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

Crystallographic data and refinement statistics: PIV5 HN ectodomain.

<p>Crystallographic data and refinement statistics: PIV5 HN ectodomain.</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

Domains of the PIV5-HN ectodomain.

<p>A) Regions of the PIV5-HN ectodomain by residue number. The known structure(s) of each segment are noted. B) Overlay of the head domains of chains A–D (colored blue, magenta, orange, and green, respectively) of the PIV5-HN ectodomain structure and the 1Z4X structure (yellow). The ordered sulfate in the active site is shown as orange and red spheres. The flexible loop containing His 188 is outlined. C) Overlay of the 4HB stalk of the PIV5-HN ectodomain structure (orange) with the isolated PIV5-HN stalk structure (PDB ID: 3TSI, yellow).</p

Crystal structures of various HN ectodomain conformations.

<p>A) Cartoon representation of the NDV HN crystal structure (PDB ID: 3TIE) in the “4-heads-down” conformation. Active site residues (E400, R415, and Y525) are shown as yellow, blue and red spheres. The locations of the second sialic acid binding sites are not shown. B) Model of the “4-heads-up” conformation of PIV5 HN comprised of separate previously solved crystal structures of the head domain (PDB ID: 1Z4X) and the 4HB stalk (PDB ID: 3TSI). Sialyllactose in the active site is shown as yellow and red spheres. Ordered glycan chains are shown as sticks. Coloring as in A. C–E) Front, side, and top views, respectively, of the “2-heads-up/2-heads-down” conformation revealed by the PIV5-HN ectodomain crystal structure. A sulfate ion coordinated by R405, R495, and Y523 in each active site, which partially overlaps with sialyllactose in the head domain of the 1Z4X structure, is shown as orange and red spheres. Ordered glycan chains are shown as sticks. Coloring as in A–B. F) Sigma-A weighted 2Fo-Fc electron density map rendered at 1.0 sigma showing the fit of the final PIV5-HN ectodomain model. D105 and E108, project charged sidechains toward the central 4HB axis and are marked with an asterisk. The region shown is the continuous density that links the stalk of chain B to its globular head domain. The PIV5-HN ectodomain structure was used to position head domains relative to the stalk in the 4-heads-up model in B.</p

Electron density at the PIV5-HN ectodomain active sites.

<p>A) Close-up view of the active site of chain B (magenta) overlaid with the 1Z4X structure (yellow). The sulfate ion in chain B is not shown, but sialyllactose (SLT) from 1Z4X is shown as sticks. The sigma-A weighted Fo-Fc map rendered at 3.8 sigma is shown as green in A–C. B) Similar view as in A but of chain D. Lactose (LAT, gray) is modeled into significant positive difference density near H188 that is found in chains A and D but not in chains B and C. The sulfate ion is shown as sticks. C) Another view of the scene in B. D) Sigma-A weighted 2Fo-Fc map rendered at 1.0 sigma resulting from refinement of lactose fit into the positive difference density in chain D (orientation as shown in B).</p

Model of paramyxovirus activation of F by HN.

<p>The heads of HN in the 4-heads down conformation block F activation by preventing access to critical residues in the mid-upper HN 4HB stalk. However, upon receptor binding HN heads move to the up position revealing residues in the HN stalk that interact with and trigger F for fusion. HN molecules were aligned by relevant domains with the 2-heads-up/2-heads-down structure to provide proper orientation of the 4-heads-up and 4-heads-down conformations with respect to one another and the exact spacing of the heads relative to the stalk in the 4-heads-up model. The distances from the membrane that missing residues from the F and HN ectodomain crystal structures would occupy if they are fully helical are indicated. Without invoking flexibility of the membrane proximal portion of the HN ectodomain, a non-canonical transmembrane domain region for F <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat.1003534-Bissonnette1" target="_blank">[53]</a>, or rearrangement of surface loops in F or HN heads - the spacing predicts limited ways in which F can contact the stalk of HN. PIV5 HN-stalk residues (red) or equivalent residues identified in MeV H (blue) and NDV HN (light pink) implicated in direct contact with F are shown as spheres. Approximately equivalent PIV5 F residues as those identified in canine distemper virus implicated in direct contact with MeV H are shown as spheres (blue). Missing portions of the ectodomain structures are shown as dotted lines, and the viral membrane is shown as a thick gray line. B) View of the F/HN interaction model shown from the bottom (view from the viral membrane) and C) top (looking down onto the viral membrane). In A–C, HN is colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat-1003534-g001" target="_blank">figure 1</a>. The cleaved form of PIV5 F (PDB ID: 4GIP) is shown. Domains I, II, III, and HRB are colored yellow, red, magenta, and blue, respectively. D) Electrostatic surface map of the F/HN interaction. The view is the same as in A above. Surfaces were calculated using the Adaptive Poisson-Bolzmann Solver <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat.1003534-Baker1" target="_blank">[54]</a> and PDB2PQR software (v1.3) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat.1003534-Dolinsky1" target="_blank">[55]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat.1003534-Dolinsky2" target="_blank">[56]</a> via the AMBER algorithm within PyMOL. The electrostatic potential ranges from −2 (red) to +2 (blue) kT/e.</p

Stalk/head domain interface in the PIV5-HN ectodomain structure.

<p>A) Cartoon rendering with transparent surface of the PIV5-HN stalk showing the locations of known category I (affect fusion only) and category II (affect fusion and NA) mutations in NDV and PIV5 HN (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat.1003534-Bose1" target="_blank">[21]</a> and expanded to include current data). The stalk residues at the stalk/head interface are shown as sticks and labeled. B) Zoomed-in view of the stalk/head interface with individual chains colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat-1003534-g001" target="_blank">Fig. 1</a>. Sidechains at the interface are shown as lighter colored sticks. C) A MONSTER contact map of the DOD₁ interface analogous to that in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003534#ppat-1003534-g003" target="_blank">Fig. 3B</a>. PIV5 residues at the stalk/head interface that align with residues present in the NDV stalk/head interface are highlighted with an asterisk. Red asterisks mark identical residues across both interfaces. D) Overlay of the head domains of a covalent dimer of NDV HN (yellow) with PIV5 HN (green and orange) showing the different angles of the stalks relative to the heads in the two structures.</p