Solution structure and neutralizing antibody binding studies of domain III of the dengue‐2 virus envelope protein

Flaviviruses are small (50 nm) positive-strand RNA viruses that contain a lipid-bilayer membrane. Forty species of the flavivirus family have been associated with human diseases and most of them are transmitted to their vertebrate hosts by infected mosquitoes or ticks.1 Among these, yellow fever (YF), Japanese encephalitis (JE), tick-borne encephalitis (TBE), and dengue (DEN) are the most important viral arboviruses in the world. DEN viral infections affect more than 100 million populations per year. There has been a marked increase of this disease over the last decades associated with significant mortality.2 Like other flaviviruses, the virion of DEN contains three structural proteins—a 12 kD nucleocapsid or core protein (C), a 8 kD nonglycosylated membrane protein (M), and a 53 kD glycosylated envelope protein (E), as well as seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).3 The E protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in viral attachment, fusion, penetration, hemagglutination, host range and cell tropism, and virus virulence and attenuation.1 The three-dimensional structures of the DENV-2, DENV-3, and TBEV E proteins have been determined by X-ray crystallography.4-6 The X-ray structures reveal that the E protein is composed of three discernible domains, Domain I, II, and III. The solution structures of the Domain III of JEV, LGTV, WNV, and DENV-4 envelope proteins have been studied recently.7-11 Monoclonal antibodies that bind to Domain III of DENV are found to be the most efficient blockers of virus adsorption to vero cells.12 However, no detailed spatial configuration of the epitopes of Domain III of DENV E protein has been provided. In here we have determined the solution structure of the domain III of the DENV-2 E protein. Moreover, we have identified the neutralizing epitopes of the DENV-2 Domain III to its specific neutralization antibody (mAb 137-22) by site-directed mutagenesis. Our results provide a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against DEN viruses. The DNA fragments encoding residues 288–397 of the Domain III of DEN virus Type 2 envelope protein were amplified by PCR. The amplified PCR products were subcloned into pET-blue 2 vector. Plasmids encoding DENV-2 Domain III were introduced into E.coli strains BL21(DE3), and transformants were cultured overnight at 37°C in SOB medium supplemented with ampicilin (100 μg/mL). The uniform 15N-labeled and/or 15N/13C-labeled samples were expressed in cells grown in M9 minimal media containing 15NH4Cl and/or (U-13C) glucose. The cell lysates were centrifuged and the supernatant from the centrifugation was applied to Ni-NTA column according to the protocols provided by the manufacturer (QIAGEN) at 4°C. Fractions containing the DENV Domain III were pooled, concentrated, and added to a 1.6 cm × 60.0 cm Sephacryl S-100 column (Pharmacia). Finally, the protein was eluted with NMR buffer containing 50 mM K2HPO4 (pH 6.5), 100 mM NaCl, 10 mM NaN3, and 0.1 mM EDTA. Purity of the protein was confirmed by HPLC and mass spectroscopy. The final protein concentration for NMR study was 0.6 mM in 90%H2O/10%D2O. All NMR data used in the structural analysis were acquired with Bruker Avance 600 or 500 MHz spectrometers equipped with triple-resonance probes at 25°C. 1H NMR data were referenced to the 1H resonance frequency of DSS; 13C and 15N resonances were referenced indirectly by multiplying the proton frequency by 0.25144953 for 13C and 0.101329118 for 15N.13, 14 The NMR experiments performed included 2D 1H-15N HSQC, 1H-13C HSQC, 3D 15N-NOESY-HSQC, HNCO, HN(CA)CO, HN(CO)CA, HNCA, CBCA(CO)NH, and HNCACB for backbone assignments, 15N-TOCSY-HSQC, HCC(CO)NH-TOCSY, HCCH-TOCSY, HCCH-COSY, HBHA(CO)NH for side chain assignments.15 All spectra were processed with the program XWIN-NMR 2.6 and NMRPipe and analyzed using NMRView 5.04. NOE-based distance restraints were collected from analysis of 3D 15N-edited NOESY-HSQC and 13C-edited NOESY-HSQC spectra recorded with mixing time of 150and 80 ms, respectively. The ϕ and ψ angles were obtained based on 3JHNHα coupling constants derived from HNHA experiment and predicted from TALOS.16 Hydrogen bond restraints were included in calculations only if the amide protons were slowly exchanging and if the β-strand interstrand NOE cross-peaks were observed. The structure calculations were carried out using X-PLOR 3.85117 program on a SuSE Linux 7.3 PC. The best 20 lowest energy structures were further analyzed with MOLMOL18 and PROCHECK-NMR.19 The coordinates of both the representative structure and the family of structures have been deposited at the Brookhaven Protein Data Bank (access number 2JSF). The percent assignment of backbone 1H, 15N, 13C, and 13CO resonances for the 109 residue DENV-2 Domain III is 96% and for side chain resonances is 91%.20 No signals were detected for the backbone amide protons of residues S297, Q315, I338, K343, V346, G348, and K393. A total of 1619 NOE-derived distance constraints, including 500 intraresidue, 422 sequential, 105 medium (1 < i − j < 5), 466 long-range (i − j ≥ 5) and 52 hydrogen bond distance restraints in conjunction with 74 backbone dihedral angles are used in the structure calculations. Multiple sequence alignments with secondary structural elements for the Domain III of the envelope proteins from various flaviviruses are shown in Figure 1(A). The overlay of the backbone atoms for the 20 lowest energy structures and the ribbon diagram of the lowest energy structure of DENV-2 Domain III are shown in Figure 1(B,C). The energetic and structural statistics are listed in Table I. The RMSD calculated from the averaged structured region for DENV-2 domain III is 0.63 Å for the backbone heavy atoms (N, C, and Cα) and 1.27 Å for all heavy atoms. (A) A structure-based multiple sequence alignment of the Domain III of JEV, WN, Dengue, Dengue-2, Dengue-3, Dengue-4, YF, and TBEV. Secondary structural elements are shown above the alignment. Black boxes denote neutralizing epitopes mapped by chemical shift changes of the JEV Domain III/mAb E3.3 complex.7 Red box denotes the sequence variations between the β4-β5 loop. Residues I335, I357, and V382 studied by site-directed mutagenesis are shown in red dots above the sequence alignment. (B) A stereoview of the backbone superimposition of the final 20 structures. (C) Ribbon diagram of the lowest energy structure of DENV-2 Domain III. The solution structure of DENV-2 Domain III forms a β-barrel similar to the Domain III of the other flaviviruses (see Fig. 1). However, there are localized differences between these proteins. Formation of an extra β strand (β5a) is found in the X-ray structure of DENV-2 Domain III4 but is not found in the current solution structure and the X-ray and NMR derived TBEV, DENV-3, DENV-4, WNV, JEV, LGTV, and OHFV Domain III structures.11 This region shows considerable sequence variation among the Domain III of flaviviruses [Fig. 1(A)]. However, the exact function of this region is not known. Strands β3 and β6 are found in the current DENV-2 Domain III structure as well as in the other X-ray and NMR-derived Domain III structures. However, they are not observed in the JEV and WNV Domain III structures.11 These β strands, except strand β5a, are all located in the loop regions on the top of the β-barrel. The structural differences may be attributed to the crystal or solution conditions or to the variable families of flaviviruses.11 Previously, the neutralizing antibody binding epitopes of the JEV Domain III are found to locate on the top portion of the β-barrel including residues in the N-termini and the loops.7 Similar results have also been found in the Domain III/antibody complex structure of WNV.21 Although the Domain III structures of the four DEN viruses display similar overall fold, each virus posses an unique electrostatic surface on the top portion of the β-barrel.11 To investigate the neutralizing antibody binding epitopes of DENV-2 Domain III, we have performed site-directed mutagenesis and western blot analysis on residues I335, I357, and V382 with a DENV-2 Domain III-specific neutralizing antibody mAb 137-22.22 Residues 335 and 382 are located in the β3-β4 and β8-β9 loops and residue 357 is located in the β6 strand [Fig. 2(B)]. All of these residues are located on the top portion of the β-barrel but have not been reported to be involved in the neutralizing antibody binding. Based on the western blot analysis, the I335A, I357A, and V382A mutations all exhibit significant reductions of binding to the neutralizing antibody mAb 137-22 [Fig. 2(A)]. The site-directed mutagenesis results correlate well with the previous mapped epitopes on the JEV and WNV domain III. (A) Western blot analysis of mAb 137-22 binding to the wild-type, I335A, I357A, and V382A proteins. The JEV Domain III is used as a control. (B) The structural location of residues performed in the site-directed mutagenesis and western blot analysis experiments. Residues 337 and 382 are located in the β3-β4 and β8-β9 loops and residue 357 is located in the β6 strand. In conclusion, NMR structural studies and site-directed mutagenesis were performed to determine and characterize the solution structure and neutralizing epitopes of the DENV-2 Domain III. Our results provide a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses. We would like to thank Dr. Yi-Ling Lin (Academia Sinica) for providing us the DNA strain of the DEN domain III. This study was carried out on the 600 MHz NMR spectrometer at the Regional Instrument Center at Hsinchu, National Science Council, Taiwan.