Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel

Li, Conggang; Qin, Huajun; Gao, Fei; Cross, Timothy A.

doi:10.1016/j.bbamem.2007.08.025

Cited by 92 publications

(145 citation statements)

References 78 publications

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“…A main finding of the present simulation study is that apo M2 TMD, in contrast to the amantadine-bound form, exhibits significant conformational heterogeneity. This finding is in agreement with solid-state NMR studies (9,23,(26)(27)(28). Membrane proteins appear to be less stabilized compared with soluble proteins, and the resulting conformational flexibility may be necessary for protein function (26,27,36).…”

Section: Discussionsupporting

confidence: 87%

“…This finding is in agreement with solid-state NMR studies (9,23,(26)(27)(28). Membrane proteins appear to be less stabilized compared with soluble proteins, and the resulting conformational flexibility may be necessary for protein function (26,27,36). For channel proteins in particular, which undergo conformational transitions upon activation, the existence of multiple low-energy conformational states is a requirement.…”

Section: Discussionsupporting

confidence: 84%

“…Strong evidence for greater conformational heterogeneity in the apo form has been presented by broadened resonances in solid-state NMR spectra as observed in multiple studies (see Fig. S1) (9,23,(26)(27)(28). To make comparison with this experimental observation, in Fig.…”

Section: Validation Of the Ensembles Of MD Conformations By Polarizationmentioning

confidence: 75%

“…Indeed, solid-state NMR (9,23,(26)(27)(28) and other studies have shown that the M2 TMD possesses significant conformational heterogeneity. For example, there is evidence that the transmembrane helix can be kinked around Gly-34, and the kink angle can be changed by the binding of amantadine (9,26,27). In the X-ray structure of the apo form, 1 helix of the M2 TMD exhibits a 15°kink around G34, whereas the other 3 helices appear straight (21).…”

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confidence: 93%

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Conformational heterogeneity of the M2 proton channel and a structural model for channel activation

Cross

Zhou

2009

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

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The M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Structure determination by solid-state and solution NMR and X-ray crystallography has contributed significantly to our understanding, but channel activation may involve conformations not captured by these studies. Indeed, solidstate NMR data demonstrate that the M2 protein possesses significant conformational heterogeneity. Here, we report molecular dynamics (MD) simulations of the M2 transmembrane domain (TMD) in the absence and presence of the antiviral drug amantadine. The ensembles of MD conformations for both apo and bound forms reproduced the NMR data well. The TMD helix was found to kink around Gly-34, where water molecules penetrated deeply into the backbone. The amantadine-bound form exhibited a single peak Ϸ10°in the distribution of helix-kink angle, but the apo form exhibited 2 peaks, Ϸ0°and 40°. Conformations of the apo form with small and large kink angles had narrow and wide pores, respectively, around the primary gate formed by His-37 and Trp-41. We propose a structural model for channel activation, in which the small-kink conformations dominate before proton uptake by His-37 from the exterior, and proton uptake makes the large-kink conformations more favorable, thereby priming His-37 for proton release to the interior.solid-state NMR ͉ MD simulations ͉ helix kink ͉ histidine tetrad ͉ amantadine T he M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Its function, proton conductance, is essential for effective replication of the virus. The transmembrane domain (TMD) of this homotetrameric protein contains a single helix (residues Ser-22 to Leu-46) from each subunit. Within the TMD, residues His-37 and Trp-41 constitute the primary gate that is critical for proton conductance and selectivity and pH activation (1-3). On the N-terminal (i.e., virus exterior) side, disulfide bonds involving Cys-17 and Cys-19 provide stabilization (4). On the C-terminal side, an amphiphilic helix may also be involved in pH activation (5). The antiviral drug amantadine and its derivatives inhibit the channel function by putatively binding to the TMD (6-8). The structures of the M2 TMD in the apo form and the amantadine-bound form were first determined by solid-state NMR (9, 10). In addition, extensive experimental (1, 2, 11-16) and computational (17-20) studies have been performed to model the structure of the M2 TMD and understand the molecular mechanisms of conductance and selectivity of the proton channel. Recently, 2 new structures were solved by X-ray crystallography (21) and solution NMR spectroscopy (22). Those 2 studies have generated controversies, especially about the mechanism of drug inhibition. Additional structural results have recently been published that add to the controversies surrounding inhibitor binding (23,24) and the closed-to open-state conformational transition (25).Channel activation may involve conformations not captured by the X-ray and NMR structures. Indeed, solid-state NMR...

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Section: Discussionsupporting

confidence: 87%

Section: Discussionsupporting

confidence: 84%

Section: Validation Of the Ensembles Of MD Conformations By Polarizationmentioning

confidence: 75%

mentioning

confidence: 93%

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Conformational heterogeneity of the M2 proton channel and a structural model for channel activation

Cross

Zhou

2009

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

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“…The function of the AP helix is to further stabilize the AM2 tetramer, in particular, when the TM helices open up during activation. The TM construct alone, without the AP helix, does not form a stable tetramer, which may explain the sample-to-sample variability in helix tilt observed by ssNMR (Li et al, 2007).…”

Section: Structures Of the Am2 Channel Of Influenza Amentioning

confidence: 99%

Flu channel drug resistance: a tale of two sites

Pielak

Chou

2010

Protein Cell

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The M2 proteins of influenza A and B virus, AM2 and BM2, respectively, are transmembrane proteins that oligomerize in the viral membrane to form proton-selective channels. Proton conductance of the M2 proteins is required for viral replication; it is believed to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. In addition to the role of M2 in proton conductance, recent mutagenesis and structural studies suggest that the cytoplasmic domains of the M2 proteins also play a role in recruiting the matrix proteins to the cell surface during virus budding. As viral ion channels of minimalist architecture, the membrane-embedded channel domain of M2 has been a model system for investigating the mechanism of proton conduction. Moreover, as a proven drug target for the treatment of influenza A infection, M2 has been the subject of intense research for developing new anti-flu therapeutics. AM2 is the target of two anti-influenza A drugs, amantadine and rimantadine, both belonging to the adamantane class of compounds. However, resistance of influenza A to adamantane is now widespread due to mutations in the channel domain of AM2. This review summarizes the structure and function of both AM2 and BM2 channels, the mechanism of drug inhibition and drug resistance of AM2, as well as the development of new M2 inhibitors as potential anti-flu drugs.

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Activation pH and Gating Dynamics of Influenza A M2 Proton Channel Revealed by Single‐Molecule Spectroscopy

et al. 2017

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Because of its importance in viral replication, the M2 proton channel of the Influenza A virus has been the focus of many studies. While we now know a great deal about the structural architecture underlying its proton conduction function, we know little about its conformational dynamics, especially those controlling the rate of this action. Herein, we employ a single-molecule fluorescence method to assess the dynamics of the inter-helical channel motion of both full-length M2 and the transmembrane domain of M2. We find that the rate of this motion depends not only on the identity of the channel and membrane composition, but also on pH in a sigmoidal manner. For the full-length M2 channel, the rate is increased from (~190 µs) −1 at high pH to (~80 µs) −1 at low pH, with a transition midpoint at pH 6.1. Because the latter is within the range reported for the conducting pK a value of the His37 tetrad, we believe that this inter-helical motion accompanies proton conduction.

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Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel

Cited by 92 publications

References 78 publications

Conformational heterogeneity of the M2 proton channel and a structural model for channel activation

Conformational heterogeneity of the M2 proton channel and a structural model for channel activation

Flu channel drug resistance: a tale of two sites

Activation pH and Gating Dynamics of Influenza A M2 Proton Channel Revealed by Single‐Molecule Spectroscopy

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