Title: X-ray crystal structures of the influenza A M2 proton channel

Abstract: Inhibition of the M2 proton channel in the influenza A virus prevents viral replication from occurring. However, because M2 is a membrane protein, structural studies characterizing the channel have been limited due to the challenging nature of the target.  Multiple crystal structures of M2 have been obtained using lipid cubic phase (LCP) crystallization techniques. Crystals of the open conformation of M2 in the absence of bound drug reveal an ordered network of hydrogen-bonded water molecules that are involved in shuttling protons down to the channel's gating His37 residues. This crystal form diffracts to 1.10 Å resolution and has been used in room temperature synchrotron and XFEL experiments to characterize temperature- and pH-dependent effects on the solvent network. Structures of amantadine and rimantadine bound to WT M2 have also been solved to atomic resolution (2.0 Å) and show that the bound drug ammonium group mimics a hydronium ion and sterically blocks the N-terminal portion of the pore water network. The binding of a novel inhibitor compound is also examined, with structures solved in co-crystallization trials for the wild type channel (2.6 Å) and the drug-resistant V27A mutant (2.5 Å). The position of the bound inhibitor within the channel shifts in the presence of the V27A mutation. Additionally, the structure of the prevalent drug-resistant S31N mutant has also been characterized in multiple conformational states (2.0 Å). These structures advance our understanding of drug binding and inhibition within the M2 proton channel and will help guide the design of compounds for the inhibition of drug-resistant mutants of M2.