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- EMDB-43480: Structure of a synthetic antibody in complex with a class I MHC p... -

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Entry
Database: EMDB / ID: EMD-43480
TitleStructure of a synthetic antibody in complex with a class I MHC presenting a hapten-peptide conjugate
Map data
Sample
  • Complex: Binary complex of Fab R023 with sotorasib-conjugated KRAS G12C peptide (7-16) presented by class I MHC having HLA-A*11:01
    • Complex: Fab R023
      • Protein or peptide: R023 Fab light chain
      • Protein or peptide: R023 Fab heavy chain
    • Complex: class I MHC, having HLA-A*11:01, with Beta-2-microglobulin
      • Protein or peptide: HLA-A antigen
      • Protein or peptide: Beta-2-microglobulin
    • Complex: sotorasib-conjugated KRAS G12C peptide (7-16)
      • Protein or peptide: GTPase KRas, N-terminally processed
  • Ligand: AMG 510 (bound form)
KeywordsComplex / Synthetic antibody / MHC class I / covalently modified peptide / IMMUNE SYSTEM
Function / homology
Function and homology information


response to mineralocorticoid / GMP binding / forebrain astrocyte development / LRR domain binding / regulation of synaptic transmission, GABAergic / negative regulation of epithelial cell differentiation / response to isolation stress / response to gravity / epithelial tube branching involved in lung morphogenesis / type I pneumocyte differentiation ...response to mineralocorticoid / GMP binding / forebrain astrocyte development / LRR domain binding / regulation of synaptic transmission, GABAergic / negative regulation of epithelial cell differentiation / response to isolation stress / response to gravity / epithelial tube branching involved in lung morphogenesis / type I pneumocyte differentiation / Rac protein signal transduction / myoblast proliferation / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells / RAS signaling downstream of NF1 loss-of-function variants / RUNX3 regulates p14-ARF / skeletal muscle cell differentiation / positive regulation of glial cell proliferation / SOS-mediated signalling / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / SHC1 events in ERBB4 signaling / cardiac muscle cell proliferation / antigen processing and presentation of peptide antigen via MHC class I / Signalling to RAS / SHC-related events triggered by IGF1R / Activated NTRK2 signals through FRS2 and FRS3 / Estrogen-stimulated signaling through PRKCZ / positive regulation of Rac protein signal transduction / SHC-mediated cascade:FGFR3 / glial cell proliferation / MET activates RAS signaling / SHC-mediated cascade:FGFR2 / SHC-mediated cascade:FGFR4 / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / Erythropoietin activates RAS / SHC-mediated cascade:FGFR1 / Signaling by FGFR4 in disease / Signaling by CSF3 (G-CSF) / FRS-mediated FGFR3 signaling / Signaling by FLT3 ITD and TKD mutants / FRS-mediated FGFR2 signaling / FRS-mediated FGFR4 signaling / p38MAPK events / Signaling by FGFR3 in disease / FRS-mediated FGFR1 signaling / striated muscle cell differentiation / Tie2 Signaling / protein-membrane adaptor activity / 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 / GRB2 events in ERBB2 signaling / Downstream signal transduction / Insulin receptor signalling cascade / SHC1 events in ERBB2 signaling / early endosome lumen / Ras activation upon Ca2+ influx through NMDA receptor / Constitutive Signaling by Overexpressed ERBB2 / Nef mediated downregulation of MHC class I complex cell surface expression / response to glucocorticoid / DAP12 interactions / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / VEGFR2 mediated cell proliferation / Endosomal/Vacuolar pathway / small monomeric GTPase / T cell mediated cytotoxicity / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / FCERI mediated MAPK activation / lumenal side of endoplasmic reticulum membrane / regulation of iron ion transport / cellular response to iron(III) ion / negative regulation of iron ion transport / negative regulation of forebrain neuron differentiation / antigen processing and presentation of exogenous protein antigen via MHC class Ib, TAP-dependent / peptide antigen assembly with MHC class I protein complex / ER to Golgi transport vesicle membrane / liver development / regulation of erythrocyte differentiation / female pregnancy / response to molecule of bacterial origin / Signaling by ERBB2 TMD/JMD mutants / HFE-transferrin receptor complex / transferrin transport / MHC class I peptide loading complex / cellular response to iron ion / negative regulation of receptor-mediated endocytosis / Signaling by SCF-KIT / RAF activation / Constitutive Signaling by EGFRvIII
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
HLA-A antigen / GTPase KRas / Beta-2-microglobulin
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsMaso L / Bang I / Koide S
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43480.png

Downloads & links

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Map

FileDownload / File: emd_43480.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 316.8 Å		0.83 Å/pix.
x 384 pix.
= 316.8 Å		0.83 Å/pix.
x 384 pix.
= 316.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.9866132 - 3.262109
Average (Standard dev.)0.00039415646 (±0.048810836)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 316.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43480_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43480_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Binary complex of Fab R023 with sotorasib-conjugated KRAS G12C pe...

EntireName: Binary complex of Fab R023 with sotorasib-conjugated KRAS G12C peptide (7-16) presented by class I MHC having HLA-A*11:01
Components
  • Complex: Binary complex of Fab R023 with sotorasib-conjugated KRAS G12C peptide (7-16) presented by class I MHC having HLA-A*11:01
    • Complex: Fab R023
      • Protein or peptide: R023 Fab light chain
      • Protein or peptide: R023 Fab heavy chain
    • Complex: class I MHC, having HLA-A*11:01, with Beta-2-microglobulin
      • Protein or peptide: HLA-A antigen
      • Protein or peptide: Beta-2-microglobulin
    • Complex: sotorasib-conjugated KRAS G12C peptide (7-16)
      • Protein or peptide: GTPase KRas, N-terminally processed
  • Ligand: AMG 510 (bound form)

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Supramolecule #1: Binary complex of Fab R023 with sotorasib-conjugated KRAS G12C pe...

SupramoleculeName: Binary complex of Fab R023 with sotorasib-conjugated KRAS G12C peptide (7-16) presented by class I MHC having HLA-A*11:01
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2, #4-#5
Molecular weightTheoretical: 92.9 KDa

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Supramolecule #2: Fab R023

SupramoleculeName: Fab R023 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: class I MHC, having HLA-A*11:01, with Beta-2-microglobulin

SupramoleculeName: class I MHC, having HLA-A*11:01, with Beta-2-microglobulin
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: sotorasib-conjugated KRAS G12C peptide (7-16)

SupramoleculeName: sotorasib-conjugated KRAS G12C peptide (7-16) / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human) / Synthetically produced: Yes

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Macromolecule #1: HLA-A antigen

MacromoleculeName: HLA-A antigen / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31.725936 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHSMRYFYT SVSRPGRGEP RFIAVGYVDD TQFVRFDSDA ASQRMEPRAP WIEQEGPEYW DQETRNVKAQ SQTDRVDLGT LRGYYNQSE DGSHTIQIMY GCDVGPDGRF LRGYRQDAYD GKDYIALNED LRSWTAADMA AQITKRKWEA AHAAEQQRAY L EGRCVEWL ...String:
GSHSMRYFYT SVSRPGRGEP RFIAVGYVDD TQFVRFDSDA ASQRMEPRAP WIEQEGPEYW DQETRNVKAQ SQTDRVDLGT LRGYYNQSE DGSHTIQIMY GCDVGPDGRF LRGYRQDAYD GKDYIALNED LRSWTAADMA AQITKRKWEA AHAAEQQRAY L EGRCVEWL RRYLENGKET LQRTDPPKTH MTHHPISDHE ATLRCWALGF YPAEITLTWQ RDGEDQTQDT ELVETRPAGD GT FQKWAAV VVPSGEEQRY TCHVQHEGLP KPLTLRW

UniProtKB: HLA-A antigen

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Macromolecule #2: Beta-2-microglobulin

MacromoleculeName: Beta-2-microglobulin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.819238 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
AIQRTPKIQV YSRHPAENGK SNFLNCYVSG FHPSDIEVDL LKNGERIEKV EHSDLSFSKD WSFYLLYYTE FTPTEKDEYA CRVNHVTLS QPKIVKWDRD M

UniProtKB: Beta-2-microglobulin

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Macromolecule #3: GTPase KRas, N-terminally processed

MacromoleculeName: GTPase KRas, N-terminally processed / type: protein_or_peptide / ID: 3 / Details: Cys12 conjugated to covalent inhibitor sotorasib / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 889.094 Da
SequenceString:
VVVGACGVGK

UniProtKB: GTPase KRas

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Macromolecule #4: R023 Fab light chain

MacromoleculeName: R023 Fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.518178 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QASYVRKTIT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QASYVRKTIT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

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Macromolecule #5: R023 Fab heavy chain

MacromoleculeName: R023 Fab heavy chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.034822 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF TFSDYSIHWV RQAPGKGLEW VASISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGGWIAAMD YWGQGTLVTV FNQIKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF TFSDYSIHWV RQAPGKGLEW VASISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGGWIAAMD YWGQGTLVTV FNQIKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT

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Macromolecule #6: AMG 510 (bound form)

MacromoleculeName: AMG 510 (bound form) / type: ligand / ID: 6 / Number of copies: 1 / Formula: MOV
Molecular weightTheoretical: 562.61 Da
Chemical component information

ChemComp-MOV:
AMG 510 (bound form)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMNaH2PO4sodium phosphate
1.8 mMKH2PO4potassium phosphate
138.0 mMNaClsodium chloride
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 2.0 sec. / Average electron dose: 57.56 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 90612
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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