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- PDB-7zqs: Cryo-EM Structure of Human Transferrin Receptor 1 bound to DNA Aptamer -

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Basic information

Entry
Database: PDB / ID: 7zqs
TitleCryo-EM Structure of Human Transferrin Receptor 1 bound to DNA Aptamer
Components
  • DNA (30-MER)
  • Transferrin receptor protein 1
KeywordsMEMBRANE PROTEIN / CD71 / Transmembrane Glycoprotein
Function / homology
Function and homology information


transferrin receptor activity / negative regulation of mitochondrial fusion / transferrin transport / Transferrin endocytosis and recycling / positive regulation of isotype switching / response to iron ion / response to copper ion / response to manganese ion / RND1 GTPase cycle / RND2 GTPase cycle ...transferrin receptor activity / negative regulation of mitochondrial fusion / transferrin transport / Transferrin endocytosis and recycling / positive regulation of isotype switching / response to iron ion / response to copper ion / response to manganese ion / RND1 GTPase cycle / RND2 GTPase cycle / RHOB GTPase cycle / Golgi Associated Vesicle Biogenesis / RHOJ GTPase cycle / RHOC GTPase cycle / RHOQ GTPase cycle / RHOH GTPase cycle / CDC42 GTPase cycle / transport across blood-brain barrier / RHOG GTPase cycle / RHOA GTPase cycle / RAC3 GTPase cycle / RAC2 GTPase cycle / positive regulation of bone resorption / response to retinoic acid / positive regulation of B cell proliferation / clathrin-coated pit / Hsp70 protein binding / positive regulation of T cell proliferation / RAC1 GTPase cycle / cellular response to leukemia inhibitory factor / osteoclast differentiation / response to nutrient / acute-phase response / positive regulation of protein-containing complex assembly / clathrin-coated endocytic vesicle membrane / receptor internalization / HFE-transferrin receptor complex / recycling endosome / positive regulation of protein localization to nucleus / recycling endosome membrane / extracellular vesicle / melanosome / cellular response to xenobiotic stimulus / double-stranded RNA binding / Cargo recognition for clathrin-mediated endocytosis / virus receptor activity / Clathrin-mediated endocytosis / positive regulation of peptidyl-serine phosphorylation / positive regulation of NF-kappaB transcription factor activity / iron ion transport / cytoplasmic vesicle / basolateral plasma membrane / blood microparticle / positive regulation of canonical NF-kappaB signal transduction / intracellular iron ion homeostasis / response to hypoxia / early endosome / endosome membrane / endosome / intracellular signal transduction / positive regulation of protein phosphorylation / external side of plasma membrane / intracellular membrane-bounded organelle / protein-containing complex binding / positive regulation of gene expression / negative regulation of apoptotic process / protein kinase binding / perinuclear region of cytoplasm / cell surface / protein homodimerization activity / extracellular space / RNA binding / extracellular exosome / extracellular region / membrane / identical protein binding / plasma membrane
Similarity search - Function
Transferrin receptor protein 1/2, PA domain / Transferrin receptor-like, dimerisation domain / Transferrin receptor-like, dimerisation domain superfamily / Glutamate carboxypeptidase 2-like / Transferrin receptor-like dimerisation domain / PA domain superfamily / PA domain / PA domain / Peptidase M28 / Peptidase family M28
Similarity search - Domain/homology
DNA / DNA (> 10) / Transferrin receptor protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
unidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsBansia, H. / Wang, T. / Gutierrez, D. / des Georges, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM133598 United States
CitationJournal: J Am Chem Soc / Year: 2022
Title: Discovery of a Transferrin Receptor 1-Binding Aptamer and Its Application in Cancer Cell Depletion for Adoptive T-Cell Therapy Manufacturing.
Authors: Emmeline L Cheng / Ian I Cardle / Nataly Kacherovsky / Harsh Bansia / Tong Wang / Yunshi Zhou / Jai Raman / Albert Yen / Dominique Gutierrez / Stephen J Salipante / Amédée des Georges / ...Authors: Emmeline L Cheng / Ian I Cardle / Nataly Kacherovsky / Harsh Bansia / Tong Wang / Yunshi Zhou / Jai Raman / Albert Yen / Dominique Gutierrez / Stephen J Salipante / Amédée des Georges / Michael C Jensen / Suzie H Pun /
Abstract: The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across ...The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient's apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and model building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.
History
DepositionMay 2, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 17, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA (30-MER)
B: Transferrin receptor protein 1
C: DNA (30-MER)
D: Transferrin receptor protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)204,14814
Polymers201,9114
Non-polymers2,23710
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11240 Å2
ΔGint-51 kcal/mol
Surface area52110 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "C"
d_2ens_1chain "A"
d_1ens_2(chain "D" and (resid 120 through 639 or resid 641 through 758 or resid 759 through 763))
d_2ens_2(chain "B" and (resid 120 through 639 or resid 641 through 758 or resid 759 through 763))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1DADGJ
d_12ens_1MGMGK
d_21ens_1DADGA
d_22ens_1MGMGB
d_11ens_2ARGLEUM1 - 520
d_12ens_2TRPASNM524 - 641
d_13ens_2NAGNAGN
d_14ens_2NAGNAGD
d_15ens_2NAGNAGD
d_16ens_2NAGNAGD
d_17ens_2NAGNAGD
d_21ens_2ARGLEUD1 - 520
d_22ens_2TRPASND524 - 641
d_23ens_2NAGNAGE
d_24ens_2NAGNAGF
d_25ens_2NAGNAGG
d_26ens_2NAGNAGH
d_27ens_2NAGNAGI

NCS ensembles :
ID
ens_1
ens_2

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Components

#1: DNA chain DNA (30-MER)


Mass: 15988.172 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: DNA aptamer / Source: (synth.) unidentified (others)
#2: Protein Transferrin receptor protein 1 / / TR / TfR / TfR1 / Trfr / T9 / p90


Mass: 84967.078 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Human Transferrin Receptor 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: TFRC / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P02786
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#5: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / 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
Has ligand of interestN

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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: Human transferrin receptor 1 in complex with DNA aptamer
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.19 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 79.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1397
Image scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 216817 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 68.21 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003911748
ELECTRON MICROSCOPYf_angle_d0.510516232
ELECTRON MICROSCOPYf_chiral_restr0.04171840
ELECTRON MICROSCOPYf_plane_restr0.0031842
ELECTRON MICROSCOPYf_dihedral_angle_d34.14162028
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000704389280906
ens_2d_2BELECTRON MICROSCOPYNCS constraints0.00070334715418

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