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Structure and Sialyllactose Binding of the Carboxy-Terminal Head Domain of the Fibre from a Siadenovirus, Turkey Adenovirus 3.

Singh AK, Berbís MÁ, Ballmann MZ, Kilcoyne M, Menéndez M, Nguyen TH, Joshi L, Cañada FJ, Jiménez-Barbero J, Benkő M, Harrach B, van Raaij MJ - PLoS ONE (2015)

Bottom Line: The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure.It binds slightly more strongly to the avirulent form.We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain.

ABSTRACT
The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.

No MeSH data available.


Related in: MedlinePlus

Sialyllactose binding to the TAdV-3 fibre head.Saturation transfer difference NMR experiments performed on 3'- and 6'-sialyllactose in the presence of the avirulent and virulent variants of TAdV-3 fibre head are shown. In panels A through D, the off-resonance reference spectrum (top), with labels indicating the assignment for a number of representative ligand signals, and the STD spectrum (bottom, up-scaled 100X) are shown. Peaks marked with P correspond to signals belonging to the protein. In panels E through H, the epitopes deduced from the STD data are shown, mapped onto the chemical structure of 3'- and 6'-sialyllactose, with labels indicating the STD intensity for each signal, relative to the STD intensity for the 5''-N-acetyl peak. Red circles: I > 50%, orange circles: 50% > I > 30%, yellow circles: 30% > I > 10%. A) and E) avirulent variant with 3'-sialyllactose. B) and F) avirulent variant with 6'-sialyllactose. C) and G) virulent variant with 3'-sialyllactose. D) and H) virulent variant with 6'-sialyllactose.
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pone.0139339.g007: Sialyllactose binding to the TAdV-3 fibre head.Saturation transfer difference NMR experiments performed on 3'- and 6'-sialyllactose in the presence of the avirulent and virulent variants of TAdV-3 fibre head are shown. In panels A through D, the off-resonance reference spectrum (top), with labels indicating the assignment for a number of representative ligand signals, and the STD spectrum (bottom, up-scaled 100X) are shown. Peaks marked with P correspond to signals belonging to the protein. In panels E through H, the epitopes deduced from the STD data are shown, mapped onto the chemical structure of 3'- and 6'-sialyllactose, with labels indicating the STD intensity for each signal, relative to the STD intensity for the 5''-N-acetyl peak. Red circles: I > 50%, orange circles: 50% > I > 30%, yellow circles: 30% > I > 10%. A) and E) avirulent variant with 3'-sialyllactose. B) and F) avirulent variant with 6'-sialyllactose. C) and G) virulent variant with 3'-sialyllactose. D) and H) virulent variant with 6'-sialyllactose.

Mentions: To validate the identified ligand binding, TAdV-3 fibre head protein binding to 3'- and 6'-sialyllactose was examined by saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR). The STD experiment is based on the transfer of magnetization between the protein, which is selectively irradiated by a train of radiofrequency pulses, and any binding ligands [77]. For complex ligands, such as oligosaccharides, those atoms lying closer in space to the protein surface receive a higher amount of magnetization, providing information on the ligand contact atoms [78]. When the carbohydrates 3'- and 6'-sialyllactose were incubated in the presence of either the virulent or the avirulent variants of TAdV-3 fibre head proteins, saturation transfer was observed (Fig 7A), which indicated that both ligands interacted with the proteins in solution. In all instances, the highest degree of saturation was observed at the sialic acid, and to a much lesser degree, at the galactose region, and almost none at the glucose (Fig 7B). These experiments not only confirmed the protein-carbohydrate interaction but also mapped the protons of the carbohydrate residues with which the proteins interact, indicating that binding of sialyllactose to TAdV-3 fibre head occurs mainly at the sialic acid residue. STD-NMR was also performed with xylose and, although interaction signals were observed in glycan profiling, no interaction with the proteins could be measured by STD-NMR. The chemical properties of slide surfaces in glycan microarrays as well as different linkers attaching glycans to their carrier molecules can affect the way a glycan is being presented and can impact target protein-ligand interaction [79]. We speculate that this disagreement between glycan microarray profiling and STD-NMR results may be due to the presentation of the carbohydrates and the linker between xylose and bovine serum albumin. The difference in pH (7.7 for STD-NMR and 7.2 for the glycan micro-arrays) may also cause some of the differences observed.


Structure and Sialyllactose Binding of the Carboxy-Terminal Head Domain of the Fibre from a Siadenovirus, Turkey Adenovirus 3.

Singh AK, Berbís MÁ, Ballmann MZ, Kilcoyne M, Menéndez M, Nguyen TH, Joshi L, Cañada FJ, Jiménez-Barbero J, Benkő M, Harrach B, van Raaij MJ - PLoS ONE (2015)

Sialyllactose binding to the TAdV-3 fibre head.Saturation transfer difference NMR experiments performed on 3'- and 6'-sialyllactose in the presence of the avirulent and virulent variants of TAdV-3 fibre head are shown. In panels A through D, the off-resonance reference spectrum (top), with labels indicating the assignment for a number of representative ligand signals, and the STD spectrum (bottom, up-scaled 100X) are shown. Peaks marked with P correspond to signals belonging to the protein. In panels E through H, the epitopes deduced from the STD data are shown, mapped onto the chemical structure of 3'- and 6'-sialyllactose, with labels indicating the STD intensity for each signal, relative to the STD intensity for the 5''-N-acetyl peak. Red circles: I > 50%, orange circles: 50% > I > 30%, yellow circles: 30% > I > 10%. A) and E) avirulent variant with 3'-sialyllactose. B) and F) avirulent variant with 6'-sialyllactose. C) and G) virulent variant with 3'-sialyllactose. D) and H) virulent variant with 6'-sialyllactose.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4587935&req=5

pone.0139339.g007: Sialyllactose binding to the TAdV-3 fibre head.Saturation transfer difference NMR experiments performed on 3'- and 6'-sialyllactose in the presence of the avirulent and virulent variants of TAdV-3 fibre head are shown. In panels A through D, the off-resonance reference spectrum (top), with labels indicating the assignment for a number of representative ligand signals, and the STD spectrum (bottom, up-scaled 100X) are shown. Peaks marked with P correspond to signals belonging to the protein. In panels E through H, the epitopes deduced from the STD data are shown, mapped onto the chemical structure of 3'- and 6'-sialyllactose, with labels indicating the STD intensity for each signal, relative to the STD intensity for the 5''-N-acetyl peak. Red circles: I > 50%, orange circles: 50% > I > 30%, yellow circles: 30% > I > 10%. A) and E) avirulent variant with 3'-sialyllactose. B) and F) avirulent variant with 6'-sialyllactose. C) and G) virulent variant with 3'-sialyllactose. D) and H) virulent variant with 6'-sialyllactose.
Mentions: To validate the identified ligand binding, TAdV-3 fibre head protein binding to 3'- and 6'-sialyllactose was examined by saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR). The STD experiment is based on the transfer of magnetization between the protein, which is selectively irradiated by a train of radiofrequency pulses, and any binding ligands [77]. For complex ligands, such as oligosaccharides, those atoms lying closer in space to the protein surface receive a higher amount of magnetization, providing information on the ligand contact atoms [78]. When the carbohydrates 3'- and 6'-sialyllactose were incubated in the presence of either the virulent or the avirulent variants of TAdV-3 fibre head proteins, saturation transfer was observed (Fig 7A), which indicated that both ligands interacted with the proteins in solution. In all instances, the highest degree of saturation was observed at the sialic acid, and to a much lesser degree, at the galactose region, and almost none at the glucose (Fig 7B). These experiments not only confirmed the protein-carbohydrate interaction but also mapped the protons of the carbohydrate residues with which the proteins interact, indicating that binding of sialyllactose to TAdV-3 fibre head occurs mainly at the sialic acid residue. STD-NMR was also performed with xylose and, although interaction signals were observed in glycan profiling, no interaction with the proteins could be measured by STD-NMR. The chemical properties of slide surfaces in glycan microarrays as well as different linkers attaching glycans to their carrier molecules can affect the way a glycan is being presented and can impact target protein-ligand interaction [79]. We speculate that this disagreement between glycan microarray profiling and STD-NMR results may be due to the presentation of the carbohydrates and the linker between xylose and bovine serum albumin. The difference in pH (7.7 for STD-NMR and 7.2 for the glycan micro-arrays) may also cause some of the differences observed.

Bottom Line: The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure.It binds slightly more strongly to the avirulent form.We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain.

ABSTRACT
The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.

No MeSH data available.


Related in: MedlinePlus