A distinct ssDNA/RNA binding interface in the Nsp9 protein from SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel, highly infectious RNA virus that belongs to the coronavirus family. Replication of the viral genome is a fundamental step in the virus life cycle and SARS-CoV-2 non-structural protein 9 (Nsp9) is shown to be essential for virus replication through its ability to bind RNA in the closely related SARS-CoV-1 strain. Two recent studies revealing the three-dimensional structure of Nsp9 from SARS-CoV-2 have demonstrated a high degree of similarity between Nsp9 proteins within the coronavirus family. However, the binding affinity to RNA is very low which, until now, has prevented the determination of the structural details of this interaction. In this study, we have utilized nuclear magnetic resonance spectroscopy (NMR) in combination with surface biolayer interferometry (BLI) to reveal a distinct binding interface for both ssDNA and RNA that is different to the one proposed in the recently solved SARS-CoV-2 replication and transcription complex (RTC) structure. Based on these data, we have proposed a structural model of a Nsp9-RNA complex, shedding light on the molecular details of these important interactions.

Keywords: COVID-19; Nsp9; RNA; SARS-CoV-2; coronavirus.

FIGURE 1

Coronavirus family Nsp9 sequence information. (A) Primary sequence of Nsp9 with structural features indicated on top (the α1 helix is part of the monomer‐monomer interface). Residues in red boxes were successfully assigned in the HSQC spectra. (B) Sequence alignment of the Nsp9 proteins from SARS‐CoV‐2, SARS‐CoV‐1, MERS‐CoV, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), human coronavirus 229E (HCoV‐229E), mouse hepatitis virus (MHV), and avian infectious bronchitis virus (IBV). Residues in bold and red boxed residues indicate residues that exhibit significant chemical shift changes (“binding residues”) and residues involved in close contacts with RNA as revealed from molecular docking calculations, respectively, whereas gray areas indicate high sequence conservation

FIGURE 2

Size exclusion chromatography of Nsp9 confirming dimeric state in solution. (A) Size exclusion chromatography (SEC) of Nsp9 as well as five different proteins standards (β‐amylase β‐AML, alcohol dehydrogenase ADH, carbonic anhydrase CA, bovine serum albumin BSA, cytochrome C) run under the same conditions. (B) Calibration curve with the five standards and Nsp9 (denoted as red dot)

FIGURE 3

Secondary‐structure prediction of Nsp9 based on backbone chemical shifts. TALOS‐N prediction of α‐helices (red) and β‐sheets (blue) in the Nsp9 structure based on NMR backbone chemical shift data. The top of the Figure depicts a schematic of the secondary structure elements as observed in the X‐ray structure (PDB 6WXD)

FIGURE 4

NMR analysis of Nsp9 protein in complex with ssDNA and RNA. Sections ofN‐HSQC spectrum of Nsp9 in the absence (gray) and presence (1:1 mixture, red) of oligo(dT)₂₃ (A) and oligo (dU)₂₃ (B), respectively. Weighted backbone chemical shift changes of HN and N atoms for Nsp9 upon binding to ssDNA (C) and RNA (D). Residues exhibiting changes larger than the average (“binding residues”) are colored in red. Note the similarity in the binding profile between ssDNA and RNA

FIGURE 5

NMR reveals distinct interaction surface. Nsp9 dimer structure (taken from PDB 6WXD) in surface and cartoon representation (colored in light blue). The RNA binding residues as determined in Figure 4C,D were mapped onto the deposited crystal structure. (red). Note that a distinct binding interface exists on one side of the dimer structure

FIGURE 6

Structural models of Nsp9‐RNA complex structures calculated using HADDOCK. (A) Surface and cartoon representation of an Nsp9‐RNA complex model structure (containing one Nsp9 monomer) calculated using the binding residues (colored in red) as determined in Figure 4C,D in HADDOCK (Nsp9 is colored in light blue, RNA in dark blue). (B) Detailed view of the surface‐exposed aromatic residues F40 and Y66 (stick representation; colored in green) that are located within the binding interface

FIGURE 7

Mutational analysis revealing critical RNA binding residues of Nsp9. (A–D) Average (±SE) steady‐state equilibrium BLI values and fits to the steady‐state binding model from three independent binding experiments of wild‐type Nsp9 and mutants (F40A, R55A, and Y66A). Adjusted R ² as well as dissociation constants are shown. (E) Summary of dissociation constants (± SE) for Nsp9 and alanine mutants, for binding to oligo(dU)₂₃, as measured by BLI. (F) ¹H NMR spectra of Nsp9 alanine mutants show that each is correctly folded. Spectra were recorded at concentrations of between 200 and 1000 μM at 25°C

FIGURE 8

Refined HADDOCK model of Nsp9‐RNA complex. (A) Surface and cartoon representation of an Nsp9‐RNA complex model structure calculated as in Figure 6 with the addition of restraints to achieve base‐stacking between F40 with the corresponding RNA base (Nsp9 is colored in light blue, RNA in dark blue). (B) Same as in A with the protein (light gray) and the RNA (dark blue) shown as cartoon and stick representation, respectively. (C) Electrostatic potential (blue = positive, red = negative) of RNA binding interface with positively charged binding residues indicated (top) as well as detailed view (as stick and cartoon representation) of П‐ П stacking interaction between F40 and U8 (middle) and the location of the two electrostatic residues R55 and K92 (colored in green; bottom)

FIGURE 9

The RNA binding interface of Nsp9 within the replication and transcription complex (RTC). (A) Structure of the RTC taken from PDB 7CYQ with Nsp9 colored in gray and Nsp7/8/12/13 proteins in salmon (cartoon representation). (B,C) Detailed view of RNA interaction surface as proposed in (B) as opposed to in this study (C). Nsp9 is shown as surface representation (light blue) and the RNA binding site is colored in red. Note that the proposed binding site (indicated by the arrow) differs from the one determined in our structural modeling