Fig 1.
SARS-CoV-2 virion and model of structural proteins.
(A) Transmission electron microscopic image of a single SARS-CoV-2 viral particle (image credit: NIH, NIAID-RML, https://www.niaid.nih.gov/news-events/novel-coronavirus-sarscov2-images). (B) Enlarged 2D model of the viral membrane showing the four structural proteins: Spike–Spike glycoprotein, M–Membrane protein, E–Envelope protein, and N–Nucleocapsid phosphoprotein along with viral membrane and the RNA genome. (C) Domain organization of the full length N-protein showing structural regions as boxes (NTD and CTD) and the intrinsically disordered regions (IDRs) as a line. (D) Schematic model of the full length N-protein dimer formed through the CTD domains (the N-NTD is shown in brown and the N-CTD in dark brown).
Fig 2.
Solution structure of the SARS-CoV-2 N-NTD RNA binding domain.
(A) Backbone representation of the 40 converged structures of N-NTD obtained by NMR spectroscopy. (B) Cartoon representation of the lowest energy structure (structural elements are highlighted in color: α1-α2 helices in yellow, β1-(β2’-β3’)-β5 in green, and loops in gray) show the overall U-shaped antiparallel β-sheet platform (the palm) and a protruding β-hairpin (the basic finger). (C) The N-NTD molecular surface electrostatic potentials revealed a basic patch extended between the finger and the palm, with a positively charged surface shown in blue and negatively charged surface in red. (D) Topology diagram of the N-NTD and protein sequence displaying the secondary structural elements.
Fig 3.
NMR-based mapping and a model of the SARS-CoV-2 N-NTD:RNA complex.
(A) Representative regions from the 2D 15N/1H HSQC titration spectra illustrating the effects of addition of the RNA-7mer (green), 10mer (blue) and dsRNA (red) on the side-chain N-NTD amide signals (arginine side-chains are labeled along with NHε). The 50 μM 15N-labeled N-NTD protein construct was titrated with an increasing concentration of RNAs. Corresponding chemical shift perturbations (CSP) of N-NTD residues upon binding ssRNA 7mer (5′-CUAAACG-3′) in green, 10mer (5′-UCUCUAAACG-3′) in blue from viral genomic 5′ UTR containing the conserved transcriptional regulatory sequence (TRS), and a random dsRNA (RNA-7mer duplex, 5′-CACUGAC-3′ and 5′-GUCAGUG-3′) in red. (B) N-NTD:RNA complex. The RNA-10mer and dsRNA are shown as a cartoon representation (yellow) over the electrostatic surface of N-NTD shown in three orientations. (C) Cartoon representation of N-NTD highlighting all the available arginine and lysine residues in the interaction interface, shown as blue sticks, and the lower panel displays the ssRNA-10mer docked model in same orientation.
Fig 4.
Mutational analysis of N-NTD:RNA interaction.
(A) Binding curves N-NTD wild type and selected mutants (R92E, R107E, E174R, I94A, Y172A, R68E and Q163A) for RNA titrations obtained using the fluorescence anisotropy assay. Other panels display the zoom in view of the mutated residues showing hydrogen bonds between these residues and the RNA. A panel showing a plot comparing Kd values for the wild type and all mutants is also included. (B) Multiple sequence alignment of N-NTDs from SARS-CoV-2 and other selected coronaviruses, arrowheads highlight the residues selected for mutational analysis.
Table 1.
NMR Constraints and Statistics for the final set of structures.