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- EMDB-70744: Cryo-EM structure of AaaA, a Pseudomonas Aeruginosa autotransporter -
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Open data
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Basic information
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Title | Cryo-EM structure of AaaA, a Pseudomonas Aeruginosa autotransporter | |||||||||
![]() | Cryo-EM map from the local refinement. | |||||||||
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![]() | Arginine aminopeptidase / Autotransporter / Pseudomonas Aeruginosa / Virulence factor / TRANSPORT PROTEIN | |||||||||
Function / homology | ![]() autotransporter activity / outer membrane / metalloexopeptidase activity / aminopeptidase activity / cell motility / proteolysis Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.87 Å | |||||||||
![]() | Arachchige EJ / Rahman MS / Singendonk K / Kim KH | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 85.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 22.1 KB 22.1 KB | Display Display | ![]() |
Images | ![]() | 42.9 KB | ||
Filedesc metadata | ![]() | 7.4 KB | ||
Others | ![]() ![]() | 84.5 MB 84.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 843 KB | Display | ![]() |
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Full document | ![]() | 842.6 KB | Display | |
Data in XML | ![]() | 13 KB | Display | |
Data in CIF | ![]() | 15.5 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9oqaMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||
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Annotation | Cryo-EM map from the local refinement. | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.886 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Cryo-EM halfmap2 of the local refinement.
File | emd_70744_half_map_1.map | ||||||||||||
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Annotation | Cryo-EM halfmap2 of the local refinement. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Cryo-EM halfmap1 of the local refinement.
File | emd_70744_half_map_2.map | ||||||||||||
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Annotation | Cryo-EM halfmap1 of the local refinement. | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : AaaA complex with Arginine
Entire | Name: AaaA complex with Arginine |
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Components |
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-Supramolecule #1: AaaA complex with Arginine
Supramolecule | Name: AaaA complex with Arginine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: The full length AaaA contain two domains: a globular passenger domain and a beta barrel translocator domain. |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 70.36 KDa |
-Macromolecule #1: Autotransporter domain-containing protein
Macromolecule | Name: Autotransporter domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 72.329414 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: ASAWSHPQFE KGGGSGGGSG GSAWSHPQFE KENLYFQGYQ YGEYAGETLE RLITDYPGRY RGTASFAGAS KLMQSRLGFG YQTSRQDFT WAGNRSSQNV IASAPGSSGK FLVLGAHYDT YYGRPTLQGL DDNASGAAVL TEIARNLGGI ALENGLEVVG F GAEEEGLR ...String: ASAWSHPQFE KGGGSGGGSG GSAWSHPQFE KENLYFQGYQ YGEYAGETLE RLITDYPGRY RGTASFAGAS KLMQSRLGFG YQTSRQDFT WAGNRSSQNV IASAPGSSGK FLVLGAHYDT YYGRPTLQGL DDNASGAAVL TEIARNLGGI ALENGLEVVG F GAEEEGLR GSRAYVESLD ASQRANLLGM INLDSLVTGD KMYAHAGSNS VSNPALGAYR EQILRIAREL DIPLFTNPGL NA EYPAGTG CCSDGESFNG MDIPVLFIEA TNWELGDLDG YEQTDNPAIP GGSTWHDPAE DNKEVLTNAL GQERIEQRMR DFS RLLTRL VLEQTNADLL ASTASGGALA RQMEDQLQRQ HQALTRLHDR RWLTLLGSNR PVGSFDGEVG AEGEVSPDSG FDMP GNPES RRAGVHLLGD YRYSEALTLG GSLAFQRSRD KLDHGGRIEG DTWQLGLFGL YNDGGPEWLA GELNLGHTRY DSKRS VYLQ AAGGPVLLDQ RLSGDTSAWS WGARLEGGYD FSFGELRSGP LAGLDYMHYR IDDFREDEAL RTALGYEKQD YDSLEA SLG WRLRGELALG ARMRLQPYAS LRWVRELADG RLDDMDLTSR GDGRVRVADM GGVDKDFGRA QLGAQLAITE QLGVFAE AN SRFAHSEGNQ AGYSLGVNWQ F UniProtKB: Autotransporter domain-containing protein |
-Macromolecule #2: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #3: ARGININE
Macromolecule | Name: ARGININE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ARG |
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Molecular weight | Theoretical: 175.209 Da |
Chemical component information | ![]() ChemComp-ARG: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 7.2 mg/mL | ||||||||||||
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Buffer | pH: 7.5 Component:
Details: During solubilization of protein from membrane, 1% DDM was used. Subsequent purification buffers contain 0.05% DDM. | ||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 1200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.034 kPa | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: During vitrification 3 ul of sample placed on a grid and then vitrified with 10s hold and 4s blot time.. | ||||||||||||
Details | The protein sample was concentrated from the single peak fractions of size exclusion chromatography. |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Temperature | Min: 77.0 K / Max: 100.0 K |
Image recording | Film or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 7084 / Average electron dose: 44.71 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model | Chain - Residue range: 23-647 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model |
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Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | ![]() PDB-9oqa: |