Journal: Hum Gene Ther / Year: 2014 Title: Human full-length coagulation factor X and a GLA domain-derived 40-mer polypeptide bind to different regions of the adenovirus serotype 5 hexon capsomer. Authors: Sudir Sumarheni / Saw See Hong / Véronique Josserand / Jean-Luc Coll / Pierre Boulanger / Guy Schoehn / Pascal Fender / Abstract: The interaction of human adenovirus (HAdV)-C5 and many other adenoviruses with blood coagulation factors (e.g., human factor X, FX) involves the binding of their GLA domain to the hexon capsomers, ...The interaction of human adenovirus (HAdV)-C5 and many other adenoviruses with blood coagulation factors (e.g., human factor X, FX) involves the binding of their GLA domain to the hexon capsomers, resulting in high levels of hepatotropism and potential hepatotoxicity. In this study, we tested the possibility of preventing these undesirable effects by using a GLA-mimicking peptide as a competitor. An FX GLA domain-derived, 40-mer polypeptide carrying 12 carboxyglutamate residues was synthesized (GLA(mim)). Surface plasmon resistance (SPR) analysis showed that GLA(mim) reacted with free and capsid-embedded hexon with a nanomolar affinity. Unexpectedly, GLA(mim) failed to compete with FX for hexon binding, and instead significantly increased the formation of FX-hexon or FX-adenovirion complexes. This observation was confirmed by in vitro cell transduction experiments using HAdV-C5-Luciferase vector (HAdV5-Luc), as preincubation of HAdV5-Luc with GLA(mim) before FX addition resulted in a higher transgene expression compared with FX alone. HAdV-C5 virions complexed with GLA(mim) were analyzed by cryoelectron microscopy. Image reconstruction demonstrated the bona fide hexon-GLA(mim) interaction, as for the full-length FX, although with considerable differences in stoichiometry and relative location on the hexon capsomer. Three extra densities were found at the periphery of each hexon, whereas one single FX molecule occupied the central cavity of the hexon trimeric capsomer. A refined analysis indicated that each extra density is found at the expected location of one highly variable loop 1 of the hexon, involved in scavenger receptor recognition. HAdV5-Luc complexed with a bifunctional GLA(mim)RGD peptide showed a lesser hepatotropism, compared with control HAdV5-Luc alone, and efficiently targeted αβ-integrin-overexpressing tumor cells in an in vivo mouse tumor model. Collectively, our findings open new perspectives in the design of adenoviral vectors for biotherapy.
History
Deposition
Jan 29, 2014
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Header (metadata) release
Mar 12, 2014
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Map release
Aug 27, 2014
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Update
Aug 27, 2014
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Current status
Aug 27, 2014
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_2568.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Only 1/8 of the map to see the hexons
Voxel size
X=Y=Z: 2.26 Å
Density
Contour Level
By AUTHOR: 160.0 / Movie #1: 160
Minimum - Maximum
-1139.0 - 1311.0
Average (Standard dev.)
7.57692862 (±161.530014039999998)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
-223
-223
-223
Dimensions
250
250
250
Spacing
250
250
250
Cell
A=B=C: 565.0 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
2.26
2.26
2.26
M x/y/z
250
250
250
origin x/y/z
0.000
0.000
0.000
length x/y/z
565.000
565.000
565.000
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-207
-207
-206
NX/NY/NZ
414
414
414
MAP C/R/S
1
2
3
start NC/NR/NS
-223
-223
-223
NC/NR/NS
250
250
250
D min/max/mean
-1139.000
1311.000
7.577
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Supplemental data
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Sample components
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Entire : HAdV-C5 virions complexed with GLAmim
Entire
Name: HAdV-C5 virions complexed with GLAmim
Components
Sample: HAdV-C5 virions complexed with GLAmim
Virus: Human adenovirus 5
Ligand: GLAmim peptide
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Supramolecule #1000: HAdV-C5 virions complexed with GLAmim
Supramolecule
Name: HAdV-C5 virions complexed with GLAmim / type: sample / ID: 1000 / Number unique components: 2
Molecular weight
Experimental: 150 MDa / Theoretical: 150 MDa
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Supramolecule #1: Human adenovirus 5
Supramolecule
Name: Human adenovirus 5 / type: virus / ID: 1 / NCBI-ID: 28285 / Sci species name: Human adenovirus 5 / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No / Sci species serotype: 5
Host (natural)
Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weight
Experimental: 150 MDa / Theoretical: 150 MDa
Virus shell
Shell ID: 1 / Diameter: 1000 Å / T number (triangulation number): 25
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Macromolecule #1: GLAmim peptide
Macromolecule
Name: GLAmim peptide / type: ligand / ID: 1 Details: GLAmim peptide contains 12 gamma-carboxyglutamate residues and carries a biotinyl group at its C-terminal end. Number of copies: 1 / Recombinant expression: No
Source (natural)
Organism: synthetic construct (others)
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Experimental details
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Structure determination
Method
negative staining, cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
1 mg/mL
Buffer
pH: 7.4 / Details: 10mM Hepes buffer 2 mm CaCl2
Staining
Type: NEGATIVE / Details: cryo
Grid
Details: Quantifoil R2/1 holey grid
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2 seconds before plunging
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Electron microscopy
Microscope
FEI POLARA 300
Date
Sep 10, 2012
Image recording
Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 33 / Average electron dose: 15 e/Å2 / Od range: 1 / Bits/pixel: 8
Tilt angle min
0
Tilt angle max
0
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Model: Tecnai Polara / Image courtesy: FEI Company
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Image processing
Details
pft2 em3dr2
CTF correction
Details: flipmix
Final reconstruction
Applied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: pft2, em3dr2 / Number images used: 4762
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