Structure-function mapping and mechanistic insights on the SARS CoV2 Nsp1

Abstract

Non-structural protein 1 (Nsp1) is a key component of the infectious process caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), responsible for the COVID-19 pandemic. Our previous data demonstrated that Nsp1 can degrade both RNA and DNA in the absence of the ribosome, a process dependent on the metal ions Mn²⁺, Ca²⁺, or Mg²⁺ (Salgueiro et al., SARS-CoV2 Nsp1 is a metal-dependent DNA and RNA endonuclease. Biometals. 2024;37:1127-1146). The protein is composed of two structural domains: the N-terminal domain (NTD) and C-terminal domain (CTD), connected by a loop. To elucidate the function of each structural domain, we generated four truncated versions of Nsp1 containing either the NTD or the CTD. Our results indicate that the Nsp1^SARS-CoV2 domains play distinct functional roles. Specifically, the NTD is involved in nucleotide binding and regulation, while the CTD acts as the catalytic domain. Moreover, a tyrosyl radical was detected during the nuclease activity, and an in-depth analysis of the different constructs suggested that Y136 could be involved in this process. Indeed, our results show that Y136F Nsp1 variant lacks DNA nuclease activity but retains its RNA nuclease activity. Furthermore, we observed that the CTD has a propensity to associate with hydrophobic environments, suggesting that it might associate with cell membranes. However, the cellular function of this association requires further investigation. This study sheds light on the functions of the individual domains of Nsp1, providing valuable insights into its mechanism of action in Coronaviruses.

Keywords: SDS; biophysical characterization; fluorescence spectroscopy; metals; nuclease; tyrosyl radical.