In a recent study published within the journal PLOS One, researchers present the crystal structure of an anti-non-structural protein 9 (nsp) specific nanobody in complex with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp9, which they termed nsp9COV19.

Study: Inside-out: Antibody-binding reveals potential folding hinge-points inside the SARS-CoV-2 replication co-factor nsp9. Image Credit: Kateryna Kon / Shutterstock.com

Background

Nsp9 is a small accessory factor that plays an important role within the coronavirus (CoV) replication and replication transcription complex (RTC). Furthermore, nsp9 associates with the N-terminal pseudokinase Nidovirales ribonucleic acid (RNA)-dependent RNA polymerase (RdRp) associated nucleotidyltransferase domain (NiRAN) of nsp12, which is produced inside host cells as a self-cleaving PP1ab polyprotein. 

Nsp12 comprises a vital viral RdRp which, jointly with nsp7 and nsp8, becomes the RTC core components. Nsp9 has a definite viral fold, RNA-binding protein, and key element for viral messenger RNA (mRNA) capping. This protein also recruits other proteins for viral 5’-mRNA capping, which is a vital aspect of viral replication, thus making nsp12 a potentially viable therapeutic goal. 

Within the SARS-CoV-2 capping model, the nsp9 and NiRAN domain act jointly as a polyribonucleotidyltransferase (PRNTase) with catalytic and adduct-accepting residues on different amino acid chains.

VHHs are camelid immunoglobulins (Ig)-derived nanobodies with variable heavy domains. Previous studies have shown that these nanobodies may very well be highly specific for nsp9. Likewise, several studies have identified many small molecules with affinity for nsp9COV19 and the potential to inhibit NiRAN engagement and, because of this, prevent SARS-CoV-2 RNAylation and capping. 

Concerning the study

In the current study, researchers describe the flexibleness of nsp9. To this end, nsp9 and anti-nsp9 VHH2nsp23 complex were purified and co-complexed on gel filtration. Thereafter, the crystal structure of this complex was examined at a resolution of two.4Å using X-ray diffraction.

After many rounds, the team finally built and refined a crystal structure of the VHH2nsp23-bound state of nsp9COV19, which had two copies of the nsp9COV19: VHH2nsp23 complex inside the asymmetric unit overlay.

Transformation-related protein 53 (Trp-53) was found to be a primary feature of the extensive antibody-binding interface of nsp9COV19, by which the CDR3-loop formed an prolonged β-sheet interaction. Nanobody binding triggered large-scale topological changes to the unique coronaviral fold of nsp9, which distorted all NiRAN-interacting elements of nsp9. 

Nonetheless, it stays unclear whether VHH binding artificially induced this binding state or whether it was an alternate pre-existing structural state or folding intermediate that remained trapped inside nsp9.

A Kleywegt plot was used to explore potential points of flexibility inside the nsp9 fold and compare sure and unbound states of nsp9COV19. Ramachandran distances were also mapped between each state onto the nsp9COV19 structure.

Results

The VHH2nsp23 binding site on nsp9COV19 was extensive, by which residues inside the VHHCDR2 and VHHCDR3 loops constituted 27% and 66% of the binding interface, respectively.

The VHHCDR3 residues 103YYFST107 ran antiparallel with the nsp9COV19 s5-strand and formed 4 β-sheet hydrogen (H)-bonds. These residues also established backbone-sidechain interactions with VHHThr-107. Moreover, the VHHCDR2 loop of VHH2nsp23 contributed substantial contacts that were entirely backbone-sidechain mediated. 

Trp-53 of nsp9COV19 was clamped betweenVHHMet-50 and VHHIle-52, each of that are hydrophobic residues. Together, eight epitope-specific H-bonds, multiple van der Waals interactions, and one salt bridge contributed to the anti-nsp9COV19specificity of VHH2nsp23.

After VHH binding, the authors noted that the native unliganded type of nsp9COV19 underwent some structural rearrangements. To this end, α-helix repositioning from outside the mini β-barrel and in between the s2-s3 and s4-s5 loops stationed 30Å apart was observed. Nevertheless, a lot of the β-structural elements of nsp9COV19 remained topologically nearby within the sure state. 

Other structural points inside the nsp9 fold that is likely to be serving as hinge points were also identified. Leu-42, for instance, resided and formed a normal straight β-strand within the VHH2nsp23-bound state, while within the apo state, Leu-42resided at a kink-point, thus allowing s3 to show away from αC. Largescale movement of αC was mainly facilitated by backbone shifts in residues Lys-92/Gly-93, which VHH2nsp23 directly contacted.

During viral 5’ mRNA-capping, the N-terminus of nsp9COV19 inserts into the nsp12 NiRAN domain, which is mediated through traits of the apo-nsp9 fold. These traits include the GxxxG interaction motif and N-terminus projecting into its lively site. 

This recent conformational state of the s2-s3 loop and realigned αC distorted elements engaged 

Conclusions

The binding epitope VHH2nsp23 was defined by resolving its crystal structure with nsp9COV19. As previously indicated in nuclear magnetic resonance (NMR) chemical peak perturbation assays, the VHH2nsp23 epitope gave the impression to be targeting residues inside the s4 and s5 strands of nsp9 and focused specifically upon Trp-53. Additional structural rearrangements gave rise to the VHH2nsp23-bound architecture of nsp9COV19.

This recent VHH2nsp23-bound state of nsp9 might appear physiologically irrelevant at present. Nonetheless, its formation allowed more freedom for the nsp9 N-terminus following the lack of the Tyr-cradle. This structure also had several structural points inside the nsp9 distinct fold that is likely to be acting as hinge points. Subsequently, despite occurring at a site farther away from the NiRAN-interaction site, these structural changes could disrupt a protein interface. 

This novel VHH-bound state of nsp9COV19 cryptically encasing the C-terminal α-helix of nsp9 needs to be investigated as a possible COVID-19 therapeutic, because it was shown to limit NiRAN engagement, which can prevent SARS-CoV-2 RNAylation and 5’mRNA capping.

Journal reference:

• Pan, Y., Chandrasekharan, I. R., Tennant, L., et al. (2023). Inside-out: Antibody-binding reveals potential folding hinge-points inside the SARS-CoV-2 replication co-factor nsp9. PLoS One.  doi:!0.1371/journal.pone.0283194