New insights into complex formation by SARS-CoV-2 nsp10 and nsp14
(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.
(Less)
- author
- 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
- organization
- publishing date
- 2024-02-29
- 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}}, }