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- PDB-8w2v: Solution Structure of the CD28 hinge used in chimeric antigen rec... -

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Basic information

Entry
Database: PDB / ID: 8w2v
TitleSolution Structure of the CD28 hinge used in chimeric antigen receptor (CAR) T-cells
ComponentsT-cell-specific surface glycoprotein CD28
KeywordsIMMUNE SYSTEM / CAR-T / CD28 / immunotherapy / intrinsic disorder
Function / homology
Function and homology information


Nef mediated downregulation of CD28 cell surface expression / positive regulation of inflammatory response to antigenic stimulus / regulatory T cell differentiation / protein complex involved in cell adhesion / regulation of regulatory T cell differentiation / CD4-positive, alpha-beta T cell proliferation / positive regulation of isotype switching to IgG isotypes / negative thymic T cell selection / Co-stimulation by CD28 / positive regulation of CD4-positive, alpha-beta T cell proliferation ...Nef mediated downregulation of CD28 cell surface expression / positive regulation of inflammatory response to antigenic stimulus / regulatory T cell differentiation / protein complex involved in cell adhesion / regulation of regulatory T cell differentiation / CD4-positive, alpha-beta T cell proliferation / positive regulation of isotype switching to IgG isotypes / negative thymic T cell selection / Co-stimulation by CD28 / positive regulation of CD4-positive, alpha-beta T cell proliferation / CD28 dependent Vav1 pathway / positive regulation of interleukin-4 production / CD28 dependent PI3K/Akt signaling / humoral immune response / positive regulation of interleukin-10 production / immunological synapse / positive regulation of viral genome replication / coreceptor activity / positive regulation of T cell proliferation / T cell costimulation / phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of interleukin-2 production / positive regulation of mitotic nuclear division / positive regulation of translation / T cell activation / positive regulation of cytokine production / apoptotic signaling pathway / Constitutive Signaling by Aberrant PI3K in Cancer / PIP3 activates AKT signaling / T cell receptor signaling pathway / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / transcription by RNA polymerase II / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / cell surface receptor signaling pathway / external side of plasma membrane / negative regulation of gene expression / positive regulation of gene expression / negative regulation of apoptotic process / protein kinase binding / cell surface / positive regulation of transcription by RNA polymerase II / identical protein binding / plasma membrane / cytosol
Similarity search - Function
T cell antigen CD28 / ICOS V-set domain / Cytotoxic T-lymphocyte protein 4/CD28 / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
T-cell-specific surface glycoprotein CD28
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsFolimonova, V. / Chen, X. / Walters, K.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)1ZIABC011627 United States
CitationJournal: Commun Biol / Year: 2024
Title: CD28 hinge used in chimeric antigen receptor (CAR) T-cells exhibits local structure and conformational exchange amidst global disorder.
Authors: Folimonova, V. / Chen, X. / Negi, H. / Schwieters, C.D. / Li, J. / Byrd, R.A. / Taylor, N. / Youkharibache, P. / Walters, K.J.
History
DepositionFeb 21, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: T-cell-specific surface glycoprotein CD28


Theoretical massNumber of molelcules
Total (without water)4,3081
Polymers4,3081
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: NMR Distance Restraints, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)25 / 50structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein/peptide T-cell-specific surface glycoprotein CD28 / TP44


Mass: 4308.027 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD28 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P10747

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-15N HSQC
121isotropic12D CON
1201isotropic42D 1H-15N HSQC
131isotropic23D HN(CA)CB
141isotropic23D CBCA(CO)NH
1191isotropic22D 1H-15N HSQC
2251isotropic42D 1H-15N HSQC
151isotropic23D HNCO
2261isotropic43D HNCO
161isotropic23D HN(CA)CO
2271isotropic43D HN(CA)CO
2301isotropic43D HNCO-COSY
1101isotropic23D HNCAN
192isotropic42D 1H-13C HSQC
2282isotropic22D 1H-13C HSQC
182isotropic43D 1H-13C NOESY
2292isotropic23D 1H-13C NOESY
1182isotropic12D 1H-13C HSQC
172isotropic13D 1H-13C CCH TOCSY
1112isotropic13D 1H-13C (H)CCH TOCSY
1213isotropic12D 1H-15N HSQC
1123isotropic12D CACO
1133isotropic13D CANCO
1174isotropic32D 1H-15N HSQC
2234isotropic22D 1H-15N HSQC
1144isotropic33D 1H-15N NOESY
2244isotropic23D 1H-15N NOESY
1155isotropic22D 1H-15N HSQC
1165isotropic23D HNHB

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution10.5 mM [U-100% 13C; U-100% 15N] CD28 hinge, 20 mM sodium phosphate, 50 mM sodium chloride, 2 mM DTT, 20 uM zinc sulphate, 1 mM pefabloc, 0.1 % sodium azide, 95% H2O/5% D2O15N, 13C-labeled CD28 hinge in 20 mM NaPO4 at pH 6.5, 50 mM NaCl, 2 mM dithiothreitol, 20 uM ZnSO4, 1mM pefabloc, 0.1% NaN3, and 95% H2O / 5% D2O15N, 13C-labeled CD28 hinge95% H2O/5% D2O
solution20.6 mM [U-100% 13C; U-100% 15N] CD28 hinge, 20 mM sodium phosphate, 50 mM sodium chloride, 2 mM DTT, 20 uM zinc sulphate, 1 mM pefabloc, 0.1 % sodium azide, 100% D2O15N, 13C-labeled CD28 hinge in 20 mM NaPO4 at pH 6.5, 50 mM NaCl, 2 mM dithiothreitol, 20 uM ZnSO4, 1mM pefabloc, 0.1% NaN3, and 100% D2O15N, 13C-labeled CD28 hinge100% D2O
solution30.45 mM [U-100% 13C; U-100% 15N] CD28 hinge, 20 mM sodium phosphate, 50 mM sodium chloride, 2 mM DTT, 20 uM zinc sulphate, 1 mM pefabloc, 0.1 % sodium azide, 95% H2O/5% D2O15N, 13C-labeled CD28 hinge in 20 mM NaPO4 at pH 6.5, 50 mM NaCl, 2 mM dithiothreitol, 20 uM ZnSO4, 1mM pefabloc, 0.1% NaN3, and 95% H2O/ 95% D2O. 450 uM concentration.15N, 13C-labeled CD28 hinge95% H2O/5% D2O
solution40.4 mM [U-100% 15N] CD28 hinge, 20 mM sodium phosphate, 50 mM sodium chloride, 2 mM DTT, 20 uM zinc sulphate, 1 mM pefabloc, 0.1 % sodium azide, 95% H2O/5% D2O15N-labeled CD28 hinge in 20 mM NaPO4 at pH 6.5, 50 mM NaCl, 2 mM dithiothreitol, 20 uM ZnSO4, 1mM pefabloc, 0.1% NaN3, and 95% H2O/ 95% D2O. 400 uM concentration.15N-labeled CD28 hinge95% H2O/5% D2O
solution50.28 mM [U-100% 15N] CD28 hinge, 20 mM sodium phosphate, 50 mM sodium chloride, 2 mM DTT, 20 uM zinc sulphate, 1 mM pefabloc, 0.1 % sodium azide, 95% H2O/5% D2O15N-labeled CD28 hinge in 20 mM NaPO4 at pH 6.5, 50 mM NaCl, 2 mM dithiothreitol, 20 uM ZnSO4, 1mM pefabloc, 0.1% NaN3, and 95% H2O/ 95% D2O. 280 uM concentration.15N-labeled CD28 hinge95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mMCD28 hinge[U-100% 13C; U-100% 15N]1
20 mMsodium phosphatenatural abundance1
50 mMsodium chloridenatural abundance1
2 mMDTTnatural abundance1
20 uMzinc sulphatenatural abundance1
1 mMpefablocnatural abundance1
0.1 %sodium azidenatural abundance1
0.6 mMCD28 hinge[U-100% 13C; U-100% 15N]2
20 mMsodium phosphatenatural abundance2
50 mMsodium chloridenatural abundance2
2 mMDTTnatural abundance2
20 uMzinc sulphatenatural abundance2
1 mMpefablocnatural abundance2
0.1 %sodium azidenatural abundance2
0.45 mMCD28 hinge[U-100% 13C; U-100% 15N]3
20 mMsodium phosphatenatural abundance3
50 mMsodium chloridenatural abundance3
2 mMDTTnatural abundance3
20 uMzinc sulphatenatural abundance3
1 mMpefablocnatural abundance3
0.1 %sodium azidenatural abundance3
0.4 mMCD28 hinge[U-100% 15N]4
20 mMsodium phosphatenatural abundance4
50 mMsodium chloridenatural abundance4
2 mMDTTnatural abundance4
20 uMzinc sulphatenatural abundance4
1 mMpefablocnatural abundance4
0.1 %sodium azidenatural abundance4
0.28 mMCD28 hinge[U-100% 15N]5
20 mMsodium phosphatenatural abundance5
50 mMsodium chloridenatural abundance5
2 mMDTTnatural abundance5
20 uMzinc sulphatenatural abundance5
1 mMpefablocnatural abundance5
0.1 %sodium azidenatural abundance5
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)
170 mM25 degree6.51 atm298 K
270 mM11 degree6.51 atm284 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE IIIBrukerAVANCE III7001
Bruker AVANCE IIIBrukerAVANCE III8002
Bruker AVANCE IIIBrukerAVANCE III8504
Bruker AVANCE IIIBrukerAVANCE III9003

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Processing

NMR software
NameDeveloperClassification
TopSpinBruker Biospincollection
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
MddNMRV. Orekhov, V. Jaravine, M. Mayzel, K. Kazimierczukprocessing
XEASYBartels et al.peak picking
XEASYBartels et al.data analysis
TALOS+Yang Shen, Frank Delaglio, Gabriel Cornilescu & Ad Baxdata analysis
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorerefinement
RefinementMethod: simulated annealing / Software ordinal: 7
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 50 / Conformers submitted total number: 25

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