M2 proton channel

3D model. (M2 labeled in white.)

The Matrix-2 (M2) protein is a proton-selective ion channel protein, integral in the viral envelope of the influenza A virus. The channel itself is a homotetramer (consists of four identical M2 units), where the units are helices stabilized by two disulfide bonds, and is activated by low pH. The M2 protein is encoded on the seventh RNA segment together with the M1 protein. Proton conductance by the M2 protein in influenza A is essential for viral replication.

Structure

Flu_M2

the closed state structure of m2 protein h+ channel by solid state nmr spectroscopy
Identifiers
Symbol Flu_M2
Pfam PF00599
InterPro IPR002089
SCOP 1mp6
SUPERFAMILY 1mp6
TCDB 1.A.19
OPM superfamily 206
OPM protein 2kqt

In influenza A virus, M2 protein unit consists of three protein segments comprising 97 amino acid residues: (i) an extracellular N-terminal domain (residues 1–23); (ii) a transmembrane segment (TMS) (residues 24–46); (iii) an intracellular C-terminal domain (residues 47–97). The TMS forms the pore of the ion channel. The important residues are the imidazole of His37 (pH sensor) and the indole of Trp41 (gate).[1] This domain is the target of the anti influenza drugs, amantadine and its ethyl derivative rimantadine, and probably also the methyl derivative of rimantadine, adapromine. The first 17 residues of the M2 cytoplasmic tail form a highly conserved amphipathic helix.[2]

The amphipathic helix residues (46–62) within the cytoplasmic tail play role in virus budding and assembly. The influenza virus utilizes these amphipathic helices in M2 to alter membrane curvature at the budding neck of the virus in a cholesterol dependent manner.[3] The residues 70–77 of cytoplasmic tail are important for binding to M1 and for the efficient production of infectious virus particles. This region also contains a caveolin binding domain (CBD). The C-terminal end of the channel extends into a loop (residues 47–50) that connects the trans membrane domain to the C-terminal amphipathic helix. (46–62). Two different high-resolution structures of truncated forms of M2 have been reported: the crystal structure of a mutated form of the M2 transmembrane region (residues 22–46),[4] as well as a longer version of the protein (residues 18–60) containing the transmembrane region and a segment of the C-terminal domain as studied by nuclear magnetic resonance (NMR).[5]

The two structures also suggest different binding sites for the adamantane class of anti-influenza drugs. According to the low pH crystal structure a single molecule of amantadine binds in the middle of the pore, surrounded by residues Val27, Ala30, Ser31 and Gly34. In contrast, the NMR structure showed four rimantadine molecules bind to the lipid facing outer surface of the pore, interacting with residues Asp44 and Arg45. However, a recent solid state NMR spectroscopy structure shows that the M2 channel has two binding sites for amantadine, one high affinity site is in the N terminal lumen, and a second low affinity site on the C terminal protein surface.[6]

M2 protein of Influenza B

The M2 protein of influenza B is 109 residue long, homo tetramer and is a functional homolog of influenza A protein. There is almost no sequence homology between influenza AM2 and BM2 except for the HXXXW sequence motif in the TMS that is essential for channel function. Its proton conductance pH profile is similar to that of AM2. However, the BM2 channel activity is higher than that of AM2, and the BM2 activity is completely insensitive to amantadine and rimantadine.[7]

Proton conductance and selectivity

The M2 ion channel of both influenza A and B are highly selective for protons. The channel is activated by low pH and has a low conductance.[8] Histidine residues at position 37 (His37) are responsible for this proton selectivity and pH modulation. When His37 is replaced with glycine, alanine, glutamic acid, serine or threonine, the proton selective activity is lost and the mutant can transport Na+ and K+ ions also. When imidazole buffer is added to cells expressing mutant proteins, the ion selectivity is partially rescued.[9]

Acharya et al. suggested that the conduction mechanism involves the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior.[10] Water molecules within the pore form hydrogen-bonded networks or 'water wires' from the channel entrance to His37. Pore-lining carbonyl groups are well situated to stabilize hydronium ions via second-shell interactions involving bridging water molecules. A collective switch of hydrogen bond orientations may contribute to the directionality of proton flux as His37 is dynamically protonated and deprotonated in the conduction cycle.[11] The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms near by. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters.

Function

The M2 channel protein is an essential component of the viral envelope because of its ability to form a highly selective, pH-regulated, proton-conducting channel. The M2 proton channel maintains pH across the viral envelope during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. As virus enters the host cell by receptor-mediated endocytosis, endosomal acidification occurs. This low pH activates the M2 channel, which brings protons into the virion core. Acidification of virus interior, leads to weakening of electrostatic interaction and leads to dissociation between M1 and viral ribonucleoprotein (RNP) complexes. Subsequent membrane fusion releases the uncoated RNPs into the cytoplasm which is imported to the nucleus to start viral replication.

After its synthesis within the infected host cell, M2 is inserted into the endoplasmic reticulum (ER) and transported to the cell surface via trans-Golgi network (TGN). Within the acidic TGN, M2 transports H+ ions out of the lumen, and maintains hemagglutinin (HA) metastable configuration.[12] At its TGN localization, M2 protein's ion channel activity has been shown to effectively activate the NLRP3 inflammasome pathway.[13]

Other important functions of M2 are its role in formation of filamentous strains of influenza, membrane scission and the release of the budding virion. M2 stabilizes the virus budding site, and mutations of M2 that prevent its binding to M1 can impair filament formation at the site of budding.

Transport reaction

The generalized transport reaction catalyzed by the M2 channel is:

H+ (out) ⇌ H+ (in)

Inhibition and resistance

The anti-influenza virus drug, amantadine, is a specific blocker of the M2 H+ channel. In the presence of amantadine, viral uncoating is incomplete, and the RNP core fails to promote infection. Aminoadamantanes, including amantadine and rimantadine have been widely abandoned due to virus resistance, but thermodynamic considerations allow design of new derivatives.[14]

Two different sites for drug interaction have been proposed. One is a lipid-facing pocket between 2 adjacent transmembrane helices (around Asp-44), at which the drug binds and inhibits proton conductance allosterically. The other is inside the pore (around Ser-31), at which the drug directly blocks proton passage.[15]

However, the M2 gene is susceptible to mutations. When one of five amino acids in the transmembrane region gets suitably substituted, the virus becomes resistant to the existing M2 inhibitors. S31N mutation is responsible for more than 90% resistance to the drugs amantadine and rimantadine. As the mutations are relatively frequent, presence of the selection factors (e.g., using amantadine for treatment of sick poultry) can lead to emergence of a resistant strain. The US CDC has released information stating that most strains are now resistant to the two drugs available, and their use should be discontinued.

See also

References

  1. ↑ Tang Y, Zaitseva F, Lamb RA, Pinto LH (October 2002). "The gate of the influenza virus M2 proton channel is formed by a single tryptophan residue". The Journal of Biological Chemistry. 277 (42): 39880–6. doi:10.1074/jbc.M206582200. PMID 12183461.
  2. ↑ Holsinger LJ, Nichani D, Pinto LH, Lamb RA (March 1994). "Influenza A virus M2 ion channel protein: a structure-function analysis". Journal of Virology. 68 (3): 1551–63. PMC 236612Freely accessible. PMID 7508997.
  3. ↑ Rossman JS, Jing X, Leser GP, Lamb RA (September 2010). "Influenza virus M2 protein mediates ESCRT-independent membrane scission". Cell. 142 (6): 902–13. doi:10.1016/j.cell.2010.08.029. PMC 3059587Freely accessible. PMID 20850012.
  4. ↑ Stouffer AL, Acharya R, Salom D, Levine AS, Di Costanzo L, Soto CS, Tereshko V, Nanda V, Stayrook S, DeGrado WF (January 2008). "Structural basis for the function and inhibition of an influenza virus proton channel". Nature. 451 (7178): 596–9. Bibcode:2008Natur.451..596S. doi:10.1038/nature06528. PMC 3889492Freely accessible. PMID 18235504.
  5. ↑ Schnell JR, Chou JJ (January 2008). "Structure and mechanism of the M2 proton channel of influenza A virus". Nature. 451 (7178): 591–5. Bibcode:2008Natur.451..591S. doi:10.1038/nature06531. PMC 3108054Freely accessible. PMID 18235503.
  6. ↑ Cady SD, Schmidt-Rohr K, Wang J, Soto CS, Degrado WF, Hong M (February 2010). "Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers". Nature. 463 (7281): 689–92. Bibcode:2010Natur.463..689C. doi:10.1038/nature08722. PMC 2818718Freely accessible. PMID 20130653.
  7. ↑ Pielak RM, Chou JJ (February 2011). "Influenza M2 proton channels". Biochimica et Biophysica Acta. 1808 (2): 522–9. doi:10.1016/j.bbamem.2010.04.015. PMC 3108042Freely accessible. PMID 20451491.
  8. ↑ Mould JA, Li HC, Dudlak CS, Lear JD, Pekosz A, Lamb RA, Pinto LH (March 2000). "Mechanism for proton conduction of the M(2) ion channel of influenza A virus". The Journal of Biological Chemistry. 275 (12): 8592–9. doi:10.1074/jbc.275.12.8592. PMID 10722698.
  9. ↑ Venkataraman P, Lamb RA, Pinto LH (June 2005). "Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus". The Journal of Biological Chemistry. 280 (22): 21463–72. doi:10.1074/jbc.M412406200. PMID 15784624.
  10. ↑ Acharya R, Carnevale V, Fiorin G, Levine BG, Polishchuk AL, Balannik V, Samish I, Lamb RA, Pinto LH, DeGrado WF, Klein ML (August 2010). "Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus". Proceedings of the National Academy of Sciences of the United States of America. 107 (34): 15075–80. doi:10.1073/pnas.1007071107. PMC 2930543Freely accessible. PMID 20689043.
  11. ↑ Thomaston JL, Alfonso-Prieto M, Woldeyes RA, Fraser JS, Klein ML, Fiorin G, DeGrado WF (November 2015). "High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction". Proceedings of the National Academy of Sciences of the United States of America. 112 (46): 14260–5. doi:10.1073/pnas.1518493112. PMC 4655559Freely accessible. PMID 26578770.
  12. ↑ Sakaguchi T, Leser GP, Lamb RA (May 1996). "The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus". The Journal of Cell Biology. 133 (4): 733–47. doi:10.1083/jcb.133.4.733. PMC 2120830Freely accessible. PMID 8666660.
  13. ↑ Ichinohe T, Pang IK, Iwasaki A (May 2010). "Influenza virus activates inflammasomes via its intracellular M2 ion channel". Nature Immunology. 11 (5): 404–10. doi:10.1038/ni.1861. PMC 2857582Freely accessible. PMID 20383149.
  14. ↑ Homeyer N, Ioannidis H, Kolarov F, Gauglitz G, Zikos C, Kolocouris A, Gohlke H (January 2016). "Interpreting Thermodynamic Profiles of Aminoadamantane Compounds Inhibiting the M2 Proton Channel of Influenza A by Free Energy Calculations". Journal of Chemical Information and Modeling. 56 (1): 110–26. doi:10.1021/acs.jcim.5b00467. PMID 26690735.
  15. ↑ Pielak RM, Schnell JR, Chou JJ (May 2009). "Mechanism of drug inhibition and drug resistance of influenza A M2 channel". Proceedings of the National Academy of Sciences of the United States of America. 106 (18): 7379–84. doi:10.1073/pnas.0902548106. PMC 2678642Freely accessible. PMID 19383794.

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