Mutation-induced loop opening and energetics for binding of tamiflu to influenza N8 neuraminidase

Tamiflu, also known as oseltamivir (OTV), binds to influenza A neuraminidase (H5N1) with very high affinity (0.32 nM). However, this inhibitor binds to other neuraminidases as well. In the present work, a systematic computational study is performed to investigate the mechanism underlying the binding of oseltamivir to N8 neuraminidase (NA) in "open" and "closed" conformations of the 150-loop through molecular dynamics simulations and the popular and well established molecular mechanics Poisson-Boltzmann (MM-PBSA) free energy calculation method. Whereas the closed conformation is stable for wild type N8, it transforms into the open conformation for the mutants Y252H, H274Y, and R292K, indicating that bound to oseltamivir these mutants are preferentially in the open conformation. Our calculations show that the binding of wild type oseltamivir to the closed conformation of N8 neuraminidase is energetically favored compared to the binding to the open conformation. We observe water mediated binding of oseltamivir to the N8 neuraminidase in both conformations which is not seen in the case of binding of the same drug to the H5N1 neuraminidase. The decomposition of the binding free energy reveals the mechanisms underlying the binding and changes in affinity due to mutations. Considering the mutant N8 variants in the open conformation adopted during the simulations, we observe a significant loss in the size of the total binding free energy for the N8(Y252H)-OTV, N8(H274Y)-OTV, and N8(R292K)-OTV complexes compared to N8(WT)-OTV, mainly due to the decrease in the size of the intermolecular electrostatic energy. For R292K, an unfavorable shift in the van der Waals interactions also contributes to the drug resistance. The mutations cause a significant expansion in the active site cavity, increasing its solvent accessible surface compared to the crystal structures of both the open and closed conformations. Our study underscores the need to consider dynamics in rationalizing the structure-function relationships of various antiviral inhibitor-NA complexes.