Electron density maps for key regions of the C-terminal extension.

<p>(A) Omit map electron density for C-terminal residues 611–615, which insert into the NA domain active site. (B) Omit map electron density for residues surrounding the interchain disulfide bond at C596. Residues associated with different chains of the observed NA domain dimers are indicated with an A: or B: prefix. (C) Omit map electron density C-terminal extension residues 583–588, which engage a second sialic acid binding site. The omit maps was calculated using the SFCHECK <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002855#ppat.1002855-Vaguine1" target="_blank">[46]</a> program and contoured at 1.57 sigma.</p

Crystallographic data and refinement statistics.

<p>Crystallographic data and refinement statistics.</p

The inhibitory C-terminal extension forms a well-defined structure in crystals of the Ulster NA domain.

<p>(A) Crystal structure of the Ulster HN NA domain dimer, shows a dimer-of-dimers tetramer. The C-terminal extension is highlighted in dark blue and red in each pair of dimers. (B) The Ulster HN dimer is shown, with the C-terminal extension in dark blue. The C-terminal extension begins at the base of the β-propeller domain, extends along the outside of the dimer interface, and then rises above the active site before inserting the C-terminus into the receptor-binding site.</p

The C-terminal extension inhibits the NA domain active site.

<p>(A) Top view of a single HN NA domain subunit, showing superpositions with the NDV Kansas low-pH and DANA-bound crystal structures. The additional residues of the C-terminal extension are highlighted in blue and overlap the ligand bound in the active site. The D198 loop is indicated, which changes conformation in the Ulster HN structure, contributing to blocking the active site. (B,C) Detailed comparison of contacts made by the Ulster HN C-terminus and DANA sialic acid within the active site. Dotted lines represent hydrogen bonds and polar contacts identified by Pymol.</p

Recombinant NDV Ulster F and HN are expressed as F₀ and HN₀ precursors.

<p>(A) Sequence alignment of C-termini of NDV HN strains, La Sota (La), Ulster and Australia-Victoria (AV). Core NA domain residues are colored red, partially conserved C-terminal residues are colored blue. The cysteine at residue 596 is indicated by an asterisk. (B) NDV Ulster F and HN expressed from cDNAs in HeLa cells were metabolically labeled, immunoprecipitated and polypeptides analyzed by SDS-PAGE. Addition of trypsin to the transfected cells led to processing of the precursor proteins (F₀ and HN₀) as shown by the cleavage of F₀ to F₁ and F₂ and the faster mobility of HN as compared to HN₀. (C) Hemadsorption assay on HeLa cells expressing Ulster HN. Cells were overlaid with chicken red blood cells and subsequently washed with PBS⁺ and incubated at. 4°C. pCAGGS is vector control. Ulster HN expressed in HeLa cells binds sialic acid on chicken red blood cells very poorly. Treatment of HN₀ with trypsin causes a large increase in red blood cell binding.</p

C596 in the HN C-terminal extension mediates NA domain dimerization.

<p>(A) HN Ulster WT NA domain and HN C596S NA domain were expressed and purified as described in Methods and analyzed by SDS-PAGE under reducing and non-reducing conditions. The gel was stained with Coomassie Brilliant Blue. (B) Sucrose density gradient sedimentation and SDS-PAGE (non-reducing conditions) of Ulster HN NA domain and HN C596S NA domain proteins. (C) EM of WT Ulster HN NA domain, indicates the protein consists primarily of dimers, and the HN C596S NA domain, consists mostly as monomers.</p

Structure of the Parainfluenza Virus 5 (PIV5) Hemagglutinin-Neuraminidase (HN) Ectodomain

<div><p>Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.</p></div

Hemadsorption of WT and C596S HN proteins.

<p>Hemadsorption was assayed by measuring the hemoglobin absorbance at 540 nM.</p><p>The values shown are the average of three experiments performed in duplicate.</p

Neuraminidase activity of the Ulster WT and C596S proteins.

<p>The neuraminidase activity of the Ulster WT and C596S proteins was assayed by measuring the fluorescence, at an emission wavelength of 440 nm, produced upon cleavage of the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid. The fluorescence shown was produced by equivalent amounts of protein.</p

A novel PIV5-HN head domain dimer-of-dimers interface.

<p>A) Cartoon representation of the PIV5-HN ectodomain structure dimer-of-dimers interface (DOD₂). Chains A and D are blue and green, respectively. Residues comprising the interface are shown as lighter color sticks, and residues that form disulfide bonds are colored tan. B) Contact map showing specific interactions within the interface. Gray, green, and red lines represent hydrophobic, electrostatic, and hydrogen bond interactions, respectively. Figure produced using the MONSTER server for analysis of macromolecular complexes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat.1003534-Salerno1" target="_blank">[52]</a>. C–D) Reducing and non-reducing SDS-PAGE gels, respectively, showing HN Cys mutants immunoprecipitated from cell extracts. The positions of monomer and dimer are indicated. The C111A mutation eliminates wt dimer formation. E) Flow cytometry reveals that each of the HN Cys mutants were expressed on the surface of cells within 2-fold of wt. MFI = mean fluorescence intensity. F) A cell-cell fusion assay indicates each of the HN Cys mutants remained fusogenic at levels similar to wt. Experiments were done in triplicate and error bars represent S.E.M in E–F.</p