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New insights into complex formation by SARS-CoV-2 nsp10 and nsp14

Sele, Céleste LU ; Krupinska, Ewa LU ; Andersson Rasmussen, Anna LU ; Ekström, Simon LU ; Hultgren, Lucas LU ; Lou, Jiaqi ; Kozielski, Frank LU ; Fisher, S Zoë LU and Knecht, Wolfgang LU (2024) In Nucleosides, Nucleotides & Nucleic Acids
Abstract

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139... (More)

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10-nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone.

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organization
publishing date
type
Contribution to journal
publication status
epub
subject
in
Nucleosides, Nucleotides & Nucleic Acids
pages
15 pages
publisher
Taylor & Francis
external identifiers
  • scopus:85186909396
  • pmid:38422227
ISSN
1525-7770
DOI
10.1080/15257770.2024.2321600
language
English
LU publication?
yes
id
26d5eb9b-4560-41fc-becf-49736747ccf2
date added to LUP
2024-03-07 10:22:32
date last changed
2024-04-17 17:13:43
@article{26d5eb9b-4560-41fc-becf-49736747ccf2,
  abstract     = {{<p>SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10-nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone.</p>}},
  author       = {{Sele, Céleste and Krupinska, Ewa and Andersson Rasmussen, Anna and Ekström, Simon and Hultgren, Lucas and Lou, Jiaqi and Kozielski, Frank and Fisher, S Zoë and Knecht, Wolfgang}},
  issn         = {{1525-7770}},
  language     = {{eng}},
  month        = {{02}},
  publisher    = {{Taylor & Francis}},
  series       = {{Nucleosides, Nucleotides & Nucleic Acids}},
  title        = {{New insights into complex formation by SARS-CoV-2 nsp10 and nsp14}},
  url          = {{http://dx.doi.org/10.1080/15257770.2024.2321600}},
  doi          = {{10.1080/15257770.2024.2321600}},
  year         = {{2024}},
}