Crystal Structure of Reovirus Attachment Protein σ1 in Complex with Sialylated Oligosaccharides

Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α -helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties

HA assay of T3D σ1 point mutants.

<p>Purified reovirus virions (10¹¹ particles) were serially diluted in 0.05 ml of PBS in 96-well U-bottom microtiter plates. Bovine erythrocytes were washed twice with PBS and resuspended at a concentration of 1% (vol/vol) in PBS. Erythrocytes (0.05 ml) were added to wells containing virus and incubated at 4°C for at least 2 h. HA titer is expressed as 10¹¹ particles divided by the number of particles/HA unit. One HA unit equals the number of particles sufficient to produce HA. <b>*</b>, <i>P</i><0.05 in comparison to T3D (Student's <i>t</i> test).</p

Interactions between σ1 and sialic acid.

<p>(<b>A</b>) Simulated annealing omit difference density map contoured at 3σ and displayed with a radius of 2.2 Å around the bound α-2,3-sialyllactose. The sugar moieties are labeled Sia (sialic acid), Gal (galactose), and Glc (glucose) here and in subsequent figures. (<b>B</b>) Detailed interactions between σ1 and the terminal sialic acid of α-2,3-sialyllactose. Residues in the binding region are drawn in ball and stick representation, while the rest of the protein is shown as a ribbon drawing. The σ1 residues forming hydrogen bonds or salt bridges with the ligand are shown in green, and residues forming van der Waals contacts are shown in cyan. The side chain of Asn189 (colored dark blue) is contributed by a neighboring σ1 monomer (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002166#ppat-1002166-g001" target="_blank">Figure 1A</a>). Sialic acid is shown in ball-and-stick representation, with carbons colored orange, oxygens colored red, and nitrogens colored blue. Bridging waters are shown as orange spheres. Hydrogen bonds and salt bridges are represented with broken lines. (<b>C</b>) Superposition of all three bound ligands into a single binding site. The superposition was performed using σ1 residues only. While the orientation of the terminal sialic acid is nearly identical, the lactose moieties are facing in different orientations as a result of their participation in different crystal contacts.</p

Structural adaption of the binding site.

<p>(<b>A</b>) Superposition of all seven β-spiral repeats. Repeat β3, which is shown in darker shading, interacts with sialic acid and deviates markedly in its structure from the other repeats. Conserved hydrophobic residues are colored in blue, the position in the β-turn that is usually occupied by proline or glycine is shown in magenta. Arg202 and Pro204, which are part of repeat β3, are highlighted in red and yellow, respectively. (<b>B</b>) Superposition of a prototypical β-spiral repeat (β6) onto the sialic acid binding repeat β3. Amino acids are shown in ball and stick representation, and residues Arg202 and Pro204 in β3 are labeled. Panel B is enlarged to provide a clearer view.</p

σ1 in complex with sialic acid in different linkages.

<p>(A) View into the carbohydrate-binding site of the complex of σ1 with α-2,6-sialyllactose. (B) View into the carbohydrate-binding site of the complex of σ1 with α-2,8-disialyllactose. The orientation in panel (A) differs by 60 degrees along a vertical axis from the orientations shown in panel (B) and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002166#ppat-1002166-g003" target="_blank">Figure 3</a> to provide a clearer view into of the binding site. In both panels, σ1 residues directly contacting the ligand are shown in green, and surrounding residues making van der Waals contacts are shown in cyan. The ligands are shown in ball-and-stick representation, with carbons colored orange, oxygens colored red, and nitrogens colored blue. Hydrogen bonds are represented with broken black lines. The maps are simulated annealing omit difference density maps contoured at 3σ and displayed with a radius of 2.2 Å around the ligands.</p

Data collection and refinement statistics.

<p>r.m.s.d., root-mean-square deviation. sl, sialyllactose.</p><p>*R_work  =  R_free  =  Σ| |F_obs(hkl) | - |F_calc (hkl) | |/Σ |F_obs (hkl). R_free was calculated with 5% of the data.</p>#<p>R_merge  =  Σ | <i>I</i> - <<i>I</i>> |/Σ<i>I</i></p

Structure of T3D σ1.

<p>(<b>A</b>) Ribbon drawing of the T3D σ1 body and head domains in complex with α-2,3-sialyllactose. The σ1 monomers are shown in red, blue, and yellow. The body domain consists of seven triple β-spiral repeats (β1–β7) and an α-helical coiled-coil domain (cc) that is inserted between β-spiral repeats β4 and β5. The bound α-2,3-sialyllactose is shown in stick representation and colored in orange. (<b>B</b>) Molecular surface of the σ1 structure, shown in semitransparent white coloring. (<b>C</b>) Model of full-length σ1, including a computer-generated trimeric α-helical coiled coil structure spanning σ1 residues 1–160 at the N-terminus.</p