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- PDB-7lzj: DpK2 bacteriophage tail spike depolymerase -

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Basic information

Entry
Database: PDB / ID: 7lzj
TitleDpK2 bacteriophage tail spike depolymerase
ComponentsDepolymerase
KeywordsSUGAR BINDING PROTEIN / bateriophage tail spike / depolymerase / klebsiella targeting
Function / homologybiological process involved in interaction with host / Pectin lyase fold/virulence factor / viral life cycle / virion component / Depolymerase
Function and homology information
Biological speciesKlebsiella phage GH-K3 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBelousoff, M.J. / Bamert, R.S. / Dunstan, R.A. / Lithgow, T.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1092262 Australia
CitationJournal: 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
DepositionMar 10, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 25, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 8, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Sep 15, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation_author.identifier_ORCID
Revision 1.3May 29, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation / Item: _citation.country

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Structure visualization

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Assembly

Deposited unit
A: Depolymerase
B: Depolymerase
C: Depolymerase


Theoretical massNumber of molelcules
Total (without water)295,3113
Polymers295,3113
Non-polymers00
Water00
1


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

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Components

#1: Protein Depolymerase


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

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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: DpK2 depolymerase tail spike complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacteriophage sp. (virus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 43 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 331000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00216994
ELECTRON MICROSCOPYf_angle_d0.43223076
ELECTRON MICROSCOPYf_dihedral_angle_d3.8799907
ELECTRON MICROSCOPYf_chiral_restr0.0442487
ELECTRON MICROSCOPYf_plane_restr0.0033037

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