Water orientation and dynamics in the closed and open influenza B virus M2 proton channels.
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ABSTRACT:
SUBMITTER: Gelenter MD
PROVIDER: S-EPMC7955094 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:M2 of the influenza virus is an intriguing transmembrane protein that forms a minuscule proton channel in the viral envelope. Its recognized function is to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. It is vital for viral replication and it is a target for the anti-influenza drugs, amantadine and rimantadine. Recently, high resolution structures of M2 channels of both flu A and B have been obtained, providing the desperately needed structural details for understanding the mechanism of proton conductance. In particular, the establishment of the functional solution NMR system of the proton channels enabled simultaneous high resolution structure characterization and measurement of channel dynamics coupled to channel activity. This review summarizes our current understanding of how protons are conducted through the M2 channel from a structural point of view, as well as the modes by which important channel gating elements function during proton conduction.
| S-EPMC3108042 | biostudies-literature
Project description:The influenza B M2 (BM2) proton channel is activated by acidic pH to mediate virus uncoating. Unlike influenza A M2 (AM2), which conducts protons with strong inward rectification, BM2 conducts protons both inward and outward. Here we report 1.4- and 1.5-Å solid-state NMR structures of the transmembrane domain of the closed and open BM2 channels in a phospholipid environment. Upon activation, the transmembrane helices increase the tilt angle by 6° and the average pore diameter enlarges by 2.1 Å. BM2 thus undergoes a scissor motion for activation, which differs from the alternating-access motion of AM2. These results indicate that asymmetric proton conduction requires a backbone hinge motion, whereas bidirectional conduction is achieved by a symmetric scissor motion. The proton-selective histidine and gating tryptophan in the open BM2 reorient on the microsecond timescale, similar to AM2, indicating that side chain dynamics are the essential driver of proton shuttling.
| S-EPMC7641042 | biostudies-literature
Project description:In this study, four possible conformations of the His-37 and Trp-41 residues for the closed state of the influenza M2 ion channel were identified by a conformation scan based on a solid-state NMR restraint. In the four conformations, the His-37 residue can be of either the t-160 or t60 rotamer, whereas Trp-41 can be of either the t-105 or t90 rotamer. These conformations were further analyzed by density functional theory calculations and molecular dynamics simulations, and the data indicate that the His-37 residue most likely adopts the t60 rotamer and should be monoprotonated at the delta-nitrogen site, whereas Trp-41 adopts the t90 rotamer. This result is consistent with published experimental data and points to a simple gating mechanism: in the closed state, the His-37 and Trp-41 residues adopt the (t60, t90) conformation, which nearly occludes the pore, preventing nonproton ions from passing through due to the steric and desolvation effects. Moreover, the His-37 tetrad interrupts the otherwise continuous hydrogen-bonding network of the pore water by forcing the water molecules above and below it to adopt opposite orientations, thus adding to the blockage of proton shuttling. The channel can be easily opened by rotating the His-37 chi2 angle from 60 to 0 degrees . This open structure allows pore water to penetrate the constrictive region and to form a continuous water wire for protons to shuttle through, while being still narrow enough to exclude other ions.
| S-EPMC1366740 | biostudies-literature
Project description:Structures of truncated versions of the influenza A virus M2 proton channel have been determined recently by x-ray crystallography in the open conformation of the channel, and by NMR in the closed state. The structures differ in the position of the bound inhibitors. The x-ray structure shows a single amantadine molecule in the middle of the channel, whereas in the NMR structure four drug molecules bind at the channel's outer surface. To study this controversy we applied computational solvent mapping, a technique developed for the identification of the most druggable binding hot spots of proteins. The method moves molecular probes--small organic molecules containing various functional groups--around the protein surface, finds favorable positions using empirical free energy functions, clusters the conformations, and ranks the clusters on the basis of the average free energy. The results of the mapping show that in both structures the primary hot spot is an internal cavity overlapping the amantadine binding site seen in the x-ray structure. However, both structures also have weaker hot spots at the exterior locations that bind rimantadine in the NMR structure, although these sites are partially due to the favorable interactions with the interfacial region of the lipid bilayer. As confirmed by docking calculations, the open channel binds amantadine at the more favorable internal site, in good agreement with the x-ray structure. In contrast, the NMR structure is based on a peptide/micelle construct that is able to accommodate the small molecular probes used for the mapping, but has a too narrow pore for the rimantadine to access the internal hot spot, and hence the drug can bind only at the exterior sites.
| S-EPMC2776279 | biostudies-literature
Project description:The M2 protein of influenza viruses forms an acid-activated tetrameric proton channel. We used solid-state nuclear magnetic resonance spectroscopy to determine the structure and functional dynamics of the pH-sensing and proton-selective histidine-37 in M2 bound to a cholesterol-containing virus-envelope-mimetic membrane so as to better understand the proton conduction mechanism. In the high-pH closed state, the four histidines form an edge-face π-stacked structure, preventing the formation of a hydrogen-bonded water chain to conduct protons. In the low-pH conducting state, the imidazoliums hydrogen-bond extensively with water and undergo microsecond ring reorientations with an energy barrier greater than 59 kilojoules per mole. This barrier is consistent with the temperature dependence of proton conductivity, suggesting that histidine-37 dynamically shuttles protons into the virion. We propose a proton conduction mechanism in which ring-flip-assisted imidazole deprotonation is the rate-limiting step.
| S-EPMC4102303 | biostudies-literature
Project description:The M2 protein of the influenza A virus is activated by low endosomal pH and performs the essential function of proton transfer into the viral interior. The resulting decrease in pH within the virion is essential for the uncoating and further replication of the viral genetic material. The x-ray crystal [Stouffer AL, et al. (2008) Nature 451:596-599] and solution NMR [Schnell JR, Chou JJ (2008) Nature 451:591-595] structures of the transmembrane region of the M2 homo-tetrameric bundle both revealed pores with narrow constrictions at one end, leaving a question as to how protons enter the channel. His-37, which is essential for proton-gating and selective conduction of protons, lies in the pore of the crystallographic and NMR structures. Here, we explore the different protonation states of the His-37 residues of the M2 bundle in a bilayer using molecular dynamics (MD) simulations. When the His-37 residues are neutral, the protein prefers an Open(out)-Closed(in) conformation in which the channel is open to the environment on the outside of the virus but closed to the interior environment of the virus. Diffusion of protons into the channel from the outside of the virus and protonation of His-37 residues in the tetramer stabilizes an oppositely gated Closed(out)-Open(in) conformation. Thus, protons might be conducted through a transporter-like mechanism, in which the protein alternates between Open(out)-Closed(in) and Closed(out)-Open(in) conformations, and His-37 is protonated/deprotonated during each turnover. The transporter-like mechanism is consistent with the known properties of the M2 bundle, including its relatively low rate of proton flux and its strong rectifying behavior.
| S-EPMC2626606 | biostudies-literature
Project description:The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K(+) or Na(+) efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K(+) to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH(in)) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH(in) first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH(in) is achieved.
| S-EPMC2932607 | biostudies-literature
Project description:The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine. 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 amino-terminal half of the pore, indicating a physical occlusion mechanism for inhibition. However, a solution NMR structure of M2(18-60) showed four rimantadines bound to the carboxy-terminal lipid-facing surface of the helices, 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 A-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 (2)H 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.
| S-EPMC2818718 | biostudies-literature
Project description:M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591-595) may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus.Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors.1) The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2) The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3) The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a "barrel hoop", holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4) The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.
| S-EPMC2826421 | biostudies-literature
Project description:Structures of the influenza A virus M2 proton channel in the open conformation have been determined by X-ray crystallography, and in the closed conformation by NMR. Whereas the X-ray structure shows a single inhibitor molecule in the middle of the channel, four inhibitor molecules bind the channel's outer surface in the NMR structure. In both structures, the strongest hot spots (i.e., regions that contribute substantially to the free energy of binding any potential ligand) lie inside the pore, and other hot spots are found at exterior locations. By considering all available models, we propose the primary drug binding site is inside the pore, but that exterior binding occurs under appropriate conditions.
| S-EPMC2919587 | biostudies-literature