title: Coexistence of two adamantane binding sites in the influenza A M2 ion channel.
abstract: The influenza A virus contains a proton-selective ion channel (M2) that is the target of the adamantane family of drug inhibitors. Two recently published studies relating to adamantane binding of the M2 ion channel using X-ray crystallography and solution NMR have reignited interest in the potential use of adamantanes in combating the spread of influenza A. However, these two studies propose different binding sites for the adamantane drugs with the X-ray M2/amantadine structure favoring an ion channel pore-binding model and the solution NMR M2/rimantadine structure suggesting the existence of a lipid-facing binding pocket. We conducted a series of surface plasmon resonance (SPR) experiments designed to accurately measure the affinity of amantadine and rimantadine to M2 ion channels embedded in 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC) liposomes. We find that this class of drug is capable of binding M2 with two different affinities in the order of 10(-4) and 10(-7) M, suggesting that both proposed binding sites are feasible. Furthermore, by examining drug binding to M2 mutant constructs (V27A, S31N, and D44A), it was possible to probe the location of the two binding sites. We show that a high-affinity binding site corresponds to the M2 ion channel pore whereas the secondary, low-affinity binding site can be attributed to the lipid face of the pore. These SPR results are in excellent agreement with the most recent solid-state NMR study of amantadine-bound M2 in lipid bilayers and provide independent support that the ion channel pore-binding site is responsible for the pharmacological activity elicited by the adamantane drugs.
authors.affiliation: Department of Structural Biology, John Curtin School of Medical Research, Australian National University, Acton, ACT 2601, Australia.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Rosenberg
authors.forename: Marco G
authors.initials: MG
authors.lastname: Casarotto
mesh_terms: D000218:Adamantane / Q000737:chemistry*; D001665:Binding Sites; D009980:Influenza A virus / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics; D000086082:Viroporin Proteins
chemical_list: D000218:Adamantane; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins