SARS-CoV-2 Nsp8 N-terminal domain dimerizes and harbors autonomously folded elements

The SARS-CoV-2 Nsp8 protein is a critical component of the RNA replicase, as its N-terminal domain (NTD) anchors Nsp12, the RNA, and Nsp13. Whereas its C-terminal domain (CTD) structure is well resolved, there is an open debate regarding the conformation adopted by the NTD as it is predicted as disordered but found in a variety of complex-dependent conformations or missing from many other structures. Using NMR spectroscopy, we show that the SARS CoV-2 Nsp8 NTD features both well folded secondary structure and disordered segments. Our results suggest that while part of this domain corresponding to two long α-helices forms autonomously, the folding of other segments would require interaction with other replicase components. When isolated, the α-helix population progressively declines towards the C-termini, and dynamics measurements indicate that the Nsp8 NTD behaves as a dimer under our conditions.
MM is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I), and a "La Caixa" Foundation (ID 100010434) Junior Leader Fellow (LCR/BQ/PR19/11700003). Funded by project COV20/00764 from the Carlos III Institute of Health and the Spanish Ministry of Science and Innovation to MM and DVL. NMR experiments were performed in the “Manuel Rico” NMR Laboratory (LMR) of the Spanish National Research Council (CSIC), a node of the Spanish Large-Scale National Facility for Biomolecular NMR (ICTS R-LRB).