Flavivirus capsid is a dimeric alpha-helical protein

Abstract

The capsid proteins of two flaviviruses, yellow fever virus and dengue virus, were expressed in Escherichia coli and purified to near homogeneity suitable for biochemical characterization and structure determination by nuclear magnetic resonance. The oligomeric properties of the capsid protein in solution were investigated. In the absence of nucleic acid, both proteins were predominantly dimeric in solution. Further analysis of both proteins with far-UV circular dichroism spectroscopy indicated that they were largely alpha-helical. The secondary structure elements of the dengue virus capsid were determined by chemical shift indexing of the sequence-specific backbone resonance assignments. The dengue virus capsid protein devoid of its C-terminal signal sequence was found to be composed of four alpha helices. The longest alpha helix, 20 residues, is located at the C terminus and has an amphipathic character. In contrast, the N terminus was found to be unstructured and could be removed without disrupting the structural integrity of the protein.

FIG. 1.

Expression and purification of DEN2C and YFC in E. coli. (Left panels) Optimized expression for DEN2C (A) and YFC (B). Shown are results of Coomassie blue-stained SDS-PAGE of uninduced (−IPTG) and induced (+IPTG) whole-cell extracts of BL21(DE3)[RIL] expressing either DEN2C or YFC protein. (Right panels) Coomassie blue-stained SDS-PAGE of DEN2C and YFC purification. Shown are insoluble (InSol) and soluble (Sol) protein extracts following cell lysis by a French pressure cell and clarification by centrifugation, pooled fractions containing partially purified capsid protein following cation-exchange chromatography with a HiTrap SP column (SP), and purified and buffer-exchanged capsid following size-exclusion chromatography with a Superose 6 column (Sizing). Arrows indicate the presence of the C protein.

FIG. 2.

Cross-linking of DEN2C and YFC. Shown are results of Coomassie blue-stained SDS-PAGE of purified DEN2C (A) and YFC (B) subjected to cross-linking (+ lanes) with the lysine-specific cross-linking reagent DSS. DSS cross-linking of DEN2C results in the appearance of dimers and trace amounts of trimers. DSS cross-linking of YFC results in dimers and tetramers. The efficiency of DSS cross-linking, estimated by the disappearance of monomer in cross-linking reactions, was routinely higher for YFC. M, 10-kDa protein molecular mass markers.

FIG. 3.

Analytical ultracentrifugation of flavivirus C proteins. (A and B) Equilibrium sedimentation analysis of DEN2C (A) and YFC (B). The change in protein concentration versus radial position is shown for each protein at two different speeds. Circles indicate the observed experimental data; solid lines indicate calculated data. Residuals are also shown. (C) Velocity sedimentation analysis of DEN2C and YFCN. The distribution of molecules with a given sedimentation coefficient is shown for DEN2C (solid line) and YFCN (dotted line). The majority of DEN2C and YFCN sediments as a single species. Additional oligomeric species with larger sedimentation coefficient values can be seen in YFCN.

FIG. 4.

Flavivirus capsid is an alpha-helical protein. (A) Comparison of the secondary structure predictions of YFC and DEN2C. Secondary structure predictions for both proteins [YFC (pred) and DEN2C (pred)] were generated individually with ProteinPredict. The secondary structure predictions that are depicted are not sequence aligned. The secondary structure of DEN2C determined by a consensus of C^α, C^β, H^α, and C′ chemical shift indexing [DEN2C (CSI)] is shown below the predicted secondary structures of YFC and DEN2C and is based on the data shown in panel B. Non-alpha-non-beta secondary structures are represented by a straight line. Alpha-helical secondary structures are represented by cylinders. The four alpha helices of DEN2C are indicated by the roman numerals I to IV. The underlined residues within the amino acid sequence of DEN2C indicate the internal hydrophobic sequence. The C-terminal signal sequence is not shown. (B) NMR analysis of the DEN C protein. The differences between observed sequence-specific chemical shift values and the corresponding predicted sequence corrected random-coil values were plotted against the corresponding residue number within DEN2C. Plots of C^α (top) and H^α (bottom) are shown.

FIG. 5.

HSQC spectrum of DEN2C. The folded state of DEN2 was accessed by NMR (see text). (A) The boxed region indicates a region of the HSQC spectrum that contains several peaks corresponding to residues within DEN2C that have random-coil chemical shift values. These residues are thought to be disordered in the solution structure of DEN2C. (B) Comparison of the boxed region in panel A with the same region within the HSQC spectrum of DEN2CΔ1-13, in which the first 13 amino acids have been removed.