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- PDB-7pqq: Structure of thermostabilised human NTCP in complex with Megabody 91 -

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Basic information

Entry
Database: PDB / ID: 7pqq
TitleStructure of thermostabilised human NTCP in complex with Megabody 91
Components
  • Anti-RON nanobody,Megabody 91,Glucosidase YgjK
  • Sodium/bile acid cotransporter
KeywordsMEMBRANE PROTEIN / bile acid transporter
Function / homology
Function and homology information


glucosidase complex / alpha,alpha-trehalase activity / trehalose catabolic process / bile acid:sodium symporter activity / glucosidase activity / bile acid transmembrane transporter activity / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / oligosaccharide catabolic process / bile acid and bile salt transport / Recycling of bile acids and salts ...glucosidase complex / alpha,alpha-trehalase activity / trehalose catabolic process / bile acid:sodium symporter activity / glucosidase activity / bile acid transmembrane transporter activity / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / oligosaccharide catabolic process / bile acid and bile salt transport / Recycling of bile acids and salts / response to nutrient levels / response to estrogen / cellular response to xenobiotic stimulus / virus receptor activity / response to ethanol / basolateral plasma membrane / DNA damage response / metal ion binding / plasma membrane
Similarity search - Function
: / Glucosidase YgjK, N-terminal / Na+/H+ antiporter like fold - #20 / Glycoside hydrolase, family 37 / Na+/H+ antiporter like fold / : / Mannosylglycerate hydrolase MGH1-like glycoside hydrolase domain / Bile acid:sodium symporter/arsenical resistance protein Acr3 / Bile acid:sodium symporter / Sodium Bile acid symporter family ...: / Glucosidase YgjK, N-terminal / Na+/H+ antiporter like fold - #20 / Glycoside hydrolase, family 37 / Na+/H+ antiporter like fold / : / Mannosylglycerate hydrolase MGH1-like glycoside hydrolase domain / Bile acid:sodium symporter/arsenical resistance protein Acr3 / Bile acid:sodium symporter / Sodium Bile acid symporter family / Sodium/solute symporter superfamily / Six-hairpin glycosidase-like superfamily / Six-hairpin glycosidase superfamily / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Glucosidase YgjK / Hepatic sodium/bile acid cotransporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGoutam, K. / Reyes, N.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)309657European Union
CitationJournal: Nature / Year: 2022
Title: Structural basis of sodium-dependent bile salt uptake into the liver.
Authors: Kapil Goutam / Francesco S Ielasi / Els Pardon / Jan Steyaert / Nicolas Reyes /
Abstract: The liver takes up bile salts from blood to generate bile, enabling absorption of lipophilic nutrients and excretion of metabolites and drugs. Human Na-taurocholate co-transporting polypeptide (NTCP) ...The liver takes up bile salts from blood to generate bile, enabling absorption of lipophilic nutrients and excretion of metabolites and drugs. Human Na-taurocholate co-transporting polypeptide (NTCP) is the main bile salt uptake system in liver. NTCP is also the cellular entry receptor of human hepatitis B and D viruses (HBV/HDV), and has emerged as an important target for antiviral drugs. However, the molecular mechanisms underlying NTCP transport and viral receptor functions remain incompletely understood. Here we present cryo-electron microscopy structures of human NTCP in complexes with nanobodies, revealing key conformations of its transport cycle. NTCP undergoes a conformational transition opening a wide transmembrane pore that serves as the transport pathway for bile salts, and exposes key determinant residues for HBV/HDV binding to the outside of the cell. A nanobody that stabilizes pore closure and inward-facing states impairs recognition of the HBV/HDV receptor-binding domain preS1, demonstrating binding selectivity of the viruses for open-to-outside over inward-facing conformations of the NTCP transport cycle. These results provide molecular insights into NTCP 'gated-pore' transport and HBV/HDV receptor recognition mechanisms, and are expected to help with development of liver disease therapies targeting NTCP.
History
DepositionSep 18, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 18, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium/bile acid cotransporter
B: Anti-RON nanobody,Megabody 91,Glucosidase YgjK


Theoretical massNumber of molelcules
Total (without water)138,2352
Polymers138,2352
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1840 Å2
ΔGint-10 kcal/mol
Surface area20240 Å2

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Components

#1: Protein Sodium/bile acid cotransporter / Cell growth-inhibiting gene 29 protein / Na(+)/bile acid cotransporter / Na(+)/taurocholate ...Cell growth-inhibiting gene 29 protein / Na(+)/bile acid cotransporter / Na(+)/taurocholate transport protein / Sodium/taurocholate cotransporting polypeptide / Solute carrier family 10 member 1 / NTCP


Mass: 36652.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC10A1, NTCP, GIG29 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q14973
#2: Antibody Anti-RON nanobody,Megabody 91,Glucosidase YgjK / Megabody 91


Mass: 101583.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The protein is a fusion with Lama Glama and Escherichia Coli K-12 as its natural source.
Source: (gene. exp.) Lama glama (llama) / Strain: K12 / Gene: ygjK, b3080, JW3051 / Production host: Escherichia coli (E. coli)
Variant (production host): It is a fusion protein having lama glama and Escherichia Coli K12 as the source.
References: UniProt: P42592, Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds
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

ComponentName: human NTCP complexed with Megabody 91 / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
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: BRIGHT FIELD
Image recordingElectron dose: 56.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 184768 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 39.62 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00383274
ELECTRON MICROSCOPYf_angle_d0.6784439
ELECTRON MICROSCOPYf_chiral_restr0.0411530
ELECTRON MICROSCOPYf_plane_restr0.0042540
ELECTRON MICROSCOPYf_dihedral_angle_d21.13311179

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