Crystal structure of ATV(ORF273), a new fold for a thermo- and acido-stable protein from the Acidianus two-tailed virus - PubMed (original) (raw)

Crystal structure of ATV(ORF273), a new fold for a thermo- and acido-stable protein from the Acidianus two-tailed virus

Catarina Felisberto-Rodrigues et al. PLoS One. 2012.

Abstract

Acidianus two-tailed virus (ATV) infects crenarchaea of the genus Acidianus living in terrestrial thermal springs at extremely high temperatures and low pH. ATV is a member of the Bicaudaviridae virus family and undergoes extra-cellular development of two tails, a process that is unique in the viral world. To understand this intriguing phenomenon, we have undertaken structural studies of ATV virion proteins and here we present the crystal structure of one of these proteins, ATV(ORF273). ATV(ORF273) forms tetramers in solution and a molecular envelope is provided for the tetramer, computed from small-angle X-ray scattering (SAXS) data. The crystal structure has properties typical of hyperthermostable proteins, including a relatively high number of salt bridges. However, the protein also exhibits flexible loops and surface pockets. Remarkably, ATV(ORF273) displays a new α + β protein fold, consistent with the absence of homologues of this protein in public sequence databases.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Crystal structure of ATV

formula image . a) Ribbon representation of the structure of a monomer of ATVformula image. Its formula image-strands are labelled. The secondary structure elements are colored from the N′- (blue) to the C′-terminus (red). The loops that were not modelled are represented by dashed, black lines. b) A view orthogonal to the previous one, showing the arrangement of the formula image-helices. This view shows part of the concave face of the monomer (top, right) c) The solvent-accessible surface of the ATVformula image monomer concave face, colored by its electrostatic potential (red: −52 mV, blue: 52 mV), calculated at pH 7 and 150 mM NaCl with APBS . Two cavities are marked with yellow open stars. d) A detail view of monomer B showing the salt bridge network formed by residues Glu29/Asp33/Lys260 as well as the disulphide bond established by cysteine residues 250 and 263. Amino acid residues are labelled in one-letter code; the secondary structure elements to which they belong are also labelled.

Figure 2

Figure 2. Oligomeric state of ATV in solution.

About 120 formula imageg of purified ATVformula image were subjected to size-exclusion chromatography coupled to MALS/RI/UV detectors as described in the Experimental section. Two chromatograms/mass analyses are combined in this figure, showing the elution of the tetrameric (left) and dimeric (right) forms of ATVformula image, obtained at pH 7.4 and 3.6, respectively. The molar mass (dotted lines), derived from refractive index measurements, and the absorption at 280 nm (full line) were plotted as functions of the elution volume around the peaks. The weight-averaged molar mass (M_w_) values determined by the ASTRA software are indicated.

Figure 3

Figure 3. Crystal packing of ATV

formula image . a) Overall view of the open and b) closed dimers found in the second crystal form. In both cases the pseudo-two-fold axis is vertical in the plane of the paper. c) View of the interface between monomer A (colored as in in Figure 1) and monomer B (grey) in the closed dimer. Amino acid residues involved in cross-monomer salt bridges are labelled in one-letter code. The secondary structure elements that support them are also labelled. d) A view of the dimers arranged as a continuous helical fiber in the crystal. The edges of a unit cell box are shown in grey color.

Figure 4

Figure 4. SAXS analysis of ATV

formula image . a) SAXS intensity as a function of the momentum transfer. This profile corresponds to the measurements taken at 4.7 mg/ml protein cocentration and pH 8.5. Average values are in red and the standard error in grey. b) The Kratky plot (see text for details) corresponds to a folded protein. c) Pair-distance distribution, P(r), function of the data shown in panel a). d) Three orthogonal views of the ab initio envelope calculated imposing orthorhombic symmetry.

Figure 5

Figure 5. Circular dichroism spectra of ATV

formula image . Mean residue ellipticity spectra recorded at pH 7.2 (black lines) and pH 0 (red lines), either at 20°C (full lines) or at 80°C (dashed lines). The content in helices and strands, as determined by deconvolution of the spectra, is shown (see text for details).

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C.F.-R. was funded by a doctoral fellowship from the Fundação para a Ciência e a Tecnologia (http://www.fct.pt/), Portugal (SFRH/BD/44380/2008). The Ville de Marseille (http://www.marseille.fr) provided a financial installation aid to M.O.-L. The Archaea Centre, Copenhagen was supported by a grant from the Danish Natural Science Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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