M2 Channel Of Influenza Research Articles

Molecular origins of asymmetric proton conduction in the influenza M2 channel.

The M2 proton channel of influenza A is embedded into the viral envelope and allows acidification of the virion when the external pH is lowered. In contrast, no outward proton conductance is observed when the internal pH is lowered, although outward current is observed at positive voltage. Residues Trp41 and Asp44 are known to play a role in preventing pH-driven outward conductance but the mechanism for this is unclear. We investigate this issue using classical molecular dynamics simulations with periodic proton hops. When all key His37 residues are neutral, inward proton movement is much more facile than outward movement if the His are allowed to shuttle the proton. The preference for inward movement increases further as the charge on the His37 increases. Analysis of the trajectories reveals three factors accounting for this asymmetry. First, in the outward direction the Asp44 trap the hydronium by strong electrostatic interactions. Secondly, Asp44 and Trp41 orient the hydronium with the protons pointing inward, hampering outward Grotthus hopping. As a result, the effective barrier is lower in the inward direction. The Trp41 add to the barrier by weakly H-bonding to potential H+ acceptors. Finally, for charged His, the H3O+ in the inner vestibule tends to get trapped at lipid-lined fenestrations of the cone-shaped channel. Simulations qualitatively reproduce the experimentally observed higher outward conductance of mutants. The ability of positive voltage, unlike proton gradient, to induce outward current appears to arise from its ability to bias H3O+ and the waters around it toward more H-outward orientations.

Molecular origins of asymmetric proton conduction in the influenza M2 channel

The M2 proton channel of influenza A is embedded into the viral envelope and allows acidification of the virion when the external pH is lowered. In contrast, no outward proton conductance is observed when the internal pH is lowered, although outward current is observed at positive voltage. Residues Trp41 and Asp44 are known to play a role in preventing pH-driven outward conductance, but the mechanism for this is unclear. We investigate this issue using classical molecular dynamics simulations with periodic proton hops. When all key His37 residues are neutral, inward proton movement is much more facile than outward movement if the His are allowed to shuttle the proton. The preference for inward movement increases further as the charge on the His37 increases. Analysis of the trajectories reveals three factors accounting for this asymmetry. First, in the outward direction, Asp44 traps the hydronium by strong electrostatic interactions. Secondly, Asp44 and Trp41 orient the hydronium with the protons pointing inward, hampering outward Grotthus hopping. As a result, the effective barrier is lower in the inward direction. Trp41 adds to the barrier by weakly H-bonding to potential H+ acceptors. Finally, for charged His, the H3O+ in the inner vestibule tends to get trapped at lipid-lined fenestrations of the cone-shaped channel. Simulations qualitatively reproduce the experimentally observed higher outward conductance of mutants. The ability of positive voltage, unlike proton gradient, to induce an outward current appears to arise from its ability to bias H3O+ and the waters around it toward more H-outward orientations.

X-Ray Crystal Structures of the Influenza a M2 Proton Channel Bound to Amantadine, Rimantadine, and Inhibiting Compounds

Inhibition of the M2 proton channel in the influenza A virus prevents viral replication from occurring. Two of the four FDA-approved drugs for the treatment of influenza infections, amantadine and rimantadine, target the M2 channel. However, because M2 is a membrane protein, structural studies of drug binding to the channel using x-ray crystallography have been limited because of the challenging nature of the target, with the one previously solved crystal structure limited to 3.5 Å resolution. Here, we have obtained multiple crystal structures of M2 in the presence of drugs and inhibiting compounds using lipidic cubic phase (LCP) crystallization techniques. We present the first crystal structures of rimantadine bound to M2 in both the Inwardclosed and Inwardopen conformations of the channel (2.0 Å, 2.5 Å), as well as amantadine bound to the Inwardclosed conformation (2.0 Å). At this resolution range the orientation of the bound drug is unambiguous, and the ammonium group of the adamantane drugs can be seen interacting with ordered water molecules present within the channel. Additionally, we report the binding of a dual-inhibiting compound to both 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. These structures further 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 the influenza M2 channel.

Mapping the Resistance Potential of Influenza's H+ Channel against an Antiviral Blocker

The development of drug resistance has long plagued our efforts to curtail viral infections in general and influenza in particular. The problem is particularly challenging since the exact mode of resistance may be difficult to predict, without waiting for untreatable strains to evolve. Herein, a different approach is taken. Using a novel genetic screen, we map the resistance options of influenza's M2 channel against its aminoadamantane antiviral inhibitors. In the process, we could identify clinically known resistant mutations in a completely unbiased manner. Additionally, novel mutations were obtained, which, while known to exist in circulating viruses, were not previously classified as drug resistant. Finally, we demonstrated the approach against an anti-influenza drug that has not seen clinical use, identifying several resistance mutations in the process. In conclusion, we present and employ a method to predict the resistance options of influenza's M2 channel to antiviral agents ahead of clinical use and without medical hazard.

Observation of Partial and Complete Block by Amantadine and Rimantadine in Influenza a M2 S31N by Electrophysiology Methods

The mutation from serine to asparagine at residue 31 of M2, which is found in the majority of influenza strains circulating in humans, inhibits amantadine (ADA-NH3+) block, at least in part due to increased exit rate (1), but from simulations it is unclear whether the inhibition results from weak binding (2) or incomplete block (3). We addressed the question of whether there is amantadine binding without block of proton conductance experimentally. Xenopus laevis oocytes were transfected with A/Udorn/72 S31N mRNA. Two-electrode voltage clamp was used to measure amantadine block of M2 proton conductance at unusually high drug concentrations. Results showed that the S31N M2 channel is completely blocked at 10 mM, proving that there is no “binding without block” for amantadine in the influenza M2 channel. More surprisingly, we found that rimantadine (ADA-CH(NH3+)-CH3), which blocks the wild-type M2 S31 channel better than amantadine, achieves a maximal, modest level of block in the S31N M2 channel at 1 mM, with no further increase in block at 10 mM drug. We conclude that rimantadine binds without block in the S31N M2 channel. These findings can further constrain simulations aimed at evaluating the mechanism of block.1. Stouffer, A. L. et al. (2008) Structure 16:1067-1076.2. Gleed, M.L. et al (2015) JPCB 119:11548-59.3. Gleed, M.L and Busath, D.D. (2015) JPCB 119:1225-31.

Studying Membrane Protein Folding by Molecular Dynamics Simulations

Membrane protein folding is a process of fundamental importance in biophysics and structural biology. Despite major advances in our understanding of translocon-mediated insertion of membrane proteins, many aspects of the underlying biophysics remain unclear.Computational methods provide a powerful tool for understanding membrane protein folding. As an initial model for a translocon tunnel, we are using a simplified pore system to investigate systematically the effects of this on helix formation. This approach is being used in parallel with enhanced sampling methods to accelerate the folding reaction. As test systems we have focused on two membrane proteins: the influenza M2 channel protein which contains a single transmembrane (TM helix plus a C-terminal amphipathic helix, and the seven TM helix protein bacteriorhodopsin, For bacteriorhodopsin we can successfully fold all seven helices inside our simple model of a translocon, starting from a fully extended conformation. For influenza M2, we observe the two helices (TM and amphipathic) folding to a state that is consistent with NMR structures.We wish to develop this model further to include the crucial step of helix insertion into the membrane. In this context we are also exploring coarse-grained approaches to help us address time-scales accessible to simulations.

2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel.

Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

Unusual architecture of the p7 channel from hepatitis C virus

The Hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations1-4. We are curious as to what structural solution has the viroporin adopted to afford selective cation conduction because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The p7 activity can be inhibited by amantadine and rimantadine2,5, which also happen to be potent blockers of the influenza M2 channel6 and licensed drugs against influenza infections7. The adamantane derivatives were subjects of HCV clinical trials8, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here, we determined the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbors, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also alludes to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter while an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording showed that these residues are indeed critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Structural Investigation of Rimantadine Inhibition of the AM2-BM2 Chimera Channel of Influenza Viruses

The M2 channel of influenza A is a target of the adamantane family antiviral drugs. Two different drug-binding sites have been reported: one inside the pore, and the other is a lipid-facing pocket. A previous study showed that a chimera of M2 variants from influenza A and B that contains only the pore-binding site is sensitive to amantadine inhibition, suggesting that the primary site of inhibition is inside the pore. To obtain atomic details of channel-drug interaction, we determined the structures of the chimeric channel with and without rimantadine. Inside the channel and near the N-terminal end, methyl groups of Val27 and Ala30 from four subunits form a hydrophobic pocket around the adamantane, and the drug amino group appears to be in polar contact with the backbone oxygen of Ala30. The structures also reveal differences between the drug-bound and -unbound states of the channel that can explain drug resistance.

How Do Aminoadamantanes Block the Influenza M2 Channel, and How Does Resistance Develop?

The interactions between channels and their cognate blockers are at the heart of numerous biomedical phenomena. Herein, we unravel one particularly important example bearing direct pharmaceutical relevance: the blockage mechanism of the influenza M2 channel by the anti-flu amino-adamantyls (amantadine and rimantadine) and how the channel and, consequently, the virus develop resistance against them. Using both computational analyses and experimental verification, we find that amino-adamantyls inhibit M2's H(+) channel activity by electrostatic hindrance due to their positively charged amino group. In contrast, the hydrophobic adamantyl moiety on its own does not impact conductivity. Additionally, we were able to uncover how mutations in M2 are capable of retaining drug binding on the one hand yet rendering the protein and the mutated virus resistant to amino-adamantyls on the other hand. We show that the mutated, drug-resistant protein has a larger binding pocket for the drug. Hence, despite binding the channel, the drug remains sufficiently mobile so as not to exert a H(+)-blocking positive electrostatic hindrance. Such insight into the blocking mechanism of amino-adamantyls, and resistance thereof, may aid in the design of next-generation anti-flu agents.

Potent Inhibitor Design Against H1N1 Swine Influenza: Structure-based and Molecular Dynamics Analysis for M2 Inhibitors from Traditional Chinese Medicine Database

The rapid spread of influenza virus subtype H1N1 poses a great threat to million lives worldwide. To search for new anti-influenza compounds, we performed molecular docking and molecular dynamics simulation to identify potential traditional Chinese medicine (TCM) constituents that could block influenza M2 channel activity. Quinic acid, genipin, syringic acid, cucurbitine, fagarine, and methyl isoferulate all have extremely well docking results as compared to control amantadine. Further de novo drug design suggests that derivatives of genipin and methyl isoferulate could have enhanced binding affinity towards M2 channel. Selected molecular dynamics simulations of M2-derivative complexes show stable hydrogen bond interactions between the derivatives and M2 residues, Ser10 and Ala9. To our best knowledge, this is the first study on the anti-viral activity of the above listed TCM compounds.

Proton Transport Through Channels: Insights and Surprises from Molecular Simulation

The behavior of hydrated excess protons passing into and through transmembrane proton channels will be presented, based on the results of a novel multi-scale computer simulation methodology. The unique electrostatics related to the dynamic delocalization of the excess proton charge defect will be elaborated, as well as its effect on the channel proton transport and selectivity properties. The often opposing and asymptotic viewpoints related to electrostatics on one hand and Grotthuss proton shuttling on the other will be reconciled and unified into a single conceptual framework. Specific simulation results will be given for various channel systems, including the M2 channel of influenza A, proton selective mutant aquaporin-1 channels, the ClC Cl-/H+ antiporter, and models of the Hv1 voltage gated proton channel. Comparison to experimental results will be discussed where possible.

Molecular Dynamics Simulations of the Dimerization of Transmembrane α-Helices

Membrane proteins account for nearly a quarter of all genes, but their structure and function remain incompletely understood. Most membrane proteins have transmembrane (TM) domains made up of bundles of hydrophobic alpha-helices. The lateral association of TM helices within the lipid bilayer is a key stage in the folding of membrane proteins. It may also play a role in signaling across cell membranes. Dimerization of TM helices is a simple example of such lateral association. Molecular dynamics (MD) simulations have been used for over a decade to study membrane proteins in a lipid bilayer environment. However, direct atomistic (AT) MD simulation of self-assembly of a TM helix bundle remains challenging. AT-MD may be complemented by coarse-grained (CG) simulations, in which small numbers of atoms are grouped together into particles. In this Account, we demonstrate how CG-MD may be used to simulate formation of dimers of TM helices. We also show how a serial combination of CG and AT simulation provides a multiscale approach for generating and refining models of TM helix dimers. The glycophorin A (GpA) TM helix dimer represents a paradigm for helix-helix packing, mediated by a GxxxG sequence motif. It is well characterized experimentally and so is a good test case for evaluating computational methods. CG-MD simulations in which two separate TM helices are inserted in a lipid bilayer result in spontaneous formation of a right-handed GpA dimer, in agreement with NMR structures. CG-MD models were evaluated via comparison with data on destabilizing mutants of GpA. Such mutants increased the conformational flexibility and the dissociation constants of helix dimers. GpA dimers have been used to evaluate a multiscale approach: A CG model is converted to an AT model, which is used as the basis of an AT-MD simulation. Comparison of three AT-MD simulations of GpA, one starting from a CG model and two starting from NMR structures, leads to convergence to a common refined structure for the dimer. CG-MD self-assembly has also been used to model dimerization of the TM domain of the syndecan-2 receptor protein. This TM helix contains a GxxxG motif, which mediates right-handed helix packing comparable to that of the GxxxG motif in GpA. The multiscale approach has been applied to a more complex system, the heterodimeric alphaIIb/beta3 integrin TM helix dimer. In CG-MD, both right-handed and left-handed structures were formed. Subsequent AT-MD simulations showed that the right-handed structure was more stable, yielding a dimer in which the GxxxG motif of the alphaIIb TM helix packed against a hydrophobic surface of the beta3 helix in a manner comparable to that observed in two recent NMR studies. This work demonstrates that the multiscale simulation approach can be used to model simple membrane proteins. The method may be applied to more complex proteins, such as the influenza M2 channel protein. Future refinements, such as extending the multiscale approach to a wider range of scales (from CG through QM/MM simulations, for example), will expand the range of applications and the accuracy of the resultant models.

Molecular strategies to inhibit the replication of RNA viruses

There are virtually no antiviral drugs available for the treatment of infections with RNA viruses. This is particularly worrisome since most of the highly pathogenic and emerging viruses are, and will likely continue to be, RNA viruses. These viruses can cause acute, severe illness, including severe respiratory disease, hemorrhagic fever and encephalitis, with a high case fatality rate. It is important to have potent and safe drugs at hand that can be used for the treatment or prophylaxis of such infections. Drugs approved for the treatment of RNA virus infections (other than HIV) are the influenza M2 channel inhibitors, amantadine and rimantadine; the influenza neuraminidase inhibitors, oseltamivir and zanamivir, and ribavirin for the treatment of infections with respiratory syncytial virus and hepatitis C virus. The molecular mechanism(s) by which ribavirin inhibits viral replication, such as depletion of intracellular GTP pools and induction of error catastrophe, may not readily allow the design of analogues that are more potent/selective than the parent drug. Highly pathogenic RNA viruses belong to a variety of virus families, each having a particular replication strategy, thus offering a wealth of potential targets to selectively inhibit viral replication. We here provide a non-exhaustive review of potential experimental strategies, using small molecules, to inhibit the replication of several RNA viruses. Other approaches, such as the use of interferon or other host-response modifiers, immune serum or neutralizing antibodies, are not addressed in this review.

The M2 channel of influenza A virus: a molecular dynamics study

Molecular dynamics simulations have been performed on a tetramer of the 25-residue (SSDPLVVAASIIGILHLILWILDRL) synthetic peptide [1]which contains the transmembrane domain of the influenza A virus M2 coat protein. The peptide bundle was initially assembled as a parallel α-helix bundle in the octane portion of a phase separated water/octane system, which provided a membrane-mimetic environment. A 4-ns dynamics trajectory identified a left-handed coiled coil state of the neutral bundle, with a water filled funnel-like structural motif at the N-terminus involving the long hydrophobic sequence. The neck of the funnel begins at V27 and terminates at H37, which blocks the channel. The C-terminus is held together by inter-helix hydrogen bonds and contains water below H37. Solvation of the S23 and D24 residues, located at the rim of the funnel, appears to be important for stability of the structure. The calculated average tilt of the helices in the neutral bundle is 27±5°, which agrees well with recent NMR data.