Biological context

Dengue 1 virus (DEN1V) is a member of the family Flaviviridae, genus Flavivirus, which contains the dengue viruses (DEN 1–4), West Nile virus, Langat virus, Omsk hemorrhagic fever virus, and many others. Receptor binding and membrane fusion of flaviviruses occurs with the envelope (E) protein (Heinze and Allison 2003). The E protein ectodomain contains three domains, ED1, ED2, and ED3. The highly immunogenic ED3 is a putative receptor-binding domain and many neutralizing antibodies bind to ED3. Thus, ED3 is an attractive target for development of vaccines, anti-viral agents, and diagnostic antigens. Chemical shift assignments and/or NMR-based structures for the ED3 proteins of Japanese encephalitis (Wu et al. 2003), West Nile virus (Volk et al. 2004a, b), Omsk hemorrhagic fever virus (Volk et al. 2006a), Dengue 4 virus (Volk et al. 2006b, 2007b), Langat virus (Mukherjee et al. 2006), and yellow fever virus (Volk et al. 2007a) have previously been reported. In addition, crystallographic structures for the extra-membrane portion of the E protein have been reported for Tick-borne encephalitis virus (Rey et al. 1995), Dengue 2 virus (Modis et al. 2003), Dengue 3 virus (Modis et al. 2005), and West Nile virus (Kanai et al. 2006; Nybakken et al. 2006).

Methods and experiments

Uniformly 13C-, 15N-labeled recombinant DEN1-ED3 (sylvatic strain P72-1244) protein encompassing residues M289–K400, with mutations N366S and E370K, was expressed using the pET-15 vector (Novagen) in which the N-terminal His-tag sequence encoded in the plasmid was removed. Cells were lysed by sonication in native buffer, the crude cell debris was centrifuged and the supernatant was removed. The pellet was resuspended in denaturing buffer (6 M guanidine HCl) and the solution was passed through a size-exclusion column (G75 Sephadex®) to provide pure protein. Pooled fractions were dialyzed to remove the denaturant. A 3 kDa centricon (Amicon, Inc.) concentrator was used to concentrate the protein. The NMR sample contained 0.25 mM protein in 25 mM Tris–HCl (pH 6.5), 0.1% NaN3 and 25 mM NaCl in 90% H2O and 10% D2O.

NMR experiments were performed at 25°C on Varian Inova 800 MHz (UTMB & Rice Univ.) or 600 MHz (Rice Univ.) spectrometers with triple-resonance coldprobes (Rice Univ.) and proton frequencies were referenced to internal DSS. The 13C and 15N dimensions were referenced indirectly using frequency ratios. Sequence-specific backbone assignments (Fig. 1) were obtained using the 2D 1H, 15N-HSQC, 3D HNCACB (Sattler et al. 1999), 3D CBCA(CO)NH, and 3D HNCO (Ikura et al. 1990; Bax and Grzesiek 1993) experiments. Non-aromatic side chain assignments were obtained using the TOCSY-[1H, 15N]-HSQC, H(C)CH-TOCSY (Clore and Gronenborn 1994), H(CCO)NH and C(CO)NH (Sattler et al. 1999) experiments. Aromatic proton assignments were obtained from the (HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE experiments (Yamazaki et al. 1993). A NOESY-[1H, 15N]-HSQC experiment (Marion et al. 1989; Wüthrich 1986) provided several missing side chain assignments. The NMR spectra were processed in VNMRJ (Varian Inc.) or Felix2000 (Felix, Inc.) software.

Fig. 1
figure 1

Proton-nitrogen HSQC spectrum of DEN1-ED3. Backbone amide resonances are indicated by the one-letter code and the sequence number. The side chain imino correlations from ASN and GLN are connected by lines

Full size image

Extent of assignments and data deposition

For the backbone atoms, 95% of the resonances have been assigned. The missing backbone atom resonances consist of one alpha carbon (N60), three carbonyl carbons (M1, N60 and P83) and 11 amide nitrogen atoms, corresponding to the six prolines, the N-terminal residues M1 and D2, and exposed residues Q28, G40 and N60.

The non-exchangeable proton side chain resonance assignments are nearly complete (99%), but many of the exchangeable proton/carbon pairs, such as the lysine HζCζ pairs and the HIS29 Hδ1/Nδ1 and Hε1/Nε1 pairs were not assigned. The aromatic carbon atoms were not assigned. The 1H, 13C, and 15N chemical shifts have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under the BMRB accession number 15782.