Figure 1.
The structure packaging of the SARS-CoV N protein and the mutation sites for probing transient molecular interaction.
(A) The domain organization of the N protein. (B) Side view of the crystal structure of 24-mer CTD molecules showing the helical packing. (C) Electrostatic surface of the crystal packing of the CTD 24-mer. Positive charges are colored in blue and negative charges are colored in red. Yellow and orange ribbons represent viral RNA strands wrapping around the helical oligomer structure. (D) A close up side view of a CTD octamer molecule shown in between of the two dashed lines on Fig. 1B. The corresponding top view is shown on (E). Spatial locations of the mutated sites are shown in orange (T264), magenta (Q290), green (R294), red (R320) or cyan (S328) in (B), (D) and (E).
Figure 2.
The CTD forms transient oligomers in solution.
(A) Chromatograms of wild-type (wt) and mutant CTDs after disulfide trapping show that some CTD mutants are forming oligomers while others are not, as evidenced by the appearance of peaks H (for high-order oligomer) and T (tetramer). Relative intensities were normalized to the major peak representing dimeric CTD (peak D). (B) Bar representation of the oligomer/dimer ratios of the CTD mutants estimated by area integration of chromatogram profiles shown in (A). The amount of oligomer is the sum of tetramer and higher oligomers. Error bars represent fitting errors calculated from the residual between fitted and experimental elution profiles. Wild-type and the T264C mutant showed negligible oligomer formation. (C) SEC profiles of NTD mutants after disulfide trapping treatment. Each reaction contained 3 mg/ml of total protein with each mutant comprising half of the concentration. Reaction conditions were the same as those used for the CTD mutants. Only very little dimer (D) formation was observed for both NTD mutation pairs with most of the protein remaining in the monomer (M) form.
Figure 3.
Increasing salt concentration leads to increased CTDQ290C self-association.
(A) Chromatogram of CTDQ290C under different salt concentrations. The systematic shift is caused by nonspecific interaction of the protein with the Superdex column matrix in the absence of salt. (B) Oligomer percentage increases with increasing salt concentration in a semi-log manner. Data points (♦) and error bars represent the average and standard error of three independent experiments. (C) Overlay of 15N-edited HSQC spectra of CTDQ290C in 50 mM NaCl (black) and 500 mM NaCl (red). Resonance patterns between the two are virtually identical, indicating that no structural changes occur at different salt concentrations.
Figure 4.
CTD acts as an oligomerization domain of DD.
(A) Representative SDS-PAGE strips of DDQ290C mutant after disulfide trapping treatment. Number on the left marks the molecular weight of the markers. The arrow denotes the position of disulfide-linked di-domain on the gel. Unlike CTDQ290C, DDQ290C did not exhibit salt-dependent oligomerization as quantified in (B). The bars represent the average results from three independent experiments.
Figure 5.
Effect of phosphomimicking negative charges on di-domain self-association.
(A) Representative SDS-PAGE strips from disulfide trapping experiments of di-domain mutated at sites known to be phosphorylated by GSK3 and SRPK1. The arrow denotes the position of disulfide-linked di-domain on the gel. Strips from other mutants are included in the supplemental material. (B) Quantification of the self-association potential of all phosphomimicking mutants from this study. Bars represent average results from three independent experiments.