Parameterization of the PA Endonuclease Bimetallic Center Reveals the Dynamics of Clinically Relevant Mutations.

Influenza A virus continues to impose a major global health and economic burden through seasonal epidemics and occasional pandemics, highlighting the critical need for continued antiviral development. As the latest addition to anti-influenza therapy, baloxavir marboxil (BXM) targets the highly conserved PA N-terminal endonuclease domain (PAN), blocking the cap-snatching process essential for viral transcription initiation. However, the rapid emergence of resistance mutations significantly reduces BXM susceptibility and compromises its clinical efficacy. Understanding the dynamics underlying resistance through computational modeling has been hindered by the complex electronic properties of the bimetallic catalytic center within the PAN active site, posing a challenge for accurate parametrization. Therefore, in this study, we systematically benchmarked metal-parametrization strategies for molecular dynamics (MD) simulations, including the nonbonded, bonded, and hybrid models, simulating wild-type PAN in both apo and drug-bound states. Identification of reliable parametrization schemes enabled MD simulations of five clinically relevant mutants, I38T/F/M, A36V, and E23K, revealing how each reshapes the conformational landscape to modulate drug-binding modes. Together, our results provide a path toward modeling complex sites in metalloenzymes and a mechanistic foundation for vulnerabilities in PAN to guide structure-based optimization of next-generation inhibitors.