collected the data and F.G. of the open form of 150-loop and illustrates that the inherent flexibility and the ligand induced flexibility of the 150-loop should be taken into consideration for future drug design. Influenza virus causes epidemics and pandemics, which severely impair 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 public health1,2,3,4,5. Two of the major pandemics of the last century were caused by N2 containing influenza viruses: H2N2 (Asian flu) and H3N2 (Hong Kong flu)1. Recent outbreaks of H3N2 and H1N2 swine influenza viruses-caused human febrile respiratory illness in the United States highlight the importance of this subtype in influenza epidemics6,7,8. Besides H1N1, the H3N2 subtype is the major causative agent of severe epidemics and is critical for vaccine development9. Therefore a comprehensive understanding of N2 containing viruses is important for preparedness against highly transmissible influenza viruses. Hemagglutinin (HA) and neuraminidase (NA) are the two major surface glycoproteins responsible for initiating influenza virus infection10,11,12,13 and virus release14,15,16, respectively. HA and NA of influenza A viruses are divided into subtypes based upon their distinct antigenic properties: seventeen for HA (H1CH17) and ten for NA (N1CN10)17,18,19. Among the influenza A viruses, only N1 and N2 have been found in human isolates responsible for pandemics and recurrent annual epidemics. With the exception of N10, recently identified in a bat influenza A virus genome, the nine NA alleles are classified into two groups according to phylogenetic analysis and structure. Group 1 NA comprises N1, N4, N5 and N8, whereas group 2 comprises N2, N3, N6, N7 and N920. 3-D structures reveal the distinct conformations of the areas adjacent to the enzymatic active site between group 1 and group 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 2 members though the active site structures are virtually identical among all the NAs20. For typical N1 subtypes (but not for the 2009 2009 H1N1 pandemic N1), the crystal structures reveal a 150-loop (formed by amino acids 147C152, N2 numbering) that adopts an open conformation forming an additional 150-cavity adjacent to the active site. Previously, no group 2 NAs have been crystallographically shown to have a 150-loop in an open conformation, although all-atom molecular dynamics simulations indicate that N2 may be able to adopt such a configuration in solution21. The recently discovered 150-cavity is currently being explored as a novel target for group 1 specific influenza NA 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 inhibitors20,22. Detailed structural analyses demonstrate that residue 147 plays an essential role in the conformation of the 150-loop. Recently, our group has shown that the N5 structure contains an extended 150-cavity resulting from the unique residue N14723. In the solved N2 structures, a salt bridge between D147 and H150 contributes to a rigid closed 150-loop24. Previous molecular dynamics simulations show that the D147-H150 salt bridge greatly stabilizes the closed Rabbit Polyclonal to TBX18 150-loop conformation and that loss of this salt bridge reduces the rigidity of the 150-loop21. D147 is prevalent in N2, but rarely found in other types of influenza NA23. Compared with N2 containing D147, the flexibility of the 150-loop of 2009 H1N1 NA (G147) is much higher21, although the structure of 2009 H1N1 NA also presents a deficient 150-cavity in its crystal structure25. Since NA plays an essential role in the release of new influenza virions from host cells, inhibition of NA compromises the ability of progeny virions to spread to uninfected cells. Oseltamivir (Tamiflu) and zanamivir (Relenza) are two commercially available NA-targeted competitive inhibitors, which act against both group 1 and group 2 enzymes as well as influenza B NA16. The open 150-loop of group 1 NAs has been found to adopt a closed conformation upon binding of zanamivir, however for oseltamivir carboxylate this effect depends on both inhibitor concentration and soaking time dependent20. The crystal structures of typical group 1 NAs in complex with oseltamivir carboxylate display two 150-loop conformations, indicating a two-step process of oseltamivir carboxylate binding. Molecular dynamics simulations of the free and oseltamivir carboxylate-bound.