This report investigates the homotetrameric membrane protein structure of the S31N M2 protein from Influenza A virus in the presence of a high molar ratio of lipid. The structured regions of this protein include a single transmembrane helix and an amphipathic helix. Two structures of the S31N M2 conductance domain from Influenza A virus have been deposited in the Protein Data Bank (PDB). These structures present different symmetries about the channel main axis. We present new magic angle spinning and oriented sample solid-state NMR spectroscopic data for S31N M2 in liquid crystalline lipid bilayers using protein tetramer:lipid molar ratios ranging from 1:120 to 1:240. The data is consistent with an essentially 4-fold-symmetric structure very similar to the M2 WT structure that also has a single conformation for the four monomers, except at the His37 and Trp41 functional sites when characterized in samples with a high molar ratio of lipid. While detergent solubilization is well recognized today as a nonideal environment for small membrane proteins, here we discuss the influence of a high lipid to protein ratio for samples of the S31N M2 protein to stabilize an essentially 4-fold-symmetric conformation of the M2 membrane protein. While it is generally accepted that the chemical and physical properties of the native environment of membrane proteins needs to be reproduced judiciously to achieve the native protein structure, here we show that not only the character of the emulated membrane environment is important but also the abundance of the environment is important for achieving the native structure. This is a critical finding as a membrane protein spectroscopist's goal is always to generate a sample with the highest possible protein sensitivity while obtaining spectra of the native-like structure.

While aminoadamantanes are well-established inhibitors of the influenza A M2 proton channel, the mechanisms by which they are rendered ineffective against M2S31N are unclear. Solid state NMR, isothermal titration calorimetry, electrophysiology, antiviral assays, and molecular dynamics simulations suggest stronger binding interactions for aminoadamantanes to M2WT compared to negligible or weak binding to M2S31N. This is due to reshaping of the M2 pore when N31 is present, which, in contrast to wild-type (WT), leads (A) to the loss of the V27 pocket for the adamantyl cage and to a predominant orientation of the ligand's ammonium group toward the N-terminus and (B) to the lack of a helical kink upon ligand binding. The kink, which reduces the tilt of the C-terminal helical domain relative to the bilayer normal, includes the W41 primary gate for proton conductance and may prevent the gate from opening, representing an alternative view for how these drugs prevent proton conductance.

While it has long been agreed that the M2 proton channel functions by shuttling protons on and off of the histidine tetrad near the mid plane of the lipid bilayer, the details have been hotly debated. Now details of the shuttling mechanism have been elucidated from solid state NMR spectroscopy. Short hydrogen bonds between the imidazole-imidazolium pairs are confirmed as well as their disruption by aqueous attack followed by N-H exchange and imidazolium-imidazole hydrogen bond reformation by either a deprotonation to the aqueous pore connected to the viral external environment (a futile proton cycle) or by deprotonation to a waters in the internal cavity between the His37 and Trp41 tetrads. For this latter option when the Trp41 gate opens the proton can be conducted into the viral interior (conductance cycle). The dynamics of the Trp41 residues and those of the secondary conductance gate (Val27) have been evaluated by wide-line solid state NMR. Solid state NMR REDOR and NCA experiments of the full length wild type M2 channel in complex with rimantadine enantiomers. The binding of these enantiomers to M2 shows a difference in binding affinity and a different set of binding interactions implying that stereospecific drugs may have biomedical benefits. M2 has multiple functions, among them is the facilitation of viral budding that takes advantage of the pyramidal shape of the M2 structure to induce membrane curvature. This shape is caused by the insertion of the juxtamembrane amphipathic helix in the lipid bilayer interface. We have now shown that the stability of this helix in the membrane is the result of cholesterol binding to this helix.

Rimantadine hydrochloride (α-methyl-1-adamantane-methalamine hydrochloride) is a chiral compound which exerts antiviral activity against the influenza A virus by inhibiting proton conductance of the M2 ion channel. In complex with M2, rimantadine has always been characterized as a racemic mixture. Here, we report the novel enantioselective synthesis of deuterium-labeled (R)- and (S)-rimantadine and the characterization of their protein-ligand interactions using solid-state NMR. Isotropic chemical shift changes strongly support differential binding of the enantiomers to the proton channel. Position restrained simulations satisfying distance restraints from (13)C-(2)H rotational-echo double-resonance NMR show marked differences in the hydrogen-bonding pattern of the two enantiomers at the binding site. Together these results suggest a complex set of interactions between (R)-rimantadine and the M2 proton channel, leading to a higher stability for this enantiomer of the drug in the channel pore.

A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

The M2 proton channel from Influenza A virus is essential for the viral lifecycle and is an important drug target. Amino acid mutations in the residues lining the pore of the channel have abrogated clinical efficacy of the previously FDA-approved antiviral agents, amantadine and rimantadine. More than 95% of the circulating strains isolated from human clinical cases bear S31N mutation and a subsequent resistance to the licensed small molecule inhibitors. Efforts in rational drug design targeting S31N M2 channel have been impeded by the limited number of experimental techniques with capabilities for structural characterization of the protein-ligand interaction in native-like membrane mimetic environments. Solution and Solid State Nuclear Magnetic Resonance (NMR) investigations of the recently introduced adamantane analogues suggest multiple orientations of the inhibitor molecules bound in the pore. Position of the substituent moiety in the channel varies for different compounds, unlike the fixed orientation in the wild type channel. Here we report a Solid State NMR investigation of the transmembrane domain of S31N M2 (TMD) proton channel bound to the novel inhibitor molecules, while also incorporated into a lipid bilayer environment. Oriented sample solid state NMR experiments of S31N M2 TMD indicate that the channel is sampling two states with helical tilts of 28° and 33° relative to the bilayer normal. The kink in the monomer between two helical fragments, observed in the wild type M2 TMD bound with amantadine, is absent for the S31N M2 TMD bound to all inhibitor molecules tested to date. Stabilizing interactions are further investigated with Rotational Echo Double Resonance Magic Angle Spinning (REDOR MAS) experiments for measuring distances from the inhibitor to the protein.

Influenza A develops amantadine resistance within days in tissue culture or infection. The amantadine-resistant mutant, M2 S31N, has become globally dominant in human isolates. Infection of MDCK cells by a pandemic 2009 H1N1 strain, bearing M2 S31N, was blocked by low-micromolar concentrations for a set of amantadine analogs previously shown to block several M2-WT influenza strains from H1N1, H2N2, and H3N2 subtypes. Three sets of variants were synthesized to explore SAR properties. One demonstrated that opening the adamantane cage reduced activity. Another showed that the adduct must be more than 2 carbons. Pre-exposing the virus to drug before inoculation showed inactivation and exposure-recovery of the virus on the ∼10-minute time scale. Resistance development is dramatically reduced. For selected compounds, 10 passages (∼5 weeks) in the presence of drug were required before the 2009 H1N1 developed resistance. However, the mechanism of action is unclear. Liposome assays indicate direct block of S31N M2 (22-62). But 2009 H1N1 M2-transfected HEK cells are not blocked, either on the 2- or the 30-minute time scales. Yet, the revertant (N31S) is well blocked. Solid state NMR suggests that drugs bind to the S31N transmembrane peptide domain. The resistant strains developed in the presence of these drugs show no mutations in M2, but a few mutations in hemagglutinin. It is possible that these hydrophobic amines function partly by neutralizing the endosome. However, the virus pre-exposure results indicate a direct effect on the virus, not just on the endosome. The A/WSN/33 virus is not blocked by these drugs in cytopathic effect assays, but the revertant (N31S) is, indicating for A/WSN/33 that the M2-block is the key effect. In summary, resistance-invulnerable drugs for the 2009 H1N1 influenza A virus have been identified and the mechanism of action is yet to be defined.

NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed - neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (Anfinsen, 1973) [106]. The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins. (C) 2013 Elsevier Inc. All rights reserved.