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- PDB-8vr9: Structure of a synthetic antibody in complex with a class I MHC p... -

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Basic information

Entry
Database: PDB / ID: 8vr9
TitleStructure of a synthetic antibody in complex with a class I MHC presenting a hapten-peptide conjugate
Components
  • Beta-2-microglobulin
  • GTPase KRas, N-terminally processed
  • HLA class I histocompatibility antigen, A alpha chain
  • R023 Fab heavy chain
  • R023 Fab light chain
KeywordsIMMUNE SYSTEM / Complex / Synthetic antibody / MHC class I / covalently modified peptide
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 / Golgi medial cisterna / positive regulation of CD8-positive, alpha-beta T cell activation / response to mineralocorticoid / CD8-positive, alpha-beta T cell activation / GMP binding / positive regulation of CD8-positive, alpha-beta T cell proliferation / forebrain astrocyte development ...positive regulation of memory T cell activation / T cell mediated cytotoxicity directed against tumor cell target / TAP complex binding / Golgi medial cisterna / positive regulation of CD8-positive, alpha-beta T cell activation / response to mineralocorticoid / CD8-positive, alpha-beta T cell activation / GMP binding / positive regulation of CD8-positive, alpha-beta T cell proliferation / forebrain astrocyte development / LRR domain binding / regulation of synaptic transmission, GABAergic / negative regulation of epithelial cell differentiation / CD8 receptor binding / response to isolation stress / antigen processing and presentation of exogenous peptide antigen via MHC class I / response to gravity / epithelial tube branching involved in lung morphogenesis / beta-2-microglobulin binding / type I pneumocyte differentiation / Rac protein signal transduction / endoplasmic reticulum exit site / TAP binding / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-dependent / positive regulation of Rac protein signal transduction / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells / protection from natural killer cell mediated cytotoxicity / myoblast proliferation / skeletal muscle cell differentiation / RAS signaling downstream of NF1 loss-of-function variants / RUNX3 regulates p14-ARF / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-independent / positive regulation of glial cell proliferation / antigen processing and presentation of endogenous peptide antigen via MHC class Ib / SOS-mediated signalling / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / SHC1 events in ERBB4 signaling / cardiac muscle cell proliferation / Signalling to RAS / detection of bacterium / Activated NTRK2 signals through FRS2 and FRS3 / SHC-related events triggered by IGF1R / Estrogen-stimulated signaling through PRKCZ / glial cell proliferation / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / T cell receptor binding / SHC-mediated cascade:FGFR2 / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / SHC-mediated cascade:FGFR4 / Erythropoietin activates RAS / SHC-mediated cascade:FGFR1 / Signaling by FGFR4 in disease / FRS-mediated FGFR3 signaling / Signaling by CSF3 (G-CSF) / Signaling by FLT3 ITD and TKD mutants / FRS-mediated FGFR2 signaling / FRS-mediated FGFR4 signaling / FRS-mediated FGFR1 signaling / p38MAPK events / Signaling by FGFR3 in disease / protein-membrane adaptor activity / Tie2 Signaling / striated muscle cell differentiation / Signaling by FGFR2 in disease / GRB2 events in EGFR signaling / Signaling by FLT3 fusion proteins / SHC1 events in EGFR signaling / FLT3 Signaling / Signaling by FGFR1 in disease / EGFR Transactivation by Gastrin / NCAM signaling for neurite out-growth / CD209 (DC-SIGN) signaling / homeostasis of number of cells within a tissue / Downstream signal transduction / GRB2 events in ERBB2 signaling / Insulin receptor signalling cascade / Ras activation upon Ca2+ influx through NMDA receptor / SHC1 events in ERBB2 signaling / response to glucocorticoid / Constitutive Signaling by Overexpressed ERBB2 / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / negative regulation of receptor binding / early endosome lumen / Nef mediated downregulation of MHC class I complex cell surface expression / DAP12 interactions / VEGFR2 mediated cell proliferation / transferrin transport / small monomeric GTPase / cellular response to iron ion / Endosomal/Vacuolar pathway / lumenal side of endoplasmic reticulum membrane / FCERI mediated MAPK activation / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / peptide antigen assembly with MHC class II protein complex
Similarity search - Function
Small GTPase, Ras-type / Small GTPase Ras domain profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / 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 / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase ...Small GTPase, Ras-type / Small GTPase Ras domain profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / 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 / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Beta-2-Microglobulin / : / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / Rab subfamily of small GTPases / MHC classes I/II-like antigen recognition protein / : / Small GTP-binding protein domain / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
AMG 510 (bound form) / GTPase KRas / HLA class I histocompatibility antigen, A alpha chain / Beta-2-microglobulin
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsMaso, L. / Bang, I. / Koide, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Molecular basis for antibody recognition of multiple drug-peptide/MHC complexes.
Authors: Lorenzo Maso / Epsa Rajak / Injin Bang / Akiko Koide / Takamitsu Hattori / Benjamin G Neel / Shohei Koide /
Abstract: The HapImmune platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with ...The HapImmune platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the V and V domains, binds HLAs in an unconventional, angled way, with V making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.
History
DepositionJan 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 9, 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: HLA class I histocompatibility antigen, A alpha chain
B: Beta-2-microglobulin
C: GTPase KRas, N-terminally processed
D: R023 Fab light chain
E: R023 Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)92,8456
Polymers92,2835
Non-polymers5631
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 , 2 types, 2 molecules AB

#1: Protein HLA class I histocompatibility antigen, A alpha chain / Human leukocyte antigen A / HLA-A


Mass: 32047.309 Da / Num. of mol.: 1 / Fragment: extracellular domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HLA-A, HLAA / Plasmid: pET28 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P04439
#2: Protein Beta-2-microglobulin


Mass: 11892.291 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: B2M, CDABP0092, HDCMA22P / Plasmid: pHFT2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P61769

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Antibody , 2 types, 2 molecules DE

#4: Antibody R023 Fab light chain


Mass: 23518.178 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#5: Antibody R023 Fab heavy chain


Mass: 24034.822 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Protein/peptide / Non-polymers , 2 types, 2 molecules C

#3: Protein/peptide GTPase KRas, N-terminally processed


Mass: 789.963 Da / Num. of mol.: 1 / Fragment: residues 8-16 / Mutation: G12C / Source method: obtained synthetically / Details: Cys12 conjugated to covalent inhibitor sotorasib / Source: (synth.) Homo sapiens (human) / References: UniProt: P01116
#6: Chemical ChemComp-MOV / AMG 510 (bound form) / 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-4-[(2S)-2-methyl-4-propanoylpiperazin-1-yl]-1-[4-methyl-2-(propan-2-yl)pyridin-3-yl]pyrido[2,3-d]pyrimidin-2(1H)-one


Mass: 562.610 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C30H32F2N6O3 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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

Component
IDNameTypeEntity IDParent-IDSource
1Binary complex of Fab R023 with sotorasib-conjugated KRAS G12C peptide (8-16) presented by class I MHC having HLA-A*03:01COMPLEX#1-#2, #4-#50MULTIPLE SOURCES
2Fab R023COMPLEX#4-#51RECOMBINANT
3class I MHC, having HLA-A*03:01, with Beta-2-microglobulinCOMPLEX#1-#21RECOMBINANT
4sotorasib-conjugated KRAS G12C peptide (8-16)COMPLEX#31SYNTHETIC
Molecular weightValue: 0.0927 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMsodium phosphateNaH2PO41
21.8 mMpotassium phosphateKH2PO41
3138 mMsodium chlorideNaCl1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2 sec. / Electron dose: 57.72 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2Leginon3.6image acquisition
4cryoSPARC4CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178888 / Symmetry type: POINT

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