1Department of Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Zhongguancun Beiyitiao, Beijing 100080, 2Laboratory of Structural Biology and MOE Laboratory of Protein Sciences, School of Life Sciences and Bio-Engineering, Tsinghua University, Beijing 100084, China, 3Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA and 5Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK 4Present address: Oxagen Ltd, Milton Park, Oxfordshire OX14 4RY, UK
6 To whom correspondence should be addressed. e-mail: ggao66{at}yahoo.com; tienpo{at}sun.im.ac.cn
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Abstract |
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Keywords: construct design/fusion/heptad repeat/6-helix/Newcastle disease virus
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Introduction |
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Fusion and entry of paramyxoviruses require two glycoproteins, fusion (F) protein and haemagglutininneuraminidase protein (HN or homologue H and G) (Lamb, 1993), although some of them in the family do not absolutely require HN or its homologues (Horvath et al., 1992
; Bagai and Lamb, 1995
; Karron et al., 1997
). Newcastle disease virus (NDV) is a member of the genus Rubulavirus in the family Paramyxoviridae, which is composed of enveloped negative-stranded RNA viruses (Morrison and Portner, 1991
). It was recently proposed that NDV and related avian paramyxoviruses should be assigned as a new genus, Avulavirus, as they have important enough differences compared with the genus Rubulavirus (Chang et al., 2001
). Like all the members in the family, the NDV F protein is synthesized initially as a precursor, F0, and then cleaved into F1 and F2 by a furin-like enzyme of the host cells (Homma and Ouchi, 1973
; Scheid and Choppin, 1974
). During the attachment and subsequent fusion process, the F protein undergoes conformational changes that expose the fusion peptide in F1 and result in the fusion peptide embedding in the host cell membrane. In the F1 of NDV, the HR1 region is located at the N-terminus, immediately followed by the fusion peptide, while the HR2 region is located adjacent to the N-terminus of the transmembrane domain. Previously, we have shown that separately expressed HR1 and HR2 of the NDV F protein assembled into a 6-helix coiled-coil bundle in vitro (Yu et al., 2002
), although the actual structure is not known. Recently, the crystal structure of the pre-fusion native state of NDV F protein has been solved (Chen et al., 2001
), although there is a lack of some important domains; however, it represents the second such complex structure after the influenza HA (Chen et al., 1998
). Therefore, there is a great need for the crystal structure and biophysical analysis of the NDV post-fusion 6-helix coiled-coil bundle for the comparison of conformational changes, which could give a clear idea of the molecular mechanism of paramyxovirus fusion in the process of virus entry.
Here we have deployed a GST fusion Escherichia coli expression system to express the HR1 and HR2 as a GST fusion protein of single-chain of 2-helix (HR12) or 6-helix (HR121212). Both the expressed, GST-removed proteins form a stable 6-helix complex, representing the post-fusion state, and X-ray diffracting crystals have been obtained. This methodology would facilitate the future crystal structure analysis of the post-fusion 6-helix bundle of this group of viruses
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Materials and methods |
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The NDV F gene was cloned from the Chinese virulent isolate F48E9 (GeneBank no. AF 079172). The HR1 region used was derived from amino acids 115152 and HR2 from 442477 (see Figure 1). The regions chosen were based on the computer program Coilscan (Lupas et al., 1991) and the known crystal structure of SV5 HR1/2 (Baker et al., 1999
). Two different constructs were made by PCR, 2-helix and 6-helix, as shown in Figure 1. The 2-helix construct was made by linking the HR1 and HR2 with a six amino acid linker (SGGRGG; single amino acid abbreviations used here). The 6-helix construct was generated by sequentially linking the HR1 and HR2 using two different linkers (linker 1 as SGGRGG or linker 2 as GGSGG) (Figure 1B). All the constructs were cloned into the BamHI and XhoI sites (introduced by synthetic PCR primers) of GST fusion expression vector pGEX-6p-1 (Pharmacia), in which there is a rhinovirus 3C protease cleavage site for the fusion protein (the same as the commercial PreScission protease cleavage site). The positive plasmids were verified by direct DNA sequencing.
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Escherichia coil strain BL21(DE3) transformed with the recombinant pGEX-6p-1 plasmid was grown at 37°C in 2x YTA medium to an optical density of 0.81.0 (OD 590 nm) before induction with 1 mM IPTG for 4 h at 37°C. Bacterial cells were harvested and lysed by sonication in phosphate-buffered saline (PBS, 10 mM sodium phosphate, pH 7.3; 150 mM NaCl). TritonX-100 was then added to a final concentration of 1% and the lysate was incubated for 30 min at 0°C and was subsequently clarified by centrifugation at 12 000 g for 30 min at 4°C. The clarified supernatants were passed through a glutathioneSepharose 4B column (equalized by PBS). The GST fusion protein-bound column was washed with PBS over 10 column volumes and eluted with reduced glutathione (5 mM) for three column volumes. The GST fusion proteins were then cleaved by GST-fusion rhinovirus 3C protease (kindly provided by Drs K.Hudson and J.Heath) at 5°C for 16 h in the cleavage buffer (50 mM TrisHCl, pH 7.0; 150 mM NaCl; 1 mM DTT; 1 mM EDTA, pH 8.0). The free GST and the GST-3C protease were removed by passage through the glutathioneSepharose 4B column again. The resultant protein 2-helix and 6-helix were dialyzed against PBS and concentrated to a suitable concentration by ultrafiltration and stored at 70°C for further analysis. Proteins were analyzed by Tristricine SDSPAGE or 12% SDSPAGE. Protein concentrations were determined by the BCA protein determination assay (Pierce Biochemicals).
Gel-filtration analysis
The GST column-purified 2-helix and 6-helix proteins were separately loaded on a Hiload Superdex G75 column with an Akta FPLC system (Pharmacia). The fractions of the peak were collected and subjected to Tristricine SDSPAGE or 12% SDSPAGE. The peak molecular weight was estimated by comparison with protein standards (Pharmacia) running on the same column.
Chemical cross-linking
The gel filtration-purified 2-helix protein was dialyzed against cross-linking buffer (50 mM HEPES, pH 8.3; 100 mM NaCl) and concentrated to 2 mg/ml by ultrafiltration (10K cut-off). Proteins were cross-linked with ethylene glycol bis(succinimidylsuccinate) (EGS) (Sigma). The reaction mixtures were incubated for 1 h on ice at concentrations of 0, 0.1, 0.5, 2.0, 4.0 and 6.0 mM EGS and stopped with 50 mM glycine. The cross-linked products were analyzed under reducing conditions by 14% SDSPAGE.
Circular dichroism (CD) spectroscopy
CD spectra were measured on a Jasco J-715 spectrophotometer in PBS (10 mM sodium phosphate, pH 7.3; 150 mM NaCl). Wavelength spectra were recorded at 25°C using a 0.1 cm pathlength cuvette. Thermodynamic stability was measured at 222 nm by recording the CD signals in the temperature range 2595°C with a scan rate of 1°C/min.
Protein crystallization
Proteins (in 20 mM TrisHCl, pH 8.0) were concentrated to 10 mg/ml. The initial crystallization conditions were screened by using the hanging-drop vapor diffusion method with sparse matrix crystallization kits (Crystal Screen I and II; Hampton Research, Riverside, CA). The 2-helix protein crystals were obtained in at least four conditions and all the conditions were optimized in order to obtain the best diffracting crystals. The 6-helix crystals were also obtained under one set of conditions. The X-ray diffraction data for 2-helix crystals were collected and analyzed.
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Results |
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The constructs of 2-helix and 6-helix are shown schematically in Figure 1 and they were both expressed as soluble GST-fusion proteins in E.coli, despite the high hydrophobicity of the HR1 amino acids. In the construct design, to avoid the insolubility problem that we encountered earlier (Yu et al., 2002), we chose shorter HR1 regions that lack some amino acids near the fusion peptide. Those amino acids would not be covered by HR2 based on the crystal structure of SV5 HR1/2 (Baker et al., 1999
). It was confirmed from this result that this construct design is important to achieve highly soluble protein. The GST affinity-purified proteins from the supernatants of cell lysates were subsequently cleaved by using the GST-3C rhinovirus protease (kindly provided by Drs K.Hudson and J.Heath) as described in Materials and methods (Figure 2). After the removal of the fusion partner GST, the cleaved proteins of both 2-helix and 6-helix are soluble at high concentrations (up to 3040 mg/ml) in PBS or Tris buffer.
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The purified 2-helix and 6-helix proteins were analyzed by gel filtration and chemical cross-linking (2-helix only) for estimation of the molecular weight as described in Materials and methods. In the gel filtration, the purified proteins of both 2-helix and 6-helix gave rise to a peak with a molecular weight of 26 kDa (Figure 3), close to the theoretical values for the trimer of HR1HR2. In chemical cross-linking of the 2-helix preparations (Figure 4), both dimers and trimers could be seen on the reducing gel. Monomers/dimers tended to fade as the cross-linker EGS concentration was increased, whereas the trimer bands became thicker. All of this indicated a trimer state of the 2-helix, although the monomer/dimer bands did not disappear even at high concentrations of the cross-linker. It also showed that the 6-helix protein preparation ran in a similar position to the 2-helix trimer (Figure 3).
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To investigate the intrinsic -helix nature of the 6-helix bundle, the secondary structure of the 2-helix and 6-helix proteins was examined by CD spectroscopy as described in Materials and methods. The resulting spectra at 25°C (Figure 5A and C) show that both 2-helix and 6-helix protein preparations were stably folded and formed typical
-helix structures, with obvious double minima at 208 and 222 nm. Regarding the thermal stability (Figure 5B and D), both 2-helix and 6-helix constructs showed the formation of a very stable complex. The melting temperatures were very high, 8990°C for 2-helix (Figure 5B) and 9091°C for 6-helix (Figure 5D).
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Both 2-helix and 6-helix protein preparations were crystallizable under several conditions (Figure 6), again indicating stable complex formation of the 6-helix bundle. X-ray diffraction data from two crystals were collected up to 2.5 Å resolution and the structure determination is under way. Similarly, we have successfully obtained 2-helix crystals with other paramyxoviruses, e.g. measles virus, Menangle virus and Nipah virus.
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Discussion |
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Previously, we have used the E.coli system to express separately the F protein HR1 and HR2 of the NDV, subsequently assembling the purified HR1 and HR2 into a 6-helix bundle (Yu et al., 2002). However, we failed to obtain crystals of good quality for structural analysis. In this study, we designed two constructs by linking together the heptad repeat regions of the NDV F protein using some flexible amino acids. By using a series of biochemical and biophysical measures, our results show that both 2-helix and 6-helix proteins form soluble 6-helix trimers and are crystallizable using this method. We have since succeeded in the crystallization of 6-helix bundle HR trimers derived from some other virus fusion proteins, including those of measles virus, Menangle virus and Nipah virus. This indicates that this is a reliable method for obtaining crystallizable proteins for crystal structure analysis of such a group of proteins and will inevitably help in the study of the molecular mechanism of virus fusion with the accumulation of more structural data.
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Acknowledgements |
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References |
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Received August 13, 2002; revised January 20, 2003; accepted March 13, 2003.