XFEL structures of the influenza M2 proton channel: Room temperature water networks and insights into proton conduction

Abstract

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inward open state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inward open state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.W ater molecules in transmembrane protein pores participate in the transport of protons across the membrane bilayer. This process has been extensively studied experimentally and through computational simulations, particularly in small channels such as gramicidin A and influenza A M2. The movement of ions through channels is coupled to the diffusion of water through the pore, but protons are transported at a rate that is faster than the diffusion of H 3 O + (1, 2). Instead of diffusing through channels, protons move concertedly across networks of hydrogen-bonded waters through what is known as the Grotthuss mechanism (3-5). This mechanism of proton transport was initially discovered by the behavior of water in solution, and it has also been proposed to occur within membrane proteins containing water-filled pores (6-9). The matrix 2 (M2) protein of influenza A is one of the smallest proton-selective channels found in nature. This makes it an ideal system for studying the involvement of water in the selective transport of protons across the membrane. The M2 protein of influenza A is a tetramer made up of four 97-residue-long monomers. M2 is multifunctional, with different functions lying in different regions of the sequence. Residues 1-22 make up a conserved N-terminal domain that assists the incorporation of M2 into the virion (10) and is absent in influenza B viruses. The transmembrane domain of M2 (residues 22-46) is necessary for tetramerization (11) and forms a proton-selective channel (12-15) that is the target of the adamantane class of drugs, amantadine and rimantadine (11,(16)(17)(18). The transme...

Put a cork in it: Plugging the M2 viral ion channel to sink influenza

“…The M2 viroporin, a proton-dependent proton channel required for virus entry and egress, has historically been an effective antiviral target for drugs like amantadine and rimantadine. However, mutations in M2, 7-10, 12, 17-18, 22, 25), inhibit viruses with major resistance to other IAV antivirals like oseltamivir (5, 8, 10, 16) and/or synergize with oseltamivir (5, 16, 28), exhibit high genetic barriers to resistance following long-term in vitro passaging (5, 8, 14-16), have supportive preclinical parameters such as good stability and low toxicity (9, 16, 23), and rescue mice from lethal infection (9 Waters are shown as spheres (Thomaston et al, 2017). -S31N (19-49) in complex with (4) (PDB: 2LY0; ).…”

Influenza A Virus M2 Protein: Roles from Ingress to Egress

“…According to the water wire model, a water column in the pore is discontinued at the gate closed by deprotonated His37 residues. However, electrostatic repulsion resulting from the protonation of two or more His37 residues opens up the gate, and makes the water column continuous through which protons are transferred [ 56 , 57 , 58 , 59 ]. According to the proton relay model, the His37 imidazole side chain binds a proton from one side (outside) of the gate and releases the already bound proton on the other side (inside) of the gate [ 53 , 54 , 60 ].…”

1,2-Αnnulated Adamantane Heterocyclic Derivatives as Anti-Influenza Α Virus Agents

“…This compound was 25-and 3-fold more effective than amantadine and ribavirin respectively and its activity was comparable to that of rimantadine (EC50 15 Table 2. Anti-influenza A virus (H1N1) activity and cytotoxicity of the 1,2-annulated adamantane lactams 15,16,18,26,28,44,58,59, 66 and of the lactone 5 in MDCK cells. [65] Compound Structure Antiviral EC50 for influenza A/H1N1 (A/PR/8/34) (a) MTS / μM…”

“…Inside the pore, ordered waters form continuous hydrogen-bonding networks that span the pore from the Val27 tetrad to His37. [43,44] The binding site of Amt and Rim is the lumen of the four-helix bundle of the M2TM interacting with the porelining residues Val27, Ala30, Ser31 and Gly34 of the Nterminus portion of the M2TM. [45] The hydrophobic adamantane moiety of Amt or Rim is positioned in Nterminus on the exterior of the virus with the ammonium group directed downwards toward His37.…”