The M2 protein of influenza viruses forms an acid-activated tetrameric proton channel. We used solid-state nuclear magnetic resonance spectroscopy to determine the structure and functional dynamics of the pH-sensing and proton-selective histidine-37 in M2 bound to a cholesterol-containing virus-envelope-mimetic membrane so as to better understand the proton conduction mechanism. In the high-pH closed state, the four histidines form an edge-face π-stacked structure, preventing the formation of a hydrogen-bonded water chain to conduct protons. In the low-pH conducting state, the imidazoliums hydrogen-bond extensively with water and undergo microsecond ring reorientations with an energy barrier greater than 59 kilojoules per mole. This barrier is consistent with the temperature dependence of proton conductivity, suggesting that histidine-37 dynamically shuttles protons into the virion. We propose a proton conduction mechanism in which ring-flip–assisted imidazole deprotonation is the rate-limiting step.

Determination of the high-resolution quaternary structure of oligomeric membrane proteins requires knowledge of both the oligomeric number and intermolecular distances. The centerband-only detection of exchange (CODEX) technique has been shown to enable the extraction of the oligomeric number through the equilibrium exchange intensity at long mixing times. To obtain quantitative distances, we now provide an analysis of the mixing-time-dependent CODEX intensities using the 1H-driven spin diffusion theory. The exchange curve is fit to a rate equation, where the rate constants are proportional to the square of the dipolar coupling and the spectral overlap integral between the exchanging spins. Using a number of 13C- and 19F-labeled crystalline model compounds with known intermolecular distances, we empirically determined the overlap integrals of 13C and 19F CODEX for specific spinning speeds and chemical shift anisotropies. These consensus overlap integral values can be applied to structurally unknown systems to determine distances. Applying the 19F CODEX experiment and analysis, we studied the transmembrane peptide of the M2 protein (M2TMP) of influenza A virus bound to 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers. The experiment proved for the first time that M2TMP associates as tetramers in lipid bilayers, similar to its oligomeric state in detergent micelles. Moreover, the nearest-neighbor interhelical F-F distance between (4-19F)Phe30 is 7.9-9.5 angstroms. This distance constrains the orientation and the packing of the helices in the tetrameric bundle and supports the structural model derived from previous solid-state NMR 15N orientational data. Thus, the CODEX technique presents a general method for determining the oligomeric number and intermolecular distances in the approximately 10 angstroms range in membrane proteins and other complex biological assemblies.

The M2 protein of influenza A viruses forms a tetrameric pH-activated proton-selective channel that is targeted by the amantadine class of anti-viral drugs. Its ion channel function has been extensively studied by electrophysiology and mutagenesis; however, the molecular mechanism of proton transport is still elusive, and the mechanism of inhibition by amantadine is controversial. We review the functional data on proton channel activity, molecular dynamics simulations of the proton conduction mechanism, and high-resolution structural and dynamical information of this membrane protein in lipid bilayers and lipid-mimetic detergents. These studies indicate that elucidation of the structural basis of M2 channel activity and inhibition requires thorough examination of the complex dynamics of the protein and the resulting conformational plasticity in different lipid bilayers and lipid-mimetic environments. A. Function of the M2 proton channel of influenza A virusesThe M2 protein of influenza A and B viruses forms tetrameric proton channels that are important for the viral life cycle. After the virus enters the infected cell by endocytosis, the M2 proton channel opens in response to the low pH of the endosome, allowing proton flux into the virus, which triggers the dissociation of the viral RNA from the matrix proteins and the fusion of the viral and endosomal membranes. These events release the viral RNA to the cytoplasm for replication by the host cell (1). In a later stage of virus replication, the M2 protein maintains the high pH of the trans-Golgi network and prevents premature conformational changes of hemagglutinin in viruses with a high pH optimum of hemagglutinin-induced fusion (2).The influenza A M2 (AM2) protein contains a short N-terminal periplasmic domain, a transmembrane (TM) domain, and a C-terminal cytoplasmic tail (Fig. 1). It is one of the smallest ion channel proteins and thus an excellent system for elucidating the structure-function relation of ion channels. Extensive mutagenesis, electrophysiology (3,4) and sedimentation equilibrium experiments (5) have been conducted to characterize the function and stability of AM2 (for recent reviews, see (6,7)). The AM2 proton channel is also the target of the amantadine class of drugs, one of only two classes of anti-influenza drugs currently available. However, the efficacy of amantadine dropped by two orders of magnitude between 2002 and 2007, although the 2008 seasonal flu strains were largely sensitive to amantadine. The resistance mainly resulted from the S31N mutation in the M2 TM domain (8). Thus, elucidating the mechanism of amantadine inhibition of AM2 has great public health relevance.Recently, several high-resolution structural studies have been carried out to determine the structural basis of AM2 proton conductance and inhibition. In this article, we summarize the main functional data of AM2 and high-resolution structural information available on the TM domain, to promote future investigations of this intriguing and far from understood me...

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). 13C and 15N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, non-ideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and non-ideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (ϕ, ψ) torsion angles for three basis states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins.

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