Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms - μs) dynamics...

The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein's backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone ¹H, ¹³C, and ¹⁵N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an ~ 70% deuterated sample and residue-specific, ¹⁵N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the "missing" amide resonances for N96-L106 in the ¹H-¹⁵N HSQC spectrum, a region that comprises almost the complete C-terminal α-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to μs timescale and/or is heterogenous. These "missing" amide resonances do not unambiguously appear in the ¹H-¹⁵N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.0007 mM. At this concentration, at the detection limit, native mass spectrometry indicates the protein is exclusively in the monomeric state, suggesting the intermediate motion in the C-terminal of Nsp9 may be due to intramolecular dynamics. Perhaps this intermediate ms to μs timescale dynamics is the physical basis for a previously suggested "fluidity" of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions.