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- PDB-8k3y: The "5+1" heteromeric structure of Lon protease consisting of a s... -

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Basic information

Entry
Database: PDB / ID: 8k3y
TitleThe "5+1" heteromeric structure of Lon protease consisting of a spiral pentamer with Y224S mutation and an N-terminal-truncated monomeric E613K mutant
Components(Lon proteaseLon protease family) x 2
KeywordsHYDROLASE / AAA+ protein / ATPase
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
ADENOSINE-5'-DIPHOSPHATE / Lon protease
Similarity search - Component
Biological speciesMeiothermus taiwanensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.42 Å
AuthorsLi, S. / Hsieh, K.Y. / Kuo, C.I. / Zhang, K. / Chang, C.I.
Funding support Taiwan, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, Taiwan)108-2320-B-001-011-MY3 Taiwan
Ministry of Science and Technology (MoST, Taiwan)111-2320-B-001-013-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
DepositionJul 17, 2023Deposition 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
F: Lon protease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)511,43110
Polymers509,7226
Non-polymers1,7094
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 89307.383 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 BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A059VAZ3, endopeptidase La
#2: Protein Lon protease / Lon protease family / ATP-dependent protease La


Mass: 63185.266 Da / Num. of mol.: 1 / Mutation: E613K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Meiothermus taiwanensis (bacteria) / Gene: lonA1, lon, lon1_1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A059VAZ3, endopeptidase La
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, 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: Spiral pentamer of Lon protease with a Y224S mutation in complex with the N-terminal-truncated monomeric E613K mutant of Lon.
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Meiothermus taiwanensis (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM Tris-HCl, 200 mM NaCl, 10 mM MgCl2, 1 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(hydroxymethyl)aminomethaneC4H11NO31
2200 mMsodium chlorideNaClSodium chloride1
310 mMMagnesium chlorideMgCl21
41 mMDithiothreitolC4H10O2S21
SpecimenConc.: 0.7 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: EPU / Version: 3.3.1.5184REL / Category: image acquisition
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 4.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70828 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00635220
ELECTRON MICROSCOPYf_angle_d1.1547751
ELECTRON MICROSCOPYf_dihedral_angle_d11.954880
ELECTRON MICROSCOPYf_chiral_restr0.0655435
ELECTRON MICROSCOPYf_plane_restr0.0086206

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