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| <table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after August 15 2011. </td></tr>
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| =='''Influenza M2 Proton Channel'''==
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| <Structure load='2KQT' size='500' frame='true' align='right' caption='M2 transmembrane peptide of the influenza A virus in DMPC lipid bilayers bound to deuterated amantadine. PDB: 2KQT' scene='Insert optional scene name here' />
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| =='''Introduction'''==
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| Influenza A, better known as the flu, an infection of the nose, throat, and lungs caused by the influenza virus. Basic symptoms include aches, chills, fever, loss of energy and dizziness, but complications can include pneumonia, encephalitis, bronchitis, and death—about 36,000 people every year die of complications from the flu (CDC).
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| The M2 protein is a proton-selective ion channel protein that plays an important role in the life cycle of the influenza A virus. The channel itself is a homotetramer with four identical M2 units, where each M2 protein is a helix stabilized by two disulfide bonds. At a low pH, the channel allows hydrogen ions to enter the viral particle form the endosome, effectively lowering the pH on the interior of the virus. This in turn causes the dissociation of the viral matrix protein M1 from the ribonucleoprotein, a critical step in “uncoating” the virus to introduce its contents to the cytoplasm of the host cell (Stouffer). The M2 protein itself consists of three major protein domains: a stretch of 24 amino acids on the N-terminal end that are exposed to the external environment, 22 (largely hydrophobic) amino acids in the transmembrane region, and 52 amino acids on the C-terminal end which are exposed to the inside of the viral particle (Schnell).
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| The function of the M2 channel can be inhibited by the antiviral drug Amantadine, an inhibition that effectively blocks the virus from taking over the host cell. Amantadine inhibits the replication of influenza A viruses by interfering with the uncoating of the virus within the cell. Amantadine is an M2 inhibitor that blocks the ion channel formed by the M2 protein that spans the viral membrane. By blocking this channel, Amantadine effectively prevents the acidification and subsequent release of viral elements into the host cell. Unfortunately, the M2 gene is susceptible to mutations. When one of five (of the 22) amino acids in the transmembrane region is suitably substituted, Amantadine no longer binds in such a way that would block the motion of the protons into the virus. As of 2009, the CDC noted that a full 100% of Influenza A viruses of types H3N2 and 2009 pandemic flu samples cultured in the United States showed a resistance to Amantadine (CDC).
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| =='''Overall Structure'''==
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| The M2 proton channel , made up of 97 residues, is a homotetramer transmembrane protein made up of 4 helices. The amino terminus of the protein is exposed to the outside environment, while the carboxy terminus is exposed to the internal environment. At pH 7.5, <scene name='Sandbox_111/Residue_18-23/1'>residues 18-23</scene> form the N-terminus. <scene name='Sandbox_111/Residue_25-46/1'>Residues 25-46</scene> create a transmembrane helix which forms a channel. Furthermore, residues 47-50 create a ‘short flexible loop,’ and residues 51-59 form a C-terminal amphipathic helix (Schnell).
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| The loop created by residues 47-50 at the C-terminus connects the amphipathic helices to the transmembrane domain. The amphipathic helices lie perpendicular to the transmembrane helices. These amphipathic helices form a base that is resistant to changes in pH and therefore acts to stabilize the protein. It should be noted that the transmembrane helices are left handed while the amphipathic helices forming the base are right handed. These amphipathic helicves are also arranged head to tail (Schnell).
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| The pore created by the four tansmembrane helices is constricted at the N-terminus by the methyl groups on Val 27. On the other end of the pore, interactions between Trp41 also create a blockage. The four helices are packed so tightly that van der Waals forces are created between the indole rings of Trp41. This forms a gate. Together, the interactions between Trp41 and Val27 block the passage of water through the pore. Furthermore, hydrogen bonds between Asp44 and Trp41 stabilize the gate. When the pH is lowered, the imidazole rings of His37 are protonated and the helices undergo electrostatic repulsion. This in turn breaks the Asp44 and Trp41 interactions and the gate will open. As previously mentioned, the base created by the amphipathic helices prevents the protein from dissociating. However, cysteins at the N-terminus create disulphide bonds that also act to prevent dissociation (Schnell).
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| =='''Drug Binding Site'''==
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| Amantadine binds with high affinity to a site in the M2 protein spanning five residues: Leu 26, Val 27, Ala 30, Ser 31, and Gly 34 (Cady). This high affinity is seen at pH’s closer to neutral. In lower pH’s the protein is only somewhat bound to amantadine. Therefore, when determining the mechanism by which amantadine blocks the channel experiments must be conducted at neutral pH. <scene name='Sandbox_111/Binding/10'>Binding of amantadine</scene> to the M2 protein is illustrated for viewing of the bonds.
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| When amantadine isn't present, the <scene name='Sandbox_111/No_amantadine/3'>pore</scene> created in the M2 complex is open, allowing viral particles to pass through. In the presence of amantadine, this pore is <scene name='Sandbox_111/Amantadine_present/1'>occluded</scene>, which prevents entry of the viral particles.
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| =='''Additional Features'''==
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| <scene name='Sandbox_111/His37/1'>His37</scene> – His37 has been found to exhibit significant proton selectivity. This suggests that His37 is involved in the opening of the conductive channel. The channel is non-conductive when His37 is not protonated, and conductive when it is in the protonated state (Pielak/Chou).
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| <scene name='Sandbox_111/Trp41_ring/1'>Trp41</scene> – Replacing Trp41 with a substitute amino acid results in increased channel current in in both directions. As such, the Trp 41 site is important to directional selectivity, in that it regulates the direction of the proton channel flow. This is accomplished in part because it forms a ring that prevents water from entering the channel and reaching the His37, and thus deprotonating if the pH is high, from the C-terminus(Pielak/Chou).
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| In addition, the proton transport channel is used to equalize the pH in the channel with that of the cytoplasm in the host cell. This prevents rearrangement of the haemmagglutanin during its transport to the host cell. (Schnell)
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| =='''References'''==
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| Cady, S.D., Schmidt-Rohr, K., Wang, J., Soto, C., DeGrado, W.F., Hong, M. "Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers," (2010) ''Nature'' '''463''': 689-692.
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| Stouffer AL, Acharya R, Salom D, Levine AS, Di Costanzo L, Soto CS, Tereshko V, Nanda V, Stayrook S, DeGrado WF (2008). "Structural basis for the function and inhibition of an influenza virus proton channel". Nature 451 (7178): 596-9
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| Schnell JR, Chou JJ (2008). "Structure and mechanism of the M2 proton channel of influenza A virus". Nature 451 (7178): 591–5.
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| Pielak RM, Schnell JR, Chou JJ: Mechanism of drug inhibition and drug resistance of influenza A M2 channel. Proc Natl Acad Sci, 106(18):7379-84 (2009).
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| "CDC Recommends against the Use of Amantadine and Rimantadine for the Treatment or Prophylaxis of Influenza in the United States during the 2005–06 Influenza Season". CDC Health Alert. Centers for Disease Control and Prevention
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| Hay AJ, Wolstenholme AJ, Skehel JJ, Smith MH. The molecular basis of the specific anti-influenza action of amantadine. EMBO J 1985; 4: 3021-4.
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| Stephenson I, Nicholson KG. Influenza: vaccination and treatment. Eur Respir J 2001; 17: 1282-93.
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| Pielak RM, Chou JJ. "Infuenza M2 proton channels". BBAMEM-80262; No. of pages: 8; 4C: 2, 3, 5, 6.
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| =='''Credits'''==
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| Introduction -- Josephine Harrington
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| Overall structure -- Andrea Simoni
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| Drug binding site -- Joshua Drolet
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| Additional features -- John Hickey
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