The structure of the N-NTD in complex with the 7mer RNA duplex was calculated using HADDOCK [17 (link)]. The RNA homology model was prepared by mutating the native 7mer RNA duplex (PDB 4U37) [19 (link)] in Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) that was subsequently subjected to an energy minimization in YASARA [18 (link)]. 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 the RNA side, all 14 nucleobases were defined as active for the experimentally driven docking protocol. In addition, in all 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 [32 (link)] 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. The structure of the N-NTD in complex with the ssRNA-10mer was calculated in YASARA [18 (link)]. The significantly perturbed backbone amide groups from residues (CSP > 0.06 ppm) N47, S51, F53, L56, G60, K61, K65, F66, A90, R93, I94, R95, G97, D98, K100, K102, D103, L104, G129, R149, A152, A156, I157, L159, Q160, T165, T166, L167, Y172, G175 and R177 outlined the U-shaped binding epitope for the ssRNA-10mer molecule. In addition, the signal from arginine side-chain NHε groups were significantly perturbed for residues 88, 89, 92 and 177 but remained at their original positions for 68, 93, 95. We combined this information in generating the inter-molecular distance restraints used in YASARA docking calculation that consisted of an initial energy minimization followed by a 100 ns of molecular dynamics using the default md_fast.mrc macro. The 3.9 Å upper distance limits were set between K65 NZ–U1 P, K61 NZ–U2 P, R88 NH1 –U5 P, R89 NH1 –U4 P, R92 NH1 –A6 P, K102 NZ–A7 P, R107 NH1 –A8 P, T166 CG2 –G10 C5, and R177 NH1 –C9 P and R177 NH2 –C10 P.
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