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- PDB-6uox: Structure of itraconazole-bound NPC1 -

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Basic information

Entry
Database: PDB / ID: 6uox
TitleStructure of itraconazole-bound NPC1
ComponentsNPC intracellular cholesterol transporter 1
KeywordsMEMBRANE PROTEIN / Niemann-Pick C disease / cholesterol transport / sterol-sensing domain
Function / homology
Function and homology information


cyclodextrin metabolic process / cholesterol storage / membrane raft organization / : / intracellular cholesterol transport / intracellular lipid transport / intestinal cholesterol absorption / LDL clearance / negative regulation of epithelial cell apoptotic process / bile acid metabolic process ...cyclodextrin metabolic process / cholesterol storage / membrane raft organization / : / intracellular cholesterol transport / intracellular lipid transport / intestinal cholesterol absorption / LDL clearance / negative regulation of epithelial cell apoptotic process / bile acid metabolic process / cholesterol transport / programmed cell death / establishment of protein localization to membrane / adult walking behavior / lysosomal transport / cholesterol efflux / cellular response to steroid hormone stimulus / negative regulation of macroautophagy / cholesterol binding / protein glycosylation / cellular response to low-density lipoprotein particle stimulus / response to cadmium ion / negative regulation of TORC1 signaling / cholesterol metabolic process / neurogenesis / liver development / cholesterol homeostasis / macroautophagy / autophagy / endocytosis / transmembrane signaling receptor activity / late endosome membrane / nuclear envelope / virus receptor activity / signaling receptor activity / gene expression / lysosome / response to xenobiotic stimulus / symbiont entry into host cell / membrane raft / lysosomal membrane / perinuclear region of cytoplasm / Golgi apparatus / endoplasmic reticulum / extracellular exosome / extracellular region / membrane / plasma membrane
Similarity search - Function
NPC1-like / Niemann-Pick C1, N-terminal / Niemann-Pick C1 N terminus / Sterol-sensing domain of SREBP cleavage-activation / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
Chem-QDG / NPC intracellular cholesterol transporter 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsLong, T. / Li, X.
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis for itraconazole-mediated NPC1 inhibition.
Authors: Tao Long / Xiaofeng Qi / Abdirahman Hassan / Qiren Liang / Jef K De Brabander / Xiaochun Li /
Abstract: Niemann-Pick C1 (NPC1), a lysosomal protein of 13 transmembrane helices (TMs) and three lumenal domains, exports low-density-lipoprotein (LDL)-derived cholesterol from lysosomes. TMs 3-7 of NPC1 ...Niemann-Pick C1 (NPC1), a lysosomal protein of 13 transmembrane helices (TMs) and three lumenal domains, exports low-density-lipoprotein (LDL)-derived cholesterol from lysosomes. TMs 3-7 of NPC1 comprise the Sterol-Sensing Domain (SSD). Previous studies suggest that mutation of the NPC1-SSD or the addition of the anti-fungal drug itraconazole abolishes NPC1 activity in cells. However, the itraconazole binding site and the mechanism of NPC1-mediated cholesterol transport remain unknown. Here, we report a cryo-EM structure of human NPC1 bound to itraconazole, which reveals how this binding site in the center of NPC1 blocks a putative lumenal tunnel linked to the SSD. Functional assays confirm that blocking this tunnel abolishes NPC1-mediated cholesterol egress. Intriguingly, the palmitate anchor of Hedgehog occupies a similar site in the homologous tunnel of Patched, suggesting a conserved mechanism for sterol transport in this family of proteins and establishing a central function of their SSDs.
History
DepositionOct 15, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: NPC intracellular cholesterol transporter 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)146,88012
Polymers143,3151
Non-polymers3,56511
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2950 Å2
ΔGint39 kcal/mol
Surface area57070 Å2

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Components

#1: Protein NPC intracellular cholesterol transporter 1 / Niemann-Pick C1 protein


Mass: 143315.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NPC1 / Production host: Homo sapiens (human) / References: UniProt: O15118
#2: 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
#3: Polysaccharide alpha-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...alpha-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2/a4-b1_b3-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(3+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 8
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
#5: Chemical ChemComp-QDG / 4-(3-bromo-4-{4-[4-({(2R,4S)-2-(2,4-dichlorophenyl)-2-[(1H-1,2,4-triazol-1-yl)methyl]-1,3-dioxolan-4-yl}methoxy)phenyl]piperazin-1-yl}phenyl)-2-[(2S)-butan-2-yl]-2,4-dihydro-3H-1,2,4-triazol-3-one


Mass: 784.529 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C35H37BrCl2N8O4
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: NPC1 and itraconazole-Br complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: DARK FIELD
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0135 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209612 / Symmetry type: POINT
RefinementResolution: 4.13→231 Å / Cor.coef. Fo:Fc: 0.832 / SU B: 59.618 / SU ML: 0.778 / ESU R: 0.305 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.44541 --
obs0.44541 367648 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 175.814 Å2
Baniso -1Baniso -2Baniso -3
1-2.03 Å23.58 Å2-2.21 Å2
2---2.99 Å23.5 Å2
3---0.96 Å2
Refinement stepCycle: 1 / Total: 9138
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.0199393
ELECTRON MICROSCOPYr_bond_other_d0.0020.028723
ELECTRON MICROSCOPYr_angle_refined_deg1.1771.98312827
ELECTRON MICROSCOPYr_angle_other_deg0.981320062
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.45451157
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.01924.212387
ELECTRON MICROSCOPYr_dihedral_angle_3_deg11.875151395
ELECTRON MICROSCOPYr_dihedral_angle_4_deg8.7681536
ELECTRON MICROSCOPYr_chiral_restr0.0640.21477
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.02110591
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022162
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it2.15817.8594640
ELECTRON MICROSCOPYr_mcbond_other2.15917.8594639
ELECTRON MICROSCOPYr_mcangle_it3.96326.7855793
ELECTRON MICROSCOPYr_mcangle_other3.96226.7855794
ELECTRON MICROSCOPYr_scbond_it3.84717.8454753
ELECTRON MICROSCOPYr_scbond_other3.84717.8454753
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other5.27726.7267035
ELECTRON MICROSCOPYr_long_range_B_refined7.54410887
ELECTRON MICROSCOPYr_long_range_B_other7.54310888
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 4.125→4.232 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.584 27037 -
Rfree-0 -
obs--100 %

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