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- PDB-7pch: Human carboxyhemoglobin bound to Staphylococcus aureus hemophore ... -

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Basic information

Entry
Database: PDB / ID: 7pch
TitleHuman carboxyhemoglobin bound to Staphylococcus aureus hemophore IsdB - 1:2 complex
Components
  • Hemoglobin subunit alpha
  • Hemoglobin subunit beta
  • Iron-regulated surface determinant protein B
KeywordsMETAL TRANSPORT / Iron acquisition / Hemophore / Hemoglobin / NEAT domain
Function / homology
Function and homology information


heme transmembrane transporter activity / nitric oxide transport / hemoglobin binding / hemoglobin alpha binding / haptoglobin binding / haptoglobin-hemoglobin complex / renal absorption / organic acid binding / hemoglobin complex / oxygen transport ...heme transmembrane transporter activity / nitric oxide transport / hemoglobin binding / hemoglobin alpha binding / haptoglobin binding / haptoglobin-hemoglobin complex / renal absorption / organic acid binding / hemoglobin complex / oxygen transport / Scavenging of heme from plasma / endocytic vesicle lumen / blood vessel diameter maintenance / hydrogen peroxide catabolic process / oxygen carrier activity / Late endosomal microautophagy / Heme signaling / carbon dioxide transport / response to hydrogen peroxide / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / Cytoprotection by HMOX1 / oxygen binding / regulation of blood pressure / platelet aggregation / Chaperone Mediated Autophagy / peroxidase activity / positive regulation of nitric oxide biosynthetic process / tertiary granule lumen / Factors involved in megakaryocyte development and platelet production / ficolin-1-rich granule lumen / blood microparticle / iron ion binding / heme binding / Neutrophil degranulation / extracellular space / extracellular exosome / extracellular region / membrane / metal ion binding / cytosol
Similarity search - Function
Iron-regulated surface determinant protein IsdB / : / Iron-regulated surface determinant protein H/B, linker domain / NEAT domain / Iron Transport-associated domain / NEAT domain profile. / NEAr Transporter domain / NEAT domain superfamily / YSIRK type signal peptide / Hemoglobin, pi ...Iron-regulated surface determinant protein IsdB / : / Iron-regulated surface determinant protein H/B, linker domain / NEAT domain / Iron Transport-associated domain / NEAT domain profile. / NEAr Transporter domain / NEAT domain superfamily / YSIRK type signal peptide / Hemoglobin, pi / YSIRK Gram-positive signal peptide / Hemoglobin, alpha-type / Hemoglobin, beta-type / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / Globin/Protoglobin / Globin domain profile. / Globin / Globin / Globin-like superfamily
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Hemoglobin subunit beta / Hemoglobin subunit alpha / Iron-regulated surface determinant protein B
Similarity search - Component
Biological speciesStaphylococcus aureus subsp. aureus MW2 (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsDe Bei, O. / Gianquinto, E. / Chirgadze, D.Y. / Hardwick, S.W. / Spyrakis, F. / Luisi, B.F. / Campanini, B.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust200873/Z/16/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Cryo-EM structures of staphylococcal IsdB bound to human hemoglobin reveal the process of heme extraction.
Authors: Omar De Bei / Marialaura Marchetti / Luca Ronda / Eleonora Gianquinto / Loretta Lazzarato / Dimitri Y Chirgadze / Steven W Hardwick / Lee R Cooper / Francesca Spyrakis / Ben F Luisi / ...Authors: Omar De Bei / Marialaura Marchetti / Luca Ronda / Eleonora Gianquinto / Loretta Lazzarato / Dimitri Y Chirgadze / Steven W Hardwick / Lee R Cooper / Francesca Spyrakis / Ben F Luisi / Barbara Campanini / Stefano Bettati /
Abstract: Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but ...Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but inefficient from Hb either bound to oxygen (oxyHb) or bound to carbon monoxide (HbCO), and encompasses a sequence of structural events that are currently poorly understood. By single-particle cryo-electron microscopy, we determined the structure of two IsdB:Hb complexes, representing key species along the heme extraction pathway. The IsdB:HbCO structure, at 2.9-Å resolution, provides a snapshot of the preextraction complex. In this early stage of IsdB:Hb interaction, the hemophore binds to the β-subunits of the Hb tetramer, exploiting a folding-upon-binding mechanism that is likely triggered by a cis/trans isomerization of Pro173. Binding of IsdB to α-subunits occurs upon dissociation of the Hb tetramer into α/β dimers. The structure of the IsdB:metHb complex reveals the final step of the extraction process, where heme transfer to IsdB is completed. The stability of the complex, both before and after heme transfer from Hb to IsdB, is influenced by isomerization of Pro173. These results greatly enhance current understanding of structural and dynamic aspects of the heme extraction mechanism by IsdB and provide insight into the interactions that stabilize the complex before the heme transfer event. This information will support future efforts to identify inhibitors of heme acquisition by S. aureus by interfering with IsdB:Hb complex formation.
History
DepositionAug 3, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 13, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hemoglobin subunit alpha
B: Hemoglobin subunit beta
E: Iron-regulated surface determinant protein B
C: Hemoglobin subunit alpha
D: Hemoglobin subunit beta
F: Iron-regulated surface determinant protein B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)151,33310
Polymers148,8676
Non-polymers2,4664
Water9,188510
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, The stoichiometry of the complex was assessed by size-exclusion chromatography and multi-angle light scattering (SEC-MALS)
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16390 Å2
ΔGint-127 kcal/mol
Surface area55280 Å2

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Components

#1: Protein Hemoglobin subunit alpha / Alpha-globin / Hemoglobin alpha chain


Mass: 15150.353 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P69905
#2: Protein Hemoglobin subunit beta / Beta-globin / Hemoglobin beta chain


Mass: 15890.198 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P68871
#3: Protein Iron-regulated surface determinant protein B / Fur-regulated protein B / Staphylococcal iron-regulated protein H / Staphylococcus aureus surface protein J


Mass: 43393.129 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus subsp. aureus MW2 (bacteria)
Gene: isdB, frpB, sasJ, sirH, MW1011 / Plasmid: pASK-IBA3plus / Production host: Escherichia coli (E. coli) / References: UniProt: Q8NX66
#4: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 510 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Human carboxyhemoglobin bound to Staphylococcus aureus hemophore IsdB - 1:2 complexCOMPLEX#1-#30MULTIPLE SOURCES
2HemoglobinCOMPLEX#1-#21NATURAL
3Iron-regulated surface determinant protein BCOMPLEX#31RECOMBINANT
Molecular weightValue: 0.151 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Staphylococcus aureus subsp. aureus MW2 (bacteria)196620
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
Details: CHAPSO was added immediately before plunge freezing to overcome preferred orientation of the particles in the vitreous ice.
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
28 mMCHAPSOC32H58N2O8S1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was discharged on both sides. / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 277.15 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 magnification: 130000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 39.59 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2873
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4WarpCTF correction
7UCSF Chimera1.14model fittingFit to map was used to align ad hoc model and cryo-EM map
9PHENIX1.19.2model refinementReal-space refinement was used to start the model refinement
10Coot0.9.4model refinementThe software was used to perform the manual refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 457000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 224937 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15VMM

5vmm

15VMM1PDBexperimental model
23P5Q

3p5q

13P5Q2PDBexperimental model

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