M2 proton channel
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The Matrix-2 (M2) protein is a
proton A proton is a stable subatomic particle, symbol , H+, or 1H+ with a positive electric charge of +1 ''e'' elementary charge. Its mass is slightly less than that of a neutron and 1,836 times the mass of an electron (the proton–electron mass ...
-selective viroporin, integral in the
viral envelope A viral envelope is the outermost layer of many types of viruses. It protects the genetic material in their life cycle when traveling between host cells. Not all viruses have envelopes. Numerous human pathogenic viruses in circulation are encase ...
of the
influenza Influenza, commonly known as "the flu", is an infectious disease caused by influenza viruses. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptom ...
A virus. The channel itself is a homotetramer (consists of four identical M2 units), where the units are helices stabilized by two
disulfide bond In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
s, 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.
Influenza Influenza, commonly known as "the flu", is an infectious disease caused by influenza viruses. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptom ...
B and C viruses encode proteins with similar function dubbed "BM2" and "CM2" respectively. They share little similarity with M2 at the sequence level, despite a similar overall structure and mechanism.


Structure

In influenza A virus, M2 protein unit consists of three protein segments comprising 97 amino acid residues: (i) an extracellular
N-terminal domain The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the ami ...
(residues 1–23); (ii) a
transmembrane segment A transmembrane domain (TMD) is a membrane-spanning protein domain. TMDs generally adopt an alpha helix topological conformation, although some TMDs such as those in porins can adopt a different conformation. Because the interior of the lipid bi ...
(TMS) (residues 24–46); (iii) an intracellular
C-terminal domain The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain ( protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is ...
(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). This domain is the target of the anti influenza drugs,
amantadine Amantadine, sold under the brand name Gocovri among others, is a medication used to treat dyskinesia associated with parkinsonism and influenza caused by type A influenzavirus, though its use for the latter is no longer recommended due to wid ...
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. 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. 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 In molecular biology, caveolins are a family of integral membrane proteins that are the principal components of caveolae membranes and involved in receptor-independent endocytosis. Caveolins may act as scaffolding proteins within caveolar ...
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), 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 Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...
(NMR). 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.


Proton conductance and selectivity

The M2 ion channel of both influenza A is highly selective for protons. The channel is activated by low pH and has a low conductance. 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. 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. 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. 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 Receptor-mediated endocytosis (RME), also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, proteins – and in some cases viruses – by the inward budding of the plasma membrane ( invagination). Th ...
,
endosomal Endosomes are a collection of intracellular sorting organelles in eukaryotic cells. They are parts of endocytic membrane transport pathway originating from the trans Golgi network. Molecules or ligands internalized from the plasma membrane can f ...
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 Electrostatics is a branch of physics that studies electric charges at rest (static electricity). Since classical times, it has been known that some materials, such as amber, attract lightweight particles after triboelectric effect, rubbing. ...
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 The endoplasmic reticulum (ER) is, in essence, the transportation system of the eukaryotic cell, and has many other important functions such as protein folding. It is a type of organelle made up of two subunits – rough 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 In molecular biology, hemagglutinins (or ''haemagglutinin'' in British English) (from the Greek , 'blood' + Latin , 'glue') are receptor-binding membrane fusion glycoproteins produced by viruses in the '' Paramyxoviridae'' family. Hemagglutinins a ...
(HA) metastable configuration. At its TGN localization, M2 protein's ion channel activity has been shown to effectively activate the
NLRP3 NLR family pyrin domain containing 3 (NLRP3) (previously known as NACHT, LRR and PYD domains-containing protein 3 ALP3and cryopyrin), is a protein that in humans is encoded by the ''NLRP3'' gene located on the long arm of chromosome 1. NLRP3 is ...
inflammasome Inflammasomes are cytosolic multiprotein oligomers of the innate immune system responsible for the activation of inflammatory responses. Activation and assembly of the inflammasome promotes proteolytic cleavage, maturation and secretion of pro-in ...
pathway. 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 Amantadine, sold under the brand name Gocovri among others, is a medication used to treat dyskinesia associated with parkinsonism and influenza caused by type A influenzavirus, though its use for the latter is no longer recommended due to wid ...
, is a specific blocker of the M2 H+ channel. The drug binds in and occludes the central pore. In the presence of amantadine, viral uncoating and disassembly is incomplete.
Mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, m ...
s conferring resistance to
adamantane Adamantane is an organic compound with a formula C10H16 or, more descriptively, (CH)4(CH2)6. Adamantane molecules can be described as the fusion of three cyclohexane rings. The molecule is both rigid and virtually stress-free. Adamantane is the ...
drugs, including amantadine and rimantadine, occur in the transmembrane region and are widespread. The large majority of resistant viruses carry the S31 N mutation. Resistance to adamantanes among circulating influenza A viruses varies by region but has globally increased significantly since the early 2000s. The
US CDC The Centers for Disease Control and Prevention (CDC) is the national public health agency of the United States. It is a United States federal agency, under the Department of Health and Human Services, and is headquartered in Atlanta, Georgi ...
has released information stating that most circulating strains are now resistant to the two drugs available, and as of June 2021, their use is not recommended.


Influenza B and C M2 proteins

Influenza Influenza, commonly known as "the flu", is an infectious disease caused by influenza viruses. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptom ...
B and C viruses encode virion proteins with similar proton-transducing function dubbed "BM2" and "CM2" respectively. They share little similarity with M2 at the sequence level, despite a similar overall structure and mechanism.


BM2

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. The structure of the influenza B channel at resolutions of 1.4–1.5 Å, published in 2020, revealed that the channel opening mechanism is different from that of the influenza A channel.


CM2

CM2 may play a role in genome packaging in virions. CM2 adjusts intracellular pH, and is able to replace influenza A M2 in this capacity.


See also

*
H5N1 genetic structure H5N1 genetic structure is the molecular structure of the H5N1 virus's RNA. H5N1 is an Influenza A virus subtype. Experts believe it might mutate into a form that transmits easily from person to person. If such a mutation occurs, it might remain ...


References


External links

* {{DEFAULTSORT:M2 Protein Influenza A virus Viral structural proteins Protein families Single-pass transmembrane proteins Transmembrane transporters Transport proteins Proton channels