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- PDB-9div: The crystal structure of de novo designed ChuA binding protein C8 -

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Basic information

Entry
Database: PDB / ID: 9div
TitleThe crystal structure of de novo designed ChuA binding protein C8
ComponentsDe novo designed ChuA binding protein C8
KeywordsTRANSPORT PROTEIN / TonB-dependent transporter / ChuA / Heme / Hemoglobin / Outer-membrane / de novo designed protein / binding protein
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.46 Å
AuthorsFox, D. / 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 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release
Revision 1.1Jul 23, 2025Group: Database references / Category: citation / citation_author
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: De novo designed ChuA binding protein C8
B: De novo designed ChuA binding protein C8
C: De novo designed ChuA binding protein C8
D: De novo designed ChuA binding protein C8
E: De novo designed ChuA binding protein C8
F: De novo designed ChuA binding protein C8
G: De novo designed ChuA binding protein C8
H: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)122,7098
Polymers122,7098
Non-polymers00
Water181
1
A: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)15,3391
Polymers15,3391
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)15,3391
Polymers15,3391
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)15,3391
Polymers15,3391
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)15,3391
Polymers15,3391
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
E: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)15,3391
Polymers15,3391
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
6
F: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)15,3391
Polymers15,3391
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
7
G: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)15,3391
Polymers15,3391
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
8
H: De novo designed ChuA binding protein C8


Theoretical massNumber of molelcules
Total (without water)15,3391
Polymers15,3391
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)85.366, 110.300, 127.338
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein
De novo designed ChuA binding protein C8


Mass: 15338.574 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / Strain (production host): C41 D3
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.44 Å3/Da / Density % sol: 49.65 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5 / Details: 0.2M Na Acet, 0.1M Tris, 30% w/v PEG 4K

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.987 Å
DetectorType: DECTRIS EIGER R 4M / Detector: PIXEL / Date: Sep 18, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 2.46→46.32 Å / Num. obs: 44074 / % possible obs: 99.4 % / Redundancy: 6.7 % / CC1/2: 0.997 / Rmerge(I) obs: 0.179 / Rpim(I) all: 0.113 / Net I/σ(I): 5.7
Reflection shellResolution: 2.46→2.55 Å / Redundancy: 6.4 % / Rmerge(I) obs: 1.478 / Mean I/σ(I) obs: 0.8 / Num. unique obs: 4383 / CC1/2: 0.648 / Rpim(I) all: 0.946 / Χ2: 0.44 / % possible all: 95

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.46→46.32 Å / SU ML: 0.42 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 39.96 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3242 2153 4.91 %
Rwork0.3006 --
obs0.3018 43839 99.17 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.46→46.32 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7524 0 0 1 7525
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0077573
X-RAY DIFFRACTIONf_angle_d1.19810213
X-RAY DIFFRACTIONf_dihedral_angle_d4.3181083
X-RAY DIFFRACTIONf_chiral_restr0.0511274
X-RAY DIFFRACTIONf_plane_restr0.0081296
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.46-2.520.4571360.40942557X-RAY DIFFRACTION93
2.52-2.580.37241290.36782787X-RAY DIFFRACTION100
2.58-2.650.33671470.33212739X-RAY DIFFRACTION100
2.65-2.730.36151520.3282743X-RAY DIFFRACTION100
2.73-2.820.32551330.33622786X-RAY DIFFRACTION100
2.82-2.920.39121370.33472781X-RAY DIFFRACTION100
2.92-3.040.35971610.32572741X-RAY DIFFRACTION100
3.04-3.180.33761530.31962762X-RAY DIFFRACTION100
3.18-3.340.34591410.31782776X-RAY DIFFRACTION100
3.34-3.550.32811400.30442798X-RAY DIFFRACTION100
3.55-3.830.33821510.27762783X-RAY DIFFRACTION100
3.83-4.210.31081300.25792828X-RAY DIFFRACTION100
4.21-4.820.27821550.27042802X-RAY DIFFRACTION99
4.82-6.070.33891600.32942858X-RAY DIFFRACTION100
6.07-46.320.25541280.27412945X-RAY DIFFRACTION98
Refinement TLS params.Method: refined / Origin x: 26.9987 Å / Origin y: -13.6091 Å / Origin z: -17.8164 Å
111213212223313233
T0.4108 Å20.0461 Å2-0.0007 Å2-0.3934 Å20.0252 Å2--0.4079 Å2
L0.1873 °20.0874 °20.1684 °2-0.2957 °20.2866 °2--0.3659 °2
S-0.0429 Å °0.0079 Å °0.0352 Å °-0.0029 Å °-0.013 Å °0.0622 Å °-0.0864 Å °-0.1093 Å °0.0429 Å °
Refinement TLS groupSelection details: all

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