Structure of SARS-CoV-2 NCTD in open and closed conformations
The crystal structure of NCTD was resolved by molecular substitution at a resolution of 1.94 Å (Table 1). The asymmetric unit of the crystal contains two NCTD homodimers with an almost identical conformation (rmsd 0.14 Å). As described above, it folds into five α (α1 – α5) helices, two 310 (η1 – η2) helices, and two β (β1 – β2) chains, presenting the following sequence from N to C for the structural elements. : η1 – α1 – α2 – α3 – α4 – β1 – β2 – α5 – η217,18,19,20,21 (Fig. 1a and Fig. 1a supplementary). Two monomers intertwine through the β fork forming a β sheet of four antiparallels on one side of the dimer (Fig. 1). In contrast to the structures described above, the superposition of the subunits in our structure shows a ≈5.5 Å movement of the β fork (Fig. 1b). A subunit presents the β fork in an extended, unseen conformation that we called “open”. While the other subunit shows a β fork in a flexed conformation that we called “closed,” similar to other structures of this protein (Fig. 1b and supplementary Fig. 1b). The alternative conformation of the β fork is associated with the structural displacement of the loop between α1 and α2 (residues 280–283) (Fig. 1b).
Table 1 Collection of crystallographic data and statistics.FIG. 1: Crystal structure of SARS-CoV-2 NCTD in open and closed conformations.
a Cartoon representation of the NCTD dimer. Each monomer is blue and orange, respectively. The β fork and the loop connecting the α1 – α2 helices stand out in dark tones. Secondary structural elements and residues are numbered and labeled in order from N to C terminal: the symbol η corresponds to the helix 310; α to α-helix and β to β-chain. b Overlay of NCTD monomers. The common helical core is gray. c, d Detailed view of the opening (c) and closed (d) conformations. The side chain of key waste is shown on sticks with colored carbon atoms according to the monomer to which they belong. Hydrophobic and polar interactions are represented as dashed black lines. The C-terminal of the symmetric molecule (sym) is colored magenta. Symmetrical Pro residue is indicated by an apostrophe. The nitrogen and oxygen atoms are colored blue and red, respectively.
In the closed conformation, the β fork interacts with the C-terminal proline residue (P364) of a symmetry-related protein (Fig. 1c, di Fig. 1b supplementary). This interdimeric interaction between W330 and P364 by π-π stacking (face-to-face ring interaction) and by making hydrogen bonds with T325 and S327 (Fig. 1d), is a common feature with seven NCTD structures determined above. But surprisingly, our structure shows that in the open conformation, the side chain of the W330 residue rotates 180 ° toward the β fork making hydrophobic contacts with the side chain of the T325 and S327 residues (Fig. 1c). Therefore, our structure shows that in a side chain arrangement, the β fork changes from an open conformation to a closed conformation that could favor interdimeric interaction.
Previous SARS-CoV-2 NCTD structures showed that the β fork that does not interact with the symmetry-related molecule has an acetate (PDB 7C22) 20 or sulfate (PDB 6WZQ) 19 molecule that interacts with the side chain of W330 (Fig. 2a, supplementary b).
Structure of GTP-linked SARS-CoV-2 NCTD
From the structural data, we hypothesized that W330 could be a suitable residue for RNA recognition, as W330 could interact with the phosphate fragment, similarly to sulfate, or by stacking π- π with the nitrogenous base. To test our hypothesis, we attempted to co-crystallize NCTD individually with the oxinucleotides UTP, ATP, CTP, or GTP. Although crystals were obtained in all mixtures, only GTP was co-crystallized with NCTD. The structure of the NCTD-GTP binary complex was resolved into two different spatial groups, P21 and P1, with a resolution of 1, 8, and 2 Å, respectively (Table 1). Both crystal forms contain two protein dimers in the asymmetric unit. However, in the P21 space group there is a GTP molecule bound to one of the dimers, while in P1 the asymmetric unit contains two GTP molecules, each bound to a dimer (Fig. 2). The electron density map is well defined for the three GTP molecules, which exhibit temperature factors that increase from the guanine part to the phosphates (Fig. 2 and Fig. 3a supplementary), suggesting that the guanine is well anchored to protein and phosphates. they are more flexible. In fact, the β and γ phosphates of the P1 crystal show two alternative arrangements, one identical to the P21 crystal (Fig. 2). In both crystal forms, GTP binds to a fissure between the two subunits of the dimer, and adjacent to the β fork in the closed conformation (Fig. 3a). The crack corresponds to a cavity adjacent and perpendicular to W330, reducing the accessibility of the tryptophan to the solvent (Figure 3a and Supplementary Figure 3b).
FIG. 2: SARS-CoV-2 NCTD structures in GTP complex.
Cartoon representation of the two dimers contained in the asymmetric crystal unit of the P21 space group (a) and P1 (b). Each monomer is black and white for one dimer, and orange and blue for the second dimer. GTP is represented in sticks with green carbon atoms. The electronic density map 2Fo − Fc (p= 1) of the GTP is represented in blue. The guanine, ribose and phosphate fragments are labeled. The nitrogen, oxygen, and phosphorus atoms are colored blue, red, and orange, respectively. Temperature factors (B-factors) of the nitrogen and oxygen atoms of the nucleoside and the phosphorus atoms of the phosphates are given below.
FIG. 3: Crystal structure of SARS-CoV-2 NCTD in GTP complex.
a The top panel, the NCTD dimmer in complex with GTP is depicted in the cartoon. Each monomer is blue and orange, respectively. The β forks of the dimer are labeled and highlighted in dark tones. The GTP molecule is shown in sticks with its 2Fo − Fc electron density map (p= 1) in green. The close view of the GTP link site is shown below. The key waste side chain and the GTP molecule are shown in sticks with colored carbon atoms according to the monomer to which they belong. The H-bond interactions of the ligand are represented as dashed black lines. The C-terminal of the symmetric molecule (sym) is colored magenta with the residues indicated with apostrophes. The secondary structural elements are numbered and labeled in order from the N end to the C terminus. The nitrogen, oxygen, and phosphorus atoms are colored blue, red, and orange, respectively. b Fluorescence extinction (Fo / F) represented against increased acrylamide concentration for NCTD and NCTD-W330A proteins in the presence or absence of GTP. The trajectories are adjusted for linear regression. Statistical differences are indicated by asterisks (****p<0.0001, ns p> 0.05). c NCTD and NCTD-W330A thermal deployment curves in the presence or absence of GTP. The trajectories fit a sigmoid. The corresponding melting temperature (Tm) is indicated. d Thermophoretically quantified GTP binding. GTP is titrated to a constant amount of fluorescently labeled NCTD and NCTD-W330A. The affinity of union (Kd) is indicated. The error bar represents the standard error of the average of at least four experiments.
One side of the guanine ring establishes a T-shaped π-π (edge-to-face) interaction with the indole ring of W330 and hydrophobic interactions with K338 (Fig. 3a). The other side of the guanine ring is stacked on the guanidine group of R259, which makes hydrogen bonds with the OH- groups of the ribose ring (Fig. 3a). R259 and R262 are responsible for multiple hydrogen bonding contacts with β and γ phosphates (Fig. 3a). At the bottom of the fissure, the guanine part interacts with the M317 and the main chain of residues K338 and A336 (Fig. 3a). These interactions suggest high specificity for guanine, as other nitrogenous bases do not have as many favorable interactions as guanine (Supplementary Figure 4). For example, adenine does not have C6 oxygen that interacts with M317, K338, and A336 in the case of guanine (Supplemental Figure 4). All protein-ligand contacts are summarized in Supplementary Table 1. It is important to note that the C-terminal tail of the symmetry-related dimer also contributes to GTP binding, covering the crack as a cap, with Van der Waals and interactions hydrophobic between T362 ‘. and ribose and with π – π stacking between P364´ and W330 (Fig. 3a). The structure suggests that GTP binding favors β-fork exchange and interdimeric interaction.
Characterization of the GTP union
To confirm our structural results, we studied the binding of GTP in solution by tryptophan intrinsic fluorescence. The protein contains two tryptophan residues: W330, located in the β fork and exposed to the solvent, and W301 partially buried in the protein nucleus (Supplementary Figure 5a). NCTD shows a fluorescence peak profile with a maximum fluorescence emission centered at 340 nm that decreases simultaneously with increasing acrylamide concentration, confirming that tryptophan fluorescence can be extinguished (Figure 3b and Supplementary Figure 5b).
We also made the NCTD-W330A mutant, substituting W330 for alanine, and confirmed that the fluorescence of the remaining W301 can also be quenched (Fig. 3b). We determined the extinction of fluorescence in the absence or presence of 0.5 mM GTP. While GTP significantly decreased the NCTD extinction slope (from 6.31 ± 0.08 to 5.17 ± 0.09; p-val ****, n= 7 i n= 4), extinction of NCTD-W330A (1.99 ± 0.06; n= 4) is not affected by the presence of GTP (1.74 ± 0.06; n= 4), indicating that only …