Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms - μs) dynamics in the C-terminal helix at the dimer interface

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 H-1, C-13, and N-15 NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an similar to 70% deuterated sample and residue-specific, N-15-labelled samples (V, L, M, F, and K). A major feature of the assignments was the missing amide resonances for N96-L106 in the H-1-N-15 HSQC spectrum, a region that comprises almost the complete C-terminal alpha-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 mu s timescale and/or is heterogenous. These missing amide resonances do not unambiguously appear in the H-1-N-15 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 mu s 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.

Automated summary

This study investigates the dynamics of the SARS-CoV-2 Nsp9 protein in solution and its potential ligand-binding surfaces through chemical shift perturbation studies. By extensively assigning NMR chemical shifts, the research reveals missing amide resonances in the C-terminal region of the protein, suggesting intermediate motion or heterogeneity in this area.