Fig. 5: The nCoV400Fab inhibits the binding of N-CTD to viral RNA by blocking the potential RNA-binding surface of N-CTD. | Communications Biology

Fig. 5: The nCoV400Fab inhibits the binding of N-CTD to viral RNA by blocking the potential RNA-binding surface of N-CTD.

From: Structural basis of a human antibody targeting SARS-CoV-2 nucleocapsid protein dimerization domain and interfering with RNA-binding

Fig. 5

a Sequence alignment of SARS-CoV-2 genomic RNA (20910-21079) between BatCoV-RaTG13, SARS-CoV, BatCoV-HKU3, HCoV-OC43, and MERS-CoV. b Native PAGE of vRNA157 with varying molar ratios of N-CTD, in the absence or presence of nCoV400Fab at an equimolar ratio to N-CTD. Lane 1 contains no protein, and lane 2 contains only nCoV400Fab, while the subsequent lanes contain increasing amounts of N-CTD protein with molar ratios of 1, 2, 4, and 8 to vRNA157. The electrophoretic result was stained with ethidium bromide for nucleic acid detection (left panel), and then the same gel was stained with Coomassie Blue for protein visualization (right panel). c Competitive binding of nCoV400Fab to vRNA157 based on SARS-CoV-2 N-CTD. The biotinylated N-CTD was captured onto the streptavidin biosensors. The nCoV400Fab (upper panel) or vRNA157 (lower panel) as the first analyte, followed by nCoV400Fab mixed with vRNA157 as the second analyte. The competitive binding model was created with BioRender.com. d BLI analysis of the interaction between nCoV400 and the K257E mutant N-CTD of SARS-CoV-2. nCoV400 (5 μg/mL) was immobilized onto AHC2 biosensors and incubated with serial concentrations (3.125–100 nM) of the N-CTD K257E mutant protein. e BLI analysis of the interaction between nCoV400Fab and the N-terminally truncated N-CTD (ΔN-CTD, residues 264–364) under the same conditions as in panel (a). f Native PAGE analysis of the RNA-binding capacities of three N-CTD variants: N-terminally truncated N-CTD (ΔN-CTD, residues 264–364), K257E mutant, and wild-type N-CTD. In each assay, 2 μg of 55-nt vRNA was used as a fixed amount (defined as 1× for molar ratio calculations). Lane 1 contains vRNA only and lane 6 contains ΔN-CTD (264–364) only, while lanes 2–5 show increasing amounts of ΔN-CTD (264–364) (0.5, 1, 2, 4, and 8× relative to vRNA). In lanes 7–10, a fixed amount of vRNA was incubated with increasing amounts of K257E N-CTD mutant (1, 2, 4, and 8× relative to vRNA), while lane 11 contains only the K257E mutant. Lanes 12–15 show wild-type N-CTD incubated with vRNA at increasing protein-to-RNA molar ratios (1, 2, 4, and 8× relative to vRNA). Gels were stained with ethidium bromide for nucleic acid detection (left panels), followed by Coomassie Blue for protein visualization (right panels). The positions of free vRNA, free N-CTD variants, and bound complexes are indicated.

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