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- PDB-9dir: Cryo-EM structure of the heme/hemoglobin transporter ChuA, in com... -

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Basic information

Entry
Database: PDB / ID: 9dir
TitleCryo-EM structure of the heme/hemoglobin transporter ChuA, in complex with de novo designed binder G7
Components
  • ChuA binding protein G7
  • Outer membrane heme/hemoglobin receptor
KeywordsTRANSPORT PROTEIN / TonB-dependent transporter / ChuA / Heme / Hemoglobin / Outer-membrane / de novo designed protein / binding protein
Function / homology
Function and homology information


heme transmembrane transporter activity / siderophore uptake transmembrane transporter activity / 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
Outer membrane heme/hemoglobin receptor
Similarity search - Component
Biological speciessynthetic construct (others)
Escherichia coli CFT073 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsFox, D. / Venugopal, H. / Lupton, C.J. / Spicer, B.A. / Grinter, R.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)APP1197376 Australia
Australian Research Council (ARC)LE200100045, LE120100090 Australia
CitationJournal: Nat Commun / Year: 2025
Title: Inhibiting heme piracy by pathogenic Escherichia coli using de novo-designed proteins.
Authors: Daniel R Fox / Kazem Asadollahi / Imogen Samuels / Bradley A Spicer / Ashleigh Kropp / Christopher J Lupton / Kevin Lim / Chunxiao Wang / Hari Venugopal / Marija Dramicanin / Gavin J Knott / Rhys Grinter /
Abstract: Iron is an essential nutrient for most bacteria and is often growth-limiting during infection, due to the host sequestering free iron as part of the innate immune response. To obtain the iron ...Iron is an essential nutrient for most bacteria and is often growth-limiting during infection, due to the host sequestering free iron as part of the innate immune response. To obtain the iron required for growth, many bacterial pathogens encode transporters capable of extracting the iron-containing cofactor heme directly from host proteins. Pathogenic E. coli and Shigella spp. produce the outer membrane transporter ChuA, which binds host hemoglobin and extracts its heme cofactor, before importing heme into the cell. Heme extraction by ChuA is a dynamic process, with the transporter capable of rapidly extracting heme from hemoglobin in the absence of an external energy source, without forming a stable ChuA-hemoglobin complex. In this work, we utilise a combination of structural modelling, Cryo-EM, X-ray crystallography, mutagenesis, and phenotypic analysis to understand the mechanistic detail of this process. Based on this understanding we utilise artificial intelligence-based protein design to create binders capable of inhibiting E. coli growth by blocking hemoglobin binding to ChuA. By screening a limited number of these designs, we identify several binders that inhibit E. coli growth at low nanomolar concentrations, without experimental optimisation. We determine the structure of a subset of these binders, alone and in complex with ChuA, demonstrating that they closely match the computational design. This work demonstrates the utility of de novo-designed proteins for inhibiting bacterial nutrient uptake and uses a workflow that could be applied to integral membrane proteins in other organisms.
History
DepositionSep 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release
Revision 1.1Jul 23, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ChuA binding protein G7
B: Outer membrane heme/hemoglobin receptor


Theoretical massNumber of molelcules
Total (without water)85,0202
Polymers85,0202
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ChuA binding protein G7


Mass: 15433.850 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / Strain (production host): C41 DE3
#2: Protein Outer membrane heme/hemoglobin receptor


Mass: 69586.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli CFT073 (bacteria) / Gene: chuA, c4308 / Production host: Escherichia coli (E. coli) / Strain (production host): C41 DE3 / References: UniProt: A0A0H2VC22
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: Complex between the heme/hemoglobin transporter ChuA and de novo designed binding protein G7
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: CFT073
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41 DE3
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
10.1 MolarTris1
20.15 MolarNaCl1
30.01 %Dodecyl Maltoside1
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: 4D-STEM / Nominal defocus max: 1400 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131924 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046005
ELECTRON MICROSCOPYf_angle_d0.5548119
ELECTRON MICROSCOPYf_dihedral_angle_d4.116828
ELECTRON MICROSCOPYf_chiral_restr0.042873
ELECTRON MICROSCOPYf_plane_restr0.0041069

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