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- PDB-8sxh: Structure of the C-terminal protease CtpA-LbcA complex of Pseudom... -

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Basic information

Entry
Database: PDB / ID: 8sxh
TitleStructure of the C-terminal protease CtpA-LbcA complex of Pseudomonas aeruginosa
Components
  • Carboxyl-terminal protease
  • TPR repeat-containing protein PA4667
  • unidentified peptide
KeywordsHYDROLASE / CtpA / LbcA / C-terminal protease / Pseudomonas aeruginosa
Function / homology
Function and homology information


C-terminal processing peptidase / serine-type peptidase activity / proteolysis
Similarity search - Function
: / Activating protease CtpB N-terminal domain / C-terminal-processing peptidase S41A / tail specific protease / Tail specific protease / Peptidase family S41 / PDZ domain 6 / PDZ domain / Tetratricopeptide repeat / Tetratricopeptide repeat ...: / Activating protease CtpB N-terminal domain / C-terminal-processing peptidase S41A / tail specific protease / Tail specific protease / Peptidase family S41 / PDZ domain 6 / PDZ domain / Tetratricopeptide repeat / Tetratricopeptide repeat / ClpP/crotonase-like domain superfamily / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
Carboxy-terminal processing protease CtpB / TPR repeat-containing protein PA4667
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Escherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.94 Å
AuthorsHsu, H.-C. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI136901 United States
CitationJournal: EMBO J / Year: 2024
Title: P. aeruginosa CtpA protease adopts a novel activation mechanism to initiate the proteolytic process.
Authors: Hao-Chi Hsu / Michelle Wang / Amanda Kovach / Andrew J Darwin / Huilin Li /
Abstract: During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal ...During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA-LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.
History
DepositionMay 22, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 24, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Carboxyl-terminal protease
B: Carboxyl-terminal protease
C: TPR repeat-containing protein PA4667
D: unidentified peptide
E: Carboxyl-terminal protease
F: Carboxyl-terminal protease
G: TPR repeat-containing protein PA4667
H: unidentified peptide
I: Carboxyl-terminal protease
J: Carboxyl-terminal protease
K: TPR repeat-containing protein PA4667
L: unidentified peptide


Theoretical massNumber of molelcules
Total (without water)444,59012
Polymers444,59012
Non-polymers00
Water00
1


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

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Components

#1: Protein
Carboxyl-terminal protease


Mass: 42920.438 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: ctpA / Production host: Escherichia coli (E. coli) / References: UniProt: A0A072ZJB8
#2: Protein TPR repeat-containing protein PA4667


Mass: 61742.066 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: PA4667 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42810
#3: Protein/peptide unidentified peptide


Mass: 613.749 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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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: CtpA-LbcA complex of Pseudomonas aeruginosa / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.42 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) / Plasmid: pET15b, pCDF
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2100 mMKClKCl1
35 mMMgCl2MgCl21
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 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: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 66 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 26257
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.8particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.14model fitting
9cryoSPARC2.8initial Euler assignment
10cryoSPARC2.8final Euler assignment
11cryoSPARC2.8classification
12cryoSPARC2.83D reconstruction
13PHENIX4788model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2090314
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1185591 / Algorithm: FOURIER SPACE / Num. of class averages: 4 / Symmetry type: POINT
Atomic model buildingB value: 196.3 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17RQH

7rqh

17RQH1PDBexperimental model
27RQF

7rqf

17RQF2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00219983
ELECTRON MICROSCOPYf_angle_d0.44527036
ELECTRON MICROSCOPYf_dihedral_angle_d3.5352823
ELECTRON MICROSCOPYf_chiral_restr0.0393120
ELECTRON MICROSCOPYf_plane_restr0.0033597

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