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- PDB-7yph: Open-spiral pentamer of the substrate-free Lon protease with a Y2... -

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Basic information

Entry
Database: PDB / ID: 7yph
TitleOpen-spiral pentamer of the substrate-free Lon protease with a Y224S mutation
ComponentsLon proteaseLon protease family
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 / identical protein binding / cytoplasm
Similarity search - Function
Lon protease, bacterial / Lon protease, bacterial/eukaryotic-type / 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, N-terminal domain superfamily / Lon N-terminal domain profile. ...Lon protease, bacterial / Lon protease, bacterial/eukaryotic-type / 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, N-terminal domain superfamily / Lon N-terminal domain profile. / Lon protease, N-terminal domain / 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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Lon protease
Similarity search - Component
Biological speciesMeiothermus taiwanensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 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
A: Lon protease
B: Lon protease
C: Lon protease
D: Lon protease
E: Lon protease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)443,4397
Polymers442,3925
Non-polymers1,0462
Water0
1


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

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Components

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


Mass: 88478.492 Da / Num. of mol.: 5 / Mutation: Y224S
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
#2: 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.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*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: Homo-pentameric complex of Lon protease with ATPgammaS
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.44 MDa / Experimental value: NO
Source (natural)Organism: Meiothermus taiwanensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethane(HOCH2)3CNH21
2150 mMsodium chlorideNaClSodium chloride1
310 mMmagnesium chlorideMgCl21
41 mMdithiothreitolC4H10O2S21
52 mMAdenosine- 5'- O- (3- thiotriphosphate)C10H16N5O12P3SLi1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: MtaLon-Y224S was incubated with ATPgammaS
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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1.34 sec. / Electron dose: 73.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 12982

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2particle selection
2EPU2.1image acquisition
4cryoSPARC3.2CTF correction
7UCSF ChimeraX1.2model fittingFit in Map was used to fit our model into the EM map
9PHENIX1.18.2_3874model refinementphenix.real_space_refine was used to refine our model against the map
10Coot0.9.6model refinementmanual rebuilding model
11cryoSPARC3.2initial Euler assignment
12cryoSPARC3.2final Euler assignment
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 4721627
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 777117 / Symmetry type: POINT

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