+Open data
-Basic information
Entry | Database: PDB / ID: 7lzj | ||||||
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Title | DpK2 bacteriophage tail spike depolymerase | ||||||
Components | Depolymerase | ||||||
Keywords | SUGAR BINDING PROTEIN / bateriophage tail spike / depolymerase / klebsiella targeting | ||||||
Function / homology | biological process involved in interaction with host / Pectin lyase fold/virulence factor / viral life cycle / virion component / Depolymerase Function and homology information | ||||||
Biological species | Klebsiella phage GH-K3 (virus) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å | ||||||
Authors | Belousoff, M.J. / Bamert, R.S. / Dunstan, R.A. / Lithgow, T. | ||||||
Funding support | Australia, 1items
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Citation | Journal: Microbiol Spectr / Year: 2021 Title: Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage. Authors: Rhys A Dunstan / Rebecca S Bamert / Matthew J Belousoff / Francesca L Short / Christopher K Barlow / Derek J Pickard / Jonathan J Wilksch / Ralf B Schittenhelm / Richard A Strugnell / Gordon ...Authors: Rhys A Dunstan / Rebecca S Bamert / Matthew J Belousoff / Francesca L Short / Christopher K Barlow / Derek J Pickard / Jonathan J Wilksch / Ralf B Schittenhelm / Richard A Strugnell / Gordon Dougan / Trevor Lithgow / Abstract: The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages ...The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae. | ||||||
History |
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-Structure visualization
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 7lzj.cif.gz | 371.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7lzj.ent.gz | 298.9 KB | Display | PDB format |
PDBx/mmJSON format | 7lzj.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7lzj_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7lzj_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7lzj_validation.xml.gz | 67.3 KB | Display | |
Data in CIF | 7lzj_validation.cif.gz | 102.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/lz/7lzj ftp://data.pdbj.org/pub/pdb/validation_reports/lz/7lzj | HTTPS FTP |
-Related structure data
Related structure data | 23608MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 98437.008 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Klebsiella phage GH-K3 (virus) / Gene: GHK3_32 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3S7W7I3 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: DpK2 depolymerase tail spike complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Bacteriophage sp. (virus) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.4 |
Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Microscopy | Model: TFS GLACIOS |
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Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 43 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.16_3549: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C3 (3 fold cyclic) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 331000 / Symmetry type: POINT | ||||||||||||||||||||||||
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