Structure of the Cleavage-Activated Prefusion Form of the Parainfluenza Virus 5 Fusion Protein

Structure of the cleavage-activated prefusion form of the parainﬂuenza virus 5 fusion protein

Brett D. Welcha,b,1, Yuanyuan Liua,b,1, Christopher A. Korsa,b, George P. Lesera,b, Theodore S. Jardetzkyc,2, and Robert A. Lamba,b,2

aHoward Hughes Medical Institute and bDepartment of Molecular Biosciences, Northwestern University, Evanston, IL 60208; and cDepartment of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Robert A. Lamb, August 9, 2012 (sent for review June 28, 2012) The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by refolding, resulting in formation of a trimeric coiled coil com- fusion of the viral envelope with the plasma membrane through posed of a heptad repeat A region that extends away from the the concerted action of the fusion (F) protein and the receptor viral membrane (18–20). binding protein hemagglutinin-neuraminidase. The F protein folds Peptide inhibitor studies and available atomic structures in- initially to form a trimeric metastable prefusion form that is trig- dicate that many of the key elements of this entry mechanism are gered to undergo large-scale irreversible conformational changes common to other class I viral fusion proteins, such as the hem- to form the trimeric postfusion conformation. It is thought that agglutinin (HA) of influenza virus, gp120/41 of HIV, S protein of F refolding couples the energy released with membrane fusion. severe acute respiratory syndrome coronavirus, and glycoprotein The F protein is synthesized as a precursor (F0) that must be (GP) of Ebola virus (reviewed in ref. 4). Although X-ray struc- cleaved by a host protease to form a biologically active molecule, tures of the six-helix bundle of many type I fusion proteins have F1,F2. Cleavage of F protein is a prerequisite for fusion and virus been determined, more complete postfusion ectodomain struc- infectivity. Cleavage creates a new N terminus on F1 that contains tures are known only for PIV3 F, NDV F, and RSV F (21–25). a hydrophobic region, known as the FP, which intercalates target Furthermore, structures of the prefusion conformation of type I membranes during F protein refolding. The crystal structure of fusion proteins have been solved only for influenza virus HA, the soluble ectodomain of the uncleaved form of PIV5 F is known; PIV5 F, and Ebola virus GP (20, 26–28). The atomic structures of here we report the crystal structure of the cleavage-activated both uncleaved and protease-cleaved prefusion forms are avail- prefusion form of PIV5 F. The structure shows minimal movement able only for influenza virus HA (26, 28). The before and after of the residues adjacent to the protease cleavage site. Most of the cleavage HA structures are largely superimposable, except for hydrophobic FP residues are buried in the uncleaved F protein, and residues near the protease cleavage site that compose a surface only F103 at the newly created N terminus becomes more solvent- loop. The structures yield valuable information that helps explain accessible after cleavage. The conformational freedom of the observations regarding the protease recognition site and provides charged arginine residues that compose the protease recognition insight into the acid lability of HA after cleavage activation (28). site increases on cleavage of F protein. Earlier work using antisera to peptides derived from the F sequence suggested considerable change in antibody reactivity cleavage activation | F protein structure | paramyxoviruses occurring on cleavage of F0 to F1,F2 (29). Here we present the crystal structure of the cleaved, prefusion form of the soluble he Paramyxoviridae are enveloped, negative-strand RNA ectodomain of PIV5 F. Similar to influenza virus HA, the Tviruses that are significant pathogens in humans and animals paramyxovirus F uncleaved and cleaved structures are largely (1). The family includes parainfluenza viruses 1–5 (PIV1–5), superimposable, except for the residues composing and sur- mumps virus, measles virus, Newcastle disease virus, Sendai vi- rounding the protease recognition site. Unlike HA, there is no rus, Hendra virus, Nipah virus, respiratory syncytial virus, and concerted movement or burying of the N-terminal residues of metapneumovirus. To enter cells, paramyxoviruses, like all the FP. However, because PIV5 F is triggered for fusion by its enveloped viruses, must fuse the viral envelope with a membrane receptor binding protein HN at neutral pH, there is no need for of a host cell. For paramyxoviruses, this process involves two an HA-like mechanism for priming sensitivity to low pH. viral spike glycoproteins: a receptor binding protein, variously called HN, H, or G, and the fusion protein, F (2, 3). Results and Discussion The paramyxovirus F protein is a class I viral fusion protein Expression and Crystallization of the Prefusion PIV5 F-GCNt Protein in that initially folds in the endoplasmic reticulum into a trimeric its Cleaved Form. PIV5 F is a type I transmembrane GP with a metastable prefusion form and on triggering undergoes major 19-residue signal sequence, a large (465-residue) ectodomain, irreversible conformational changes (refolding) to form the tri- a C-terminal transmembrane anchor, and a short cytoplasmic meric postfusion conformation. F protein refolding couples the tail. F is synthesized as a precursor (F0) that must be cleaved for energy released with membrane fusion (4). The F protein is synthesized as a precursor (F0) that must be cleaved either by a host protease (furin or furin-like protease) in the trans Golgi Author contributions: B.D.W., Y.L., T.S.J., and R.A.L. designed research; B.D.W., Y.L., C.A.K., and G.P.L. performed research; Y.L. and C.A.K. contributed new reagents/analytic tools; apparatus or by an extracellular trypsin-like enzyme to form the B.D.W., G.P.L., T.S.J., and R.A.L. analyzed data; and B.D.W., T.S.J., and R.A.L. wrote biologically active molecule F1,F2. Cleavage creates a new N the paper. terminus on F1 that contains a highly conserved hydrophobic The authors declare no conflict of interest. region known as the fusion peptide (FP) (5). Cleavage of F is Database deposition: The atomic coordinates and structure factors have been deposited a prerequisite for fusion and virus infectivity (6, 7), and in- in the Protein Data Bank, www.pdb.org (PDB ID code: 4GIP). tracellular cleavage of F correlates with virus pathogenicity (8). See Commentary on page 16404. For PIV5, the receptor-binding protein hemagglutinin-neur- 1B.D.W. and Y.L. contributed equally to this work. aminidase (HN) binds to sialic acid moieties on the cell surface 2To whom correspondence may be addressed. E-mail: [email protected] or ralamb@ and is required for the activation of F occurring at the plasma northwestern.edu. membrane and at neutral pH. It is thought that F interacts with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the stalk domain of HN (9–17). On fusion activation, F undergoes 1073/pnas.1213802109/-/DCSupplemental.

16672–16677 | PNAS | October 9, 2012 | vol. 109 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1213802109 Downloaded by guest on September 26, 2021 F to be biologically active. Proteolytic cleavage of F0 to form the the cleavage protocol, thereby facilitating a homogenous pop- disulfide-linked chains F1 and F2 generates an N terminus on F1 ulation of cleaved prefusion F-GCNt. After inactivation of SEE COMMENTARY that contains the hydrophobic FP. WT PIV5 (strain W3A) F trypsin with protease inhibitors and repurification of F-CGNt by contains five arginine (Arg) residues at the cleavage site, and nickel chromatography (Ni-NTA), protein gel analysis (SDS/ cleavage occurs intracellularly in the trans Golgi network by furin PAGE) indicated the protein was cleaved into F1-GCNt and F2 or a furin-like protease during transport of F to the cell surface and was >95% pure (Fig. 1B). Electron microscopy revealed that (1). The atomic structure of uncleaved prefusion F, a mutant F the cleaved F-GCNt was stable and remained in the prefusion containing three Arg residues at the cleavage site (residues Arg- form when stored at 4 °C for days, as demonstrated by the “ ” C 98, -99, and -100, with Arg-101 and -102 deleted), indicates that characteristic tree-like morphology (Fig. 1 ). However, heat- the cleavage site residues form a small surface-exposed loop (20). ing the sample to 60 °C for 10 min (a surrogate for HN activation) converted cleaved F to the postfusion conformation, with To express a soluble ectodomain of F0 protein suitable for “ ” crystallization, we used an F protein (F-GCNt) that contains its characteristic golf tee morphology with rosette formation (30) (Fig. 1D). three Arg residues at the cleavage site and a coiled-coil trimerization domain fused in a helical frame with the C-terminal Structure of Cleaved Prefusion PIV5 F-GCNt. The cleaved prefusion heptad repeat B (HRB) region in place of the transmembrane PIV5 F-GCNt crystallized in the space group C2 and diffracted anchor and cytoplasmic tail (20). This trimerization domain is X-rays anisotropically up to 2.0–3.0 Å resolution (Table S1). The necessary for stabilizing the PIV5 F-soluble protein in its pre- structure was solved by molecular replacement using the fusion form (20, 22) (Fig. 1A). uncleaved prefusion PIV5 F-GCNt structure [Protein Data Bank F-GCNt was expressed from insect cells using a recombinant (PDB) ID code: 2B9B], and a single trimer was found in the baculovirus expression system, secreted into serum-free medium, asymmetric unit. and affinity-purified via a 6× His tag that was appended to the C Each F-GCNt monomer consists of four domains—DI, DII, terminus of the GCNt domain. Exogenous trypsin was used to DIII, and HRB regions—along with connecting loops. The three cleave F-GCNt to allow temporal control and optimization of monomers are highly interconnected, and together DI, DII, and DIII form a globular head, whereas HRB forms a trimeric coiled- coil stalk (Fig. 1 E and F). The overall shape of the molecule resembles a “tree” and is remarkably similar to the uncleaved prefusion PIV5 F-GCNt structure (20), which superimposes with an all-atom rmsd of 0.473 Å over 1,096 atoms (Fig. S1). Notably, most residues of the FP remain stationary after protease cleavage, likely because many of the FP residues are buried at the interchain boundary where DII and DIII interact (Fig. 1F and Fig. S2). The model contains all residues of the secreted protein N- terminal to the GCNt domain for all three chains. The GCNt region exhibited weak electron density, and attempts to fitor build GCNt into the available density increased the Rfree value MICROBIOLOGY slightly; thus, GCNt was omitted from the final model. An omit map was generated by deleting residues near the cleavage site (Arg-88 to -107) of the uncleaved F model used for molecular replacement. A strong difference in electron density was detected for the missing residues up to residue 97 before the cleavage site and starting with residue 106 after the cleavage site in each monomer. Missing residues were built into the cleaved model independently for each monomer, and noncrystallographic symmetry restraints were not used during refinement. Electron density is relatively weak for the residues immediately adjacent to the cleavage site (Arg-98 to -100 and -103 to -105; Arg-101 and -102 are deleted from the construct). However, in each case, the Rfree value increased when these residues were Fig. 1. Biochemical and structural characterization of PIV5 F-GCNt. (A) The separately deleted from the final model. Thus, these residues major F-GCNt domains are highlighted by color. Residue ranges are also were included in the final model. indicated. (B) SDS/PAGE analysis under reducing conditions of uncleaved (F0) The relatively high B-factors and weak electron density of + and trypsin-cleaved (F1 F2) PIV5 F-GCNt. The gel indicates the complete- residues bracketing the cleavage sites show that these residues ness of protease cleavage and purity of protein used for crystallization trials. can adopt multiple conformations after cleavage. Indeed, the The F1-GCNt and F2 fragments are the expected sizes, indicating that the fi model differs slightly near the cleavage sites for each chain (Fig. trypsin cleavage is speci c for the multibasic protease cleavage/activation A C D E site. (C and D) Electron microscopy of cleaved prefusion (C) and postfusion 2 , , , and ). A portion of heptad repeat C (HRC) near the (D) FGCNt reveals characteristic “tree-like” and “golf tee” structures, re- cleavage loop is shown in Fig. 2B to demonstrate the agreement spectively. Heat was used as a surrogate for HN activation to convert F-GCNt between the final model and the electron density map (2mFo- to the postfusion conformation, which assembles into rosettes. Arrowheads DFc) for this region. indicate molecules viewed from the side and in C point to the boundary between the treetop and trunk. (E) Cartoon representation of the crystal Comparison of the Cleaved and Uncleaved Prefusion PIV5-FGCNt structure of the cleaved prefusion F-GCNt trimer viewed from the side and Structures. After cleavage of F-GCNt, the surface loop contain- colored by domains as in A. Arrowheads indicate the locations of the termini ing the cleavage site relaxes relative to the more kinked con- resulting from protease cleavage. (F) Surface representation of the trimer formation in the uncleaved structure (Fig. 2B). This action oriented as in E with color-coded chains (A, yellow; B, red; C, orange) and the hydrophobic FP highlighted in blue. On cleavage, the FP remained sand- directs the Arg residues immediately preceding the cleavage site wiched between DII and DIII of adjacent chains as in the uncleaved structure. away from the globular structure. Before cleavage, F103 is buried The GCNt domain was omitted from the model because of insufficient against the globular head in a hydrophobic patch comprising electron density. I95, P96, V106, V125, V128, and K129 from the same chain;

Welch et al. PNAS | October 9, 2012 | vol. 109 | no. 41 | 16673 Downloaded by guest on September 26, 2021 structure; however, its side chain forms intramolecular interactions with T97, R98, A104, and V106 (Fig. 2D). In contrast to chains A and B, in chain C, F103 packs into the same hydrophobic patch as in the uncleaved structure, albeit with a different orientation (Fig. 2E). The calculated surface electrostatic values of PIV5 F-GCNt reveals several interesting features. The stalk domain is almost entirely negatively charged. Another large negative patch lies at the boundary where the FP interacts with the HRC helix. There is also a large positively charged patch formed by surface residues in DIII, the FP, and the heptad repeat A helix at the boundary where these intersect (Fig. 3 A and C). Although subtle differences are observable in the electrostatic surfaces when comparing the uncleaved and cleaved structures, the differences likely are not signiﬁcant, but rather represent small variations among the structures. Importantly, except for exposure of the newly generated termini of F2/F1-GCNt, the conformational changes occurring near the cleavage site after protease cleavage exhibit no substantial net burying or exposure of hydrophobicity or charge (Fig. 3).

PIV5 F Hydrophobic FP Residues Are Buried Before Cleavage and Do Not Undergo a Concerted Conformation Change on Cleavage Activation. Inﬂuenza virus HA and Ebola virus GP have many similarities to the F protein of paramyxoviruses in that these proteins initially fold to a metastable prefusion form, require proteolytic activation to become fusogenic, and mediate virus– cell membrane fusion through dramatic conformational rear- rangements. These proteins have several important differences, however, including the fact that HA and GP bind cellular receptors directly (31, 32), whereas most F proteins are not thought to engage receptors. In addition HA and GP are

Fig. 2. Cleavage site in the cleaved prefusion PIV5 F-GCNt structure. (A) Overlay of chains A (magenta), B (yellow), and C (blue) of the cleaved F-GCNt structure zoomed-in on the protease cleavage site. The F103 side chain, at the N terminus of F1 after cleavage, is shown as sticks, and the C-terminal alpha carbon of F2 is shown as a sphere for each chain. (B) Ribbon diagram overlay of chain A from the uncleaved (green) and cleaved (magenta) structures zoomed-in as in A. The cleaved structure is shown as a trimer making DII from the adjacent chain C (light red) visible. F103 side chains are shown as sticks. (C) Detailed view of interactions involving F103 of chain A (magenta). An intermolecular hydrogen bond is apparent between the carbonyl carbon of F103 and the NH1 nitrogen of R419 in DII (light red) of chain C. F103 of chain A is further stabilized by several intermolecular hydrophobic interactions with DII of chain C, including interactions with Q389, M388, and S419, as well as an intramolecular hydrophobic interaction with V106. (D) Detailed view of interactions involving F103 of chain B (yellow). Here intermolecular interactions with DII of the adjacent chain are minimal. Instead, F103 packs against nearby residues in chain B, including T97, R98, A104, and V106. The carbonyl carbon of F103 forms an intramolecular hydrogen bond with the nitrogen of G105, possibly shared with the nitrogen of V106. (E) Detailed view of interactions involving F103 of chain C (blue). F103 packs against the globular structure, making hydrophobic contact with P96, V107, V125, V128, and K129. A water-mediated hydrogen bond is observed between the NH2-terminal nitrogen and the NZ nitrogen of K129. (F) A portion of the model from the cleaved prefusion PIV5 F-GCNt crystal structure near the protease cleavage site showing the ﬁnal ﬁt to the 2mFo- Fig. 3. Electrostatic surface renderings of uncleaved and cleaved PIV5 DFc electron density map. F-GCNt. (A) Side view of uncleaved PIV5 F-GCNt trimer with electrostatic surface shown. (B) Close-up view of the furin cleavage loop (chain A) with a cartoon representation (gray) visible through the partially transparent however, after cleavage, F103 of chain A moves outward to form surface. The F103 side chains are shown as sticks. (C and D) Similar views of the cleaved structure as shown in A and B. Electrostatic solvent-accessible hydrophobic intramolecular interactions with V106 as well as surfaces were calculated at a salt concentration of 0.15 M using the Adaptive intermolecular interactions with Q384, M388, and S412, and Poisson–Bolzmann Solver and PDB2PQR software (v1.3) via the CHARM al- a hydrogen bond with R419 of an adjacent chain (chain C; Fig. gorithm and mapped onto the vdw surface in Pymol (v1.3). The electrostatic 2C). F103 of chain B is also projected away from the globular potential ranges from −2 (red) to +2 (blue) kT/e.

16674 | www.pnas.org/cgi/doi/10.1073/pnas.1213802109 Welch et al. Downloaded by guest on September 26, 2021 triggered to cause fusion in the low pH environment found in the terminus of HA2 bind in a nearby cavity and project into the lumen of endosome/lysosome compartments (33, 34). In con- interior of the trimer (Fig. 4B and Fig. S2B). Protonation of SEE COMMENTARY trast, F is activated by a separate receptor binding protein (HN, these buried residues is thought to help destabilize the prefusion H, or G) at neutral pH. structure, inducing the conformational rearrangement to the The atomic structures of uncleaved and cleaved influenza HA postfusion form. In addition, the HA structures reveal that provide a structural basis for understanding the pH sensitivity interactions between the core structure and residues in the and proteolytic activation of the cleaved prefusion form of HA (26, cleavage loop may reduce the accessibility of proteases and limit 28). These structures are superimposable except for the 19 residues virus pathogenicity, particularly when the cleavage loop is short. composing and adjacent to the protease cleavage site. In uncleaved Strains with longer cleavage loops, especially those with multi- HA, these residues form a surface-exposed loop (Fig. 4A and basic residues recognized by ubiquitous proteases, are more Fig. S2A). On cleavage, residues 1–10 of the newly formed N pathogenic (reviewed in ref. 35). The uncleaved and cleaved prefusion structures of PIV5 show major differences between influenza virus HA and paramyxovirus F cleavage activation. The cleavage site of PIV5 (W3A) is the C-terminal side of the R-R-R-R-R↓ recognition sequence that lies in a surface loop at the junction between the HRC and the FP (Fig. 4C and Fig. S2C). For PIV5 F-GCNt, all three Arg residues in the F cleavage site mutant are surface- exposed and protease-accessible, and all five Arg residues are assumed to be surface-exposed in WT F. After cleavage of F- GCNt, the distance between the newly created N- and C-termini of F1 and F2 is an average of 9.9 Å (Cα−Cα distance), much shorter than that for influenza virus HA1 and HA2 (22.2 Å). Furthermore, there is no concerted movement of the N- and C- termini after cleavage, no burying of exposed hydrophobic residues of the FP, and no setting of a pH-sensitive trigger (Fig. 4 C and D and Fig. S2 C and D). These observations are consistent with the findings that the FP of PIV5 F-GCNt is already mostly buried in the prefusion uncleaved structure and that PIV5 fusion is triggered by HN at neutral pH. The crystal structure of Ebola virus GP in its furin-cleaved (GP1/GP2) prefusion form was solved in complex with a neutralizing antibody (27). Unlike paramyxovirus F and influenza HA, the hydrophobic FP resides in an internal fusion loop rather than at the N- terminus of GP2 following furin cleavage. Like

PIV5 F, the FP residues of Ebola virus GP pack against an ad- MICROBIOLOGY jacent monomer within a trimer, although the Ebola FP is sub- stantially more exposed on the exterior of the trimer surface (Fig. 4 E and F and Fig. S2E). However, unlike PIV5 F, in which FP residues are deeply buried at the interface between chains, GP FP residues bind the surface of the adjacent chain. Although additional cleavage of GP by cathepsins to remove the mucin- like domain is necessary for GP activation (36), which occurs in the low-pH environment of the endosome/lysosome, evidence suggests that an additional unidentified trigger is required to initiate refolding to the postfusion conformation (34, 37). It is interesting, however, that of the three examples of FP packing in the prefusion conformation discussed, PIV5 FP residues are mostly solvent-inaccessible (Figs. 1F and 4G and Fig. S2F), and for the fusion proteins described, PIV5 F is the only one triggered at neutral pH. Fig. 4. Comparison of FPs in prefusion PIV5 F-GCNt, influenza HA, and Ebola virus GP crystal structures. (A and B) Top-down view of ribbon dia- Conclusions fl grams of the uncleaved and cleaved in uenza HA crystal structures, re- Here we present the crystal structure of the cleaved prefusion spectively (PDB ID codes: 1HA0 and 2HMG) (28, 44) [Drawn from PDB ID: 1HA0 (45).] (C and D) A similar view of uncleaved and cleaved PIV5 F crystal form of PIV5 F-GCNt, the stabilized ectodomain of the F protein. structures, respectively (PDB ID code: 2B9B) (20). In A–D, the residues that This structure, together with the previously reported structures of move after protease cleavage are highlighted. Residues N-terminal to the uncleaved prefusion F-GCNt and the postfusion structures of cleavage site are highlighted in blue, and residues C-terminal to the cleavage hPIV3 F, NDV F, and RSV F, provide a detailed high-resolution site are shown in red. Additional residues composing the FP are shown in view of the various static forms of paramyxovirus fusion proteins orange, and all FP residues have side chains, shown as sticks. (E) A similar (Fig. 5). The cleaved prefusion structures of PIV5 F, influenza view of cleaved prefusion Ebola GP (PDB ID code: 3CSY) (27). The entire HA, and Ebola virus GP exhibit different paradigms of cleavage fusion loop of Ebola virus is highlighted in orange with FP residue sidechains activation for type I fusion proteins. For Ebola virus GP, the fu- shown as sticks. (F) Close-up view of Ebola virus GP FP shown packed against sion activation trigger remains unknown (34, 37). For influenza the surface of an adjacent chain in the trimer. Color-coding is as in E.(G) Close-up view of PIV5 F FP buried at the interface between adjacent chains HA, the details of the structure help explain how the length of the of the trimer. Color-coding is as in B, except the entire FP is colored orange. cleavage loop is a determinant of pathogenicity and how burying The C-alpha carbons at each terminus are shown as spheres, colored yellow of hydrophobic residues helps set a low pH trigger. For PIV5 F, for the new N- and C-termini created by protease cleavage (B, D, E, and G fusion occurs at neutral pH on activation by the HN receptor only). Ovals illustrate the area of FP. binding protein, and the hydrophobic residues that compose the

Welch et al. PNAS | October 9, 2012 | vol. 109 | no. 41 | 16675 Downloaded by guest on September 26, 2021 Fig. 5. Structure of distinct conformational states of the F protein ectodomain. Side-view ribbon diagrams of prefusion PIV5 F-GCNt protein in its uncleaved (PDB ID code: 2B9B) and protease-cleaved states, as well as the uncleaved postfusion structure of the hPIV3 F ectodomain (PDB ID code: 1ZTM). Arrowheads indicate the position of protease cleavage sites in the prefusion structures for two of the three chains (the third is hidden from view). Arrows indicate the progression of F through its various conformational states. An exception for the uncleaved postfusion hPIV3 F structure is noted by square brackets as cleavage activation is a biological requirement for fusion activity, although not for refolding of the majority of the F ectodomain into the postfusion form. The hPIV3 F protein lacked a GCNt trimerization domain, and it folded directly into a postfusion conformation even in the absence of protease cleavage. Each structure is color-coded by domain (DI, yellow; DII, red; DIII, magenta; HRB, blue).

FP are mostly prepacked in the uncleaved structure and remain so a Mosquito robot (TTP LabTech) at Northwestern University’s High- in the cleaved form. Throughput Analysis Laboratory using a PEG/Ion screen (Hampton). The Although the conformational changes are not as dramatic as optimized crystals were grown at 20 °C by the sitting-drop vapor diffusion those observed for HA, the changes occurring on protease method with an equal-volume mixture of protein (9.6 mg/mL) and pre- fi fi cipitant [0.15 M ammonium citrate dibasic (pH 5.5) and 15% wt/vol PEG cleavage of F are not benign. Earlier ndings using F-speci c 3350]. Crystals were harvested and transferred to the precipitant plus 20% antipeptide sera were interpreted as indicating an extensive glycerol for flash-freezing in liquid nitrogen. conformational change on cleavage (29), but in retrospect this conclusion might have been affected by the possible conversion Data Collection, Structure Determination, and Refinement. A native dataset of cleaved F into postfusion F on detergent solubilization. In (400 frames spanning 240 degrees) was collected at the Life Sciences Col- previous work, we found that the anti-PIV5 F mAb F1a recog- laborative Access Team beamline at the Argonne National Laboratory Ad- nizes membrane-bound cleaved PIV5 F better than membrane- vanced Photon Source. The data were processed to 2.0 Å using the HKL-2000 bound uncleaved F (29, 38). Subtle structural changes near the program package (HKL Research) (39); however, because of anisotropy, the cleavage site might be important for interaction with HN and in data were subsequently submitted to the UCLA Molecular Biology Institute’s triggering of F. Diffraction Anisotropy Server for ellipsoidal truncation and anisotropic scaling (40). Using a cutoff of Fσ ≥3, the server recommended resolution −1 Materials and Methods limits with ellipsoid dimensions of 1/3.0, 1/2.0, and 1/2.2 Å along the three principle axes a*, b*, and c*, respectively. The server also applied an iso- Cells. Hi5 insect cells were maintained in Express 5 serum-free medium (Gibco) tropic B-factor of −22.75 Å2. Electron density maps generated with the an- supplemented with 10% GlutaMax (Gibco). Sf9 insect cell lines (for gener- isotropically processed data appeared to be of noticeably higher resolution ating baculovirus stocks) were maintained in SF900 II medium (Invitrogen) and contained more features than those generated by processing the data containing 10% FBS. to 2.55 Å in HKL-2000 [completeness = 99.9% (100%), redundancy = 5.1

(4.9), Rmerge (linear) = 0.118 (0.765), Rmerge (square) = 0.084 (0.532)] with no Cloning and Mutagenesis. The cloning and expression of PIV5 F-GCNt, which anisotropic scaling. includes ectodomain residues Arg-20 to -100 and -103 to -477 (with two Arg The uncleaved prefusion PIV5 F-GCNt structure (PDB ID code: 2B9B) was residues in the protease recognition sequence deleted to prevent intracellular used as a search model for molecular replacement to determine initial cleavage) fused to the GCNt trimerization domain, have been described phases in the C2 space group. Three monomers comprising the biological previously (20). However, to optimize protein expression, the construct used trimer were found in the asymmetric unit (rotation function z-score = 46.7, in this study was subcloned into pBACgus-11, and the factor Xa cleavage site translation function z-score = 80.8, log-likelihood gain = 8,781). Molecular between the GCNt domain and the His tag was replaced with a single replacement with postfusion F models was not successful. Model building, Ala residue. structure refinement, and validation were performed with Coot (41), PHE- NIX Refine (42), and MolProbity (43), respectively. The use of translation, Protein Expression, Digestion, and Purification. A recombinant baculovirus libration, screw motion (TLS) parameters (as recommended by PHENIX) and that expresses F-GCNt was generated using the BacVector-2000 Transfection individual B-factor restraints during late stages of refinement helped lower

Kit (Novagen). Sf9 cells were used for the production of virus stocks. The the Rfree value; however, the use of noncrystallographic symmetry restraints virus titer was determined by plaque assay. High Five cells (Invitrogen) were increased the Rfree value. Data collection and final refinement statistics are infected at a multiplicity of infection of five plaque-forming units. At 65 h provided in Table S1. Atomic coordinates and structure factors have been postinfection, the medium was harvested, and soluble F protein was purified deposited in the PDB (www.pdb.org). by affinity chromatography using Ni-NTA resin (Qiagen). Purified F protein was treated with tosyl phenylalanyl chloromethyl ketone-treated trypsin ACKNOWLEDGMENTS. We thank Professors Alfonso Mondragon and (Worthington) at a ratio of 1 unit of trypsin per 10 μg of protein. After a 1-h Heather Pinkett for useful advice and discussions. This research was incubation at 4 °C, the reaction was quenched with a molar excess of soy- supported in part by the National Institutes of Health (Research Grants AI- bean trypsin inhibitor (Worthington). The cleaved protein was again puri- 23173, to R.A.L., and GM-61050, to T.S.J.). B.D.W. was and Y.L. is an Associate fied with Ni-NTA resin and then dialyzed against 50 mM Tris and 100 mM and R.A.L. is an Investigator of the Howard Hughes Medical Institute. Our NaCl (pH 7.4). use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CHI1357. Our work at Life Sciences Collaborative Access Team fi Cleaved F Protein Crystallization. The puri ed cleaved F protein was heated to Sector 21 was supported by the Michigan Economic Development Corpora- 40 °C for 40 min to reduce the amount of soluble aggregates. The crystals tion and Michigan Technology Tri-Corridor for the support of this research were initially obtained by the hanging-drop vapor diffusion method using program (Grant 085PI000817).

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