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- PDB-6s59: Structure of ovine transhydrogenase in the apo state -

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Basic information

Entry
Database: PDB / ID: 6s59
TitleStructure of ovine transhydrogenase in the apo state
ComponentsNicotinamide nucleotide transhydrogenase
KeywordsMEMBRANE PROTEIN / mitochondrial / proton-translocating / nicotinamide nucleotide transhydrogenase
Function / homology
Function and homology information


: / proton-translocating NAD(P)+ transhydrogenase / proton transmembrane transport / mitochondrial inner membrane / identical protein binding
Similarity search - Function
NAD(P) transhydrogenase, alpha subunit / NAD(P) transhydrogenase, alpha subunit, C-terminal / 4TM region of pyridine nucleotide transhydrogenase, mitoch / NADP transhydrogenase beta-like domain / NAD(P) transhydrogenase beta subunit / Alanine dehydrogenase/NAD(P) transhydrogenase, conserved site-1 / Alanine dehydrogenase & pyridine nucleotide transhydrogenase signature 1. / Alanine dehydrogenase/pyridine nucleotide transhydrogenase, conserved site-2 / Alanine dehydrogenase & pyridine nucleotide transhydrogenase signature 2. / Alanine dehydrogenase/PNT, C-terminal domain ...NAD(P) transhydrogenase, alpha subunit / NAD(P) transhydrogenase, alpha subunit, C-terminal / 4TM region of pyridine nucleotide transhydrogenase, mitoch / NADP transhydrogenase beta-like domain / NAD(P) transhydrogenase beta subunit / Alanine dehydrogenase/NAD(P) transhydrogenase, conserved site-1 / Alanine dehydrogenase & pyridine nucleotide transhydrogenase signature 1. / Alanine dehydrogenase/pyridine nucleotide transhydrogenase, conserved site-2 / Alanine dehydrogenase & pyridine nucleotide transhydrogenase signature 2. / Alanine dehydrogenase/PNT, C-terminal domain / Alanine dehydrogenase/pyridine nucleotide transhydrogenase, N-terminal / Alanine dehydrogenase/PNT, N-terminal domain / Alanine dehydrogenase/PNT, C-terminal domain / Alanine dehydrogenase/PNT, N-terminal domain / Alanine dehydrogenase/pyridine nucleotide transhydrogenase, NAD(H)-binding domain / DHS-like NAD/FAD-binding domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / NAD(P) transhydrogenase, mitochondrial
Similarity search - Component
Biological speciesOvis aries (sheep)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsKampjut, D. / Sazanov, L.A.
Funding support Austria, 1items
OrganizationGrant numberCountry
European Union665385 Austria
CitationJournal: Nature / Year: 2019
Title: Structure and mechanism of mitochondrial proton-translocating transhydrogenase.
Authors: Domen Kampjut / Leonid A Sazanov /
Abstract: Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. ...Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyses the transfer of a hydride between NADH and NADP, coupled to the translocation of one proton across the membrane. Its main physiological function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those observed in type II diabetes. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resolution structures of intact NNT, the details of this process remain unclear. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the soluble domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochemical work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischaemia reperfusion injury, metabolic syndrome and some cancers.
History
DepositionJul 1, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 28, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2019Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Sep 25, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
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

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Structure visualization

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Assembly

Deposited unit
A: Nicotinamide nucleotide transhydrogenase
B: Nicotinamide nucleotide transhydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)234,34210
Polymers228,0212
Non-polymers6,3218
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14390 Å2
ΔGint-142 kcal/mol
Surface area92270 Å2
MethodPISA

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Components

#1: Protein Nicotinamide nucleotide transhydrogenase /


Mass: 114010.305 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Ovis aries (sheep) / Organ: heart / References: UniProt: W5PFI3
#2: Chemical
ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE / Phosphatidylcholine


Mass: 790.145 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
Has ligand of interestN

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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: Nicotinamide nucleotide transhydrogenase in the apo state
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Ovis aries (sheep)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
250 mMSodium chlorideNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was purified in LMNG (final conc. 0.06%) and contained 0.05% CHAPS as a secondary detergent.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 4-6 s blotting time

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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 FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.25 sec. / Electron dose: 90 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 760
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.5CTF correction
7Coot0.8.9model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIX1.13model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 500001
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67908 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 137 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient, EMRinger score
Atomic model buildingPDB-ID: 6QTI

6qti


Accession code: 6QTI / Source name: PDB / Type: experimental model

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