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Sele C, Krupinska E, Andersson Rasmussen A, Ekström S, Hultgren L, Lou J, Kozielski F, Fisher SZ, Knecht W
Nucleosides Nucleotides Nucleic Acids - (-) 1-15 [2024-02-29; online 2024-02-29]
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.
Structural Proteomics [Service]
PubMed 38422227
DOI 10.1080/15257770.2024.2321600
Crossref 10.1080/15257770.2024.2321600