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- PDB-7stf: Structure of KRAS G12V/HLA-A*03:01 in complex with antibody fragm... -

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Basic information

Entry
Database: PDB / ID: 7stf
TitleStructure of KRAS G12V/HLA-A*03:01 in complex with antibody fragment V2
Components
  • Beta-2-microglobulin
  • HLA class I histocompatibility antigen, A alpha chain
  • IgG, Fab Heavy Chain V2
  • IgG, Fab Light Chain V2
  • KRAS G12V (7-16)
KeywordsIMMUNE SYSTEM / Complex / MHC-I / KRAS / HLA-A3
Function / homology
Function and homology information


positive regulation of memory T cell activation / T cell mediated cytotoxicity directed against tumor cell target / TAP complex binding / positive regulation of CD8-positive, alpha-beta T cell activation / CD8-positive, alpha-beta T cell activation / Golgi medial cisterna / positive regulation of CD8-positive, alpha-beta T cell proliferation / CD8 receptor binding / antigen processing and presentation of exogenous peptide antigen via MHC class I / endoplasmic reticulum exit site ...positive regulation of memory T cell activation / T cell mediated cytotoxicity directed against tumor cell target / TAP complex binding / positive regulation of CD8-positive, alpha-beta T cell activation / CD8-positive, alpha-beta T cell activation / Golgi medial cisterna / positive regulation of CD8-positive, alpha-beta T cell proliferation / CD8 receptor binding / antigen processing and presentation of exogenous peptide antigen via MHC class I / endoplasmic reticulum exit site / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-dependent / TAP binding / protection from natural killer cell mediated cytotoxicity / beta-2-microglobulin binding / T cell receptor binding / detection of bacterium / positive regulation of ferrous iron binding / positive regulation of transferrin receptor binding / positive regulation of receptor binding / early endosome lumen / Nef mediated downregulation of MHC class I complex cell surface expression / negative regulation of receptor binding / DAP12 interactions / antigen processing and presentation of endogenous peptide antigen via MHC class Ib / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-independent / cellular response to iron ion / Endosomal/Vacuolar pathway / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / lumenal side of endoplasmic reticulum membrane / cellular response to iron(III) ion / antigen processing and presentation of exogenous protein antigen via MHC class Ib, TAP-dependent / negative regulation of forebrain neuron differentiation / regulation of erythrocyte differentiation / peptide antigen assembly with MHC class I protein complex / ER to Golgi transport vesicle membrane / regulation of iron ion transport / response to molecule of bacterial origin / MHC class I peptide loading complex / HFE-transferrin receptor complex / T cell mediated cytotoxicity / positive regulation of T cell cytokine production / antigen processing and presentation of endogenous peptide antigen via MHC class I / MHC class I protein complex / negative regulation of neurogenesis / positive regulation of receptor-mediated endocytosis / peptide antigen assembly with MHC class II protein complex / multicellular organismal-level iron ion homeostasis / MHC class II protein complex / cellular response to nicotine / specific granule lumen / positive regulation of cellular senescence / positive regulation of T cell mediated cytotoxicity / positive regulation of type II interferon production / recycling endosome membrane / phagocytic vesicle membrane / peptide antigen binding / antigen processing and presentation of exogenous peptide antigen via MHC class II / negative regulation of epithelial cell proliferation / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / positive regulation of immune response / Interferon gamma signaling / Modulation by Mtb of host immune system / positive regulation of T cell activation / Interferon alpha/beta signaling / sensory perception of smell / negative regulation of neuron projection development / positive regulation of protein binding / tertiary granule lumen / E3 ubiquitin ligases ubiquitinate target proteins / DAP12 signaling / antibacterial humoral response / MHC class II protein complex binding / late endosome membrane / T cell receptor signaling pathway / iron ion transport / ER-Phagosome pathway / T cell differentiation in thymus / early endosome membrane / protein refolding / protein homotetramerization / intracellular iron ion homeostasis / amyloid fibril formation / learning or memory / defense response to Gram-positive bacterium / immune response / Amyloid fiber formation / endoplasmic reticulum lumen / lysosomal membrane / Golgi membrane / external side of plasma membrane / innate immune response / signaling receptor binding / focal adhesion / Neutrophil degranulation / endoplasmic reticulum membrane / SARS-CoV-2 activates/modulates innate and adaptive immune responses / structural molecule activity / Golgi apparatus / cell surface / endoplasmic reticulum
Similarity search - Function
MHC class I, alpha chain, C-terminal / MHC_I C-terminus / MHC class I alpha chain, alpha1 alpha2 domains / Class I Histocompatibility antigen, domains alpha 1 and 2 / Beta-2-Microglobulin / : / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / MHC classes I/II-like antigen recognition protein / : ...MHC class I, alpha chain, C-terminal / MHC_I C-terminus / MHC class I alpha chain, alpha1 alpha2 domains / Class I Histocompatibility antigen, domains alpha 1 and 2 / Beta-2-Microglobulin / : / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / MHC classes I/II-like antigen recognition protein / : / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
HLA class I histocompatibility antigen, A alpha chain / Beta-2-microglobulin
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsWright, K.M. / Gabelli, S.B. / Miller, M.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2023
Title: Hydrophobic interactions dominate the recognition of a KRAS G12V neoantigen.
Authors: Katharine M Wright / Sarah R DiNapoli / Michelle S Miller / P Aitana Azurmendi / Xiaowei Zhao / Zhiheng Yu / Mayukh Chakrabarti / WuXian Shi / Jacqueline Douglass / Michael S Hwang / Emily ...Authors: Katharine M Wright / Sarah R DiNapoli / Michelle S Miller / P Aitana Azurmendi / Xiaowei Zhao / Zhiheng Yu / Mayukh Chakrabarti / WuXian Shi / Jacqueline Douglass / Michael S Hwang / Emily Han-Chung Hsiue / Brian J Mog / Alexander H Pearlman / Suman Paul / Maximilian F Konig / Drew M Pardoll / Chetan Bettegowda / Nickolas Papadopoulos / Kenneth W Kinzler / Bert Vogelstein / Shibin Zhou / Sandra B Gabelli /
Abstract: Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific ...Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRAS peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.
History
DepositionNov 12, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: IgG, Fab Heavy Chain V2
C: KRAS G12V (7-16)
L: IgG, Fab Light Chain V2
A: HLA class I histocompatibility antigen, A alpha chain
B: Beta-2-microglobulin


Theoretical massNumber of molelcules
Total (without water)92,1395
Polymers92,1395
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody IgG, Fab Heavy Chain V2


Mass: 23175.861 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pcDNA3.1 / Cell line (production host): HEK293F
Production host: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
#2: Protein/peptide KRAS G12V (7-16)


Mass: 885.082 Da / Num. of mol.: 1 / Mutation: G12V / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Antibody IgG, Fab Light Chain V2


Mass: 23859.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pcDNA3.1 / Cell line (production host): HEK293F
Production host: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
#4: Protein HLA class I histocompatibility antigen, A alpha chain / Human leukocyte antigen A / HLA-A


Mass: 32270.510 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: P04439 / Source: (gene. exp.) Homo sapiens (human) / Gene: HLA-A, HLAA / Plasmid: pET3a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P04439
#5: Protein Beta-2-microglobulin


Mass: 11948.353 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: P61769 / Source: (gene. exp.) Homo sapiens (human) / Gene: B2M, CDABP0092, HDCMA22P / Plasmid: pET3a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P61769

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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 antibody-fragment V2 with KRAS G12V/HLA-A*03:01
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Buffer solutionpH: 7
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4CTFFIND4.1.13CTF correction
9RELIONmodel refinement
10RELION3.1betainitial Euler assignment
11RELION3.1betafinal Euler assignment
13RELION3.1beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 367584 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient

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