Structure insights, thermodynamic profiles, dsDNA melting activity, and liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid N-terminal domain binding to DNA
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Abstract
The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD), either with or without the SR-rich motif (SR), upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on specificity for N-NTD/N-NTD-SR interaction with TRS, including an unfavorable energetic contribution to binding along with hydrogen bonds between the triple-thymidine (TTT) motif in the dsTRS and β-sheet II due to the defined position and orientation of the DNA duplex, a well-defined pattern (ΔH > 0 and ΔS > 0 for ssTRS, and ΔH < 0 and ΔS < 0 for dsTRS) for the thermodynamic profile of binding, and a preference for TRS in the formation of liquid condensates when compared to a non-specific sequence. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.
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SciScore for 10.1101/2021.07.21.453232: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Recombinant DNA Sentences Resources Protein expression and purification: The gene sequences encoding the SARS-CoV-2 (NC_045512.2) nucleocapsid protein N-terminal domain (N-NTD; residues 44–180) and the N-NTD containing the C-terminal SR-rich motif (N-NTD-SR; residues 44–212) were codon-optimized, synthesized, and subcloned into pET28a by GenScript (Piscataway, USA). pET28asuggested: RRID:Addgene_114156)Briefly, pET28a-N-NTD and pET28a-N-NTD-SR were transformed into Escherichia coli BL21 (DE3). pET28a-N-NTDsuggested: NonepET28a-N-NTD-SRsuggested: NoneSoftware and Algorithms Sentences Resources The Kd values were calculated from the … SciScore for 10.1101/2021.07.21.453232: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Recombinant DNA Sentences Resources Protein expression and purification: The gene sequences encoding the SARS-CoV-2 (NC_045512.2) nucleocapsid protein N-terminal domain (N-NTD; residues 44–180) and the N-NTD containing the C-terminal SR-rich motif (N-NTD-SR; residues 44–212) were codon-optimized, synthesized, and subcloned into pET28a by GenScript (Piscataway, USA). pET28asuggested: RRID:Addgene_114156)Briefly, pET28a-N-NTD and pET28a-N-NTD-SR were transformed into Escherichia coli BL21 (DE3). pET28a-N-NTDsuggested: NonepET28a-N-NTD-SRsuggested: NoneSoftware and Algorithms Sentences Resources The Kd values were calculated from the fitting process by nonlinear least-squares optimization using Levenberg–Marquardt interactions in the software OriginPro 2021. OriginProsuggested: NoneThe tool g_cluster of GROMACS package (40) was used to perform a cluster analysis in the 2.0 μs MD trajectories of the N-NTD:DNA complexes. GROMACSsuggested: (GROMACS, RRID:SCR_014565)The structural representations of the constructed models were displayed using PyMOL (42). PyMOLsuggested: (PyMOL, RRID:SCR_000305)The working concentration of the fluorescent dye DAPI (BioRad, #135-1303, stock in Milli-Q water) was 0.25 μg/mL and the emission recorded using a LED light fluorescent cube (excitation centered at 357/44 and emission at 447/60 nm). BioRadsuggested: NoneAll quantification and image processing steps were performed in Fiji. Fijisuggested: (Fiji, RRID:SCR_002285)Data were analyzed with GraphPad Prism (version 8.1.1; GraphPad Software). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Data were analyzed with GraphPad Prism (version 8.1.1; GraphPad Software). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 48, 50, 51 and 47. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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