Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness

Hu, Fanghao; Luo, Wenbin; Cady, Sarah D.; Hong, Mei

doi:10.1016/j.bbamem.2010.09.014

Cited by 81 publications

(146 citation statements)

References 62 publications

(102 reference statements)

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“…This conformational transition provides a pathway for proton transfer past the Trp41 gate into the viral interior. In NMR studies at low pH, both conformations are observed in equilibrium with one another (40,43,44 (Fig. S1, Bottom).…”

Section: Significancementioning

confidence: 98%

“…The Inward closed state has also been characterized by solution NMR (39,40) and solid-state NMR (41,42) studies under high pH conditions. The Inward closed conformation can transition to the Inward open conformation by straightening a kink in the TM helix near Gly34 (40,43,44). This conformational transition provides a pathway for proton transfer past the Trp41 gate into the viral interior.…”

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

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

Thomaston

Woldeyes

Nakane

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Significancementioning

confidence: 98%

Section: Significancementioning

confidence: 99%

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

Thomaston

Woldeyes

Nakane

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…1) with tight packing of His37 and Trp41 (10,19,23,26). At lower pH the channel is more dynamic (26)(27)(28), which has precluded structure determination by solution or SSNMR. However, a series of crystallographic structures of the C open states ( Fig.…”

Section: Significancementioning

confidence: 99%

Acid activation mechanism of the influenza A M2 proton channel

Liang

Swanson

Madsen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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146

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The homotetrameric influenza A M2 channel (AM2) is an acidactivated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.ion channel | proton conduction | multiscale modeling | QM/MM | free-energy sampling V isualizing the vectorial flow of protons through membrane proteins is a long-standing challenge in biophysics and chemistry, with manifold implications for understanding bioenergetics, active transport, and proton channel function. Multiscale modeling has become a full partner with experimental structural biology in addressing proton transport (PT), because it can connect the dots between the high-resolution but static pictures obtained from crystallography and the lower-resolution but dynamically informed structures seen in the ensemble averages by NMR and other spectroscopic approaches. In general, multiscale modeling connects three or more disparate but coupled scales of behavior. In the case of PT in proteins, there are usually four pertinent scales: (i) the quantum mechanical scale of bond breaking and bond making inherent in the Grotthuss proton shuttle mechanism; (ii) the molecular scale of the dynamical motions of the protein, membrane, ions, and water molecules; (iii) the "free energy" scale that arises from the statistical ensemble averaging of those molecular motions; and (iv) the "transport" scale that manifests as a macroscopic conductance, with associated gating and other possible behaviors tied to macroscopic experimental variables. The proper and rigorous connection of these various scales, in an overall multiscale computational simulation, is often a substantial challenge. Here, we turn our attention to understanding the me...

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“…Higher membrane fluidity and negatively charged lipids favor H37 protonation [31–33]. Cholesterol promotes the α-helical conformation [34], immobilizes tetramer rotational diffusion [35], and stabilizes tetramer assembly [36, 37].…”

Section: Introductionmentioning

confidence: 99%

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

Mandala

Liao

Kwon

et al. 2017

Journal of Molecular Biology

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The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH (pHout) while W41 ensures proton conduction only from the N-terminus to the C-terminus and prevents reverse current under low internal pH (pHin). Here we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low pHin. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type channel, but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted towards the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C-terminus. At low pH, the mutant channel contains more cationic tetrads than the wild-type channel, consistent with faster reverse protonation from the C-terminus. 15N NMR spectra allow the extraction of four H37 pKa’s and show that the pKa’s are more clustered in the mutant channel compared to wild-type M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in wild-type M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in wild-type M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants.

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Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness

Cited by 81 publications

References 62 publications

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

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

Acid activation mechanism of the influenza A M2 proton channel

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

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