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- PDB-9oqa: Cryo-EM structure of AaaA, a Pseudomonas Aeruginosa autotransporter -

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Basic information

Entry
Database: PDB / ID: 9oqa
TitleCryo-EM structure of AaaA, a Pseudomonas Aeruginosa autotransporter
ComponentsAutotransporter domain-containing protein
KeywordsTRANSPORT PROTEIN / Arginine aminopeptidase / Autotransporter / Pseudomonas Aeruginosa / Virulence factor
Function / homology
Function and homology information


autotransporter activity / outer membrane / metalloexopeptidase activity / aminopeptidase activity / cell motility / proteolysis
Similarity search - Function
Peptidase M28 family / Autotransporter beta-domain / Outer membrane autotransporter barrel / Autotransporter beta-domain / Autotransporter beta-domain profile. / Autotransporter beta-domain / Autotransporter beta-domain superfamily / Peptidase M28 / Peptidase family M28
Similarity search - Domain/homology
ARGININE / Autotransporter domain-containing protein
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsArachchige, E.J. / Rahman, M.S. / Singendonk, K. / Kim, K.H.
Funding support United States, 1items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: J Mol Biol / Year: 2025
Title: Structural and Functional Characterization of Pseudomonas aeruginosa Virulence Factor AaaA, an Autotransporter with Arginine-Specific Aminopeptidase Activity.
Authors: Erandi Jayawardana Arachchige / Md Shafiqur Rahman / Katharina S Singendonk / Kelly H Kim /
Abstract: AaaA is a virulence-associated outer membrane protein found in the Gram-negative pathogen Pseudomonas aeruginosa. Classified as both an autotransporter and a member of the M28 family of ...AaaA is a virulence-associated outer membrane protein found in the Gram-negative pathogen Pseudomonas aeruginosa. Classified as both an autotransporter and a member of the M28 family of aminopeptidases, AaaA has been shown to cleave N-terminal arginine residues from host-derived peptides. This activity has been demonstrated to enhance bacterial survival and suppress host immune responses by increasing local arginine availability. Here, we report the first successful purification and combined structural and biochemical characterization of full-length AaaA. We resolved its cryo-EM structure at 3.87 Å resolution, revealing the canonical three-domain architecture of autotransporters: a signal peptide, a passenger domain, and a translocator domain. Notably, the passenger domain adopts a compact globular fold characteristic of M28 aminopeptidases, which is less common than the extended or β-helical structures observed in the majority of autotransporters structurally characterized to date. The structure reveals a zinc-coordinated catalytic site and a negatively charged substrate binding pocket, consistent with specificity for positively charged N-terminal arginine residues. Mutagenesis of active site residues confirmed the molecular basis for arginine recognition. Functional assays demonstrated that AaaA exhibits zinc-dependent aminopeptidase activity across a broad pH (6-10) and temperature (20-60 °C) range. Together, these findings provide fundamental insights into the structure and function of AaaA and establish a framework for future efforts to develop targeted inhibitors that may attenuate P. aeruginosa virulence.
History
DepositionMay 20, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 13, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Autotransporter domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,6354
Polymers72,3291
Non-polymers3063
Water00
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 Autotransporter domain-containing protein / Arginine specific aminopeptidase


Mass: 72329.414 Da / Num. of mol.: 1
Fragment: N-terminal strep tag II + TEV cleavage site + AaaA (UNP residues 23-647)
Source method: isolated from a genetically manipulated source
Details: Gene synthesized / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Strain: ATCC 15692 / Gene: PA0328 / Plasmid: pET28a(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: Q9I6G3
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-ARG / ARGININE


Type: L-peptide linking / Mass: 175.209 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H15N4O2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: AaaA complex with Arginine / Type: COMPLEX
Details: The full length AaaA contain two domains: a globular passenger domain and a beta barrel translocator domain.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.07036 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Strain: ATCC 15692 / Cellular location: Membrane
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Rosetta2 / Cell: E. coli / Plasmid: pET28a(+)
Buffer solutionpH: 7.5
Details: During solubilization of protein from membrane, 1% DDM was used. Subsequent purification buffers contain 0.05% DDM.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid1
2150 mMSodium ChlorideNaCl1
30.05 %n-dodecyl -D-maltoside1
SpecimenConc.: 7.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The protein sample was concentrated from the single peak fractions of size exclusion chromatography.
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 % / Chamber temperature: 277.15 K
Details: During vitrification 3 ul of sample placed on a grid and then vitrified with 10s hold and 4s blot time.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 77 K
Image recordingElectron dose: 44.71 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7084

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.4.1particle selection
2Topaz2.4particle selection
3EPU3.8image acquisitionFEI/ Thermo Fisher)
5cryoSPARC4.4.1CTF correction
8UCSF Chimera1.17.3model fitting
10cryoSPARC4.4.1initial Euler assignment
11cryoSPARC4.4.1final Euler assignment
12RELION4.0.1classification
13cryoSPARC4.4.13D reconstruction
14PHENIX1.21.2-5419model refinement
15Coot0.9.8.96model refinement
CTF correctionDetails: Patch CTF in CryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3009424
Details: Particles picked using topaz train following blob and template picking in cryoSPARC.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 241938 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT)
Details: Final map reconstructed from local refinement following non-uniform refinement to improve the region of interest excluding the detergent belt in cryoSPARC. Mask of the region of interest ...Details: Final map reconstructed from local refinement following non-uniform refinement to improve the region of interest excluding the detergent belt in cryoSPARC. Mask of the region of interest used to do the local refinement.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingChain residue range: 23-647 / Source name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.87 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034906
ELECTRON MICROSCOPYf_angle_d0.4846631
ELECTRON MICROSCOPYf_dihedral_angle_d12.2021791
ELECTRON MICROSCOPYf_chiral_restr0.037685
ELECTRON MICROSCOPYf_plane_restr0.003899

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