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- PDB-7t0r: Crystal structure of the anti-CD4 adnectin 6940_B01 as a complex ... -

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Basic information

Entry
Database: PDB / ID: 7t0r
TitleCrystal structure of the anti-CD4 adnectin 6940_B01 as a complex with the extracellular domains of CD4 and ibalizumab fAb
Components
  • Adnectin 6940_B01
  • Ibalizumab Heavy Chain
  • Ibalizumab Light Chain
  • T-cell surface glycoprotein CD4
KeywordsANTIVIRAL PROTEIN / Inhibitor / HIV gp140 / CD4 / adnectin / ibalizumab / HIV Env / combinectin
Function / homology
Function and homology information


helper T cell enhancement of adaptive immune response / interleukin-16 binding / interleukin-16 receptor activity / maintenance of protein location in cell / T cell selection / MHC class II protein binding / cellular response to granulocyte macrophage colony-stimulating factor stimulus / interleukin-15-mediated signaling pathway / positive regulation of monocyte differentiation / Nef Mediated CD4 Down-regulation ...helper T cell enhancement of adaptive immune response / interleukin-16 binding / interleukin-16 receptor activity / maintenance of protein location in cell / T cell selection / MHC class II protein binding / cellular response to granulocyte macrophage colony-stimulating factor stimulus / interleukin-15-mediated signaling pathway / positive regulation of monocyte differentiation / Nef Mediated CD4 Down-regulation / Alpha-defensins / positive regulation of kinase activity / regulation of T cell activation / T cell receptor complex / extracellular matrix structural constituent / Other interleukin signaling / enzyme-linked receptor protein signaling pathway / Translocation of ZAP-70 to Immunological synapse / Phosphorylation of CD3 and TCR zeta chains / regulation of calcium ion transport / macrophage differentiation / Generation of second messenger molecules / T cell differentiation / PD-1 signaling / positive regulation of protein kinase activity / Binding and entry of HIV virion / coreceptor activity / positive regulation of calcium-mediated signaling / cell surface receptor protein tyrosine kinase signaling pathway / T cell activation / positive regulation of interleukin-2 production / protein tyrosine kinase binding / Vpu mediated degradation of CD4 / calcium-mediated signaling / clathrin-coated endocytic vesicle membrane / transmembrane signaling receptor activity / positive regulation of peptidyl-tyrosine phosphorylation / Downstream TCR signaling / Cargo recognition for clathrin-mediated endocytosis / positive regulation of T cell activation / MHC class II protein complex binding / Clathrin-mediated endocytosis / virus receptor activity / signaling receptor activity / positive regulation of canonical NF-kappaB signal transduction / defense response to Gram-negative bacterium / adaptive immune response / positive regulation of MAPK cascade / positive regulation of viral entry into host cell / positive regulation of ERK1 and ERK2 cascade / cell surface receptor signaling pathway / early endosome / cell adhesion / positive regulation of protein phosphorylation / immune response / membrane raft / endoplasmic reticulum lumen / external side of plasma membrane / lipid binding / endoplasmic reticulum membrane / protein kinase binding / positive regulation of DNA-templated transcription / enzyme binding / signal transduction / protein homodimerization activity / zinc ion binding / identical protein binding / plasma membrane
Similarity search - Function
CD4, extracellular / T cell CD4 receptor C-terminal region / CD4, extracellular / T cell CD4 receptor C terminal region / T-cell surface antigen CD4 / Immunoglobulin C2-set / Immunoglobulin C2-set domain / Immunoglobulin / Immunoglobulin domain / Immunoglobulin subtype 2 ...CD4, extracellular / T cell CD4 receptor C-terminal region / CD4, extracellular / T cell CD4 receptor C terminal region / T-cell surface antigen CD4 / Immunoglobulin C2-set / Immunoglobulin C2-set domain / Immunoglobulin / Immunoglobulin domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
T-cell surface glycoprotein CD4
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.65 Å
AuthorsWilliams, S.P. / Concha, N.O. / Wensel, D.L. / Hong, X.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: J Mol Biol / Year: 2022
Title: Novel Bent Conformation of CD4 Induced by HIV-1 Inhibitor Indirectly Prevents Productive Viral Attachment.
Authors: David Wensel / Shawn Williams / David P Dixon / Paris Ward / Patti McCormick / Nestor Concha / Eugene Stewart / Xuan Hong / Charles Mazzucco / Shreya Pal / Bo Ding / Christoph Fellinger / Mark Krystal /
Abstract: GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits ...GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen-deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4's flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.
History
DepositionNov 30, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 12, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: Ibalizumab Light Chain
H: Ibalizumab Heavy Chain
A: T-cell surface glycoprotein CD4
B: Ibalizumab Light Chain
C: Ibalizumab Heavy Chain
D: T-cell surface glycoprotein CD4
G: Adnectin 6940_B01
I: Adnectin 6940_B01
hetero molecules


Theoretical massNumber of molelcules
Total (without water)202,56611
Polymers202,0278
Non-polymers5383
Water00
1
L: Ibalizumab Light Chain
H: Ibalizumab Heavy Chain
A: T-cell surface glycoprotein CD4
G: Adnectin 6940_B01
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,3316
Polymers101,0144
Non-polymers3172
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Ibalizumab Light Chain
C: Ibalizumab Heavy Chain
D: T-cell surface glycoprotein CD4
I: Adnectin 6940_B01
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,2355
Polymers101,0144
Non-polymers2211
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)83.510, 164.014, 167.053
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP22121
Space group name HallP22ab(z,x,y)
Components on special symmetry positions
IDModelComponents
11A-402-

SO4

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 1 through 28 or (resid 29...
d_2ens_1(chain "D" and (resid 1 through 19 or (resid 20...
d_1ens_2chain "B"
d_2ens_2(chain "L" and ((resid 1 and (name N or name...
d_1ens_3(chain "C" and resid 1 through 221)
d_2ens_3(chain "H" and (resid 1 through 2 or (resid 3...
d_1ens_4(chain "G" and (resid 0 through 42 or (resid 43...
d_2ens_4(chain "I" and (resid 0 through 43 or (resid 44...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LYSSERC1 - 104
d_12ens_1GLNVALC106 - 355
d_13ens_1NAGNAGD
d_21ens_1LYSSERG1 - 104
d_22ens_1GLNVALG106 - 355
d_23ens_1NAGNAGH
d_11ens_2ASPASNE1 - 215
d_21ens_2ASPASNA1 - 215
d_11ens_3GLNGLUF1 - 221
d_21ens_3GLNGLUB1 - 221
d_11ens_4GLYGLUI1 - 93
d_21ens_4GLYALAJ1 - 80
d_22ens_4GLNGLUJ82 - 94

NCS ensembles :
ID
ens_1
ens_2
ens_3
ens_4

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Components

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Protein , 2 types, 4 molecules ADGI

#3: Protein T-cell surface glycoprotein CD4 / T-cell surface antigen T4/Leu-3


Mass: 41667.727 Da / Num. of mol.: 2 / Fragment: Extra-cellular domains D1-D4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD4 / Cell line (production host): HEK / Production host: Homo sapiens (human) / References: UniProt: P01730
#4: Protein Adnectin 6940_B01


Mass: 10314.485 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21[DE3]

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Antibody , 2 types, 4 molecules LBHC

#1: Antibody Ibalizumab Light Chain


Mass: 24245.900 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): CHO / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody Ibalizumab Heavy Chain


Mass: 24785.529 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): CHO / Production host: Cricetulus griseus (Chinese hamster)

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Sugars / Non-polymers , 2 types, 3 molecules

#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


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
#6: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.83 Å3/Da / Density % sol: 56.56 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop
Details: 0.01 M Na3 Citrate, 15.5% PEG3350, 0.3 M (NH4)2H Citrate, 0.1 M AmSO4
PH range: 7.0 - 7.6 / Temp details: room temperature

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Data collection

DiffractionMean temperature: 270 K / Ambient temp details: Throughout / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 0.987 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 29, 2019
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 3.5→100 Å / Num. obs: 27334 / % possible obs: 92.2 % / Redundancy: 4.7 % / Biso Wilson estimate: 70.88 Å2 / CC1/2: 0.98 / CC star: 0.995 / Rmerge(I) obs: 0.196 / Rpim(I) all: 0.098 / Rrim(I) all: 0.22 / Χ2: 0.91 / Net I/σ(I): 4.6
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
3.5-3.564.20.79211480.6670.4020.8930.86879.8
3.56-3.634.40.71711640.7220.3550.8040.87879.4
3.63-3.694.30.67511780.7920.3440.7610.98882.2
3.69-3.774.40.60812740.810.3060.6840.87786.1
3.77-3.854.40.55212440.8020.2820.6220.8686.9
3.85-3.944.40.51112850.8660.2610.5770.97287.6
3.94-4.044.50.4113080.8870.2090.4620.9189.5
4.04-4.154.60.32813320.9230.1660.3690.990.5
4.15-4.274.50.26613680.9510.1350.2990.93693.1
4.27-4.414.50.2213780.9640.1130.2480.91493.6
4.41-4.574.40.19313710.970.10.2190.99493.7
4.57-4.754.30.16213750.9790.0860.1850.97794
4.75-4.974.20.17414420.9790.0920.1980.96396.3
4.97-5.234.90.16914670.9850.0840.190.94199.3
5.23-5.565.60.18314780.9820.0840.2020.94399.6
5.56-5.985.60.16914840.9840.0780.1860.95499.7
5.98-6.595.50.15414910.9840.0720.1710.86599.9
6.59-7.545.30.10715190.9930.050.1190.81499.7
7.54-9.54.50.05515100.9960.0280.0620.80298.6
9.5-1004.50.03715180.9930.0190.0420.8992.5

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Phasing

PhasingMethod: molecular replacement
Phasing MRR rigid body: 0.476
Highest resolutionLowest resolution
Rotation29.53 Å3.96 Å

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Processing

Software
NameVersionClassification
Coot0.9.5 ELmodel building
PHENIX1.19.2_4158refinement
HKL-2000data reduction
HKL-2000data scaling
MOLREP11.6.04phasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3O2D

3o2d


Resolution: 3.65→29.53 Å / SU ML: 0.4947 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 28.4107
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2923 1587 7.05 %RANDOM
Rwork0.2258 20919 --
obs0.2304 22506 86.02 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 69 Å2
Refinement stepCycle: LAST / Resolution: 3.65→29.53 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12930 0 33 0 12963
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00413245
X-RAY DIFFRACTIONf_angle_d0.833518143
X-RAY DIFFRACTIONf_chiral_restr0.0492159
X-RAY DIFFRACTIONf_plane_restr0.00612307
X-RAY DIFFRACTIONf_dihedral_angle_d5.3791875
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2DX-RAY DIFFRACTIONTorsion NCS1.14831224882
ens_2d_2LX-RAY DIFFRACTIONTorsion NCS2.72642336745
ens_3d_2HX-RAY DIFFRACTIONTorsion NCS3.64626328409
ens_4d_2IX-RAY DIFFRACTIONTorsion NCS0.747473744279
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.65-3.770.3464910.2641250X-RAY DIFFRACTION57.41
3.77-3.90.35211110.27261380X-RAY DIFFRACTION64.02
3.9-4.060.3011120.26631624X-RAY DIFFRACTION74.47
4.06-4.240.35311380.23931793X-RAY DIFFRACTION82.42
4.24-4.470.26691470.21911894X-RAY DIFFRACTION87.37
4.47-4.740.29261690.19911953X-RAY DIFFRACTION89.39
4.74-5.110.24771600.21122102X-RAY DIFFRACTION95.32
5.11-5.620.27051600.21932199X-RAY DIFFRACTION99.28
5.62-6.430.31271760.24462227X-RAY DIFFRACTION99.79
6.43-8.070.31321540.23552246X-RAY DIFFRACTION99.75
8.07-29.530.26291690.19942251X-RAY DIFFRACTION95.16

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