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- PDB-7upt: Human mitochondrial AAA protein ATAD1 (with a catalytic dead muta... -

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Basic information

Entry
Database: PDB / ID: 7upt
TitleHuman mitochondrial AAA protein ATAD1 (with a catalytic dead mutation) in complex with a peptide substrate (open conformation)
Components
  • Outer mitochondrial transmembrane helix translocase
  • Unknown peptide substrate
KeywordsPROTEIN TRANSPORT / AAA protein / mitochondria / tail-anchored protein / membrane protein
Function / homology
Function and homology information


extraction of mislocalized protein from mitochondrial outer membrane / membrane protein dislocase activity / Class I peroxisomal membrane protein import / Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate / negative regulation of synaptic transmission, glutamatergic / regulation of postsynaptic neurotransmitter receptor internalization / peroxisomal membrane / positive regulation of receptor internalization / learning / memory ...extraction of mislocalized protein from mitochondrial outer membrane / membrane protein dislocase activity / Class I peroxisomal membrane protein import / Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate / negative regulation of synaptic transmission, glutamatergic / regulation of postsynaptic neurotransmitter receptor internalization / peroxisomal membrane / positive regulation of receptor internalization / learning / memory / postsynaptic membrane / mitochondrial outer membrane / glutamatergic synapse / ATP hydrolysis activity / ATP binding / membrane / cytosol
Similarity search - Function
AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Outer mitochondrial transmembrane helix translocase
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsWang, L. / Toutkoushian, H. / Belyy, V. / Kokontis, C. / Walter, P.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM032384 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021741 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD020054 United States
CitationJournal: Elife / Year: 2022
Title: Conserved structural elements specialize ATAD1 as a membrane protein extraction machine.
Authors: Lan Wang / Hannah Toutkoushian / Vladislav Belyy / Claire Y Kokontis / Peter Walter /
Abstract: The mitochondrial AAA (TPase ssociated with diverse cellular ctivities) protein ATAD1 (in humans; Msp1 in yeast) removes mislocalized membrane proteins, as well as stuck import substrates from the ...The mitochondrial AAA (TPase ssociated with diverse cellular ctivities) protein ATAD1 (in humans; Msp1 in yeast) removes mislocalized membrane proteins, as well as stuck import substrates from the mitochondrial outer membrane, facilitating their re-insertion into their cognate organelles and maintaining mitochondria's protein import capacity. In doing so, it helps to maintain proteostasis in mitochondria. How ATAD1 tackles the energetic challenge to extract hydrophobic membrane proteins from the lipid bilayer and what structural features adapt ATAD1 for its particular function has remained a mystery. Previously, we determined the structure of Msp1 in complex with a peptide substrate (Wang et al., 2020). The structure showed that Msp1's mechanism follows the general principle established for AAA proteins while adopting several structural features that specialize it for its function. Among these features in Msp1 was the utilization of multiple aromatic amino acids to firmly grip the substrate in the central pore. However, it was not clear whether the aromatic nature of these amino acids were required, or if they could be functionally replaced by aliphatic amino acids. In this work, we determined the cryo-EM structures of the human ATAD1 in complex with a peptide substrate at near atomic resolution. The structures show that phylogenetically conserved structural elements adapt ATAD1 for its function while generally adopting a conserved mechanism shared by many AAA proteins. We developed a microscopy-based assay reporting on protein mislocalization, with which we directly assessed ATAD1's activity in live cells and showed that both aromatic amino acids in pore-loop 1 are required for ATAD1's function and cannot be substituted by aliphatic amino acids. A short α-helix at the C-terminus strongly facilitates ATAD1's oligomerization, a structural feature that distinguishes ATAD1 from its closely related proteins.
History
DepositionApr 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Outer mitochondrial transmembrane helix translocase
B: Outer mitochondrial transmembrane helix translocase
C: Outer mitochondrial transmembrane helix translocase
D: Outer mitochondrial transmembrane helix translocase
E: Outer mitochondrial transmembrane helix translocase
F: Outer mitochondrial transmembrane helix translocase
G: Unknown peptide substrate
hetero molecules


Theoretical massNumber of molelcules
Total (without water)233,69318
Polymers230,6087
Non-polymers3,08511
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Outer mitochondrial transmembrane helix translocase / ATPase family AAA domain-containing protein 1 / hATAD1 / Thorase


Mass: 38289.855 Da / Num. of mol.: 6 / Mutation: E193Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATAD1, FNP001 / Production host: Escherichia coli (E. coli)
References: UniProt: Q8NBU5, Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate
#2: Protein/peptide Unknown peptide substrate


Mass: 869.063 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
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: ATAD1-substrate complex in open conformation / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.2 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Escherichia coli (E. coli)562
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21DE3
Buffer solutionpH: 7.5
SpecimenConc.: 4 mg/ml / Embedding 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: SPOT SCAN
Electron lensMode: DIFFRACTION / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 67 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45003 / Symmetry type: POINT

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