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- PDB-8q6j: Atomic structure and conformational variability of the HER2-Trast... -

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Basic information

Entry
Database: PDB / ID: 8q6j
TitleAtomic structure and conformational variability of the HER2-Trastuzumab-Pertuzumab complex
Components
  • (Pertuzumab Fab ...) x 2
  • Receptor tyrosine-protein kinase erbB-2
  • Trastuzumab Fab heavy chain
  • Trastuzumab fab light chain
KeywordsSTRUCTURAL PROTEIN / ErbB-2 / Pertuzumab / Trastuzumab / Ternary complex / Protein / Flexibility / Continuous conformation / Cryo-EM / Single particle analysis
Function / homology
Function and homology information


negative regulation of immature T cell proliferation in thymus / ERBB3:ERBB2 complex / ERBB2-ERBB4 signaling pathway / GRB7 events in ERBB2 signaling / immature T cell proliferation in thymus / RNA polymerase I core binding / semaphorin receptor complex / regulation of microtubule-based process / ErbB-3 class receptor binding / Sema4D induced cell migration and growth-cone collapse ...negative regulation of immature T cell proliferation in thymus / ERBB3:ERBB2 complex / ERBB2-ERBB4 signaling pathway / GRB7 events in ERBB2 signaling / immature T cell proliferation in thymus / RNA polymerase I core binding / semaphorin receptor complex / regulation of microtubule-based process / ErbB-3 class receptor binding / Sema4D induced cell migration and growth-cone collapse / motor neuron axon guidance / PLCG1 events in ERBB2 signaling / neurotransmitter receptor localization to postsynaptic specialization membrane / ERBB2-EGFR signaling pathway / neuromuscular junction development / ERBB2 Activates PTK6 Signaling / enzyme-linked receptor protein signaling pathway / positive regulation of Rho protein signal transduction / ERBB2-ERBB3 signaling pathway / positive regulation of transcription by RNA polymerase I / Drug-mediated inhibition of ERBB2 signaling / Resistance of ERBB2 KD mutants to trastuzumab / Resistance of ERBB2 KD mutants to sapitinib / Resistance of ERBB2 KD mutants to tesevatinib / Resistance of ERBB2 KD mutants to neratinib / Resistance of ERBB2 KD mutants to osimertinib / Resistance of ERBB2 KD mutants to afatinib / Resistance of ERBB2 KD mutants to AEE788 / Resistance of ERBB2 KD mutants to lapatinib / Drug resistance in ERBB2 TMD/JMD mutants / oligodendrocyte differentiation / ERBB2 Regulates Cell Motility / semaphorin-plexin signaling pathway / PI3K events in ERBB2 signaling / positive regulation of protein targeting to membrane / regulation of angiogenesis / regulation of ERK1 and ERK2 cascade / Schwann cell development / coreceptor activity / Signaling by ERBB2 / transmembrane receptor protein tyrosine kinase activity / phosphatidylinositol 3-kinase/protein kinase B signal transduction / TFAP2 (AP-2) family regulates transcription of growth factors and their receptors / myelination / GRB2 events in ERBB2 signaling / cellular response to epidermal growth factor stimulus / positive regulation of cell adhesion / SHC1 events in ERBB2 signaling / Downregulation of ERBB2:ERBB3 signaling / positive regulation of MAP kinase activity / Constitutive Signaling by Overexpressed ERBB2 / positive regulation of epithelial cell proliferation / basal plasma membrane / positive regulation of translation / neurogenesis / Signaling by ERBB2 TMD/JMD mutants / wound healing / placental growth factor receptor activity / insulin receptor activity / vascular endothelial growth factor receptor activity / hepatocyte growth factor receptor activity / macrophage colony-stimulating factor receptor activity / platelet-derived growth factor alpha-receptor activity / platelet-derived growth factor beta-receptor activity / stem cell factor receptor activity / boss receptor activity / protein tyrosine kinase collagen receptor activity / brain-derived neurotrophic factor receptor activity / GPI-linked ephrin receptor activity / transmembrane-ephrin receptor activity / epidermal growth factor receptor activity / fibroblast growth factor receptor activity / insulin-like growth factor receptor activity / cellular response to growth factor stimulus / receptor protein-tyrosine kinase / Signaling by ERBB2 ECD mutants / neuromuscular junction / peptidyl-tyrosine phosphorylation / Signaling by ERBB2 KD Mutants / receptor tyrosine kinase binding / neuron differentiation / ruffle membrane / Downregulation of ERBB2 signaling / cell surface receptor protein tyrosine kinase signaling pathway / Constitutive Signaling by Aberrant PI3K in Cancer / transmembrane signaling receptor activity / PIP3 activates AKT signaling / myelin sheath / presynaptic membrane / heart development / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / protein tyrosine kinase activity / RAF/MAP kinase cascade / positive regulation of cell growth / basolateral plasma membrane / histone H3Y41 kinase activity / histone H2AXY142 kinase activity / positive regulation of MAPK cascade / positive regulation of protein phosphorylation / early endosome
Similarity search - Function
: / Epidermal growth factor receptor transmembrane-juxtamembrane segment / Tyrosine protein kinase, EGF/ERB/XmrK receptor / Growth factor receptor domain 4 / Growth factor receptor domain IV / Receptor L-domain / Furin-like cysteine-rich domain / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain ...: / Epidermal growth factor receptor transmembrane-juxtamembrane segment / Tyrosine protein kinase, EGF/ERB/XmrK receptor / Growth factor receptor domain 4 / Growth factor receptor domain IV / Receptor L-domain / Furin-like cysteine-rich domain / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain / Furin-like repeat / Furin-like repeats / Growth factor receptor cysteine-rich domain superfamily / : / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Receptor tyrosine-protein kinase erbB-2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRuedas, R. / Vuillemot, R. / Tubiana, T. / Winter, J.M. / Pieri, L. / Arteni, A.A. / Samson, C. / Jonic, J. / Mathieu, M. / Bressanelli, S.
Funding support France, 2items
OrganizationGrant numberCountry
Centre National de la Recherche Scientifique (CNRS) France
Other private
CitationJournal: J Struct Biol / Year: 2024
Title: Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex.
Authors: Rémi Ruedas / Rémi Vuillemot / Thibault Tubiana / Jean-Marie Winter / Laura Pieri / Ana-Andreea Arteni / Camille Samson / Slavica Jonic / Magali Mathieu / Stéphane Bressanelli /
Abstract: Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody- ...Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.
History
DepositionAug 11, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 20, 2023Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Database references / Refinement description / Category: citation / citation_author / em_3d_fitting_list
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_3d_fitting_list.initial_refinement_model_id
Revision 1.2Oct 23, 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: Trastuzumab fab light chain
B: Trastuzumab Fab heavy chain
C: Pertuzumab Fab light chain
D: Pertuzumab Fab heavy chain
E: Receptor tyrosine-protein kinase erbB-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,74510
Polymers162,9085
Non-polymers1,8375
Water00
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 E

#5: Protein Receptor tyrosine-protein kinase erbB-2


Mass: 68794.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ERBB2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P04626

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Antibody , 4 types, 4 molecules ABCD

#1: Antibody Trastuzumab fab light chain


Mass: 23466.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody Trastuzumab Fab heavy chain


Mass: 23425.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody Pertuzumab Fab light chain


Mass: 23548.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#4: Antibody Pertuzumab Fab heavy chain


Mass: 23674.486 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): CHO / Production host: Cricetulus griseus (Chinese hamster)

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Sugars , 2 types, 5 molecules

#6: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#7: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN
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: Ternary complex of her2-trastuzumab-pertuzumab Fabs / Type: COMPLEX
Details: Fab fragment from trastuzumab and pertuzumab linked to the extracellular domain of erbb2 (her2)
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.171364 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 / Cell: human
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 240000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.1 mm / Alignment procedure: BASIC
Specimen holderCryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.71 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8928
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.2particle selectiontemplate picking
2EPUimage acquisition
4cryoSPARC4.1.2CTF correction
7UCSF ChimeraX1.4model fitting
8PHENIX1.20.1model fitting
9Coot0.8.9.2model fitting
11PHENIX1.20.1model refinement
12Coot0.8.9.2model refinement
13cryoSPARC4.1.2initial Euler assignment
14cryoSPARC4.1.2final Euler assignment
15cryoSPARC4.1.2classification
16cryoSPARC4.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2954825
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 711008 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDSource nameTypeAccession codeInitial refinement model-IDChain-ID
1AlphaFoldin silico model
21n8z

1n8z

PDBexperimental model1n8z2
31s78

1s78

CPDBexperimental model1s783C
41s78

1s78

DPDBexperimental model1s783D
56oge

6oge

PDBexperimental model6oge4
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00711809
ELECTRON MICROSCOPYf_angle_d0.70216078
ELECTRON MICROSCOPYf_dihedral_angle_d13.1344289
ELECTRON MICROSCOPYf_chiral_restr0.0461817
ELECTRON MICROSCOPYf_plane_restr0.0062085

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