Scientists explore guanine-specific pocket in SARS-CoV-2 nucleocapsid protein of therapeutic value against multiple coronaviruses

By Neha MathurReviewed by Aimee MolineuxJul 19 2022

In a recent study published in Communications Biology, researchers studied open and closed conformations of the C-terminal of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein termed the N^CTD. Additionally, they derived the crystal structure of the N^CTD in complex with guanine triphosphate (GTP).

Background

One-third of the SARS-CoV-2 genome contains open-reading frames (ORFs) for spike (S), membrane (M), envelope (E), N, and other accessory proteins. Of these SARS-CoV-2 proteins, N boosts the efficacy of its transcription and assembly. Its N^CTD binds genomic ribonucleic acid (gRNA) and interacts with SARS-CoV-2 M protein; hence, studies have implicated the N^CTD for interacting with a conserved packaging signal (PS) required for SARS-CoV-2 RNA packaging. However, previous studies have not elucidated the specificity of the PS, the RNA constituents of the N^CTD and their recognition process.

About the study      

In the present study, researchers described SARS-CoV-2 N^CTD structures showing its alpha-helical core with a protruding β-hairpin used for dimerization. Additionally, they characterized the SARS-CoV-2 N^CTD-GTP binary complex.

The authors hypothesized that a highly conserved tryptophan residue W330 in the N^CTD could be a suitable amino acid (AA) residue for RNA recognition. It could interact with phosphate moieties via π–π stacking with the nitrogen base. Further, the team characterized the GTP binding in solution using the intrinsic fluorescence of tryptophan.

To confirm that the fluorescence of the W301 AA residue was also quenchable, the team made a mutant N^CTD-W330A, replacing W330 with alanine. Further, they determined the quenching of the fluorescence in the absence or presence of 0.5 millimolar (mM) of GTP. They also performed differential scanning fluorimetry (DSF) experiments with N^CTD and N^CTD-W330A in the presence or absence of five mM GTP. Furthermore, the researchers used microscale thermophoresis (MST) to measure the binding affinity (K_d) for GTP of the N^CTD and N^CTD-W330A.  Lastly, they analyzed the binding of the N^CTD to an RNA oligonucleotide derived from the apical region of the stem-loop 5a (SL5a) of SARS-CoV-2 gRNA.

Study findings

The crystallographic asymmetric unit of SARS-CoV-2 N^CTD had two homodimers and five α-helices (α1–α5). A superimposition of its subunits showed a ≈5.5 angstroms (Å) β-hairpin movement. One subunit presented the β-hairpin in an extended conformation, termed 'open', and the other in a flexed conformation, termed 'closed'. The third β-hairpin conformation was implicated in the structural loop shift between AA residues 280 to 283 lying between α1 and α2. A 180° rotation of the side chain of the W330 AA residue towards the β-hairpin made hydrophobic interactions with the side chain of the AA residues S327 and T325, and that changed β-hairpin conformation from open to closed in favor of the interdimeric interaction.

Only the GTP co-crystallized with N^CTD , forming a binary structure resolved in two different space groups, P21, and P1, with both having two protein dimers in the asymmetric unit. In closed conformation, GTP was bound to a cleft between the dimer subunits and adjoining the β-hairpin. The cleft adjacent and perpendicular to W330 residue reduced the tryptophan accessibility to the solvent. The electron density map of the binary complex showed that the guanine (G) was anchored strongly to the N^CTD, and the phosphate moietes were more flexible. Notably, other oxynucleotides, such as uridine triphosphate (UTP), adenosine triphosphate (ATP), cytidine triphosphate (CTP), etc., did not co-crystallized with N^CTD.

W301 AA residue partly hidden in the protein core showed a fluorescence peak. Its maximum fluorescence emission occurred at ~340 nm and decreased in parallel with the increasing acrylamide concentration, confirming the tryptophan fluorescence was quenchable. The results showed that only GTP affected the accessibility of W330 while ATP or UTP did not. Moreover, the N^CTD presented a sigmoidal trajectory with the changes in temperature while the mutant remained unaffected.

The N^CTD and its mutant had a K_d value of 196 μM and 858 μM, respectively, in the presence of GTP. These values indicated the affinity of one nucleotide of a GTP molecule that binds RNA, not the synergistic effect of several nucleotide interactions. Phylogenetic studies have pointed to the conservation of SL5a-c across alpha- and beta-coronaviruses, including SARS-CoV-2. The N^CTD specifically recognize the SL5a apical sequence from SARS-CoV-2, supporting the notion that this protein domain might be that region of the N protein that recognizes specific features of coronavirus gRNA.

Further, the authors identified fifteen invariant residues in the SARS-CoV-2 N^CTD among human coronaviruses. Three of these residues, R259, R262, and F274, belonged to GTP binding pocket and R259 and R262 were responsible for multiple hydrogen-bonding contacts with the β- and γ-phosphates. Since this GTP-binding pocket remains conserved across SARS-CoV-1, SARS-CoV-2, Middle Eastern Respiratory Syndrome (MERS), and NL63, there is a possibility of using the GTP binding pocket as a therapeutic target. In fact, it is feasible to design specific compounds against the SARS-CoV-2 N^CTD.

Conclusions

The current crystal structure and biochemical assays revealed a specific interaction between the G, a nucleotide enriched in the PS regions of alpha- and beta-coronaviruses, and W330 AA residue. In addition, SARS-CoV-2 derived RNA hairpin SLs showed a preferential interaction of the N^CTD to G-containing RNA oligonucleotides and the loss of specificity in the mutant W330A. Since these N^CTD interactions facilitate the SARS-CoV-2 assembly process, the study results indicate that the GTP-binding pocket in the viral N protein might help design SARS-CoV-2 assembly inhibitors.