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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

Saturation transfer difference NMR experiments performed on 3'-sialyllactose in the presence of different TAdV-3 fibre head mutants.A) Off-resonance (reference) spectrum. B) STD spectrum with wild-type virulent protein. C) STD spectrum with R368A mutant protein. D) STD spectrum with E392A mutant protein. E) STD spectrum with N407A mutant protein. F) STD spectrum with K421A mutant protein. G) STD spectrum with K439A mutant protein.
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pone.0139339.g009: Saturation transfer difference NMR experiments performed on 3'-sialyllactose in the presence of different TAdV-3 fibre head mutants.A) Off-resonance (reference) spectrum. B) STD spectrum with wild-type virulent protein. C) STD spectrum with R368A mutant protein. D) STD spectrum with E392A mutant protein. E) STD spectrum with N407A mutant protein. F) STD spectrum with K421A mutant protein. G) STD spectrum with K439A mutant protein.

Mentions: Due to the binding of the ligand between two trimers in the crystal lattice, two potential binding sites were identified. The first potential binding site involves residues Glu392, Thr419, Val420, Lys421 and Asn422, while the other potential binding site involves residues Arg368, Asn407 and Lys439. To discriminate between the two possible binding sites, five independent point mutations were introduced, the resulting proteins were expressed and their capacity for binding sialyllactose was analyzed by ITC. Substitution of residues Arg368, Asn407 or Lys439 by alanine had no significant impact on the binding affinity (Table 2). In contrast, substitution of Glu392 by alanine drastically reduced the affinity of the mutant for 3'-sialyllactose and a Lys421Ala mutant rendered 3'-sialyllactose binding undetectable. This indicates that sialyllactose binds to the first site, and not to the second. The orientation of the sialyllactose is consistent with the STD-NMR results: the protons that show most saturation transfer (Fig 7) are closest to the protein. The same mutant proteins were also analyzed by STD-NMR, with the same results, i.e. no effect for the site 2 mutants and strong effects for the site 1 mutants analyzed (Fig 9).


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)

Saturation transfer difference NMR experiments performed on 3'-sialyllactose in the presence of different TAdV-3 fibre head mutants.A) Off-resonance (reference) spectrum. B) STD spectrum with wild-type virulent protein. C) STD spectrum with R368A mutant protein. D) STD spectrum with E392A mutant protein. E) STD spectrum with N407A mutant protein. F) STD spectrum with K421A mutant protein. G) STD spectrum with K439A mutant protein.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4587935&req=5

pone.0139339.g009: Saturation transfer difference NMR experiments performed on 3'-sialyllactose in the presence of different TAdV-3 fibre head mutants.A) Off-resonance (reference) spectrum. B) STD spectrum with wild-type virulent protein. C) STD spectrum with R368A mutant protein. D) STD spectrum with E392A mutant protein. E) STD spectrum with N407A mutant protein. F) STD spectrum with K421A mutant protein. G) STD spectrum with K439A mutant protein.
Mentions: Due to the binding of the ligand between two trimers in the crystal lattice, two potential binding sites were identified. The first potential binding site involves residues Glu392, Thr419, Val420, Lys421 and Asn422, while the other potential binding site involves residues Arg368, Asn407 and Lys439. To discriminate between the two possible binding sites, five independent point mutations were introduced, the resulting proteins were expressed and their capacity for binding sialyllactose was analyzed by ITC. Substitution of residues Arg368, Asn407 or Lys439 by alanine had no significant impact on the binding affinity (Table 2). In contrast, substitution of Glu392 by alanine drastically reduced the affinity of the mutant for 3'-sialyllactose and a Lys421Ala mutant rendered 3'-sialyllactose binding undetectable. This indicates that sialyllactose binds to the first site, and not to the second. The orientation of the sialyllactose is consistent with the STD-NMR results: the protons that show most saturation transfer (Fig 7) are closest to the protein. The same mutant proteins were also analyzed by STD-NMR, with the same results, i.e. no effect for the site 2 mutants and strong effects for the site 1 mutants analyzed (Fig 9).

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