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

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

To follow changes in the chemical shifts of a protein upon RNA binding, we calculated chemical shift perturbations (CSPs). The CSP of each assigned resonance in the 2D 15 N/ 1 H HSQC spectra of the protein in the free state is calculated as the geometrical distance in ppm to the peak in the 2D 15 N/ 1 H HSQC spectra acquired under different conditions using the formula: Δ = √ Δ 2 + (Δ • ) 2 , where  is a weighing factor of 0.2 used to account for differences in the proton and nitrogen spectral widths. (14) Molecular docking -The structure for the N-NTD in complex with the 7mer RNA duplex was calculated using HADDOCK (4) . The RNA homology model was prepared by mutating the native 7mer RNA duplex (PDB 4U37) (5) in Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) that was subsequently subjected to an energy minimization in YASARA (13) . For the actual docking, we used a representative structure from the set of obtained structures and followed a standard protocol. As active were selected those N-NTD residues with CSP > 0.05 ppm and at least 20% solvent accessibility (A50, T57, H59, R92, I94, S105, R107, R149, A152 and Y172), while as passive were additionally selected adjacent solvent exposed residues (T49, T54, L55, R88, A90, K102, L104, Y109, Y111, P151, A155, A156, E174 and G175). On RNA side, all 14 nucleobases were defined as active for the experimentally driven . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 docking protocol. In addition, in total three regions within the N-NTD were defined as fully flexible segments for the advanced stages of the docking calculation (the N-terminal G1-T9, the central loop I54-M61 and the C-terminal S136-S140). The final set of 200 water-refined structures was clustered using a Fraction of Common Contacts approach (15) with a default cut-off 0.75 and a minimal cluster size = 4. The resulting structures were sorted into 7 clusters and the most populated cluster (n = 30) that also exhibited the lowest interaction energy was selected for detailed analysis.