Structural insights into the substrate specificity of the endonuclease activity of the influenza virus cap-snatching mechanism

Figure 1.. RNA and DNA oligonucleotides bound to the PAN endonuclease. RNA oligomers have orange carbon atoms and DNA oligomers have magenta carbon atoms. (A) Active site mutant E119D bound to RNA oligomer AG*CA, where * indicates the location of the uncleavable sulfate linkage. (B) Mutant E119D bound to DNA oligomer TAGCAT. One metal is visible at the active site and was refined as a Mn2+ (yellow sphere). (C) Wild type PAN endonuclease bound to DNA oligomer TAGC. Two metal sites are visible at the active site; a Mg2+ (green sphere), and a shared Mg2+/Mn2+ (overlapping green/yellow spheres) with 70% and 30% occupancies, respectively. (D) Wild type PAN endonuclease bound to DNA oligomer TGAGC. In this structure, the oligomer is cleaved into a 5′ TG (light pink carbon atoms) and a 3′ AGC. There is a single Mn2+ (yellow sphere) at the active site. In each figure, the letter indicating the bound 5′ nucleotide is colored according to the pocket it occupies: P-2, magenta; P-1, red; P0 (5′ phosphate at the two-metal site), blue; P1, green; P2, yellow; and P3, orange. The orientation of each figure is the same apart from D to more clearly show the cleaved DNA. In each structure, the interacting…

Figure 2.. Baloxavir bound to the PAN endonuclease. (A) Wild type PAN. (B) I38T PAN. (C) E119D PAN. In each figure, baloxavir is represented as sticks with black carbon atoms, the PAN cartoon and key residues are colored cyan, the active site Mn2+ ions are large yellow spheres and the water molecules are small red spheres. Note in B that the side chain of the Thr38 point mutant is oriented such that the hydroxyl group can make hydrogen bond interactions with the main chain carbonyl oxygens of Lys34 and Phe35 (dotted black lines) while the methyl group optimizes its apolar interaction with baloxavir.

Figure 3.. Energy minimized models of RNA oligonucleotide GCAU bound to PAN endonuclease. (A) Overview of the wild type model in the same orientation as Figure 1. (B) Close up of the GC dinucleotide at the two-metal active site center of the wild type model showing the key hydrogen bonding interactions. (C) The interactions of the 2′ OH groups of the oligonucleotide sugar-phosphate backbone in the wild type model. (D) Close up of the Thr38 interactions in the I38T mutant model. To distinguish these as models rather than actual crystal structures, the RNA oligomer has orange carbon atoms and the protein and side chains are in light green. The metals are modeled as Mn2+ (yellow) and water molecules are shown as red spheres.

Figure 4.. Implications for PAN endonuclease inhibitor development. (A) Overlap of the bound P-1/P0 RNA dinucleotide with the previously determined bound mononucleotide (pdb file 5CCY). Note that the mononucleotide actually spans the P-1 and P0 sites. The dinucleotide is colored orange and the mononucleotide is colored magenta and shown in ball-and-sticks. The Mn2+ ions at the active site are shown as yellow spheres. Note that Tyr24 in the mononucleotide complex (magenta sticks) moves to maintain the stacking interaction with the base. (B) Spatial comparison of baloxavir and the RNA dinucleotide bound at the P-1 and P0 sites. (C) Spatial comparison of L-742,001 and the RNA dinucleotide bound at the P-1 and P0 sites. In (B) and (C), the RNA dinucleotide is shown as an orange van der Waal surface, the PAN cartoon and key residues are colored lime green, and baloxavir and L-742,001 are shown as black and magenta van der Waal surfaces, respectively. In all panels, the RNA dinucleotide and the PAN endonuclease are derived from the wild type RNA-bound model shown in Figure 3B with GC occupying the P-1 and P0 pockets.