173
21LYSLYSALAALABB842 - 93580 - 173
12LYSLYSALAALAAA842 - 93580 - 173
22LYSLYSALAALACC842 - 93580 - 173
13LYSLYSALAALABB842 - 93580 - 173
23LYSLYSALAALACC842 - 93580 - 173
14GLNGLNSERSERDD3 - 1233 - 123
24GLNGLNSERSEREE3 - 1233 - 123
15GLNGLNSERSERDD3 - 1233 - 123
25GLNGLNSERSERFF3 - 1233 - 123
16GLNGLNSERSEREE3 - 1243 - 124
26GLNGLNSERSERFF3 - 1243 - 124

NCS ensembles :
ID
1
2
3
4
5
6

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Components

#1: Protein Protein patched homolog 1 / PTC1


Mass: 30659.311 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Human PTCH1-ECD2, containing a N-terminal His6-Tag for IMAC purification.
Source: (gene. exp.) Homo sapiens (human) / Gene: PTCH1, PTCH / Plasmid: pHLsec / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q13635
#2: Antibody Nanobody NB75


Mass: 14820.158 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Llama nanobody NB75 containing a a C-terminal His6-Tag for IMAC purification.
Source: (gene. exp.) Lama glama (llama) / Plasmid: pMES4y / Production host: Escherichia coli (E. coli)
#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.: 2
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-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 202 / 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

Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5 / Details: 0.2 M sodium nitrate, 20% (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 29, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9686 Å / Relative weight: 1
ReflectionResolution: 2.2→39.16 Å / Num. obs: 42185 / % possible obs: 99.7 % / Redundancy: 8.6 % / CC1/2: 0.997 / Rmerge(I) obs: 0.164 / Rpim(I) all: 0.059 / Net I/σ(I): 9.8
Reflection shellResolution: 2.2→2.26 Å / Redundancy: 8.3 % / Mean I/σ(I) obs: 1.1 / Num. unique obs: 3069 / CC1/2: 0.391 / Rpim(I) all: 0.722 / % possible all: 99.3

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Processing

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

6dmy


Resolution: 2.2→39.16 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.94 / SU B: 7.193 / SU ML: 0.169 / Cross valid method: THROUGHOUT / ESU R: 0.244 / ESU R Free: 0.197 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.24073 2027 4.8 %RANDOM
Rwork0.20376 ---
obs0.20562 40108 99.47 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 56.492 Å2
Baniso -1Baniso -2Baniso -3
1-1.37 Å20 Å20 Å2
2---0.63 Å20 Å2
3----0.74 Å2
Refinement stepCycle: LAST / Resolution: 2.2→39.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5112 0 33 202 5347
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0135270
X-RAY DIFFRACTIONr_bond_other_d0.0010.0184608
X-RAY DIFFRACTIONr_angle_refined_deg1.4551.6527146
X-RAY DIFFRACTIONr_angle_other_deg1.291.59410702
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4395644
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.25222.816277
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.22115838
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.9581527
X-RAY DIFFRACTIONr_chiral_restr0.0630.2670
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.025975
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021178
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_it4.535.9092594
X-RAY DIFFRACTIONr_mcbond_other4.5285.9082593
X-RAY DIFFRACTIONr_mcangle_it6.2188.8543232
X-RAY DIFFRACTIONr_mcangle_other6.2188.8563233
X-RAY DIFFRACTIONr_scbond_it5.1476.2762676
X-RAY DIFFRACTIONr_scbond_other5.1396.2762673
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other7.179.2383909
X-RAY DIFFRACTIONr_long_range_B_refined9.75121410
X-RAY DIFFRACTIONr_long_range_B_other9.75321322
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A29120.11
12B29120.11
21A29540.1
22C29540.1
31B29170.11
32C29170.11
41D35440.12
42E35440.12
51D37250.08
52F37250.08
61E35650.12
62F35650.12
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.368 155 -
Rwork0.326 2909 -
obs--98.81 %

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