Crystal structures of influenza nucleoprotein complexed with nucleic acid provide insights into the mechanism of RNA interaction

Figure 1.. (A) Apo-NP (PDB 2Q06) and the obligate monomeric NP-DL and DLDC2 shown in the same orientation. Three RNA nucleotides are modeled onto the body domain near the 74–88 loop. The proteins share highly similar tertiary folds demonstrating neither RNA binding nor tail-loop deletion distorts the overall structure. G1 residues (R74, R75, R174, R175), R65 and K87 are marked in blue in the structures. (B) 2Fo – Fc omit maps were generated for the nucleotides modeled on NP-DL (black: simple omit map; pink: simulated-annealing omit map) and contoured at 1σ as shown. (C) The RNA moieties on NP-DL and DLDC2 are situated in a cavity defined by R65, R74/75 and K87. The conformations of two RNA moieties do not deviate significantly. Protein cores are shown as ribbons (NP-DL: green; DLDC2: magenta). RNA is shown as cartoon in green (NP-DL/RNA) and pink (DLDC2/RNA).

Figure 2.. Three nucleotides built onto (A) NP-DL and (B) DLDC2, respectively, with side chains of nearby interacting residues shown. Numerous side-chain interactions can be identified as detailed in Table 3, including R65 and K87 which form hydrogen bonds with RNA and are previously uncharacterized. The RNA can possibly interact with both R74 or R75 depending on loop flexibility.

Figure 3.. (A) Here we align the 74–88 loops on all RNA-bound chains in panel (1) and non-RNA bound chains from our structures to apo-NP (PDB 2Q06) in panel (2). Peptides are colored according to colors of labels. When bound with RNA, the loop adopts a ‘down’ conformation, in contrast to the ‘up’ conformation when RNA-free. Due to the weak density, part of the loops could not be modeled confidently and is indicated by dashes here. Panel (3) is an overlay of panels (1) and (2) and the possible clash between the loop in ‘up’ conformation with the 5′ RNA is indicated. (B) 2mFo – DFc map (black, contoured at 1σ) for residues F71-R75 (green) on NP-DL complex is shown. The peptide is overlaid with the corresponding residues from apo-NP (PDB 2Q06, orange). The two peptides align with each other until D72 where they deviate to give two different conformations. (C) The 74–88 loop from apo-NP (PDB 2Q06), NP-DL and DLDC2 are shown with positive residues R74, R75, R174 and R175 marked blue. The ‘down’ conformation of the 74–88 loop in NP-DL and DLDC2 lead to opening up of G1 groove as indicated by increased distances between R174 to R74.

Figure 4.. (A) Alignment of NP sequences near the C-terminal region extracted from influenza A, B, C and D nucleoproteins. Conserved YFF motif is highlighted in box. (B) In H5 NP, the C-terminal tail of obligate monomers flips onto the protein core to cover the RNA-groove and as such Y496 is brought to vicinity to both Y148 and R150 of the positive G1 groove. D497-R152 salt-bridge is one of the stabilizing forces for this conformation. Shown in Figure 4B is an overlay of wild-type NP (orange) and the obligate monomer NP-DL (green). Panel A highlights the rearrangement of the YFF motif during this conformation change while panel B shows the local environment around the D497-R152 salt-bridge when NP is in monomeric state.

Figure 5.. Label-free MST was performed to determine the affinity between DLDC4 and DLDC8 NP variants towards 9-mer RNA (top-left and bottom-left panels, respectively). By saturating DLDC8 mutant with a synthetic tail peptide, RNA binding was attenuated by 8-fold. MST curve is expressed as Fnorm (0/00) versus concentration. Data represent mean values from at least three independent measurements. Error bars represent standard deviation. In the bottom right panel, for comparison, MST curves were expressed as fraction of protein bound vs ligand (RNA) concentration (Blue: DLDC8; Green: peptide-saturated DLDC8).