Sele, C; Krupinska, E; Andersson Rasmussen, A; Ekström, S; Hultgren, L; Lou, J; Kozielski, F; ... Knecht, W; + view all Sele, C; Krupinska, E; Andersson Rasmussen, A; Ekström, S; Hultgren, L; Lou, J; Kozielski, F; Fisher, SZ; Knecht, W; - view fewer (2024) New insights into complex formation by SARS-CoV-2 nsp10 and nsp14. Nucleosides, Nucleotides and Nucleic Acids 10.1080/15257770.2024.2321600. (In press).

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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 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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