Activation and Proton Transport Mechanism in Influenza A M2 Channel

Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His³⁷ tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His³⁷ provide insight into the mechanism of proton transport. The channel is closed at both His³⁷ and Trp⁴¹ sites in the singly and doubly protonated states, but it opens at Trp⁴¹ upon further protonation. Anions access the charged His³⁷ and by doing so stabilize the protonated states of the channel. The narrow opening at the His³⁷ site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His³⁷ correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val²⁷ remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations.     

Activation and Proton Transport Mechanism in Influenza A M2 Channel

Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His³⁷ tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His³⁷ provide insight into the mechanism of proton transport. The channel is closed at both His³⁷ and Trp⁴¹ sites in the singly and doubly protonated states, but it opens at Trp⁴¹ upon further protonation. Anions access the charged His³⁷ and by doing so stabilize the protonated states of the channel. The narrow opening at the His³⁷ site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His³⁷ correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val²⁷ remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations.     

pH-driven helix rotations in the influenza M2 H+ channel: a potential gating mechanism

The pH activated M2 H⁺ channel from influenza A has been a subject of numerous studies due to following: (1) It serves as a target for the aminoadamantane drugs that block its channel activity. (2) M2’s small size makes it amenable to biophysical scrutiny. (3) A single histidine residue is thought to control the pH gating of the channel. Recent FTIR analysis proposed that the helices of the channel rotate about their directors during pH activation. Herein, we report on molecular dynamics simulations of the X-ray structure of the protein with three charged histidine residues, representing the open form of the protein and two rotated forms with neutral histidines, representing its closed form. We compare the channel stability, convergence, interaction with water and hydration of the histidine residues that have been implicated in channel gating. Taken together, we show that both forms of the protein are stable during the course of the MD simulation and that indeed a rotation of the helices leads to channel closure. Finally, we propose a mechanism for channel gating that involves protonation of the histidine residues that necessities their increased solvation.     

Computer simulation of ion channel gating: the M(2) channel of influenza A virus in a lipid bilayer

The transmembrane fragment of the influenza virus M(2) protein forms a homotetrameric channel that transports protons. In this paper, we use molecular dynamics simulations to help elucidate the mechanism of channel gating by four histidines that occlude the channel lumen in the closed state. We test two competing hypotheses. In the "shuttle" mechanism, the delta nitrogen atom on the extracellular side of one histidine is protonated by the incoming proton, and, subsequently, the proton on the epsilon nitrogen atom is released on the opposite side. In the "water-wire" mechanism, the gate opens because of electrostatic repulsion between four simultaneously biprotonated histidines. This allows for proton transport along the water wire that penetrates the gate. For each system, composed of the channel embedded in a hydrated phospholipid bilayer, a 1.3-ns trajectory was obtained. It is found that the states involved in the shuttle mechanism, which contain either single-protonated histidines or a mixture of single-protonated histidines plus one biprotonated residue, are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfer. In contrast, the fully biprotonated state is not stable. Additional simulations show that if only two histidines are biprotonated, the channel deforms but the gate remains closed. These results support the shuttle mechanism but not the gate-opening mechanism of proton gating in M(2).     

Evaluating how rimantadines control the proton gating of the influenza A M2-proton port via allosteric binding outside of the M2-channel: MD simulations

In order to understand how rimantadine (RMT) inhibits the proton conductance in the influenza A M2 channel via the recently proposed “allosteric mechanism”, molecular dynamics simulations were applied to the M2-tetrameric protein with four RMTs bound outside the channel at the three protonation states: the 0H-closed, 1H-intermediate and 3H-open situations. In the 0H-closed state, a narrow channel with the RMT-Asp44-Trp41 H-bond network was formed, therefore the water penetration through the channel was completely blocked. The Trp41-Asp44 interaction was absent in the 1H-intermediate state, whilst the binding of RMT to Asp44 remained, which resulted in a weakened helix-helix packing, therefore the channel was partially prevented. In the 3H-open state it was found that the electrostatic repulsion from the three charged His37 residues allowed the Trp41 gate to open, permitting water to penetrate through the channel. This agreed well with the potential of the means force which is in the following order: 0H > 1H > 3H.     

Protonation dynamics of the alpha-toxin ion channel from spectral analysis of pH-dependent current fluctuations.

To probe protonation dynamics inside the fully open alpha-toxin ion channel, we measured the pH-dependent fluctuations in its current. In the presence of 1 M NaCl dissolved in H2O and positive applied potentials (from the side of protein addition), the low frequency noise exhibited a single well defined peak between pH 4.5 and 7.5. A simple model in which the current is assumed to change by equal amounts upon the reversible protonation of each of N identical ionizable residues inside the channel describes the data well. These results, and the frequency dependence of the spectral density at higher frequencies, allow us to evaluate the effective pK = 5.5, as well as the rate constants for the reversible protonation reactions: kon = 8 x 10(9) M-1 s-1 and koff = 2.5 x 10(4) s-1. The estimate of kon is only slightly less than the diffusion-limited values measured by others for protonation reactions for free carboxyl or imidazole residues. Substitution of H2O by D2O caused a 3.8-fold decrease in the dissociation rate constant and shifted the pK to 6.0. The decrease in the ionization rate constants caused by H2O/D2O substitution permitted the reliable measurement of the characteristic relaxation time over a wide range of D+ concentrations and voltages. The dependence of the relaxation time on D+ concentration strongly supports the first order reaction model. The voltage dependence of the low frequency spectral density suggests that the protonation dynamics are virtually insensitive to the applied potential while the rate-limiting barriers for NaCl transport are voltage dependent. The number of ionizable residues deduced from experiments in H2O (N = 4.2) and D2O (N = 4.1) is in good agreement.     

Design of an Efficient Inhibitor for the Influenza A Virus M2 Ion Channel

Influenza A virus is capable of rapidly infecting large human populations, warranting the development of novel drugs to efficiently inhibit virus replication. A transmembrane ion channel formed by the M2 protein plays an important role in influenza virus replication. A reasonable approach to designing an effective antivirus drug is constructing a molecule that binds in the M2 transmembrane proton channel, blocks H+ proton diffusion through the channel, and thus the influenza A virus cycle. The known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus replication. A new class of positively charged molecules, diazabicyclooctane derivatives with a constant charge of +2, was proposed to block proton diffusion through the M2 ion channel. Molecular dynamics simulations were performed to study the temperature fluctuations in the M2 structure, and ionization states of histidine residues were established at physiological pH values. Two types of diazabicyclooctane derivatives were analyzed for binding with the M2 ion channel. An optimal structure was determined for a blocker to most efficiently bind with the M2 ion channel and block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport through the M2 ion channel in addition to a steric barrier.     

Structure and dynamics of the influenza A M2 Channel: a comparison of three structures

The M2 protein is an essential component of the Influenza virus’ infectivity cycle. It is a homo-tetrameric bundle forming a pH-gated H⁺ channel. The structure of M2 was solved by three different groups, using different techniques, protein sequences and pH environment. For example, solid-state NMR spectroscopy was used on a protein in lipid bilayers, while X-ray crystallography and solution NMR spectroscopy were applied on a protein in detergent micelles. The resulting structures from the above efforts are rather distinct. Herein, we examine the different structures under uniform conditions such as a lipid bilayer and specified protonation state. We employ extensive molecular dynamics simulations, in several protonation states, representing both closed and open forms of the channel. Exploring the properties of each of these structures has shown that the X-ray structure is more stable than the other structures according to various criteria, although its water conductance and water-wire formation do not correlate to the protonation state of the channel.     

Two possible conducting states of the influenza A virus M2 ion channel

Molecular dynamics simulations have been performed on protonated four-helix bundles based on the 25-residue Duff-Ashley transmembrane sequence of the M2 channel of the influenza A virus. Well-equilibrated tetrameric channels, with one, two and four of the H37 residues protonated, were investigated. The protonated peptide bundles were immersed in the octane portion of a phase-separated water/octane system, which provided a membrane-mimetic environment. The simulations suggest that there could be two conducting states of the M2 channel corresponding to tetramers containing one or two protonated histidines. The more open structure of the doubly protonated state suggests it would have the higher conductance.     

Assessing the acid-base and conformational properties of histidine residues in human prion protein (125-228) by means of pK(a) calculations and molecular dynamics simulations

A thorough study of the acid-base behavior of the four histidines and the other titratable residues of the structured domain of human prion protein (125-228) is presented. By using multi-tautomer electrostatic calculations, average titration curves have been built for all titratable residues, using the whole bundles of NMR structures determined at pH 4.5 and 7.0. According to our results, (1) only histidine residues are likely to be involved in the first steps of the pH-driven conformational transition of prion protein; (2) the pK(a)'s of His140 and His177 are approximately 7.0, whereas those of His155 and His187 are < 5.5. 10-ns long molecular dynamics simulations have been performed on five different models, corresponding to the most significant combinations of histidine protonation states. A critical comparison between the available NMR structures and our computational results (1) confirms that His155 and His187 are the residues whose protonation is involved in the conformational rearrangement of huPrP in mildly acidic condition, and (2) shows how their protonation leads to the destructuration of the C-terminal part of HB and to the loss of the last turn of HA that represent the crucial microscopic steps of the rearrangement.     

Active-site serine phosphate and histidine residues of phosphoglucomutase: pH titration studies monitored by 1H and 31P NMR spectroscopy

1H and 31P NMR pH titrations were conducted to monitor changes in the environment and protonation state of the histidine residues and phosphoserine group of rabbit muscle phosphoglucomutase on binding of metal ions at the activating site and of substrate (glucose phosphate) at the catalytic site. Imidazole C epsilon-H signals from 8 of the 10 histidines present in the free enzyme were observed in 1H NMR spectra obtained by a spin-echo pulse sequence at 470 MHz; their pH (uncorrected pH meter reading of a 2H2O solution measured with a glass electrode standardized with H2O buffer) titration properties (in 99% 2H2O) were determined. Three of these histidine residues, which have pKa values ranging from 6.5 to 7.9, exhibited an atypical pH-dependent perturbation of their chemical shifts with a pHmid of 5.8 and a Hill coefficient of about 2. Since none of the observed histidines has a pKa near 5.8, it appears that these three histidines interact with a cluster consisting of two or more groups which become protonated cooperatively at this pH. Binding of Cd2+ at the activating site of the enzyme abolishes the pH-dependent transition of these histidines; hence, the putative anion cluster may constitute the metal ion binding site, or part of it. Two separate 31P NMR peaks from phosphoserine-116 of the phosphoenzyme were observed between pH 6 and 9. Apparently, the metal-free enzyme exists as a pH-dependent mixture of conformers that provide two different environments, I and II, for the enzymic phosphate group; the transition of the phosphate group between these two environments is slow on the NMR time scale.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence and kinetic properties of Ru(III) (NH3)5 modified transferrin

Diferric transferrin was modified using aquopentaammine ruthenium(II), a reagent for surface-accessible uncoordinated histidines. Introduction of the cationic Ru(III) (NH3)3 + 5 group on the imidazole of only 5.5 of the 17 uncoordinated histidines enhances the rates of pyrophosphate-assisted iron removal from the N-terminal and C-terminal binding sites by 16- and 2-fold, respectively. This differential effect on the kinetics of the two sites may partially explain why in the native protein the N-terminal site is more labile than the C-terminal site in acidic solutions where histidine residues become positively charged through protonation. The distance between the metal site and nearby uncoordinated histidines was estimated from fluorescence energy transfer measurements using Tb (III) as the donor and pentaammine ruthenium(III)-labeled imidazole of histidine as the acceptor chromophore. A Tsou Chen-Lu statistical analysis of the fluorescence quenching data suggest that two residues in each lobe of the protein are involved in quenching the fluorescence. By using estimates for the index of refraction and the quantum yield and assuming the energy transfer follows parallel first-order kinetics, an upper limit for the donor-acceptor distance of about 1.4 nm was obtained, assuming two uncoordinated histidine residues equidistant from the metal. His-207 and His-242 in the N-terminal lobe of transferrin and His-535 and His-577 in the C-terminal lobe are within this distance, based on information from the lactoferrin crystal structure. It is postulated that His-207 in the N-terminal lobe and His-535 in the C-terminal lobe are the uncoordinated residues that, when protonated or modified with Ru(III) (NH3)3 + 5, lead to accelerated loss of iron from the two binding sites of the protein.     

Susceptibility of the guard-cell K+-uptake channel KST1 to Zn2+ requires histidine residues in the S3-S4 linker and in the channel pore

 Potassium channels are inhibited by several mono- and divalent cations. To identify sites involved in the interaction between K⁺ channels and cationic effectors, we expressed the potato (Solanum tuberosum L.) guard-cell K⁺-uptake channel KST1 in Xenopus oocytes. This channel was reversibly blocked by extracellular Zn²⁺ in the micromolar range. In the presence of this heavy metal, steady-state currents were reduced in a pH-dependent but voltage-independent manner. Since Zn²⁺-inhibition was less effective at elevated external proton concentrations, we generated alanine mutants with respect to both extracellular histidines in KST1. Whereas substitution of the pore histidine H271 resulted in a reduced blockade by Zn²⁺, the channel mutant KST1-H160A in the S3-S4 linker lost most of its Zn²⁺ sensitivity. Since both histidines alter the susceptibility of KST1 to Zn²⁺, the block may predominantly result from these two sites. We thus conclude that the S3-S4 linker is involved in the formation of the outer pore. Received: 3 May 1999 / Accepted: 8 July 1999     

Ionization states of residues in OmpF and mutants: effects of dielectric constant and interactions between residues

To understand ion permeation, one must assign correct ionization states to titratable amino acid residues in protein channels. We report on the effects of physical and methodological assumptions in calculating the protonation states at neutral bulk pH of titratable residues lining the lumen of the native Escherichia coli OmpF channel, and five mutants. We systematically considered a wide range of assumed protein dielectric constants and all plausible combinations of protonation states for electrostatically interacting side chains, and three different levels of accounting for solute shielding: 1), full nonlinear Poisson-Boltzmann; 2), linearized Poisson-Boltzmann; and 3), neglect of solute shielding. For this system we found it acceptable to neglect solute shielding, a result we postulate to be generalizable to narrow lumens of other protein channels. For the large majority of residues, the protonation state at neutral bulk pH was found to be independent of the assumed dielectric constant of the protein, and unambiguously determined by the calculation; for native OmpF only Asp-127 has a protonation state that is sensitive to the assumed protein dielectric constant. Our results are significant for understanding two published experimental observations: the structure of the narrow part of the channel, and the ionic selectivity of OmpF mutants.     

External tetraethylammonium as a molecular caliper for sensing the shape of the outer vestibule of potassium channels

External tetraethylammonium (TEA+) blocked currents through Kv1.1 channels in a voltage-independent manner between 0 and 100 mV. Lowering extracellular pH (pHo) increased the Kd for TEA+ block. A histidine at position 355 in the Kv1.1 channel protein (homologous to Shaker 425) was responsible for this pH-dependent reduction of TEA+ sensitivity, since the TEA+ effect became independent of pHo after chemical modification of the Kv1.1 channel at H355 and in the H355G and H355K mutant Kv1.1 channels. The Kd values for TEA+ block of the two mutant channels (0.34 +/- 0.06 mM, n = 7 and 0.84 +/- 0. 09 mM, n = 13, respectively) were as expected for a vestibule containing either no or a total of four positive charges at position 355. In addition, the pH-dependent TEA+ effect in the wt Kv1.1 channel was sensitive to the ionic strength of the solution. All our observations are consistent with the idea that lowering pHo increased protonation of H355. This increase in positive charge at H355 will repel TEA+ electrostatically, resulting in a reduction of the effective [TEA+]o at the receptor site. From this reduction we can estimate the distance between TEA+ and each of the four histidines at position 355 to be approximately 10 A, assuming fourfold symmetry of the channel and assuming that TEA+ binds in the central axis of the pore. This determination of the dimensions of the outer vestibule of Kv1.1 channels confirms and extends earlier reports on K+ channels using crystal structure data as well as peptide toxin/channel interactions and points out a striking similarity between vestibules of Kv1.1 and KcsA channels.     

Analysis of site-specific histidine protonation in human prolactin

The structural and functional properties of human prolactin (hPRL), a 23 kDa protein hormone and cytokine, are pH-dependent. The dissociation rate constant for binding to the extracellular domain of the hPRL receptor increases nearly 500-fold over the relatively narrow and physiologic range from pH 8 to 6. As the apparent midpoint for this transition occurs around pH 6.5, we have looked toward histidine residues as a potential biophysical origin of the behavior. hPRL has a surprising number of nine histidines, nearly all of which are present on the protein surface. Using NMR spectroscopy, we have monitored site-specific proton binding to eight of these nine residues and derived equilibrium dissociation constants. During this analysis, a thermodynamic interaction between a localized triplet of three histidines (H27, H30, and H180) became apparent, which was subsequently confirmed by site-directed mutagenesis. After consideration of multiple potential models, we present statistical support for the existence of two negative cooperativity constants, one linking protonation of residues H30 and H180 with a magnitude of approximately 0.1 and the other weaker interaction between residues H27 and H30. Additionally, mutation of any of these three histidines to alanine stabilizes the folded protein relative to the chemically denatured state. A detailed understanding of these complex protonation reactions will aid in elucidating the biophysical mechanism of pH-dependent regulation of hPRL's structural and functional properties.     

Protonation states of important acidic residues in the central Ca²⁺ ion binding sites of the Ca²⁺-ATPase: a molecular modeling study

The P-type ATPases are responsible for the transport of cations across cell membranes. The sarco(endo)plasmic reticulum Ca2?-ATPase (SERCA) transports two Ca2? ions from the cytoplasm to the lumen of the sarco(endo)plasmic reticulum and countertransports two or three protons per catalytic cycle. Two binding sites for Ca2? ions have been located via protein crystallography, including four acidic amino acid residues that are essential to the ion coordination. In this study, we present molecular dynamics (MD) simulations examining the protonation states of these amino acid residues in a Ca2?-free conformation of SERCA. Such knowledge will be important for an improved understanding of atomistic details of the transport mechanism of protons and Ca2? ions. Eight combinations of the protonation of four central acidic residues, Glu309, Glu771, Asp800, and Glu908, are tested from 10 ns MD simulations with respect to protein stability and ability to maintain a structure similar to the crystal structure. The trajectories for the most prospective combinations of protonation states were elongated to 50 ns and subjected to more detailed analysis, including prediction of pK(a) values of the four acidic residues over the trajectories. From the simulations we find that the combination leaving only Asp800 as charged is most likely. The results are compared to available experimental data and explain the observed destabilization upon full deprotonation, resulting in the entry of cytoplasmic K? ions into the Ca2? binding sites during the simulation in which Ca2? ions are absent. Furthermore, a hypothesis for the exchange of protons from the central binding cavity is proposed.     

The influence of amino acid protonation states on molecular dynamics simulations of the bacterial porin OmpF

Several groups, including our own, have found molecular dynamics (MD) calculations to result in the size of the pore of an outer membrane bacterial porin, OmpF, to be reduced relative to its size in the x-ray crystal structure. At the narrowest portion of its pore, loop L3 was found to move toward the opposite face of the pore, resulting in decreasing the cross-section area by a factor of approximately 2. In an earlier work, we computed the protonation states of titratable residues for this system and obtained values different from those that had been used in previous MD simulations. Here, we show that MD simulations carried out with these recently computed protonation states accurately reproduce the cross-sectional area profile of the channel lumen in agreement with the x-ray structure. Our calculations include the investigation of the effect of assigning different protonation states to the one residue, D(127), whose protonation state could not be modeled in our earlier calculation. We found that both assumptions of charge states for D(127) reproduced the lumen size profile of the x-ray structure. We also found that the charged state of D(127) had a higher degree of hydration and it induced greater mobility of polar side chains in its vicinity, indicating that the apparent polarizability of the D(127) microenvironment is a function of the D(127) protonation state.     

Are aromatic carbon donor hydrogen bonds linear in proteins?

Proteins fold and maintain structure through the collective contributions of a large number of weak, noncovalent interactions. The hydrogen bond is one important category of forces that acts on very short distances. As our knowledge of protein structure continues to expand, we are beginning to appreciate the role that weak carbon-donor hydrogen bonds play in structure and function. One property that differentiates hydrogen bonds from other packing forces is propensity for forming a linear donor-hydrogen-acceptor orientation. To ascertain if carbon-donor hydrogen bonds are able to direct acceptor linearity, we surveyed the geometry of interactions specifically involving aromatic sidechain ring carbons in a data set of high resolution protein structures. We found that while donor-acceptor distances for most carbon donor hydrogen bonds were tighter than expected for van der Waals packing, only the carbons of histidine showed a significant bias for linear geometry. By categorizing histidines in the data set into charged and neutral sidechains, we found only the charged subset of histidines participated in linear interactions. B3LYP/6-31G**++ level optimizations of imidazole and indole-water interactions at various fixed angles demonstrates a clear orientation dependence of hydrogen bonding capacity for both charged and neutral sidechains. We suggest that while all aromatic carbons can participate in hydrogen bonding, only charged histidines are able to overcome protein packing forces and enforce linear interactions. The implications for protein modeling and design are discussed.     

Ionization of His 55 at the dimer interface of dynein light-chain LC8 is coupled to dimer dissociation

LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that interacts with a wide variety of cellular proteins and is presumed to play a fundamental role in dynein assembly and cargo recruitment and in the assembly of protein complexes unrelated to dynein. LC8 is a dimer at physiological pH but dissociates to a folded monomer at pH < 4.8. We have suggested that acid-induced dimer dissociation is due to protonation of His 55, which is stacked against His 55' and completely buried in the dimer interface. In this work, we show that the pH-induced dissociation is reversible and indeed governed by the ionization state of His 55. Mutagenesis of His 55 to Lys results in a monomer in the pH range of 3-8, while the mutation to Ala results in a dimer in the same pH range. Mutations that disrupt intermolecular hydrogen bonds between Tyr 65 and Lys 44' and His 55 and Thr 67' do not change the association state of the dimer. Titration curves for His 55 and the two other histidines, His 72 and 68, were determined by (13)C-(1)H NMR for H55K and for WT-LC8 in the monomeric and dimeric states. The pK(a) values of His 72 and His 68 are 6 in the WT dimer and 6.2-6.5 in monomeric H55K, while the pK(a) of His 55 is about 4.5 in the WT dimer. These results indicate that deprotonation of His 55 is linked to dimer formation and that mutation of His 55 to a small neutral residue or to a positively charged residue uncouples the protonation and dissociation processes.