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- PDB-9dhe: The crystal structure on the heme/hemoglobin transporter ChuA, in... -

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Basic information

Entry
Database: PDB / ID: 9dhe
TitleThe crystal structure on the heme/hemoglobin transporter ChuA, in complex with heme
ComponentsTonB-dependent receptor
KeywordsTRANSPORT PROTEIN / TonB-dependent transporter / ChuA / Heme / Hemoglobin / Outer-membrane
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 (TBDR) proteins signature 1. / TonB box, conserved site / 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 haem/haemoglobin receptor / TonB-dependent haemoglobin/transferrin/lactoferrin receptor / TonB-dependent receptor (TBDR) proteins signature 1. / TonB box, conserved site / 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
PROTOPORPHYRIN IX CONTAINING FE / Hemin TonB-dependent receptor
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 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 3, 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

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Assembly

Deposited unit
A: TonB-dependent receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)70,1752
Polymers69,5581
Non-polymers6161
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)78.200, 116.112, 123.289
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein TonB-dependent receptor / Ligand-gated channel protein / TonB-dependent heme/hemoglobin receptor ChuA/ShuA / TonB-dependent ...Ligand-gated channel protein / TonB-dependent heme/hemoglobin receptor ChuA/ShuA / TonB-dependent hemoglobin/transferrin/lactoferrin family receptor


Mass: 69558.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: CTF073
Gene: chuA, shuA, B6R15_003762, BE932_16525, D4N09_20470, D9E49_06195, DNX30_14835, EIA08_11240, EPS76_04355, EWK56_15360, ExPECSC038_04966, F7F11_22070, FPI65_21585, G5603_10405, GFY48_03270, GP711_ ...Gene: chuA, shuA, B6R15_003762, BE932_16525, D4N09_20470, D9E49_06195, DNX30_14835, EIA08_11240, EPS76_04355, EWK56_15360, ExPECSC038_04966, F7F11_22070, FPI65_21585, G5603_10405, GFY48_03270, GP711_16795, GQN34_12665, GRC73_08695, HEP34_004102, HI055_004051, HIE29_002171, HL601_09740, HVV39_10295, HVW04_16595, NCTC8621_00323, OGM49_23355
Production host: Escherichia coli (E. coli) / Strain (production host): C41 DE3 / References: UniProt: A0A023L3G7
#2: 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
Has ligand of interestY
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: 4.02 Å3/Da / Density % sol: 69.43 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7 / Details: 20 % PEG 1500, 0.1 M MIB Buffer

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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 X 4M / Detector: PIXEL / Date: Jun 11, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 2.8→48.43 Å / Num. obs: 28374 / % possible obs: 99.9 % / Redundancy: 10 % / Biso Wilson estimate: 88.87 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.187 / Rpim(I) all: 0.065 / Net I/σ(I): 8.9
Reflection shellResolution: 2.8→2.95 Å / Rmerge(I) obs: 3.67 / Num. unique obs: 4040 / CC1/2: 0.442 / Rpim(I) all: 1.286

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.2→48.41 Å / SU ML: 0.4781 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 33.2877
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2992 967 5.06 %
Rwork0.2506 18131 -
obs0.253 19098 99.85 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 104.19 Å2
Refinement stepCycle: LAST / Resolution: 3.2→48.41 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4782 0 43 0 4825
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00274940
X-RAY DIFFRACTIONf_angle_d0.6476723
X-RAY DIFFRACTIONf_chiral_restr0.046709
X-RAY DIFFRACTIONf_plane_restr0.0053884
X-RAY DIFFRACTIONf_dihedral_angle_d7.1881684
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2-3.370.33931580.29732509X-RAY DIFFRACTION99.74
3.37-3.580.30361220.25792557X-RAY DIFFRACTION99.78
3.58-3.860.32981380.2462564X-RAY DIFFRACTION99.93
3.86-4.240.28541350.22172576X-RAY DIFFRACTION99.89
4.24-4.860.25531340.19552578X-RAY DIFFRACTION99.96
4.86-6.120.27271520.23392599X-RAY DIFFRACTION100
6.12-48.410.32821280.29362748X-RAY DIFFRACTION99.69
Refinement TLS params.Method: refined / Origin x: 2.3723709092 Å / Origin y: -21.227887992 Å / Origin z: -18.9944875714 Å
111213212223313233
T0.708706497794 Å2-0.0404222924407 Å2-0.0127302102609 Å2-0.79960534975 Å2-0.0762673240468 Å2--0.658370543077 Å2
L1.03786951608 °2-0.50244120616 °2-0.0844374391203 °2-2.70022472452 °2-0.513744176042 °2--1.70219966695 °2
S0.0853595397759 Å °-0.135335773295 Å °-0.028740287754 Å °-0.240271477357 Å °0.085017929857 Å °0.0854592307027 Å °0.0626647449635 Å °0.0825113752134 Å °-0.182278027339 Å °
Refinement TLS groupSelection details: all

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