The preference signature of the SARS-CoV-2 Nucleocapsid NTD for its 5’-genomic RNA elements

Abstract

The nucleocapsid protein (N) of SARS-CoV-2 plays a pivotal role during the viral life cycle. It is involved in RNA transcription and accounts for packaging of the large genome into virus particles. N manages the enigmatic balance of bulk RNA-coating versus precise RNA-binding to designated cis-regulatory elements. Numerous studies report the involvement of its disordered segments in non-selective RNA-recognition, but how N organizes the inevitable recognition of specific motifs remains unanswered. We here use NMR spectroscopy to systematically analyze the interactions of N’s N-terminal RNA-binding domain (NTD) with individual cis RNA elements clustering in the SARS-CoV-2 regulatory 5’-genomic end. Supported by broad solution-based biophysical data, we unravel the NTD RNA-binding preferences in the natural genome context. We show that the domain uses a set of flexible sensory residues to read the intrinsic signature of preferred RNA elements for selective and stable complex formation within the large pool of available motifs.

The Role of Disordered Regions in Orchestrating the Properties of Multidomain Proteins: The SARS-CoV-2 Nucleocapsid Protein and Its Interaction with Enoxaparin

“…On the other hand, several regions show higher flexibility (red, low R 2 / R 1 ratios). These comprise the finger (residues 92-106), a few external loops, and the residues at the edges of the construct, as also previously reported in the literature [ 24 , 57 , 58 ].…”

“…It is interesting to compare our results with recent studies focusing on the interaction of NTD with different fragments of nucleic acids. Indeed, NMR spectra were used to follow CSPs of NTD upon the addition of increasing concentrations of nucleic acid fragments to map interaction surfaces [ 19 , 22 , 25 , 58 , 72 ]. The region of the basic finger is generally extensively perturbed.…”

“…The interaction with EP significantly resembles this general behavior, indicating that it shares common features with the interplay of NTD with different kinds of polyanions, such as nucleic acid fragments. The identification of specific features linked to the different types of partner molecules (RNA, DNA), to whether they are single or double strand, to how the length of the fragment and its conformation affect the interaction are still a matter of debate [ 22 , 25 , 58 ].…”

The disordered N-terminal tail of SARS CoV-2 Nucleocapsid protein forms a dynamic complex with RNA

“…To test whether sequence specificity can affect affinity and mode of binding of the specific RNA with the disordered region, we studied the interactions with a 21 nucleotide sequence (V21) from the 5’ UTR of the viral genome. This region of the genome was previously found interacting with the N protein in in cell crosslinking studies [10] and has been confirmed to adopt no secondary structure at room temperature [39] .…”

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2

“…The N-terminal RBD (NTD) interacts with RNA as a monomer and its interactions with ss and double-stranded (ds) RNA have been modelled 7 . Recent studies support the idea that despite being a promiscuous RNA binder, NTD binds to a variety of RNA structures with different signatures, suggesting a possible mechanism for regulation and RNA processing 8,9 . On the other hand, the C-terminal RBD (CTD) is present in solution as a swapped dimer as determined by x-ray crystallography, however little is known about the structural details of RNA recognition 10,11 .…”