Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein

Dhurvas Chandrasekaran Dinesh; Dominika Chalupska; Jan Silhan; Vaclav Veverka; Evzen Boura

Effective drugs are urgently needed to combat the COVID-19 disease. Most patients are not given any drug and the treatment relies on curing the symptoms. The most promising drug is remdesivir, a nucleotide analog that targets the viral RNA-dependent RNA-polymerase (RdRp). Viral polymerases are certainly good targets for antiviral compounds as these enzymes are absolutely vital for any +RNA virus. However, every viral protein is a potential target for antiviral compounds and an effective treatment may require several active compounds, each targeting a different protein at the same time. This approach, known as HAART (highly active antiretroviral therapy), has proven effective in the case of HIV, which is another virus with an RNA genome. In this study, we structurally characterized the N protein, a structural protein that is essential for assembly of the virion. We obtained an atomic model of the N protein N-terminal domain in an unliganded form and in complex with an RNA duplex. The structure revealed a right hand fold featuring a prominent basic finger protruding from the palm. Analysis of its electrostatic potential . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.02.022194 doi: bioRxiv preprint ( Figure 2C ) revealed highly positively charged canyon that is situated in the interface between the basic finger and the palm subdomain and constitutes a putative RNA binding site. We performed an NMR titration experiment to obtain experimental proof of the RNA binding site. An overlay of the 15 N/ 13 C labeled protein NMR spectrum in the absence of ligand and in complex with RNA revealed amino acid residues with large chemical shifts upon the addition of RNA (Figure 3 ). Not surprisingly, all these residues are located in or in close proximity to the basic canyon, confirming the canyon as the RNA binding site. To illustrate how the coronaviral N-NTD recognizes RNA we built an atomic model of the N-NTD:RNA complex using the NMR titration data as a restraint for computer simulations in HADDOCK. The model reveals a hotspot on the surface of the N-NTD between the finger and palm subdomains that is essential for RNA binding and suggests this site as an ideal place for targeting by small molecules.