Kinetic analysis of the M2 proton conduction of the influenza virus

The M2 protein of the flu virus forms a proton selective channel that is necessary for viral replication. The channel has a slow rate of conduction but attains near perfect selectivity for protons. Many models have been proposed to explain the mechanism of proton conduction based on whole cell channel recordings and molecular dynamics simulations, but a detailed kinetic analysis of the channel activity has not yet been performed. We obtained detailed conduction vs pH measurements for M2 and a number of its variants using a sensitive and reproducible liposome proton flux assay. The proton transport follows Michaelis-Menten-like kinetics with two saturation steps: one pseudosaturation at pH ～5.5, and another full saturation at pH ～4. The heart of the mechanism is the pore-lining His37 and Trp41. NMR measurements suggest that histidine and tryptophan act in unison to transport protons down the concentration gradient. The log of apparent K(m) derived from the kinetics data matches closely to the histidine pK(a) and correlates with chemical shift perturbation of the Trp41 gate, indicating that histidine protonation and opening of the channel gate are synchronized events. Finally, mutagenesis and structural analysis identified key residues that affect the rate of conduction.     

Free energy calculations on the two drug binding sites in the M2 proton channel

Two alternative binding sites of adamantane-type drugs in the influenza A M2 channel have been suggested, one with the drug binding inside the channel pore and the other with four drug molecule S-binding to the C-terminal surface of the transmembrane domain. Recent computational and experimental studies have suggested that the pore binding site is more energetically favorable but the external surface binding site may also exist. Nonetheless, which drug binding site leads to channel inhibition in vivo and how drug-resistant mutations affect these sites are not completely understood. We applied molecular dynamics simulations and potential of mean force calculations to examine the structures and the free energies associated with these putative drug binding sites in an M2-lipid bilayer system. We found that, at biological pH (~7.4), the pore binding site is more thermodynamically favorable than the surface binding site by ~7 kcal/mol and, hence, would lead to more stable drug binding and channel inhibition. This result is in excellent agreement with several recent studies. More importantly, a novel finding of ours is that binding to the channel pore requires overcoming a much higher energy barrier of ~10 kcal/mol than binding to the C-terminal channel surface, indicating that the latter site is more kinetically favorable. Our study is the first computational work that provides both kinetic and thermodynamic energy information on these drug binding sites. Our results provide a theoretical framework to interpret and reconcile existing and often conflicting results regarding these two binding sites, thus helping to expand our understanding of M2-drug binding, and may help guide the design and screening of novel drugs to combat the virus.     

Formation of the layer of influenza A virus M1 matrix protein on lipid membranes at pH 7.0

Russian Chemical Bulletin - The kinetics of interaction of influenza A virus M1 matrix protein with hydrophilic polymer-supported lipid bilayers formed by...     

Exploring organosilane amines as potent inhibitors and structural probes of influenza a virus M2 proton channel

We describe the use of organosilanes as inhibitors and structural probes of a membrane protein, the M2 proton channel from influenza A virus. Organosilane amine inhibitors were found to be generally as potent as their carbon analogues in targeting WT A/M2 and more potent against the drug-resistant A/M2-V27A mutant. In addition, intermolecular NOESY spectra with dimethyl-substituted organosilane amine inhibitors clearly located the drug binding site at the N-terminal lumen of the A/M2 channel close to V27.     

Synthesis and membrane binding properties of a lipopeptide fragment from influenza virus a hemagglutinin

Hemagglutinin from influenza virus A is a S-palmitoylated lipoglycoprotein in which the lipid groups are thought to influence the interaction between cell membrane and capsid during budding of viral offspring as well as fusion processes of the viral membrane with the endosome after entry of the viral particle into the cell. The paper describes the development of a method for the synthesis of characteristic lipidated hemagglutinin derived peptides which additionally carry the fluorescent 7-nitrobenz-2oxa-1,3-diazole (NBD) group. To achieve this goal the enzyme-sensitive para-phenylacetoxybenzyloxycarbonyl (PAOB) ester was developed. It is cleaved from the peptides and lipidated peptides under very mild conditions and with complete selectivity by treatment with the enzyme penicillin G acylase; this results in the formation of a phenolate. This intermediate spontaneously undergoes fragmentation thereby releasing the desired carboxylates. The combined use of this enzyme-labile fragmenting ester with the acid-labile Boc group, the Pd(0)-sensitive allyl ester and the corresponding Aloc urethane gave access to a mono-S-palmitoylated and a doubly S-palmitoylated NBD-labelled hemagglutinin peptide. The binding of these lipopeptides to model membranes was analyzed in a biophysical setup monitoring the transfer of fluorescent-labelled lipopeptide from vesicles containing the non-exchangeable fluorescence quencher Rho-DHPE to quencher-free vesicles. The experiments demonstrate that one lipid group is not sufficient for quasi-irreversible membrane insertion of lipidated peptides. This is, however, achieved by introduction of the bis-palmitoyl anchor. The intervesicle transfer always implies release of peptides localized at the outer face of the vesicles into solution followed by diffusion to and insertion into acceptor vesicles. For peptides bound at the inner face of the vesicle membrane, however, an additional flip-flop diffusion to the outer face has to occur beforehand. The kinetics of these processes were estimated by fast chemical quench of the outside fluorophores by sodium dithionite.     

Side-chain conformation of the M2 transmembrane peptide proton channel of influenza a virus from 19F solid-state NMR

The M2 transmembrane peptide (M2TMP) of the influenza A virus forms a tetrameric helical bundle that acts as a proton-selective channel important in the viral life cycle. The side-chain conformation of the peptide is largely unknown and is important for elucidating the proton-conducting mechanism and the channel stability. Using a 19F spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain 19F-19F distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. 19F CODEX data at a key residue of the proton channel, Trp41, confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of approximately 11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous 15N NMR experiments and the backbone channel diameter has a narrow allowed range, this 19F distance constrains the Trp41 side-chain conformation to t90 (chi1 approximately 180 degrees , chi2 approximately 90 degrees ). This Trp41 rotamer, combined with a previously measured 15N-13C distance between His37 and Trp411, suggests that the His37 rotamer is t-160. The implication of the proposed (His37, Trp41) rotamers to the gating mechanism of the M2 proton channel is discussed. Binding of the antiviral drug amantadine to the peptide does not affect the F-F distance at Trp41. Interhelical 19F-19F distances are also measured at residues 27 and 38, each mutated to 4-19F-Phe. For V27F-M2TMP, the 19F-19F distances suggest a mixture of dimers and tetramers, whereas the L38F-M2TMP data indicate two tetramers of different sizes, suggesting side chain conformational heterogeneity at this lipid-facing residue. This work shows that 19F spin diffusion NMR is a valuable tool for determining long-range intermolecular distances that shed light on the mechanism of action and conformational heterogeneity of membrane protein oligomers.     

Molecular dynamics simulation of a hydrated diphytanol phosphatidylcholine lipid bilayer containing an alpha-helical bundle of four transmembrane domains of the influenza A virus M2 protein

An alpha-helical bundle composed of four transmembrane portions of the M2 protein from the Influenza A virus has been studied in a hydrated diphytanol phosphatidylcholine bilayer using molecular dynamics (MD) calculations. Experimentally, the sequence utilized is known to aggregate as a four-helix bundle and act as a pH-gated proton-selective ion channel, which is blocked by the drug amantadine hydrochloride. In the presented simulation, the ion channel was initially set up as a parallel four-helix bundle. The all-atom simulation consisted of almost 16,000 atoms, described classically, using a forcefield from the CHARMM22 database. Bilayers with and without the bundle were shown to be stable throughout the nanosecond timescale of the MD simulation. Structural and dynamical properties of the bilayer both with and without the transmembrane protein are reported.     

Magic-angle-spinning NMR of the drug resistant S31N M2 proton transporter from influenza A

We report chemical shift assignments of the drug-resistant S31N mutant of M2(18-60) determined using 3D magic-angle-spinning (MAS) NMR spectra acquired with a (15)N-(13)C ZF-TEDOR transfer followed by (13)C-(13)C mixing by RFDR. The MAS spectra reveal two sets of resonances, indicating that the tetramer assembles as a dimer of dimers, similar to the wild-type channel. Helicies from the two sets of chemical shifts are shown to be in close proximity at residue H37, and the assignments reveal a difference in the helix torsion angles, as predicted by TALOS+, for the key resistance residue N31. In contrast to wild-type M2(18-60), chemical shift changes are minimal upon addition of the inhibitor rimantadine, suggesting that the drug does not bind to S31N M2.     

Water-soluble peptide-coated nanoparticles: control of the helix structure and enhanced differential binding to immune cells

The stabilizing action of C(α)-tetrasubstituted α-amino acids inserted into a sequence of short peptides allowed for the first time the preparation of water-soluble nanoparticles of different materials coated with a helix-structured undecapeptide. This peptide coating strongly favors nanoparticle uptake by human immune system cells.     

A structural and energetics analysis of the binding of a series of N-acetylneuraminic-acid-based inhibitors to influenza virus sialidase

Summary A molecular dynamics/energy-minimisation protocol has been used to analyse the structural and energetic effects of functional group substitution on the binding of a series of C4-modified 2-deoxy-2,3-didehydro-N-acetylneuraminic acid inhibitors to influenza virus sialidase. Based on the crystal structure of sialidase, a conformational searching protocol, incorporating multiple randomisation steps in a molecular dynamics simulation was used to generate a range of minimum-energy structures. The calculations were useful for predicting the number, location, and orientation of structural water molecules within protein-ligand complexes. Relative binding energies were calculated for the series of complexes using several empirical molecular modelling approaches. Energies were computed using molecular-mechanics-derived interactions as the sum of pairwise atomic nonbonded energies, and in a more rigorous manner including solvation effects as the change in total electrostatic energy of complexation, using a continuum-electrostatics (CE) approach. The CE approach exhibited the superior correlation with observed affinities. Both methods showed definite trends in observed and calculated binding affinities; in both cases inhibitors with a positively charged C4 substituent formed the tightest binding to the enzyme, as observed experimentally.This paper is based on a presentation given at the 14th Molecular Graphics and Modelling Society Conference, held in Cairns, Australia, August 27–September 1, 1995.Presently on a visiting postdoctoral fellowship in the Department of Biomolecular Structure, Glaxo Research & Development Ltd, Greenford, Middlesex UB6 OHE, U.K.     

Magic angle spinning and oriented sample solid-state NMR structural restraints combine for influenza a M2 protein functional insights

As a small tetrameric helical membrane protein, the M2 proton channel structure is highly sensitive to its environment. As a result, structural data from a lipid bilayer environment have proven to be essential for describing the conductance mechanism. While oriented sample solid-state NMR has provided a high-resolution backbone structure in lipid bilayers, quaternary packing of the helices and many of the side-chain conformations have been poorly restrained. Furthermore, the quaternary structural stability has remained a mystery. Here, the isotropic chemical shift data and interhelical cross peaks from magic angle spinning solid-state NMR of a liposomal preparation strongly support the quaternary structure of the transmembrane helical bundle as a dimer-of-dimers structure. The data also explain how the tetrameric stability is enhanced once two charges are absorbed by the His37 tetrad prior to activation of this proton channel. The combination of these two solid-state NMR techniques appears to be a powerful approach for characterizing helical membrane protein structure.     

Molecular modeling of a putative antagonist binding site on helix III of the β-adrenoceptor

Summary In recent biochemical studies it was demonstrated that residue Asp¹¹³ of the-adrenoceptor (-AR) is an indispensable amino acid for the binding of-AR antagonists. Earlier fluorescence studies showed that a tryptophan-rich region of the-AR is involved in the binding of propranolol, the prototype-AR antagonist. Bearing these two biochemical findings in mind, we explored the-AR part containing Asp¹¹³, for an energetically favorable antagonist binding site. This was done by performing molecular docking studies with the antagonist propranolol and a specific-AR peptide which included, besides Asp¹¹³, two possibly relevant tryptophan residues. In the docking calculations, the propranolol molecule was allowed to vary all its internal torsional angles. The receptor peptide was kept in an-helix conformation, while side chains relevant to ligand binding were flexible to enable optimal adaptations to the ligand's binding conformation. By means of force-field calculations the total energy was minimized, consisting of the intramolecular energies of both ligand and receptor peptide, and the intermolecular energy. We found an antagonist binding site, consisting of amino acids Asp¹¹³ and Trp¹⁰⁹, which enabled energetically favorable interactions with the receptor-binding groups of propranolol. According to these results, binding involves three main interaction points: (i) a reinforced ionic bond; (ii) a hydrogen bond; and (iii) a hydrophobic/charge transfer interaction. The deduced binding site shows a difference in affinity between the levo- and dextrorotatory isomers of propranolol caused by a difference in ability to form a hydrogen bond, which is in conformity with the experimentally observed stereoselectivity. Moreover, it also provides an explanation for the ₁-selectivity ofp-phenyl substituted phenoxypropanolamines like betaxolol. Thep-phenyl substituent of betaxolol was shown to be sterically hindered upon binding to the ₂-AR peptide, whereas this hindrance is very likely to be much less with the ₁-AR peptide. Finally, the proposed antagonist binding site is discussed in the light of some recent biochemical findings and theories.Abbreviations -AR -adrenergic receptor - cDNA complementary DNA - H-bond hydrogen bond - VdW van der Waals - QSAR quantitative structure-activity relationship - ¹²⁵I-pBABC p-(bromoacetamido)benzyl-1-[¹²⁵I]iodocarazol     

Molecular dynamics simulation directed rational design of inhibitors targeting drug-resistant mutants of influenza A virus M2

Influenza A virus M2 (A/M2) forms a homotetrameric proton selective channel in the viral membrane. It has been the drug target of antiviral drugs such as amantadine and rimantadine. However, most of the current virulent influenza A viruses carry drug-resistant mutations alongside the drug binding site, such as S31N, V27A, and L26F, etc., each of which might be dominant in a given flu season. Among these mutations, the V27A mutation was prevalent among transmissible viruses under drug selection pressure. Until now, V27A has not been successfully targeted by small molecule inhibitors, despite years of extensive medicinal chemistry research efforts and high throughput screening. Guided by molecular dynamics (MD) simulation of drug binding and the influence of drug binding on the dynamics of A/M2 from earlier experimental studies, we designed a series of potent spirane amine inhibitors targeting not only WT, but also both A/M2-27A and L26F mutants with IC(50)s similar to that seen for amantadine's inhibition of the WT channel. The potencies of these inhibitors were further demonstrated in experimental binding and plaque reduction assays. These results demonstrate the power of MD simulations to probe the mechanism of drug binding as well as the ability to guide design of inhibitors of targets that had previously appeared to be undruggable.     

Helical hairpin structure of influenza hemagglutinin fusion peptide stabilized by charge-dipole interactions between the N-terminal amino group and the second helix

The fusion domain of the influenza coat protein hemagglutinin HA2, bound to dodecyl phosphocholine micelles, was recently shown to adopt a structure consisting of two antiparallel α-helices, packed in an exceptionally tight hairpin configuration. Four interhelical H(α) to C═O aliphatic H-bonds were identified as factors stabilizing this fold. Here, we report evidence for an additional stabilizing force: a strong charge-dipole interaction between the N-terminal Gly(1) amino group and the dipole moment of helix 2. pH titration of the amino-terminal (15)N resonance, using a methylene-TROSY-based 3D NMR experiment, and observation of Gly(1 13)C' show a strongly elevated pK = 8.8, considerably higher than expected for an N-terminal amino group in a lipophilic environment. Chemical shifts of three C-terminal carbonyl carbons of helix 2 titrate with the protonation state of Gly(1)-N, indicative of a close proximity between the N-terminal amino group and the axis of helix 2, providing an optimal charge-dipole stabilization of the antiparallel hairpin fold. pK values of the side-chain carboxylate groups of Glu(11) and Asp(19) are higher by about 1 and 0.5 unit, respectively, than commonly seen for solvent-exposed side chains in water-soluble proteins, indicative of dielectric constants of ε = ～30 (Glu(11)) and ～60 (Asp(19)), placing these groups in the headgroup region of the phospholipid micelle.     

Recent progress in computational approaches to studying the M2 proton channel and its implication to drug design against influenza viruses

For quite a long period of time in history, many intense efforts have been made to determine the 3D (three-dimensional) structure of the M2 proton channel. The reason why the M2 proton channel has attracted so many attentions is because (1) it is the key for really understanding the life cycle of influenza viruses, and (2) it is indispensable for conducting rational drug design against the flu viruses. Recently, the long-sough 3D structures of the M2 proton channels for both influenza A and B viruses were consecutively successfully determined by the high-resolution NMR spectroscopy (Schnell J.R. and Chou, J.J., Nature, 2008, 451: 591-595; Wang, J., Pielak, R.M., McClintock, M.A., and Chou, J.J., Nature Structural & Molecular Biology, 2009,16: 1267-1271). Such a milestone work has provided a solid structural basis for in-depth understanding the action mechanism of the M2 channel and rationally designing effective drugs against influenza viruses. This review is devoted to, with the focus on the M2 proton channel of influenza A, addressing a series of relevant problems, such as how to correctly understand the novel allosteric inhibition mechanism inferred from the NMR structure that is completely different from the traditional view, what the possible impacts are to the previous functional studies in this area, and what kind of new strategy can be stimulated for drug development against influenza.     

Synthetic model of the phosphate binding protein: solid-state structure and solution-phase anion binding properties of a large oligopyrrolic macrocycle

The macrocyclic receptors 4-6 were synthesized via the anion-templated condensation of appropriately chosen dialdehyde and diamine building blocks. Whereas all three products could be obtained directly via the appropriate choice of reaction conditions, the larger [3+3] product, 6, which incorporates three of each precursor subunit, could also be obtained conveniently via an indirect procedure involving ring expansion of the smaller [2+2] macrocycle 4. As detailed earlier (Sessler, J. L.; Katayev, E. A.; Pantos, G. D.; Reshetova, M. D.; Khrustalev, V. N.; Lynch, V. M.; Ustynyuk, Y. A. Angew. Chem. 2005, 117, 7552-7556; Angew. Chem., Int. Ed. 2005, 44, 7386-7390), this ring expansion occurs under thermodynamic control in the presence of HSO4- and H2PO4- anions in acetonitrile solution and serves to effect the conversion of 4 to 6. An analysis of the X-ray crystal structure of complex 6H22+.HPO42- revealed a strong resemblance to the active site of the phosphate binding protein (PBP) with similar structural analogies being drawn between the active site of the sulfate binding protein (SBP) and the corresponding hydrogensulfate anion complex. In both cases, the anions are bound in a 1:1 fashion in the solid state through a complementary hydrogen bond network involving both the receptor 6 and the anions. UV-vis spectroscopic titrations provide support for the conclusion that macrocycle 6 binds the hydrogensulfate and dihydrogenphosphate anion (studied as the corresponding tetrabutylammonium salts) with high selectivity and affinity in acetonitrile (log Ka for the first binding interaction approaching 7), albeit with different receptor-to-anion binding stoichiometries (1:1 vs 1:3 for HSO4- and H2PO4-, respectively).     

Energetics of binding and protein unfolding upon amphiphilic drug complexation with a globular protein in different aqueous media

The interactions and complexation process of the structurally related amphiphilic phenothiazines promazine and triflupromazine hydrochlorides with horse myoglobin in aqueous buffered solutions of pH 2.5, 5.5 and 9.0 have been examined by zeta-potential, isothermal titration calorimetry (ITC), UV-vis spectroscopy and dynamic light-scattering techniques with the aim of analyzing the effect of hydrophobic and electrostatic forces, the alteration of protein conformation and the effect of substituents in the drug molecular structure on the binding mechanism and structure of the resulting complexes. The energetics and stoichiometry of the binding process was derived from ITC. The enthalpies of binding obtained are small and exothermic, and the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. Binding isotherms were obtained from microcalorimetric data by using a theoretical model based on the Langmuir isotherm. zeta-Potential data showed a reversal in the sign of the protein charge at pH 9.0 as a consequence of drug binding. Gibbs energies of drug binding per mole of drug were also derived from zeta-potential data. On the other hand, binding of the phenothiazines causes a conformational transition on protein structure which was followed as a function of drug concentration by using UV-vis spectroscopy. These data were analyzed to obtain the Gibbs energy of the transition in water (DeltaG(w)(degrees)) and in a hydrophobic environment (DeltaG(hc)(degrees)). Finally, the population distribution of the different species in solution and their size was analyzed through dynamic light scattering. The existence of an aggregation process of drug/protein complexes, mainly at pH 2.5, was observed. We think this is a consequence of the already expanded structure of the protein at this pH and the subsequent binding of drug molecules to the protein.     

Substituent effects on the binding of natural product anthocyanidin inhibitors to influenza neuraminidase with mass spectrometry

The binding of three closely related anthocyanins within the 430-cavity of influenza neuraminidase is studied using a combination of mass spectrometry and molecular docking. Despite their similar structures, which differ only in the number and position of the hydroxyl substituents on the phenyl group attached to the chromenylium ring, subtle differences in their binding characteristics are revealed by mass spectrometry and molecular docking that are in accord with their inhibitory properties by neuraminidase inhibition assays. The cyanidin and delphinidin, with the greatest number of hydroxyl groups, bind more strongly and are better inhibitors than pelargonidin that contains a lone hydroxyl group at the 4′ position. The study demonstrates, for the first time, the sensitivity of the mass spectrometry based approach for investigating the molecular basis and relative affinity of antiviral inhibitors, with subtly different structures, to their target protein. It has broader application for the screening of other protein interactions more generally with reasonable high-throughput.