Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6032–6036, May 1998 Biochemistry The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil WINFRIED WEISSENHORN*, LESLEY J. CALDER†,STEPHEN A. WHARTON†,JOHN J. SKEHEL†, AND DON C. WILEY*‡§ *Laboratory of Molecular Medicine, Howard Hughes Medical Institute, The Children’s Hospital, 320 Longwood Avenue Boston, MA 02215; †National Institute for Medical Research, Mill Hill, The Ridgeway, London, NW7 1AA, United Kingdom; and ‡Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138 Contributed by Don C. Wiley, March 27, 1998 ABSTRACT The ectodomain of the Ebola virus Gp2 gly- Here, we report that the intact extracellular part of the coprotein was solubilized with a trimeric, isoleucine zipper Ebola virus subunit Gp2, lacking the N-terminal fusion pep- derived from GCN4 (pIIGCN4) in place of the hydrophobic tide, can be expressed and solubilized by adding a trimeric fusion peptide at the N terminus. This chimeric molecule zipper in-frame with the predicted coiled-coil region of Gp2. forms a trimeric, highly a-helical, and very thermostable The oligomeric state of the chimeric molecule was character- molecule, as determined by chemical crosslinking and circular ized by chemical crosslinking and secondary structure analysis dichroism. Electron microscopy indicates that Gp2 folds into and showed a high a-helical content. Most strikingly, electron a rod-like structure like influenza HA2 and HIV-1 gp41, micrographs (EM) indicate a long rod-shaped structure similar providing further evidence that viral fusion proteins from to EM images observed of the low-pH-induced conformation diverse families such as Orthomyxoviridae (Influenza), Ret- of influenza virus HA2 (20, 21) and of fragments of HIV-1 env roviridae (HIV-1), and Filoviridae (Ebola) share common gp41 (22, 23), suggesting a similar role for Ebola Gp2 in structural features, and suggesting a common membrane membrane fusion. fusion mechanism. METHODS The filovirus, Ebola virus, has been linked to a number of lethal outbreaks of hemorrhagic fever (1, 2). The virus genome Cloning, Protein Expression, and Purification. The Ebola is negative-stranded and encodes for seven structural and gp2 gene sequence encoding residues 552–650 (Zaire subtype) regulatory proteins (3, 4), including a surface glycoprotein (4) was amplified with synthetic oligonucleotides, and Cys-556 (Gp) that is synthesized as a precursor molecule and then and Cys-609 were mutated to serines by standard PCR meth- cleaved into two subunits (5, 6), Gp1 and Gp2, the latter of ods. DNA encoding GCN4 residues 250–280 with both the a which is anchored in the membrane. Ebola Gp is encoded in and d positions of the coiled coil mutated to isoleucine (pII) two ORFs (7, 8), which produce a secreted and a membrane- (24) was synthesized as two overlapping oligonucleotides. The anchored form of Gp, whereas all other filovirus genomes DNA fragments encoding pII and Gp2 were subcloned into the encode only the membrane-anchored Gp. The secreted Ebola expression vector pRSET (Invitrogen) and transformed into Gp dimer interacts with neutrophils through a Fc g receptor III Escherichia coli cells BL21 DE3ypUBS (25). The DNA se- (CD16b) (9) and the membrane-anchored form binds to a quence was verified by sequencing. After induction of protein number of target cells, including endothelial cells (9, 10) and expression by isopropyl b-D-thiogalactoside (Sigma), bacterial liver cells (11), and is thought to mediate viral entry. Infection pellets were lysed in 50 mM TriszHCl, pH 8.8y100 mM NaCl and replication in endothelial cells was proposed to contribute by sonication, and the supernatant was cleared by centrifuga- to the severe hemorrhagic character of the late stages of tion at 40,000 rpm for 1 h. The soluble fraction was loaded onto disease (9, 10). a DEAE-Sepharose (Pharmacia) column (5 3 25 cm) and Amino acid sequences with the potential to form coiled coils protein was eluted with a 0.1–0.4 M NaCl gradient. Fractions have been recognized adjacent to N-terminal fusion peptides y a containing pIIgp were identified by SDS PAGE (26), concen- in many viral Gps (12–14) and similar -helical models have trated in centriprep-30 (Amicon), and further purified by gel been proposed for the HIV-gp41, Avian sarcoma virus, and filtration chromatography with Superdex 200 (Pharmacia) (20 Ebola virus transmembrane (TM) Gp subunits (12, 14). The mM Tris, pH 8.8y100 mM NaCl). x-ray crystal structures of the low-pH induced conformation of Chemical Crosslinking. pIIGp(552–650) (2 mgyml) in 50 influenza virus hemagglutinin (HA) 2 (15) of a protease mM Hepes, pH 8.3y100 mM NaCl was crosslinked with resistant fragment of HIV-1 env gp41 (16–18), and of a small ethyleneglycol bis(-succinimidylsuccinate) (EGS) (Pierce). fragment of the Moloney murine leukemia virus (MoMuLV) The reactions were incubated for1honiceatconcentrations TM protein (19) revealed that the central part of all of these of 0.1, 0.5, 2.0, and 5.0 mM EGS and then quenched with 50 molecules is a similar, long, triple-stranded coiled coil, in the mM glycine. Crosslinked products were analyzed under reduc- former two cases surrounded by an outer layer of antiparallel ing conditions on SDSyPAGE (26). a-helices. This structural organization that places the mem- Circular Dichroism. CD spectra of pIIGp(552–650) (0.15 brane anchor in close proximity to the hydrophobic fusion mgyml; 10 mM phosphate, pH 8.0y100 mM NaCl) were peptide, at the same end of a long rod-shaped molecule, was recorded at 20°C and 95°C by using a 1-mm cell on an AVIV proposed to facilitate the membrane fusion process (17) and 62DS spectropolarimeter and averaging five measurements. thus viral entry. Thermodynamic stability was measured at 222 nm by moni- The publication costs of this article were defrayed in part by page charge Abbreviations: HA, hemagglutinin; Gp, glycoprotein; MoMuLV, payment. This article must therefore be hereby marked ‘‘advertisement’’ in Moloney murine leukemia virus; TM, transmembrane. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed at: Department of © 1998 by The National Academy of Sciences 0027-8424y98y956032-5$2.00y0 Molecular and Cellular Biology, Harvard University, 7 Divinity PNAS is available online at http:yywww.pnas.org. Avenue, Cambridge, MA 02138-2092. 6032 Downloaded by guest on September 29, 2021 Biochemistry: Weissenhorn et al. Proc. Natl. Acad. Sci. USA 95 (1998) 6033 toring the CD signal between 20°C and 95°C with a scan rate is highly overexpressed in E. coli and was purified from the soluble of 1° per min. The protein concentration was calculated by fraction of bacterial lysates. Constructs lacking pIIGCN4 were measuring the OD280, with an extinction coefficient of expressed only as insoluble inclusion bodies. In addition, the 29,910yM per cm. The percentage of a-helical content was solubility was dependent on the inclusion of residues 644–650 U 2 estimated from [ ]222 by assuming that a value of 33,000 (data not shown). A difference in mobility of reduced and degree cm2 dmol21 corresponds to 100% a-helix content (27). nonreduced pIIGp2(552–650) (Fig. 1b, lanes 1 and 2) indicated U 2 2 21 The baseline value of [ ]222, equal to 2,500 deg cm dmol that the disulfide bond between Cys-601 and Cys-608 had formed of unfolded pIIGp2(552–650) was considered to be 0% a-helix properly. Cysteine residues 556 and 609 were mutated to Ser to content. avoid nonspecific disulfide crosslinking. Chemical crosslinking To follow the unfolding in guanidine hydrochloride, pH 8.0, indicates a trimeric oligomeric state. After crosslinking two new pIIGp2(552–650) was incubated in the denaturant at concentra- bands appear (Fig. 1b, lanes 3–6), migrating at 26 and approxi- U tions of 0.5–8.0 M for 30 min at room temperature, and the [ ]222 mately 44 kDa, corresponding to a dimeric and trimeric form of was plotted versus the guanidine hydrochloride concentration. pIIGp2(552–650). Electron Microscopy. Samples were adsorbed onto carbon High a-Helical Content and Thermostability. The CD films, negatively stained with 1% sodium silcotungstate (pH spectrum of pII pIIGp2(552–650) measured from 195 nm to 7.0), and examined with a JEOL 1200EX microscope at 100 kV 250 nm had a maximum at 194 nm and minima at 208 and 222 as described previously (28, 29). nm characteristic of a-helices (Fig. 2a). The estimated a-he- lical content is approximately 84%. Thermal unfolding is RESULTS observed by monitoring the changes in ellipticity at 222 nm. No clear unfolding transition occurs; instead, the increasing tem- Expression and Oligomeric State of an Ebola Virus Gp2 perature slightly, but steadily, reduces the overall helical Subunit Chimera. Residues 552–650 of the extracellular region of content and shows some cooperative transition at approxi- Ebola Gp2, without the N-terminal fusion peptide (Fig. 1a), were mately 90°C. No complete unfolding is observed when the expressed as a fusion protein in E. coli. The heptad positions a and temperature is raised to 98°C (Fig. 2b). This partial unfolding d in the GCN4 coiled coil were mutated to isoleucine, which has also is shown in a spectrum recorded at 95°C (Fig. 2a), which been shown to induce a trimeric coiled-coil conformation (24) indicates that the a-helical content is reduced to only 56%. and placed in register with a proposed coiled-coil segment in Refolding is not complete as the a-helical content is restored Ebola Gp2 (14) (Fig. 1a). This Gp2-chimera [pIIGp2(552–650)] to only 71% (Fig.