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- PDB-6wqh: Molecular basis for the ATPase-powered substrate translocation by... -

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Basic information

Entry
Database: PDB / ID: 6wqh
TitleMolecular basis for the ATPase-powered substrate translocation by the Lon AAA+ protease
Components
  • Ig2 substrate
  • Lon protease
KeywordsMOTOR PROTEIN / Lon AAA+ / protease / cryo-EM / dual pore-loops
Function / homology
Function and homology information


endopeptidase La / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / cellular response to heat / sequence-specific DNA binding / serine-type endopeptidase activity / ATP hydrolysis activity / ATP binding / identical protein binding / metal ion binding / cytoplasm
Similarity search - Function
Lon protease, bacterial / Lon protease, bacterial/eukaryotic-type / Lon protease AAA+ ATPase lid domain / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon N-terminal domain profile. ...Lon protease, bacterial / Lon protease, bacterial/eukaryotic-type / Lon protease AAA+ ATPase lid domain / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon N-terminal domain profile. / Lon protease, N-terminal domain / Lon protease, N-terminal domain superfamily / ATP-dependent protease La (LON) substrate-binding domain / Found in ATP-dependent protease La (LON) / PUA-like superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-4KZ / ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Lon protease
Similarity search - Component
Biological speciesMeiothermus taiwanensis (bacteria)
Dictyostelium discoideum (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZhang, K. / Li, S. / Hsiehb, K. / Sub, S. / Pintilie, G. / Chiu, W. / Chang, C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
CitationJournal: J Biol Chem / Year: 2021
Title: Molecular basis for ATPase-powered substrate translocation by the Lon AAA+ protease.
Authors: Shanshan Li / Kan-Yen Hsieh / Shih-Chieh Su / Grigore D Pintilie / Kaiming Zhang / Chung-I Chang /
Abstract: The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive ...The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 Å resolution. Our data indicate that substrate interactions are mediated by the dual pore loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.
History
DepositionApr 28, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 9, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Nov 17, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Oct 16, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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  • Deposited structure unit
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  • EMDB-21870
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Lon protease
B: Lon protease
C: Lon protease
F: Lon protease
D: Lon protease
E: Lon protease
S: Ig2 substrate
hetero molecules


Theoretical massNumber of molelcules
Total (without water)538,09818
Polymers533,1657
Non-polymers4,93411
Water00
1


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

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Components

#1: Protein
Lon protease / ATP-dependent protease La


Mass: 88701.766 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Meiothermus taiwanensis (bacteria) / Gene: lonA1, lon
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A059VAZ3, endopeptidase La
#2: Protein/peptide Ig2 substrate


Mass: 954.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Unfolded substate / Source: (gene. exp.) Dictyostelium discoideum (eukaryote)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#4: Chemical
ChemComp-4KZ / N-[(1R)-1-(dihydroxyboranyl)-2-phenylethyl]-Nalpha-(pyrazin-2-ylcarbonyl)-L-phenylalaninamide


Mass: 418.253 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C22H23BN4O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationY
Sequence detailsThe substrate Ig2 is in the unfolded state, the side chains can not be modeled and are listed as ...The substrate Ig2 is in the unfolded state, the side chains can not be modeled and are listed as UNK in the sequence and coordinates. The complete studied sequence is the following: MTVKPAPSAEHSYAEGEGLVKVFDNAPAEFTIFAVDTKGVARTDGGDP FEVAINGPDGLVVDAKVTDNNDGTYGVVYDAPVEGNYNVNVTLRGNPI KNMPIDVKAIEGANGEDSSFGSFTFTVAAKNKKGEVKTYGGDKFEVSI TGPAEEITLDACDNQDGTYTAAYSLVGNGRFSTGVKLNGKHIEGSPFK QVLGNPGKKNPEVKSFTTTRTAN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged stateCOMPLEX#1-#20MULTIPLE SOURCES
2Lon proteaseCOMPLEX#11RECOMBINANT
3Ig2 substrateCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.36 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Meiothermus taiwanensis (bacteria)172827
23Dictyostelium discoideum (eukaryote)44689
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
23Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 8
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 8.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1800
Image scansMovie frames/image: 30

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Processing

EM software
IDNameVersionCategory
1EMAN22.3particle selection
2EPU2image acquisition
4CTFFIND4.13CTF correction
7UCSF Chimera1.13.1model fitting
11RELION3.0.2classification
12RELION3.0.23D reconstruction
13PHENIX1.18model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23487 / Symmetry type: POINT
Atomic model buildingPDB-ID: 4YPL

4ypl


Accession code: 4YPL / Source name: PDB / Type: experimental model

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