CONFORMATIONAL EQUILIBRIUM AND WATER DESOLVATION DELINEATE THE ENCOUNTER COMPLEX BETWEEN SARS-COV-2 N-TERMINAL DOMAIN OF NUCLEOCAPSID PROTEIN AND RNA.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in over 7 million deaths globally, prompting extensive research into the molecular mechanisms of the disease. Coronaviruses possess unique replication and transcription systems due to their large RNA genomes and proofreading machinery. The nucleocapsid protein (N) is highly conserved among coronaviruses, and is by far the most abundant structural protein during viral infection. N plays key roles in regulating viral gene expression and nucleocapsid assembly, and is essential for viral RNA replication, transcription, and packaging. However, several aspects of its molecular function, including RNA binding specificity, remain incompletely understood.

Here, we experimentally dissect the structural and dynamical properties of the N-terminal domain of the N protein (N-NTD) of SARS-CoV-2, which contributes to binding specificity for transcription regulatory sequences (TRS), which are genomic RNA elements that mediate discontinuous transcription in coronaviruses. Previous studies have shown that N-NTD interacts with TRS RNA with nanomolar affinity. Moreover, N-NTD displays melting activity toward double-stranded TRS RNA (dsTRS). To better understand the details of this protein-RNA interaction and to gain insights into the formation of the encounter complex, we measured the ¹⁵N relaxation dispersion via Carr-Purcell-Meiboom Gill (CPMG) of N-NTD in the presence of TRS RNA. We also characterized N-NTD conformational exchange using ¹⁵N and ¹⁹F Chemical Exchange Saturation Transfer (CEST) and assessed water exchange by CLEANEX-NMR.

Relaxation dispersion experiments revealed a two-state conformational equilibrium on the micro- to millisecond timescale, along with exposure of hydrophobic residues in the excited state. CEST experiments confirmed conformational exchange involving residues critical for RNA interaction. The water exchange experiments enabled the mapping of the solvent exposure across the entire finger region and solvent-accessible loops of N-NTD. These results highlight residues directly involved in the solvation layer and the salt dependence of the interaction, shedding light on their roles in the formation of the initial encounter complex with RNA. These early binding steps may be fundamental for selecting specific binding pathways and mediating affinity toward different RNA sequences, ultimately influencing the differential expression of mRNAs encoding structural and accessory viral proteins during infection.