Magnesium-dependent RNA binding to the PA endonuclease domain of the avian influenza polymerase

Influenza A viruses are highly pathogenic and pose an unpredictable public health danger to humans. An attractive target for developing new antiviral drugs is the PA N-terminal domain (PA_N) of influenza polymerase, which is responsible for the endonuclease activity and essential for viral replication. Recently, the crystal structures of the holo form of PA_N as well as PA_N bound to different inhibitors have been reported, but the potency and selectivity of these inhibitors still need to be improved. New drug design can be guided by a better understanding of the endonuclease activity of PA _N. However, this requires the structure of PA_N in complex with the host mRNA, which has not been determined yet. In particular, divalent metal ions are known to be essential for RNA cleavage, but it is not clear whether there is either one or two Mg ions in the PA_N active site. In the present work, we have modeled the complex of the PA_N endonuclease domain with the host mRNA in the presence of either one or two Mg²⁺ by using all-atom molecular dynamics. These simulations identify crucial interactions between the enzyme and the nucleic acid. Moreover, they validate a previous hypothesis that a second metal ion binds in the presence of the RNA substrate and therefore support a two-metal ion mechanism, in which K134 decreases the pK_a of the nucleophilic water. Nevertheless, at low Mg concentrations an alternative, one-metal ion mechanism is possible, with K137 as the catalytic lysine and H41 as the general base, rationalizing previous unexpected mutagenesis results. The RNA-enzyme interactions determined here could likely be used to design more specific endonuclease inhibitors to fight influenza viral infections.