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- PDB-7ypk: Close-ring hexamer of the substrate-bound Lon protease with an S6... -

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Basic information

Entry
Database: PDB / ID: 7ypk
TitleClose-ring hexamer of the substrate-bound Lon protease with an S678A mutation
Components
  • Lon protease
  • alpha-S1-casein
KeywordsHYDROLASE / Lon protease / hydrolysis / AAA proteins
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 / metal ion binding / identical protein 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 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
ADENOSINE-5'-DIPHOSPHATE / Lon protease
Similarity search - Component
Biological speciesMeiothermus taiwanensis (bacteria)
Bos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLi, S. / Hsieh, K.Y. / Kuo, C.I. / Lee, S.H. / Ho, M.R. / Wang, C.H. / Zhang, K. / Chang, C.I.
Funding support Taiwan, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, Taiwan)108-2320-B-001-011-MY3 Taiwan
CitationJournal: Nat Commun / Year: 2023
Title: A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.
Authors: Shanshan Li / Kan-Yen Hsieh / Chiao-I Kuo / Tzu-Chi Lin / Szu-Hui Lee / Yi-Ru Chen / Chun-Hsiung Wang / Meng-Ru Ho / See-Yeun Ting / Kaiming Zhang / Chung-I Chang /
Abstract: Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In ...Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In this ATP-dependent translocating state, the substrate is gripped by the pore loops of the ATPase domains arranged in a universal right-handed spiral staircase organization. However, the process by which a AAA+ protein is activated to adopt this substrate-pore-loop arrangement remains unknown. We show here, using cryo-electron microscopy (cryo-EM), that the activation process of the Lon AAA+ protease may involve a pentameric assembly and a substrate-dependent incorporation of the sixth protomer to form the substrate-pore-loop contacts seen in the translocating state. Based on the structural results, we design truncated monomeric mutants that inhibit Lon activity by binding to the native pentamer and demonstrated that expressing these monomeric mutants in Escherichia coli cells containing functional Lon elicits specific phenotypes associated with lon deficiency, including the inhibition of persister cell formation. These findings uncover a substrate-dependent assembly process for the activation of a AAA+ protein and demonstrate a targeted approach to selectively inhibit its function within cells.
History
DepositionAug 3, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: 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_DOI / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
S: alpha-S1-casein
F: Lon protease
A: Lon protease
B: Lon protease
C: Lon protease
D: Lon protease
E: Lon protease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)535,43013
Polymers532,8677
Non-polymers2,5636
Water00
1


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

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Components

#1: Protein/peptide alpha-S1-casein


Mass: 1635.006 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Unfolded substate / Source: (natural) Bos taurus (domestic cattle)
#2: Protein
Lon protease / ATP-dependent protease La


Mass: 88538.594 Da / Num. of mol.: 6 / Mutation: S678A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Meiothermus taiwanensis (bacteria) / Gene: lonA1, lon, lon1_1 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A059VAZ3, endopeptidase La
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Sequence detailsThe sequence of alpha-S1-casein is corresponded to the UniProt entry P02662. However, the authors ...The sequence of alpha-S1-casein is corresponded to the UniProt entry P02662. However, the authors do not know how the coordinates align with the sequence and the residue numbering is arbitrary.

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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
1Close-ring hexamer of MtaLon-S678A in a substrate-engaged stateCOMPLEX#1-#20MULTIPLE SOURCES
2alpha-S1-caseinCOMPLEX1NATURAL
3Close-ring hexamer of Lon proteaseCOMPLEX1RECOMBINANT
Molecular weightValue: 0.53 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Bos taurus (domestic cattle)9913
32Meiothermus taiwanensis (bacteria)172827
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethane(HOCH2)3CNH21
2150 mMsodium chlorideNaCl1
310 mMmagnesium chlorideMgCl21
41 mMdithiothreitolC4H10O2S21
51 mMAdenosine diphosphateC10H15N5O10P21
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Lon protease was incubated with alpha-S1-casein
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.52 sec. / Electron dose: 59 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 12546

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Processing

EM software
IDNameVersionCategoryDetails
2EPU2.1image acquisition
4cryoSPARC3.2CTF correction
7UCSF ChimeraX1.2model fittingFit in Map was used to fit our model into the EM map
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
12cryoSPARC3.23D reconstruction
13PHENIX1.18.2_3874model refinementphenix.real_space_refine was used to refine our model against the map
14Coot0.9.6model refinementmanual rebuilding model
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1756270
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 207760 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7FID

7fid


Accession code: 7FID / Source name: PDB / Type: experimental model

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