- Open Access
Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus
© Nguyen et al.; licensee BioMed Central. 2015
- Received: 2 March 2015
- Accepted: 11 May 2015
- Published: 22 May 2015
In adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus.
We constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414–535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution.
Like in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed.
Chimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of these fibre head domains.
- Anomalous dispersion
- Fibre protein
- Host-cell recognition
- Isomorphous replacement
Atadenovirus is one of the five genera of the family Adenoviridae [1, 2, 25]. They are serologically and phylogenetically distinct from the other adenovirus genera, and their genomic organization also differs [1, 8, 14, 26]. Their capsids contain an extra protein (LH3), important for capsid stability . Four trimeric LH3 knobs are present on each facet, with the LH3 protein being wedged in-between three hexon trimers. Atadenoviruses have been detected in a broad range of hosts, including predominantly scaled reptiles (order Squamata), as well as birds, ruminants and a marsupial [16, 26, 28–35]. So far, five species of atadenovirus have been confirmed: Snake atadenovirus A, Duck atadenovirus A, Bovine atadenovirus D, Ovine atadenovirus D and Possum atadenovirus A [2, 3, 36].
About half of identified ruminant adenoviruses are mastadenoviruses, while the remainder are atadenoviruses . From the bovine adenoviruses, serotypes 1, 2, 3, 9 and 10 are mastadenoviruses [37, 38], while bovine adenoviruses 4, 5, 6, 7 and 8 are atadenoviruses [39, 40]. Bovine adenovirus 4 (BAdV-4; strain THT/62) is the reference strain for the Bovine atadenovirus D species and was first isolated and characterized in Hungary (GenBank accession number AF036092) [39, 41, 42]. BAdV-4 contains a single fibre gene, encoding a protein of 535 amino acids in length with low sequence identity to known adenovirus fibres (15–19 %). Like other adenovirus fibres, it is predicted to consist of three domains: an N-terminal penton-base binding domain, a shaft domain and a C-terminal fibre head domain (Fig. 1b). Based on the location of the triple beta-spiral shaft repeats [43, 44], the N-terminal penton-base-binding domain of the fibre is proposed to contain amino acids 1–80, while the C-terminal fibre head domain was expected to start around residue 414. Here, we report the expression, purification, crystallization and structure solution of the fibre head domain of BAdV-4 at 1.2 Å (0.12 nm) resolution. It is the second atadenovirus fibre head structure that has been solved, after that of the SnAdV-1 fibre head domain ; and the first of an atadenovirus which infects mammalian cells. Although the secondary structure topology is the same as for other adenovirus fibre heads, differences are observed in the loops connecting the beta-strands and in the predicted surface charge. Our structure is a first step towards a better understanding of BAdV-4 host cell interaction, which, in turn, will have implications for the use of mammalian atadenovirus for medical purposes, be it in the use of whole viruses , or in chimeric fibres with atadenovirus fibre heads .
Purification, crystallization and structure solution of the BAdV-4 fibre head
Crystallographic data collection, phase determination, solvent flattening and refinement statistics (all values in parenthesis are for the highest resolution bin)
Cell parameters (a, b, c) (Å)
43.3, 48.2, 52.1
43.4, 48.4, 52.6
Cell parameters (α, β, γ) (°)
117.1, 95.6, 110.2
116.9, 95.8, 110.2
Wilson B (Å2)
Number of heavy atom sites
Phasing power (isomorphous/anomalous)
Figure of merit
Solvent flattening (40.8 % solvent)
Hand score (original/inverted)
Overall correlation on |E|2/contrast
Resolution range (Å)
No. reflections used in refinement
No. reflections used for R-free
No. of protein/solvent atoms
Average B protein/solvent atoms (Å2)
Ramachandran plot (favoured/allowed) (%)
R.m.s. deviation of bonds (Å) and angles (°)
The two beta-sheets of the BAdV-4 fibre head monomer face each other at an angle of around 120°. The ABCJ-sheet is mostly on the “inside”, and only a part of the AB beta-hairpin is solvent-accessible. The rest of the AB beta-hairpin and the whole C- and J-strands are buried and extensively involved in inter-monomer contacts. In contrast, most of the GHID-sheet is solvent-accessible (Fig. 3b). Together with the alpha-helix, the two beta-sheets from each monomer form a compact globular trimeric beta-propellor.
Comparison with other adenovirus fibre heads
Mastadenovirus fibre heads are larger than atadenovirus fibre heads, as previously described , mainly due to the surface loops being longer. The AB-, CD-, DG-, HI- and IJ-loops are all shorter in the BAdV-4 fibre head than in the prototype mastadenovirus fibre head structure, HAdV-5. Exceptions are strands G and H and the BC-loop, which are longer in the BAdV-4 fibre head. The low similarity of the structures makes it impossible to make meaningful speculations about receptor binding.
The CD- and IJ-loops of the BAdV-4 fibre head are longer than those in the SnAdV-1 fibre head. In contrast, the DG-loop of the BAdV-4 fibre head is two amino acids shorter than its SnAdV-1 counterpart. A noticeable difference is observed in the length of the G- and H-strands, which are quite a bit longer in the BAdV-4 fibre head (9 vs. 5 for the G-strand and 13 vs. 8 residues for the H-strand). As mentioned before, the CD-, IJ- and GH-loops are all located on the top of the molecule, while the DG-loop is on the side, making all of them potentially important for receptor interaction. In a structure-based alignment (Fig. 5c), the lack of sequence similarity becomes even more evident, except for the very C-terminal part of the protein (end of the H-strand and the I- and J-strands and the HI-turn). It may be speculated that this conserved part of the protein is important for initiation of protein folding and has therefore diverged less. When calculated electrostatic surfaces are considered, the BAdV-4 fibre head has distinct electro-negatively charged patches on its surface (Fig. 4a), while the SnAdV-1 surface is more electro-positive (Fig. 4b) . This may have implications for receptor-binding and suggest they do not bind the same receptor, although the nature of the receptors for neither of the viruses is known.
Potential inter-monomer interactions of atadenovirus fibre heads
SnAdV-1 fibre head
BAdV-4 fibre head
Hydrophobic interactions (<5 Å)
Leu265 - Ala245
Ile445 - Val246
Phe268 - Ala245
Tyr484 - Ile496
Phe268 - Ala257
Tyr484 - Met523
Pro301 - Tyr276
Leu306 - Pro310
Leu306 - Leu311
Ala308 - Pro310
Phe340 - Phe274
Phe340 - Tyr276
Tyr341 - Tyr276
Leu342 - Ala257
Leu342 - Phe274
Leu342 - Tyr276
Lys239NZ - Ser241O
Arg446NH2 - Leu436O
Lys239NZ - Asp243OD2
Pro482O - Gln482NE2
Lys270NZ - Gln259OE1
Tyr484N - Gln455OE1
Lys302NZ - Gly278O
Tyr484OH - Gln490N
Arg304NH1 - Glu333OE1
Tyr484OH - Thr488OG1
Arg304NH1 - Glu333OE2
Ser530O - Gln455N
Arg304NH2 - Glu333OE1
Ser530OG - Ser454OG
Phe340N - Ser336OG
Thr532OG1 - Ser454OG
Ionic interactions (<6 Å)
Lys239 - Asp243
Glu263 - Arg261
Lys302 - Asp279
Lys302 - Glu333
Arg304 - Glu333
Aromatic interactions (4.5–7 Å)
Phe340 - Phe274
Phe340 - Tyr276
Tyr341 - Tyr276
Cation-pi interactions (<6 Å)
Lys270 - Phe274
Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for a gene therapy vector , but the structure of its fibre head is unknown. Our BAdV-4 fibre head structure is likely similar to that of the OAdV-7 fibre head , with which it shares 44 % sequence identity (51 out of 116 amino acids; Fig. 5d). It should now be possible to build a reliable structural model of the OAdV-7 fibre head in order to identify surface residues possibly involved in receptor binding and mutating them. Our cloning, expression and crystallization strategy may also be used to obtain crystals for the OAdV-7 fibre head, and the structure determined experimentally by molecular replacement using data collected on these crystals.
The high resolution structure of BAdV-4 fibre head is the second solved structure of an atadenovirus fibre head domain. However, it is the first fibre head structure of an atadenovirus which infects a mammalian host. The structure showed that the atadenovirus fibre head structure is conserved, including the alpha-helix in the DG-loops, between two species infecting very different hosts, even though the sequence identity is very low. Differences in the conformation of surface loops and in the predicted surface charge may be of importance for primary receptor recognition.
Cloning, expression and purification
BAdV-4 strain THT/62  was propagated on primary or low-passage-number calf testis cell cultures, then the virus was purified by ultracentrifugation, and the DNA extracted as described earlier . Three genome fragments, coding for the full BAdV-4 fibre protein or parts of it containing the putative head domain (UniProt Q997H2) were amplified from the extracted viral DNA (GenBank accession No AF036092)  by polymerase chain reaction (PCR) using three forward primers including a BamHI restriction site and a reverse primer with a HindIII restriction site. The amplified PCR products were cloned into the expression vector pET28a(+) (Novagen, Merck, Darmstadt, Germany), previously digested with the same restriction enzymes. The inserts of the resulting plasmids were sequenced and found to be correct. The pET28a(+) vector provides an N-terminal six-histidine tag.
For protein expression, E. coli strain JM109(DE3) was transformed with the respective expression vector and bacteria were grown aerobically at 37 °C until the optical density at 600 nm reached 0.5–0.8. At this point, the culture was cooled on ice for 30 min, IPTG was added to a final concentration of 0.5–1 mM and aerobic growth was continued for 18–20 h at 16 °C. Cells from 2 l of culture were harvested by centrifugation (10 min 5000 × g), re-suspended in buffer A (10 mM Tris–HCl pH 7.5, 0.5 M sodium chloride, 10 % (v/v) glycerol) including 20 mM imidazole and stored at −20 °C. After thawing, cells were lysed by two passes through a French press at about 7 MPa. Cell rests were removed by centrifugation for 30 min at 20000 × g.
For purification, 2 ml of nickel-NTA resin slurry (BioRad, Madrid, Spain) was added to the protein-containing supernatant and incubated with occasional gentle shaking for 30 min on ice. The resin was then transferred to a column and washed with 30 ml of buffer A with 20 mM imidazole. BAdV-4 fibre protein variants were eluted using a step-gradient of imidazole in buffer A (50 mM, 100 mM, 250 mM and 500 mM imidazole; steps of 5 ml). After analysis by denaturing gel electrophoresis, fractions containing 100–500 mM imidazole were pooled, dialysed against 20 mM MES pH 6.5 and loaded onto a Resource S6 column (GE-Healthcare Biosciences, Uppsala, Sweden). The protein was eluted with a linear gradient of 0–1 M sodium chloride in 20 mM MES pH 6.5. Fractions containing pure protein were concentrated to 18 mg/ml using an Amicon Ultra concentrator with a molecular weight cut-off of 10 kDa (Millipore, Madrid, Spain). Three washes with 10 ml 10 mM Tris–HCl pH 7.5, 50 mM sodium chloride and 5 % (v/v) glycerol were applied. The sample was stored at 4 °C prior to crystallization trials.
A thermal shift assay  was carried out in an iCycler iQ PCR Thermal Cycler (Bio-Rad, Hercules CA, USA) in the presence of the fluorescent dye SYPRO Orange (Life Technologies SA, Madrid, Spain). Reaction volumes of 30 μl were prepared in 200 μl with 30 μg of protein and 5X SYPRO Orange from the supplied 5000X stock solution. Thermal denaturation curves were obtained by heating the samples from 20 °C to 95 °C with a ramp rate of 1 °C/min and monitoring the fluorescence at every 0.5 °C increment. The melting temperature Tm is defined as the point where the slope of the fluorescence increase is maximal.
Crystallization, crystallographic data collection and structure solution
The BAdV-4 fibre head protein was crystallized using the sitting drop vapour diffusion method (robotic setup with a Genesis RSP 150 workstation; Tecan, Männedorf, Switzerland or by manual setup). In either case, 50 μl of reservoirs were employed, and drops were prepared containing 0.2 μl of protein sample and 0.2 μl of the respective reservoir solution for robotic setups and 0.6 μl of protein plus 0.6 μl of reservoir for manual setups. Crystals were harvested with Litholoops (Molecular Dimensions, Newmarket, England) or Micromounts (Mitegen, Ithaca, New York, USA), transferred to cryo-protection solution (reservoir solution containing 20 % (v/v) glycerol) and flash-cooled in liquid nitrogen.
A heavy atom derivative was prepared by adding a few grains of methylmercury chloride to the reservoir of one drop and letting the drop equilibrate with the reservoir overnight. Two μl of reservoir solution was then added to the drop and incubated for about 5 min. The crystal was harvested in cryo-solution without methylmercury chloride as described for the native.
Crystallographic data were collected from a native and a methylmercury chloride derivative crystal at the BL13-XALOC beamline of the ALBA synchrotron , using a wavelength at which significant anomalous signal from the added mercury atoms was expected (1.0023 Å). Later, a higher-resolution native dataset was collected at beamline ID29 of the ESRF . Crystallographic data collected at BL13-XALOC were integrated using iMosflm  and further processed using POINTLESS, AIMLESS and TRUNCATE  from the CCP4-suite . Data collected at ID29 were processed using XDS [65, 66], analyzed using AIMLESS and processed by TRUNCATE to obtain structure factor amplitudes. A self-rotation function was calculated with MOLREP .
Structure solution was done using AUTOSHARP , which employs SHELX for heavy atom positions substructure determination , SHARP for phase determination  and SOLOMON for solvent flattening . Reflections were selected in thin shells for calculation of Rfree . Phases from AUTOSHARP were combined with the structure factor amplitudes from the high-resolution native dataset and input into ARPWARP for automated model building . This obtained model was completed using COOT  and structure refined using REFMAC5 , including anisotropic temperature factor refinement. Validation was done with MOLPROBITY . Structure comparisons, including r.m.s.d. and Z-score calculations, were performed using the DALI server . Figures were made using PYMOL (The PYMOL Molecular Graphics System, Version 220.127.116.11. Schrödinger, LLC). Protein assembly parameters were calculated using PISA  and individual interactions identified with PIC .
We thank Javier Varela (CIB-CSIC) for N-terminal sequence analysis, Sergio Ciordia, Mari Carmen Mena and Alberto Paradela for mass spectrometry, Carmen San Martín for careful reading of the manuscript and Jordi Benach (ALBA beamline BL13/XALOC) and Christoph Mueller-Dieckmann (ESRF beamline ID29) for help with using synchrotron data collection facilities. We acknowledge ALBA/CELLS and the European Synchrotron Radiation Facility for access, which contributed to the results presented here. The research leading to these results was sponsored by grant BFU2011-24843 (to MJvR, THN, MSG and AKS) from the Spanish Ministry of Economy and Competitiveness, a VAST-CSIC PhD fellowship to THN, a FEMS short-term Research Fellowship award to MZB, a RISAM fellowship to MSG, a La Caixa fellowship to AKS, and by grant OTKA NN107632 from the Hungarian Scientific Research Fund (to BH).
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