Selected article for: "protein complex and rna duplex"
Author: Dhurvas Chandrasekaran Dinesh; Dominika Chalupska; Jan Silhan; Vaclav Veverka; Evzen Boura
Title: Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein
  • Document date: 2020_4_5
    • ID: ezpjdz55_8
    Snippet: The structure of the N-NTD suggests a putative RNA binding site. Intrigued, we aimed to obtain an experimental model of the N-NTD in a complex with RNA. We performed an NMR-based titration experiment using a short double-stranded RNA (5'-CACUGAC-3' and 5'-GUCAGUG-3'). Basically, we were adding isotopically unlabeled RNA to the 15 N/ 13 C labeled protein and we followed changes in positions of assigned signals in NMR spectra ( Figure 3 ) to reveal.....
    Document: The structure of the N-NTD suggests a putative RNA binding site. Intrigued, we aimed to obtain an experimental model of the N-NTD in a complex with RNA. We performed an NMR-based titration experiment using a short double-stranded RNA (5'-CACUGAC-3' and 5'-GUCAGUG-3'). Basically, we were adding isotopically unlabeled RNA to the 15 N/ 13 C labeled protein and we followed changes in positions of assigned signals in NMR spectra ( Figure 3 ) to reveal the molecular interface of the N-NTD:RNA complex. Overlay of the 2D 15 N/ 1 H HSQC and 2D 13 C/ 1 H HSQC spectra of a free and RNA bound N-NTD revealed residues that were significantly perturbed by RNA binding. All these significantly affected residues (A50, T57, H59, R92, I94, S105, R107, R149, Y172) are located in the basic finger or close to the junction between the basic finger and the palm as expected based on the analysis of the electrostatic potential. The copyright holder for this preprint (which was not peer-reviewed) is the .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 We next used the experimental data to build an atomic model of the protein:RNA complex. We used the HADDOCK protocol for NMR-restraint driven docking simulations (4) . As a starting conformation of the RNA we chose a short double helix based on the published crystal structure of a short RNA duplex (5) . A detailed analysis of the chemical shift perturbations (CSP) mapped onto the solution structure of N-NTD provided a set of 'active' solvent-accessible residues on N-NTD that were expanded to surrounding 'passive' residues. The selection criteria for active residues were that their CSP values were more than one standard deviation from the average value (0.05 ppm) calculated for the entire set of CSPs and that their accessibility to solvent was more than 20% (6) . Restraints for the RNA molecule were kept ambiguous to avoid potential bias. A standard docking protocol yielded a set of water-refined conformations for the protein:RNA complex that were clustered into several distinct classes. As expected, the RNA duplex molecule was bound in the positively charged cleft in all clusters. The most populated cluster also contained the fewest violations of experimental restraints and was therefore selected as a representative conformation for the N-NTD: RNA complex.

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