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

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.

Fig 1. Structure of the TAdV-3 fibre head.

A) Side view of the fibre head trimer. B) Top view of the fibre head trimer. Between panels A and B the structure has been turned approximately 90° towards the reader. Each monomer is coloured differently. C) Side view of a monomer with the amino-terminus, carboxy-terminus and beta-strands labelled. D) Topology diagram of the monomer labelled as in panel C, but with the start and end residue of each strand also indicated. In panels A and B, Met354 and Met376 are shown in space-filled representation and labelled.

Fig 2. Thermal stability assay of virulent and avirulent TAdV-3 fibre head proteins.

The relative fluorescence emission intensity (RFU, arbitrary unit) is plotted as a function of the temperature. Data for virulent (open circles) and avirulent (filled squares) TAdV-3 fibre head proteins are shown. A melting temperature of around 80°C was estimated for both proteins.

Fig 3. Comparison of the structures of the fibre head trimers of the virulent and avirulent variants of TAdV-3.

One monomer of the virulent form is shown in red secondary structure cartoon representation, the other two in gray, while the avirulent fibre head trimer is shown in yellow and white, respectively. Residues Ile354 and Thr376 of the virulent form and Met354 and Met376 of the avirulent form are shown in stick representation and labelled.

Fig 4. Superposition of the TAdV-3 fibre head monomer onto the structural homologues.

(A) Superposition of the TAdV-3 fibre head monomer onto the mammalian reovirus sigma1 head domain and the porcine adenovirus 4 NADC-1 strain fibre head domain (B). Superposition of the TAdV-3 fibre head monomer onto the mammalian reovirus sigma1 head domain and the porcine adenovirus 4 NADC-1 strain fibre head domain The TAdV-3 fibre head monomer is shown in green, the sigma1 head domain in orange and the porcine adenovirus 4 NADC-1 strain fibre head domain in blue. An asterisk shows the location of the DG-loops, it can be seen that the DG-loop of the TAdV-3 fibre head is shorter than its reovirus sigma 1 and porcine adenovirus 4 NADC-1 strain fibre head counterparts.

Fig 5. Surface properties of the TAdV-3 fibre head.

A) Side view. B) Top view. Calculated negatively charged regions are shown in red and positive regions in blue. The location of some of the polar, electropositive and electronegative amino acid side-chains that contribute to these charged regions are indicated.

Fig 6. TAdV-3 fibre head binding to a glycan microarray.

A histogram representing the fluorescence intensity of TAdV-3 bound to presented glycans on the microarray surface detected by fluorescently-labelled anti-His antibody is shown. Histograms represent the average of three replicate experiments and error bars depict one standard deviation of the mean calculated over three microarray slides. The broken line represents the binding threshold (approximately five times background).

Fig 7. 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.

Fig 8. Structure of the TAdV-3 fibre head protein (avirulent form) bound to 3'-sialyllactose.

A) Difference density for 3'-sialyllactose obtained after refining the protein model only, before introducing solvent or the ligand, contoured at 2.5 sigma. The main interactions of the sialyllactose are with the magenta monomer shown towards the back. The beta-hairpin from the neighbouring monomer (of the same trimer) is shown in green. A monomer of a neighbouring trimer that approaches the sialyllactose closely in the crystal structure is shown in white. Residues that were selected for site-directed mutagenesis are labelled. B) Location of the ligand between two trimers in the crystal structure. The ligand is shown in yellow, the TAdV-3 fibre head trimer in magenta, green and cyan, and the neighbouring trimer in white, gray and black. In panels C and D, the refined structure of the complex is shown. C) The TAdV-3 fibre head trimer is shown in cartoon representation, in the same colour scheme and orientation as in Fig 1, while sialyllactose ligand is shown in stick representation (carbons yellow). Fig 8C is in the same orientation as the electrostatic representation in Fig 5A. D) Close-up of the binding site for sialyllactose on the fibre head. Interacting residues and relevant atoms of the ligand are labelled (Ac, acetyl; CO2, carboxy). Potential hydrogen bonds are indicated. An arrow indicates the oxygen atom to which the lactose group should be attached. The orientations of panels A, B, and D are the same for clarity.

Fig 9. 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.