Biochemical analysis of SARS-CoV-2 Nsp13 helicase implicated in COVID-19 and factors that regulate its catalytic functions

Figures

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  Figure 1. Biochemical analysis of RNA helicase activity by purified recombinant Nsp13 protein as a function of nucleotide and duplex length.A, purified recombinant Nsp13 protein (5000 ng) after final dialysis (see Experimental procedures), resolved by SDS-PAGE and detected by Coomassie staining. B, nucleoside triphosphate requirements for Nsp13 helicase activity. Nsp13 (2.6 nM) was incubated with a partial duplex (13-bp) RNA substrate (0.25 nM) and the indicated nucleotide (2 mM) and 5 mM Mg2+ for 15 min at 37 °C. Reaction products were analyzed by native polyacrylamide gel electrophoresis and quantified as described under Experimental procedures. C and D, Nsp13 helicase activity as a function of protein concentration under multiple-turnover conditions. C, representative native polyacrylamide gel analysis of products from Nsp13 reaction mixtures with a 13-bp RNA partial duplex substrate conducted in the presence of 2 mM ATP and 5 mM Mg2+ for 15 min at 37 °C. Filled triangle represents heat-denatured RNA substrate control. D, quantitative assessment of Nsp13 helicase activity as a function of protein concentration on increasing-length duplex RNA substrates, as indicated. Data represent the…

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  Figure 2. Nsp13 helicase activity as a function of ATP and Mg2+ concentration.A, quantitative assessment of helicase activity by Nsp13 (0.325 nM) as a function of ATP concentration at 1 mM MgCl2. B, quantitative assessment of helicase activity by Nsp13 (0.325 nM) as a function of MgCl2 concentration at 2 mM ATP. C, quantitative assessment of helicase activity by Nsp13 (0.325 nM) at the indicated ATP concentration in the presence of 1 mM MgCl2 or 5 mM MgCl2. D, quantitative assessment of helicase activity by Nsp13 (0.325 nM) as a function of ATP-MgCl2, fixed at a 1:1 molar ratio. All experiments were performed using a partial duplex 13-bp RNA substrate (0.25 nM) incubated with Nsp13 for 15 min at 37 °C. E, single-turnover kinetic analysis of Nsp13 helicase (0.1625 nM) as a function of RNA duplex length using reaction conditions of 2 mM ATP and 1 mM MgCl2. Quantitative assessment of Nsp13 helicase activity is shown. F, single-turnover kinetic analysis of reaction mixtures containing Nsp13 (0.1625 nM) and the 30-bp partial duplex RNA substrate (0.25 nM) using 2 mM ATP and either 1 mM MgCl2 or 5 mM MgCl2 incubated at 37 °C for the indicated time points.

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  Figure 3. Strand-specific effect of backbone discontinuity on Nsp13 helicase activity.A, depiction of the partial duplex RNA substrates with a polyglycol linker (PEG) in the top or bottom strand. B, quantitative assessment of Nsp13 helicase activity from a 15-min incubation at 37 °C with 1 mM MgCl2 and 2 mM ATP as a function of protein concentration on the backbone-modified substrates (0.25 nM) shown in (A). Data represent the average of at least three independent experiments with SD indicated by error bars. C, representative native polyacrylamide gel analyses of reactions products from Nsp13 reaction mixtures designed to assess sequestration by RNA partial duplex molecules containing the PEG linker in the top or bottom strand, as indicated. Nsp13 was preincubated with the specified RNA partial duplex molecules in the presence of 2 mM ATP and 1 mM Mg2+ followed by the addition of a radiolabeled forked duplex tracker substrate as described in Experimental procedures. Filled triangle represents heat-denatured RNA substrate control. D, quantitative assessment of Nsp13 helicase activity as a function of the indicated concentration of the competitor RNA partial duplex with PEG in the top or…

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  Figure 4. Chemical identity of nucleic acid translocating strand dictates Nsp13 helicase substrate preference.A, depiction of the partial duplex substrates with DNA or RNA in the top or bottom strand. B, quantitative assessment of Nsp13 helicase activity from a 15-min incubation at 37 °C with 1 mM Mg2+ and 2 mM ATP as a function of protein concentration on the nucleic acid substrates (0.25 nM) shown in (A). Inset shows the lower Nsp13 concentrations depicted in the larger graph. C and D, representative native polyacrylamide gel analyses of Nsp13 binding mixtures from Nsp13 reaction mixtures designed to assess binding of protein to DNA and RNA forked substrates conducted in the presence and absence of nucleotide. The indicated concentrations of Nsp13 were incubated with the specified forked duplex substrate (0.25 nM) for 15-min incubation at RT with no nucleotide or ATPγS (2 mM). E, quantitative assessment of Nsp13 binding to DNA (C) and RNA (D). Data represent the average of at least three independent experiments with SD indicated by error bars.

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  Figure 5. RNA/DNA chimeric substrates reveal that the chemistry of the preexisting 5ʹ single-stranded tail of the top strand is the key determinant for Nsp13 helicase activity on forked duplex substrates.A, depiction of the partial duplex substrates harboring chimeric RNA/DNA nucleotides in the 5ʹ tail of the top strand. B, quantitative assessment of Nsp13 helicase activity from a 15-min incubation at 37 °C with 1 mM Mg2+ and 2 mM ATP as a function of protein concentration on the nucleic acid substrates (0.25 nM) shown in (A). Data represent the average of at least three independent experiments with SD indicated by error bars.

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  Figure 6. Nsp13 disrupts the high-affinity interaction of streptavidin bound to biotinylated RNA in a unidirectional manner.A, streptavidin displacement as a function of Nsp13 concentration under multi-turnover conditions. Representative polyacrylamide gel analysis of Nsp13 enzyme titration in streptavidin displacement reactions incubated for 15 min at 37 °C in the presence of 1 mM MgCl2 and 2 mM ATP using a 39-mer RNA substrate (0.25 nM) with a biotin moiety at the 3ʹ end (A) or 5ʹ end (B). Biotinylated oligonucleotide was preincubated with 3 nM streptavidin monomer for 10 min, and reactions were initiated by the addition of 1 μM biotin immediately followed by the indicated Nsp13 concentration. C, quantitative assessment of Nsp13-catalyzed streptavidin displacement. Data represent the average of three independent experiments with SD indicated by error bars.

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  Figure 7. Nsp13-catalyzed streptavidin displacement as a function of MgCl2 concentration. Representative polyacrylamide gel analysis of Nsp13 titration in streptavidin displacement reactions incubated for 15 min at 37 °C using 39-mer RNA substrate (0.25 nM) with a 3ʹ biotin moiety in the presence of 2 mM ATP and 1 mM MgCl2 (A) or 5 mM MgCl2 (B). Biotinylated oligonucleotide was preincubated with 3 nM streptavidin monomer for 10 min and reactions were initiated by the addition of 1 μM biotin immediately followed by the indicated Nsp13 concentration. C, quantitative assessment of Nsp13-catalyzed streptavidin displacement. Data represent the average of three independent experiments with SD indicated by error bars.

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  Figure 8. Effect of MgCl2 on Nsp13’s binding to the RNA substrate. Nsp13 (12.8 nM) was incubated with the 16-bp RNA forked duplex substrate (0.25 nM) for 15 min at RT. A, binding mixtures were assessed by electrophoretic mobility shift assay, as described in Experimental procedures. B, quantitative analysis is shown. Data represent the average of three independent experiments with SD indicated by error bars.

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  Figure 9. Chemical energy requirements for Nsp13 to disrupt a high-affinity RNA–protein interaction compared with RNA helicase activity. Representative polyacrylamide gel analysis of Nsp13 enzyme titration using the indicated nucleotide (2 mM) in the presence of 1 mM Mg2+ for streptavidin displacement reactions using 3ʹ-biotinylated RNA substrate preincubated with streptavidin (3 nM monomer) for 10 min and initiated by addition of biotin (1 μM) and Nsp13 (A) or helicase reactions using 16-bp RNA partial duplex substrate (0.25 nM) (C). Note in (B), filled triangle represents heat-denatured RNA substrate control. In both helicase and streptavidin displacement experiments, reaction mixtures were incubated for 15 min at 37 °C. Data for streptavidin displacement (C) or RNA helicase activity (D) represent the average of three independent experiments with SD indicated by error bars.