Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers

Abstract

The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine 1. Resistance to these drugs has compromised their effectiveness against many influenza strains, including pandemic H1N1. A recent crystal structure of M2(22-46) showed electron densities attributed to a single amantadine in the N-terminal half of the pore 2, suggesting a physical occlusion mechanism for inhibition. However, a solution NMR structure of M2(18-60) showed four rimantadines bound to the C-terminal lipid-facing surface of the helices 3, suggesting an allosteric mechanism. Here we show by solid-state NMR spectroscopy that two amantadine-binding sites exist in M2 in phospholipid bilayers. The high-affinity site, occupied by a single amantadine, is located in the N-terminal channel lumen, surrounded by residues mutated in amantadine-resistant viruses. Quantification of the protein – amantadine distances resulted in a 0.3 Å-resolution structure of the high-affinity binding site. The second, low-affinity, site was observed on the C-terminal protein surface, but only when the drug reaches high concentrations in the bilayer. The orientation and dynamics of the drug are distinct in the two sites, as shown by 2H NMR. These results indicate that amantadine physically occludes the M2 channel, thus paving the way for developing new antiviral drugs against influenza viruses. The study demonstrates the ability of solid-state NMR to elucidate small-molecule interactions with membrane proteins and determine high-resolution structures of their complexes.

Viroporins: structure, function and potential as antiviral targets

“…Several adamantane-resistant variants occur within the channel lumen, consistent with the majority of structural studies that place a single adamantane moiety at this position, physically occluding the channel (Cady et al, 2010;Duff et al, 1994;Hu et al, 2007;Stouffer et al, 2008;Wang et al, 2001). However, in 2008, a drug-bound M2 CD solution structure identified rimantadine molecules bound at four membrane-exposed sites defined by Asp44 on the channel periphery (Schnell & Chou, 2008).…”

“…Perhaps the most biologically relevant M2 structure comprises CD peptides in a DOPC/DOPE (dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine) bilayer at pH 7.5 [Protein Data Bank (PDB) ID: 2LOJ] (Sharma et al, 2010), although no drug molecule was bound. Recent drug-bound studies include a solid-state NMR structure in DMPC (dimyristoylphosphatidylcholine) bilayers with amantadine bound to the channel lumen (PDB ID: 2KQT) (Cady et al, 2009(Cady et al, , 2010, as well as solution structures of CD peptides in detergent micelles with four rimantadine molecules bound to a peripheral, membraneexposed binding site (PDB ID: 2RLF) (Schnell & Chou, 2008). Generally, solution structures show more compacted lumenal domains and a varied orientation of the C-terminal basic helices compared with solid-state structures.…”

Exploring the Proton Conductance and Drug Resistance of BM2 Channel through Molecular Dynamics Simulations and Free Energy Calculations at Different pH Conditions

“…The solid-state NMR spectroscopy has confirmed that two drug binding sites could exist in the M2 proton channel and that the drug AMT exhibits different orientations at the two binding sites. 32 By performing a series of surface plasmon resonance (SPR) experiments measuring the affinity of AMT and RMT to the AM2 channel, Rosenberg et al 33 probed the two possible binding sites. To investigate the two alternative binding sites of AMT and RMT inside the AM2 proton channel, Gu et al 35 carried out MD simulations and PMF calculations and discovered that at intermediate pH conditions (pH ≈ 7.4), the surface binding site (S-binding) was less thermodynamically favorable than the pore binding (P-binding).…”

Docking and Molecular Dynamics (MD) Simulations in Potential Drugs Discovery: An Application to Influenza Virus M2 Protein

“…The sites of binding for rimantadine and amantadine are well known from crystallographic data (Schnell and Chou, 2008;Cady et al, 2010), for omeprazole we chose the same site of binding as for amantadine. Docking procedure was provided with the usage of DOCK6 program (Brozell et al, 2012).…”