pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus

The M2 proton channel from the influenza A virus is a small protein with a single transmembrane helix that associates to form a tetramer in vivo. This protein forms proton-selective ion channels, which are the target of the drug amantadine. Here, we propose a mechanism for the pH-dependent association, and amantadine binding of M2, based on studies of a peptide representing the M2 transmembrane segment in dodecylphosphocholine micelles. Using analytical ultracentrifugation, we find that the sedimentation curves for the peptide depend on its concentration in the micellar phase. The data are well-described by a monomer-tetramer equilibrium, and the binding of amantadine shifts the monomer-tetramer equilibrium toward tetrameric species. Both tetramerization and the binding of amantadine lead to increases in the magnitude of the ellipticity at 223 nm in the circular dichroism spectrum of the peptide. The tetramerization and binding of amantadine are more favorable at elevated pH, with a pK(a) that is assigned to a His side chain, the only ionizable residue within the transmembrane helix. Our results, interpreted quantitatively in terms of a reversible monomer and tetramer protonation equilibrium model, suggest that amantadine competes with protons for binding to the deprotonated tetramer, thereby stabilizing the tetramer in a slightly altered conformation. This model accounts for the observed inhibition of proton flux by amantadine. Additionally, our measurements suggest that the M2 tetramer is substantially protonated at neutral pH and that both singly and doubly protonated states could be involved in M2's proton conduction at more acidic pHs.     

A pH-dependent conformational ensemble mediates proton transport through the influenza A/M2 protein

The influenza A/M2 protein exhibits inwardly rectifying, pH-activated proton transport that saturates at low pH. A comparison of high-resolution structures of the transmembrane domain at high and low pH suggests that pH-dependent conformational changes may facilitate proton conduction by alternately changing the accessibility of the N-terminal and C-terminal regions of the channel as a proton transits through the transmembrane domain. Here, we show that M2 functionally reconstituted in liposomes populates at least three different conformational states over a physiologically relevant pH range, with transition midpoints that are consistent with previously reported His37 pK(a) values. We then develop and test two similar, quantitative mechanistic models of proton transport, where protonation shifts the equilibrium between structural states having different proton affinities and solvent accessibilities. The models account well for a collection of experimental data sets over a wide range of pH values and voltages and require only a small number of adjustable parameters to accurately describe the data. While the kinetic models do not require any specific conformation for the protein, they nevertheless are consistent with a large body of structural information based on high-resolution nuclear magnetic resonance and crystallographic structures, optical spectroscopy, and molecular dynamics calculations.     

Structural and dynamic mechanisms for the function and inhibition of the M2 proton channel from influenza A virus

The M2 proton channel from influenza A virus, a prototype for a class of viral ion channels known as viroporins, conducts protons along a chain of water molecules and ionizable sidechains, including His37. Recent studies highlight a delicate interplay between protein folding, proton binding, and proton conduction through the channel. Drugs inhibit proton conduction by binding to an aqueous cavity adjacent to M2's proton-selective filter, thereby blocking access of proton to the filter, and altering the energetic landscape of the channel and the energetics of proton-binding to His37.     

Structural basis for proton conduction and inhibition by the influenza M2 protein

The influenza M2 protein forms an acid‐activated and drug‐sensitive proton channel in the virus envelope that is important for the virus lifecycle. The functional properties and high‐resolution structures of this proton channel have been extensively studied to understand the mechanisms of proton conduction and drug inhibition. We review biochemical and electrophysiological studies of M2 and discuss how high‐resolution structures have transformed our understanding of this proton channel. Comparison of structures obtained in different membrane‐mimetic solvents and under different pH using X‐ray crystallography, solution NMR, and solid‐state NMR spectroscopy revealed how the M2 structure depends on the environment and showed that the pharmacologically relevant drug‐binding site lies in the transmembrane (TM) pore. Competing models of proton conduction have been evaluated using biochemical experiments, high‐resolution structural methods, and computational modeling. These results are converging to a model in which a histidine residue in the TM domain mediates proton relay with water, aided by microsecond conformational dynamics of the imidazole ring. These mechanistic insights are guiding the design of new inhibitors that target drug‐resistant M2 variants and may be relevant for other proton channels.     

Acid–base titration across the membrane system of rat-liver mitochondria: Catalysis by uncouplers

1. Pulsed acid–base titrations of suspensions of rat-liver mitochondria under anaerobic equilibrium conditions show fast and slow titration processes. 2. The fast process is the titration of the outer aqueous phase of the mitochondria, which is continuous with the suspension medium, and the slow process can be identified with the titration of the inner aqueous phase of the mitochondria, which is separated from the outer aqueous phase by the non-aqueous osmotic barrier or M phase of the cristae membrane system. 3. The buffering power of the outer and inner phases have been separately measured over a range of pH values. 4. The rate of titration of the inner aqueous phase under a known protonmotive force across the M phase has been characterized by an effective proton conductance coefficient, which, near pH7 and at 25°, is only 0·45μmho/cm.² of the M-phase membrane. 5. The low effective proton conductance of the M phase will account quantitatively for the observed respiratory control in state 4, assuming that oxidoreduction and phosphorylation are coupled by a circulating proton current as required by the chemi-osmotic hypothesis. 6. The addition of 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) at normal uncoupling concentrations causes a large increase in the effective proton conductance of the M phase of the cristae membrane. 7. The increase of the effective proton conductance of the M phase by 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) will account quantitatively for the short-circuiting effect of the uncoupling agent on the proton current and for the observed rise of the rate of respiration to that characteristic of state 3 or higher.     

A theory for the proton transport of the influenza virus M2 protein: extensive test against conductance data

A theory for calculating the proton flux through the influenza virus M2 channel is tested here against an extensive set of conductance data. The flux is determined by the rate constants for binding to the His³⁷ tetrad from the two sides of the membrane and the corresponding unbinding rate constants. The rate constants are calculated by explicitly treating the structure and dynamics of the protein. Important features revealed by previous studies, such as a gating role for Val²⁷ at the entrance to the channel pore, and channel activation by viral exterior pH, are incorporated in this theory. This study demonstrates that the conductance function of the M2 proton channel can now be quantitatively rationalized by the structure and dynamics of the protein.     

Breaking the Carboxyl Rule: LYSINE 96 FACILITATES REPROTONATION OF THE SCHIFF BASE IN THE PHOTOCYCLE OF A RETINAL PROTEIN FROM EXIGUOBACTERIUM SIBIRICUM*?

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D₂O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pK_a of 8.5 ± 0.3, which reflects the pK_a of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pK_a of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.     

Mechanistic Insight into the H<Subscript>2</Subscript>O/D<Subscript>2</Subscript>O Isotope Effect in the Proton Transport of the Influenza Virus M2 Protein

The M2 proton channel is essential for the replication of the flu virus and is a known drug target. The functional mechanism of channel activation and conductance is key to both the basic biology of viral replication and the design of drugs that can withstand mutations. A quantitative model was previously developed for calculating the rate of proton transport through the M2 channel. The permeant proton was assumed to diffuse to the pore, obligatorily bind to the His37 tetrad, and then dissociate and be released to either side of the tetrad. Here the model is used to calculate the effect of a change in solvent from H₂O to D₂O on the rate of proton transport. The solvent substitution affects two parameters in the model: the proton diffusion constant and the pK _a for proton binding to the His37 tetrad. When the known effects on these two parameters are included, the deuterium isotope effect calculated from the model is in quantitatively agreement with experimental results. This strict test of the theoretical model provides strong support for the hypothesis that the permeant proton obligatorily binds to and then unbinds from the His37 tetrad. This putatively essential role of the His37 tetrad in the functional mechanism of the M2 channel makes it a promising target for designing mutation-tolerant drugs.     

Proton conduction in phosphatidylethanolamine

The dc conductivity of polycrystalline phosphatidylethanolamine (PE) was measured in the temperature range 60–120°C. Since no conclusive evidence had so far been obtained for the presence of proton conduction in this phospholipid, hydrogen gas was shown in the present experiment to evolve during the electrolysis in its premelted state between 91 and 124°C. In this temperature range molecules assume rotation around the molecular axes and proton conduction of the Grotthus type takes place possibly along two chains of intermolecular hydrogen bonds running in parallel. Zwitter-ions behave cooperatively as proton donors and acceptors in transferring proton from molecule to molecule via the hydrogen bond networks. This efficient push-pull way of proton transferring seems to account for the fact that no polarization was observed in the dc conduction experiments. The amount of evolved gas appears to be not exactly in accordance with Faraday's law and discussions are made on possible causes for this slight deviation.     

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

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). (13)C and (15)N 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, nonideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and nonideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (?, 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.     

Binding and internalization of alpha 2-microglobulin by cultured fibroblasts. Effects of monovalent ionophores

Receptor-bound alpha 2-macroglobulin (alpha 2M) undergoes a two-step process in its internalization by cultured fibroblasts. First, the receptor- alpha 2M complexes concentrate in coated pits on the cell surface. Second, the alpha 2M is internalized into endocytic vesicles we have termed receptosomes. Using a variety of monovalent ionophores and inhibitors of ATP synthesis, the present report provides data that discriminates between these two steps. Appearance of alpha 2M-receptor complexes in coated pits occurs at 4 degrees C and is inhibited by primary amines as well as some other drugs and chemical reagents [1, 2]. Internalization of alpha 2M-receptor complexes into receptosomes is inhibited by monovalent ionophores that disrupt proton gradients (monensin, nigericin, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and 3,3',4',5-tetrachlorosalicyanilide), but not the Na+ specific ionophore antamanide or the K+ specific ionophore valinomycin. Using electron microscopy, the proton ionophores appear to interfere with the transfer of alpha 2M from coated pits to receptosomes. Prolonged incubation with monensin in the presence of alpha 2M also decreases the number of alpha 2M receptors on the cell surface, but this did not appear sufficient to account for the extensive inhibition of internalization. Monensin also inhibited the internalization of vesicular stomatitis virus and epidermal growth factor (EGF). Our data suggest that a proton gradient may be necessary for receptor-mediated endocytosis of alpha 2M and some other ligands.     

Quantitative determination of anomeric forms of sugar produced by amylases. V. Anomeric forms of maltose produced in the hydrolytic reaction of substituted phenyl alpha-maltosides catalyzed by saccharifying alpha-amylase from B. subtilis.

1. Hydrolyses of phenyl alpha-maltoside and its derivatives with various substituents (p-NO2, p-C1, p-CH3, p-C2H5, and p-C(CH3)3) catalyzed by saccharifying alpha-amylase from B. subtilis3 [EC 3.2.1.1] were studied under conditions such that the products were only maltose and the corresponding phenols (1), in order to determine quantitatively the anomeric form of the sugar produced from each substrate. 2. At the optimum pH of this enzyme (pH-5.4), maltose released from all the substituted substrates studied was entirely in the beta-form. These results are in remarkable contrast to the previous finding that alpha-maltose is exclusively produced from unsubstituted phenyl alpha-maltoside by this enzyme (2). 3. At pH 6.18 and 6.73, maltose produced from unsubstituted phenyl alpha-maltoside (?M) or p-tert-butylphenyl alpha-maltoside (PTB?M) was a mixture of alpha- and beta-anomers, the ratio being dependent on pH as follows: For ?M, the percentage of alpha-anomer was 100% (pH 5.4), 80 (pH 6.18), and 55% (pH 6.73), whereas for PTB?M, the percentage of beta-anomer was 100% (pH 5.4), 75% (pH 6.18), and 60% (pH 6.73).     

Acid proteases from species of Mucor. IV. Hydrogen-tritium exchange of Mucor miehei protease.

The kinetics of hydrogen-tritium exchange were studied in the range pH-3 for both the fully and partially tritiated protein. Exchange constants for an intermediate class and slow class of hydrogens were determined and found to give a parabolic curve characteristic of acid and base catalysis about the observed pHmin of 4.03. The anomalous rate retardation on the acid portion of the curve was attributed to electrostatic interactions which could be evaluated quantitatively from the titration data. Partial tritation and pH cross-over experiments indicated that the rank order was pH-independent thus eliminating the possiblitity of a major conformational change. Consequently, the data are most likely explicable in terms of restricted solvent accessibility.     

Flu channel drug resistance: a tale of two sites

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.     

Discrimination of tertiary and quaternary Bohr effect in the O2 binding of Helix pomatia beta-hemocyanin

Simultaneous determination of proton uptake and oxygen binding has been carried out on Helix pomatia beta-hemocyanin under equilibrium conditions in the absence of buffer and at different initial pH values. Oxygen-binding isotherms of unbuffered H. pomatia beta-hemocyanin, in the presence of phenol red as pH indicator, have been determined employing a thin-layer apparatus. Application of this very accurate technique allows monitoring of proton uptake (or release) coupled to O2-binding also at extremes of saturation which are often difficult to explore and analyze. The data have been analyzed within the framework of the cooperon model (M. Brunori, M. Coletta and E. Di Cera, Biophys. Chem. 23 (1986) 215) and compared with those obtained in the presence of buffer. Comparison of pH changes with ligand binding of the T state over all the saturation range has allowed us to discriminate and obtain quantitative estimates of the Bohr protons associated with both oxygenation of the T state and quaternary allosteric transition; no protons are taken up or released during oxygenation of the R state. These results differ quantitatively from those obtained in the presence of buffer, which alters significantly the T state contribution to the overall Bohr effect.     

Protonation and deprotonation of the M, N, and O intermediates during the bacteriorhodopsin photocycle

Transient pH changes were measured with phenol red and chlorophenol red in the 30-microseconds-50-ms time range during the photocycle of bacteriorhodopsin (BR), the light-driven proton pump. At pH greater than or equal to 7, the results confirmed earlier data and suggestions that one proton is released during the L----M reaction, and taken up again during the decay of N. These are likely to be steps in the proton transport process. At pH less than 7, however, the time-resolved pH traces were complex and indicated additional protonation reactions. The data were explained by a model which assumed pH-dependent protonation states for M and N which varied from -1 to 0, and for O which varied from 0 to + 2, relative to BR. If the kinetics of the vectorial proton translocation process were taken as pH independent, this treatment of the data suggested that a residue with a pKa of 5.9 was made protonable in M and N and two residues with pKa's of 6.5 were made cooperatively protonable in O. The additional protons detected are not necessarily in the vectorial proton transfer pathway (i.e., they are probably "Bohr protons"), and while they must reflect conformational and/or neighboring ionization changes in the BR as it passes through the M, N, and O states, their role, if any, in the transport is uncertain.     

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function

The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.     

Lateral proton conduction at a lipid/water interface. Effect of lipid nature and ionic content of the aqueous phase

Fast lateral proton conduction was observed along the lipid/water interface using a fluorescence technique. This conduction can be detected for a large number of lipids, both phospholipids and glycolipids. The efficiency of the proton transfer is dependent on the molecular packing of the host lipid at a given surface pressure. The proton conduction which is present in the liquid expanded state is abolished by the transition to the liquid condensed state. The proton transfer is affected slightly by the ionic content of the aqueous subphase except in the case of calcium which can inhibit the conduction along phosphatidylglyceroethanolamine. We suggest that the transfer of the protons occurs along a bidimensional hydrogen-bond network formed from the polar head groups, their water molecules of hydration and the water molecules which are intercalated between the lipid molecules.     

Time dependence of the proton relaxation times of regenerating rat liver

The proton longitudinal relaxation time of regenerating rat liver has been found to increase during the first 24 hours after hepatectomy and to drop back to normal in the following hours. The decreased relaxation rate may be related to the increase of the water mobility due to the expansion of the intercellular spaces during the massive proliferation of the first day, or to the increased cell hydration which is known to occur during active cell proliferation. Equations have been derived for the proliferation process, and the competing inhibition process, active from the 24th hour, which can quantitatively account for the proton relaxation behaviour.