Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms – μs) dynamics in the C-terminal helix at the dimer interface

Buchko, Garry W.; Zhou, Mowei; Craig, Jonathan M.; Voorhis, Wesley C. Van; Myler, Peter J.

doi:10.1007/s12104-020-09992-1

Cited by 17 publications

(47 citation statements)

References 28 publications

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“…Notably, one of the two studies (Buchko et al) 17 revealed that Nsp9 exists predominantly as a dimer at protein concentrations >100 μM, in excellent agreement with our size exclusion data (Figure 2 ). However, although not observed at concentrations used in our NMR experiments, Buchko et al suggested that dissociation into monomers could still play a functional role for these proteins, for example through binding to other proteins (such as Nsp12 in the context of RTC formation; see next section).…”

Section: Discussionsupporting

confidence: 91%

“…A very recent study has already established that Nsp9 exists as a dimer in solution at concentrations as low as 1 μM with dissociation into monomers in the nM range. 17 After expression, we purified Nsp9 (for sequence information refer to Figure 1 ) for the purpose of NMR spectroscopy, size exclusion chromatography (SEC) and biolayer interferometry (BLI) experiments at concentrations between 50 and 2600 μM. To confirm that Nsp9 is indeed a dimer in solution at the lowest concentration used (50 μM), we carried out SEC experiments with a set of protein standards of known molecular weight (Figure 2 ).…”

Section: Resultsmentioning

confidence: 99%

“…Using standard triple resonance approaches we have been able to assign the backbone chain atoms of 77 out of the 113 residues of Nsp9 (red boxed in Figure 1A and listed in Table S1 ) and have identified 27 out of 29 residues that exhibit significant chemical shift changes upon binding of ssDNA/RNA (Figure 4 ). During the course of our work, another two groups published near‐complete solution‐state backbone assignments of Nsp9, 17 , 24 one of which was available in the BMRB database (50513), along with our data (50725). Overall, our assignments are in good agreement with the published data with only minor differences observed, which could be explained by the different pH value used in our final NMR buffer (6) compared to the other two studies (7).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…[ 5–7 ] This result is compatible with the recently reported NMR studies of SARS‐CoV‐2 Nsp9. [ 11,12 ] 2 H, 15 N, 13 C triply‐labeled and selectively labeled samples were in fact necessary to improve coherence transfer in 3D experiments for backbone assignment. [ 11 ] Our 3D data confirmed poor coherence transfer for both wild‐type Nsp9 and mutated triSer‐Nsp9.…”

Section: Resultsmentioning

confidence: 99%

NMR‐Based Analysis of Nanobodies to SARS‐CoV‐2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID‐19

et al. 2021

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Following the entry into the host cell, SARS‐CoV‐2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B‐cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID‐19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild‐type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication.

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“…This was different for the subsequently eluting dimeric fl nsp9 fraction, which had a A260/280 ratio of below 0.7 and could be concentrated to >5 mg/ml (Table 2). The excellent protein quality and stability are supported by the available HSQC (Figure 2), and a near-complete backbone assignment (Dudas et al, 2021).…”

Section: Nsp7 and Nsp8mentioning

confidence: 82%

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

Altincekic

Korn

Qureshi

et al. 2021

Front. Mol. Biosci.

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The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

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Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms – μs) dynamics in the C-terminal helix at the dimer interface

Cited by 17 publications

References 28 publications

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

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

NMR‐Based Analysis of Nanobodies to SARS‐CoV‐2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID‐19

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

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