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- PDB-8the: Cryo-EM structure of Pseudomonas aeruginosa TonB-dependent transp... -

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Basic information

Entry
Database: PDB / ID: 8the
TitleCryo-EM structure of Pseudomonas aeruginosa TonB-dependent transporter PhuR in complex with synthetic antibody and heme
Components
  • (Synthetic Antibody ...) x 2
  • Heme/hemoglobin uptake outer membrane receptor PhuR
KeywordsMEMBRANE PROTEIN / beta barrel / outer membrane protein / heme binding protein / heme transport
Function / homology
Function and homology information


heme transport / siderophore transmembrane transport / heme transmembrane transporter activity / siderophore uptake transmembrane transporter activity / outer membrane / cellular response to iron ion / cell outer membrane
Similarity search - Function
TonB-dependent haem/haemoglobin receptor / TonB-dependent haemoglobin/transferrin/lactoferrin receptor / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor-like, beta-barrel / TonB-dependent receptor (TBDR) proteins profile. / Vitamin B12 transporter BtuB-like / TonB-dependent receptor, plug domain superfamily / TonB-dependent receptor, plug domain / TonB-dependent Receptor Plug Domain / TonB-dependent receptor-like, beta-barrel domain superfamily
Similarity search - Domain/homology
Chem-CTQ / PROTOPORPHYRIN IX CONTAINING FE / Heme/hemoglobin uptake outer membrane receptor PhuR
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsKnejski, P. / Erramilli, S.K. / Kossiakoff, A.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117372 United States
CitationJournal: Structure / Year: 2024
Title: Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding.
Authors: Paweł P Knejski / Satchal K Erramilli / Anthony A Kossiakoff /
Abstract: Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake ...Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.
History
DepositionJul 15, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 17, 2024Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Heme/hemoglobin uptake outer membrane receptor PhuR
H: Synthetic Antibody Heavy Chain
L: Synthetic Antibody Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,7445
Polymers135,1083
Non-polymers2,6352
Water1,45981
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Heme/hemoglobin uptake outer membrane receptor PhuR


Mass: 86370.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: phuR, PA4710 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9HV88

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Antibody , 2 types, 2 molecules HL

#2: Antibody Synthetic Antibody Heavy Chain


Mass: 25525.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Synthetic Antibody Light Chain


Mass: 23212.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Non-polymers , 3 types, 83 molecules

#4: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CTQ / (2~{R},4~{R},5~{S},6~{S})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[[(2~{R},3~{S},4~{S},5~{R},6~{S})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{R},4~{R},5~{S},6~{S})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{S})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-4,5-bis(oxidanyl)oxane-2-carboxylic acid


Mass: 2018.542 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C102H190N2O32P2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 81 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: Heme bound P. aeruginosa PhuR with bound synthetic antibody
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 5 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: 281 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 defocus max: 2500 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 5031

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
4RELIONCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3final Euler assignment
13cryoSPARC3.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 292512 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model

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