The Functional Heart of the M2 Channel

The equilibration of pH between external environment and viral interior is an essential part of the influenza virus lifecycle. After infection of a eukaryotic cell, the virus needs to transmit protons across its lipidic envelope from the acidic exterior, an endosome, to the interior, eventually triggering unpacking of the viral genome (1). This essential function is fulfilled by the viral protein M2 channel, a tetrameric assembly of four transmembrane helices. The proton pore is formed in the center of the four helices. Two amino-acid residues in the pore are crucial for function, forming a HxxxW motif: Histidine 37 provides for the selectivity and Tryptophan 41 for the pH-dependent gating. Structure, conformation, and mechanism of the M2 channel and in particular of these two residues are subject to intense ongoing research (2).

In this issue of Biophysical Journal, Williams et al. (3) use solid-state NMR spectroscopy to determine the orientation of the Trp⁴¹ side chain in the low and the high pH states of the M2 channel. So far, 10 different structures have been deposited in the Protein Data Bank database, determined by different techniques: solid-state NMR spectroscopy, solution NMR spectroscopy, and x-ray crystallography (4–13). These 10 structures feature, however, a total of three different rotamers for the Trp⁴¹ chain. Williams et al. (3) now employ solid-state NMR distance measurements between selected nuclei in specifically isotope-labeled samples to distinguish between the rotameric Trp⁴¹ orientations. Importantly, the channel is reconstituted in native-like lipid bilayers for the solid-state magic-angle spinning NMR experiments.

The proton conductance mechanism of the M2 channel had previously been determined. In the high pH closed state, the four His³⁷ side chains block the channel by forming a π-stacked structure. At low pH, however, the His³⁷ cluster is conductive, due to fast protonation and deprotonation as part of a proton wire (14,15). Together with these previous experiments, the orientation of the Trp side chain can now further explain the conducting and gating mechanism of the M2. Trp⁴¹ is found to adopt a t90 rotamer in both pH states, with 20° difference in χ₁ and χ₂ between the high and low pH states. Additional measurements determine the amplitudes of the Trp side-chain dynamics, showing increased fluctuations at low pH. Importantly, His³⁷ and Trp⁴¹ approach each other closely at low pH by a cation-π interaction. This close interaction provides the structural basis for the gating by Trp, as it reduces the proton dissociation rate from His³⁷ to water from 4.5 × 10⁵ s⁻¹ to <1% thereof. Furthermore, the Trp gate, which is located toward the virus interior, blocks the accessibility of protons to the His gate and thus results in the overall unidirectional proton conductance. Overall, the visualization of the measured amplitudes of the two side chains pumping protons through the M2 channel by their reorientation evokes the visual analogy of a stroking heart that pumps blood through its valves (Fig. 7 of Williams et al. (3)).

Determination of amino-acid side-chain rotamers is a fundamental biophysical challenge, often necessary to understand protein function at atomic detail. Specific solid-state NMR experiments, such as the REDOR (16) and CODEX (17) experiments used here, in combination with suitable isotope labeling schemes are central techniques in the ongoing work of Luo and Hong (18), Luo et al. (19), Hong et al. (20), and Wang et al. (21). This article demonstrates the potential of solid-state NMR to determine conformation and dynamics of amino-acid side chains at atomic resolution in the functional center of a membrane protein, outlining a procedure to be carried out also on larger and more complex membrane protein systems.