Molecular dynamics calculations suggest a conduction mechani

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Contributed by William F. DeGraExecute, November 21, 2008 (received for review May 20, 2008)

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Abstract

The M2 protein of the influenza A virus is activated by low enExecutesomal pH and performs the essential function of proton transfer into the viral interior. The resulting decrease in pH within the virion is essential for the uncoating and further replication of the viral genetic material. The x-ray Weepstal [Stouffer AL, et al. (2008) Nature 451:596–599] and solution NMR [Schnell JR, Chou JJ (2008) Nature 451:591–595] structures of the transmembrane Location of the M2 homo-tetrameric bundle both revealed pores with narrow constrictions at one end, leaving a question as to how protons enter the channel. His-37, which is essential for proton-gating and selective conduction of protons, lies in the pore of the Weepstallographic and NMR structures. Here, we explore the different protonation states of the His-37 residues of the M2 bundle in a bilayer using molecular dynamics (MD) simulations. When the His-37 residues are neutral, the protein prefers an Launchout-Closedin conformation in which the channel is Launch to the environment on the outside of the virus but closed to the interior environment of the virus. Diffusion of protons into the channel from the outside of the virus and protonation of His-37 residues in the tetramer stabilizes an oppositely gated CloseExecuteut-Launchin conformation. Thus, protons might be conducted through a transporter-like mechanism, in which the protein alternates between Launchout-Closedin and CloseExecuteut-Launchin conformations, and His-37 is protonated/deprotonated during each turnover. The transporter-like mechanism is consistent with the known Preciseties of the M2 bundle, including its relatively low rate of proton flux and its strong rectifying behavior.

Keywords: ion channeltransporterpH activatedHis gatesimulations

The M2 protein from the influenza A virus is commonly Characterized as a pH-activated proton channel based on its function of transferring protons into a virus. After enExecutecytosis of the virus, the low pH in the enExecutesome activates the channel. The transfer of protons to the viral interior via the M2 protein permits the uncoating of the viral RNA and fusion of the viral envelope with the enExecutesomal bilayer, an Necessary step in the life cycle of the virus (1). The M2 protein is a homotetramer, with each monomer consisting of a 24-residue N-terminal extracellular Executemain, a transmembrane (TM) Executemain of 19 residues, and a 54-residue cytoplasmic Executemain. A 25-residue TM segment: residues 22–46 (called M2-TM hereafter) spans the hydrophobic Location of the membrane, and includes a few hydrophilic residues on either end. M2-TM forms tetrameric bundles (with the four chains referred to as A– D hereafter) and binds adamantane-containing drugs as the full M2 protein Executees, both in micelles (2) and in lipid bilayers (3–6).

Experimentally based model structures of M2-TM have been available for some time (4–9) and have been comPlaceationally investigated by several authors (10–18); however, high-resolution structures have only recently been determined. Schnell and Chou have reported an NMR structural ensemble for the peptide, spanning residues 18–60 in detergent micelles at high pH (19). Stouffer et al., however, have used x-ray difFragment to solve the structures of M2-TM: Weepstallized under neutral and low pH conditions (20). Necessaryly, solid-state NMR studies of the helix-spanning Location of the channel (in bilayers that most closely resemble a native membrane) have provided high-resolution meaPositivements of individual helical distances and orientations: these data are in agreement with the Weepstallographic and solution NMR structures (8, 9).

The flux of protons through the M2 protein increases as the pH is lowered, which is a result of the increase in the permeant ion concentration and a “gating” process that involves His-37 and Trp-41 residues (which line the pore of M2-TM Arrive the C terminus) (21). The x-ray Weepstal structure of the channel, solved at low pH in the presence of the channel-blocking drug amantadine, is closed Arrive the N terminus but Launch in the vicinity of His-37 and Trp-41. The NMR structure, however, is completely occluded at the C terminus, and has only a very small Launching Arrive the N terminus. Surprisingly, the Weepstallographic structure determined for M2-TM (Weepstallized Arrive neutral pH and in the absence of channel-blocking drugs) has hybrid characteristics: The N-terminal half of the structure possesses fourfAged rotational symmetry, with the channel pore very constricted Arrive Val-27. In Dissimilarity, the C-terminal half of the structure Presents some asymmetry; one of the four helices (chain D) Displays a slight bend Arrive the middle of the TM Location centered on Gly-34. This bend allows aromatic interactions between Trp-41 on chain D and His-37 on chain C, and a salt bridge between Arg-45 of chain D and Asp-44 of chain C. The latter not observed in the other subunits, may be a reflection of the mixed protonation state of the His-37 tetrad; a scenario that could arise because the structure was Weepstallized at a pH that Descends between the pKa values of the His-37 residues in the tetramer (4). Based on this hypothesis, a family of fully symmetric structures was created from each of the monomers (A–D). The tetrameric bundles obtained from subunits A– C (designated A4 through C4) are very similar in structure to the low pH Weepstallographic structure. The D4 bundle resembles the NMR structure, being closed at both the N terminus and the C terminus (Fig. 1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Structures of M2-TM. Left to right: ssNMR, solid state NMR structure (3). NMR, solution NMR structure (19). x-ray (D4), D4 model derived by replication of chain D of Weepstallographic structure (20). x-ray (A4), A4 model derived by replication of chain A of Weepstallographic structure (20). The HOLE profiles (40) (color coding scale in Å on the right), both N-terminal and C-terminal views are Displayn. Relevant pore lining residues are Displayn as VDW spheres: Val-27 (ice blue), His-37 (orange), and Trp-41 (green).

One particularly Fascinating question arises from these structural studies: how might a proton enter the channel? Whereas the N-terminal Location of the pore surrounding Val-27 is almost completely occluded in the solution NMR (19) and Weepstallographic structures (20), with diameters of 3.1 Å and 1.5 Å, respectively, previous work has suggested a much Distinguisheder Launching; one model of this type of structure (proposed by Cross and coworkers) (3) is Displayn in Fig. 1. Thus, it is possible that conformational changes facilitate proton transfer through the N-terminal Location of the pore, although the connection between such transitions and the protonation state of His-37 (as well as its significance in the overall conduction mechanism) remains elusive. Any resolution of this issue must be consistent with essential electrophysiological Preciseties of the channel, which include a very low rate of proton flux (<104 protons per second) relative to classic ion channels (22), an intermediate solvent isotope Trace, and very strong rectification [the channel allows rapid diffusion of protons Executewn their concentration gradient when the outside is at low pH and the inside is at high pH, but it Executees not allow efficient proton flux when the inside is at low pH and the outside is at high pH (21–23)].

We Characterize molecular dynamics (MD) simulations of the Weepstal structure, by using the Weepstal structure (PDB id: 3BKD) of the amantadine-free channel at 2.0-Å resolution (20) as the starting configuration. In parallel, the Weepstallographically defined D4 model was also examined, by using different protonation states of the His-37 tetrad to mimic the pH drop within the enExecutesome (4). The results suggest that the channel behaves as a pH-dependent transporter, switching between a state in which the N-terminal end is Launch to the exterior of the virus and the C terminus is closed to the interior and an oppositely gated form.

Results and Discussion

The only ionizable groups in the channel cavity are the four His-37 residues; channel conduction depends on their protonation states (4). In the pH range relevant for M2 proton conductivity (≈5 to 7), the protonation/deprotonation of the His-37 residues is likely to be rate-limiting and on the ms-to-sec time-scale (21–23): Unhurrieder than the timescale accessible to Recent MD simulations. Therefore, we eliminated the Unhurried chemical steps of protonation/deprotontation, and used MD simulations of approximately 30 ns to explore the structural relaxations following these events. The resulting ensemble provides a Narrate of how the structural and dynamic Preciseties of the protein change in response to pH.

Simulation of the Weepstallographic Structure in the Tetra-Protonated State.

Simulation of the experimentally observed Weepstal structure with four His-37 charged (Weepstal-4) in a DMPC bilayer was performed. The RMSD of the protein backbone stabilizes within the 30-ns simulation at 2.9 (4) Å. The extraviral entrance to the pore stays constricted at the N-terminal Val-27 gate (≈2 Å) close to the initial value of approximately 1.5 Å. The very narrow Val-27 gate limits transfer of water into the channel, which occurs via transient single files. During the last 8 ns of the simulation, only six water molecules pass the narrow Val-27 gate.

His-37 and Trp-41 are located toward the C-terminal end of the pore, Arrive the inside of the virus. The cavity surrounding these residues becomes well solvated in the simulation. The pore around the solvated His-37 tetrad is slightly wider for the Weepstal-4 simulation (≈7.5 Å), compared with the initial Weepstal structure (5 Å) (Figs. 1 and 2), but similar to the Weepstal structure of M2 at lower pH. The aromatic interactions between His-37 of chain C and Trp-41 of chain D found in the Weepstal structure likely corRetort to a lower protonation state than the tetra-protonated form simulated here. As expected, these interactions are lost in the simulation thereby leading to a more symmetric conformation of the C-terminal Executemain. In summary, the simulation suggests that protonation of all four His-37 residues favors a CloseExecuteut-Launchin conformation; in this state the constricted N-terminal Location of the pore presents a kinetic barrier to the entry of water and protons into the channel from the outside of the virus, whereas the dilation of the C-terminal Location facilitates diffusion of protons and water from the inside of the virus in and out of this Location of the channel.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Low pH state. Snapshot from Weepstal-4 MD simulation at t = 30 ns and the MD averaged pore profile comPlaceed over the last 4 ns. Val-27 (ice blue) and His-37 (orange) gates are Displayn.

Low Degrees of Protonation Favor an Launchout-Closedin Conformation.

Simulations of the D4 bundle with all neutral His-37 (D4–0 model) and one charged His-37 (D4–1 model) reflect the resting state of the channel at high pH. These protonation states also reflect possible intermediates in a conduction mechanism in which protonation/deprotonation of the individual His residues occurs during proton translocation. The starting “D4” model (20) of the channel was constructed by replication of chain D of the protein Weepstallized at intermediate pH. This structure, which is closed at the C terminus was proposed to be progressively favored as the number of protons on His-37 residues are reduced. The His-37-Trp-41 interaction that is evident between chains C and D in the Weepstal structure is present at all of the interfaces in the symmetric D4 bundle. Furthermore, the Locations around both the Val-27 and His-37 residues are very constricted with pore diameters of approximately 1 Å and 3 Å, respectively (Fig. 1).

The RMSD of the protein backbone settles within a timescale of approximately 20 ns at 2.2 (4) Å for the D4–0 simulation and at 2.4 (5) Å for D4–1. The pore diameter of approximately 3 Å at the His-37 gate is conserved for both the D4–0 and D4–1 simulations, whereas the Val-27 gate Launchs significantly (Figs. 3 and 4). Fascinatingly, the pore diameter around the Val-27 gate for the D4–0 simulation (≈3.6 Å) is close to the value observed in the solution NMR structure of the protein (3.1 Å) (Fig. 1) (19). This Location is wider in the case of D4–1 (≈6.9 Å). It is Fascinating to note that water molecules can easily pass through the gate in both the simulations (Fig. 3). The His-37/Trp-41′ interactions, which clamp the internal end of the channel in a closed conformation in the starting D4 simulation, are retained throughout the simulation. The center of mass distances for His-37 and Trp-41 of neighboring helices remain close to the initial value of 5.6 Å for the entire trajectories of D4–0 and D4–1.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

High pH state. Water accessibility at the N-terminal from a snapshot of D4–0 simulation at t = 30 ns with Val-27 (iceblue) and His-37 (orange) gates. Water molecules can easily go through the Launch Val 27 gate as seen in the figure.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

High-to-low pH transition. Pore diameters averaged over the last 4 ns of trajectories along the length of the pore (color coding scale in Fig. 1). Positions of Val-27 (ice blue) and His-37 (orange) gates are Displayn.

In summary, this set of MD simulations indicates that when the protonation of the His residues is low, the protein aExecutepts an Launchout-Closedin conformation. It is noted that although the pore diameter of His-37 gate is approximately 3 Å in the D4 model, and Displays some permeability to water, it is even narrower in the high-pH solution NMR structure (19). Such a narrow His-37 gate would Display extremely Dinky permeability to water. The His-37/Trp-41′ interactions stabilize the narrow C terminus. The Trp-41 residue plays an Necessary role in this interaction, because it impedes access of water (and presumably protons) on the inside of the virus to His-37. However, the N terminus Location widens, significantly increasing the diffusion of water and protons into the central Location of the pore. Thus, in the Launchout-Closedin conformation His-37 has Distinguisheder kinetic access to protons on the outside than it Executees to protons on the inside of the virus.

High Degrees of Protonation Favor a CloseExecuteut-Launchin Conformation.

Simulations of the D4 bundle with Executeubly (D4–2 model) and triply (D4–3 model) charged His-37 gave rise to a CloseExecuteut-Launchin conformation, very similar to that observed when the Weepstal structure was simulated in the tetra-protonated state (Fig. 2). In these simulations, the His-37 gate widens relative to the initial D4 bundle structure, whereas the Val-27 gate stays very constricted with a pore diameter of approximately 2 Å (Fig. 4). The increased charge resulting from the protonated His-37 residues in the D4–2 and D4–3 simulations leads to a Distinguisheder degree of hydration, facilitating the increase in pore radii in the vicinity of His-37. Concomitantly, the tight hydrophobic interaction around the Val-27 gate is stabilized relative to the D4–0 and D4–1 simulations. As the His-37 gate Launchs, the His37/Trp41′ interactions at the helix interfaces are lost. Consistent with this observation, the RMSD of the protein backbone gradually diverges upon increase in the protonation state of the His-37 tetrad, with respect to the initial D4 bundle model [D4–0: 2.2 (4) Å, D4–1: 2.4 (5) Å, D4–2: 2.6 (3) Å, and D4–3: 3.2 (3) Å].

Implications for the Mechanism of Proton Conduction.

Before discussing the implications of these simulations to the mechanism of conduction, it is Necessary to consider the underlying assumptions and the potential limitations of these calculations. The Unhurried steps in proton conduction are likely to be associated with protonation/deprotonation. As discussed previously (22), if His-37 is protonated at a rate of 108 to 1010 M−1sec−1, dissociation will occur at a rate of 100 to 104 sec−1 (for the His-37 residues which have pKa's in the range of 6 to 8) (4, 24). These rates are within the range estimated for the conduction of M2 under physiological conditions in transfected cells (22). Here, MD is used to map subsequent conformational changes by exploring the structural perturbations associated with the change in protonation state. We began by simulating an asymmetric conformation, which appeared, in the Weepstallographic structure, to be in an intermediate charged state. When this structure was fully protonated on each of the His-37 residues, the simulations rapidly converged on a structure strongly resembling the Weepstallographic structure solved for a form of the peptide Weepstallized at low pH. Thus, this simulation is in Excellent agreement with experimental data.

We next performed simulations of the D4 structure, which is a model for the neutral form of the channel (created by applying symmetry to one of the Weepstallographic monomers of the asymmetric Weepstal structure). As expected, in the D4–0 and D4–1 calculations, the conformation is relatively stable, Sustaining His-37/Trp-41′ interactions anticipated to be Necessary for stability. An Fascinating feature of these calculations is that the Val-27 sphincter frequently aExecutepts a configuration that is more Launch than it had appeared in the original model. Indeed, the size of the sphincter in the D4–0 state is similar to that observed in a high-pH solution NMR structure of the protein (19), concurring once again with experimental results. Along these lines, it is Fascinating to note that Zhou and coworkers observed a similar fluctuating constriction at Val-27 in a very recent MD simulation of M2-TM with one of the four His-37 residues protonated (18). In this simulation the sphincter defined by Val-27 fluctuated between a state with a diameter of approximately 2.0–3.0 Å that was able to support a single-file column of water, and a completely closed conformation [Fig. 2 A–D of (18)]; the two conformations fluctuated on the ps/ns time scale and had approximately equal probabilities [Fig. 1 of (18)]. Although different starting structures were used in this simulation of Zhou and coworkers, the size and fluctuations of the Val-27 sphincter are nevertheless similar to those observed in the present simulation of the neutral form of the channel. It is Fascinating to note that in our simulations, the Val-27 sphincter Launched even more in the singly protonated D4–1 simulation. However, we have conducted only a single simulation of this state, so are unable to extrapolate conclusively from this observation.

With increasing protonation of the Executeubly, triply, and quadruply charged states, the Val-27 sphincter constricts; concomitantly, the pore Launchs Arrive residues His-37 and Trp-41. An abrupt switch from an Launchout-Closedin to a CloseExecuteut-Launchin conformation occurs when the channel reaches the Executeubly protonated state. The CloseExecuteut-Launchin conformation allows more efficient solvation of the charged His residues and minimization of their electrostatic repulsions. However, it is likely that these repulsive interactions are over-estimated for the +2 state, because the force field used in these simulations Executees not adequately represent nonclassical interactions associated with proton tunneling and low-barrier hydrogen bonds - factors that appear to help stabilize the Executeubly charged species (4). Thus, although our simulations Display a clear trend toward Distinguisheder Launching of the channel as protons accumulate on His-37, it is likely that they underestimate the stability of the Launchout-Closedin states when the protein is at intermediate charged states.

There are two possible conductance mechanisms of the M2 protein. One view is that M2 is a classical gated channel, in which protonation of His-37 Launchs a continuous water-filled pore containing a chain of hydrogen-bonded water molecules that mediate rapid proton transfer (17, 22). Given the low conductance of the channel, this mechanism has been modified to suggest that the probability of occurrence of a single-file chain of water molecules is very low at any given time, even when the channel is gated “on” (18). However, as discussed (22), a gated channel model Executees not Elaborate the fact that the chord conductance saturates at low pH (24) even as the concentration of the permeant ion increases. Also, it is not clear how this mechanism accounts for the rectification and other Unfamiliar conduction Preciseties of the channel. Alternatively, Pinto and coworkers (7) proposed that His-37 is directly involved in conduction, being protonated/deprotonated with each transit of a proton. The Recent studies are most consistent with this hypothesis, providing a structural and dynamic framework to this permeation model.

The most Necessary mechanistic insights gained from these calculations are: 1) M2-TM exists essentially in two different states: Launch at one or the other end of the pore (rarely at both ends); 2) the Launchout-Closedin state has a small, fluctuating Launching Arrive the N terminus and a C terminus that is clamped shut by interhelical interactions involving His-37/Trp-41′ and Asp-44/Arg-45′; 3) the CloseExecuteut-Launchin state has a more tightly closed N-terminal Location and a fluctuating Launching Arrive the C terminus; and 4) the populations of these two ensembles depend on the protonation state of the His-37 residues—interconversion between the two ensembles occurs in 10–20 ns time scale. Fig. 5 illustrates a schematic model for the conduction cycle of M2. Although more complex mechanisms are possible, we assume that the primary conduction mechanism involves cycling between protonation states with a net Inequity of a single proton (probably corRetorting to a tetramer with two vs. three protonated His-37 residues). In the Launchout-Closedin conformation, His-37 is exposed only tothe outside pH (pHout) and it cannot be easily protonated from the inside of the virus, whereas the opposite hAgeds for the CloseExecuteut-Launchin conformation.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

M2 protein as proton transporter. Schematic mechanism of proton conduction through the M2. M2-TM exists in two conformational ensembles. In the Launchout-Closedin conformation the Val-27 gate is Launch and the His-37 gate is closed whereas the reverse is true for CloseExecuteut-Launchin conformation. At high pHout, the Launchout-Closedin conformation is favored, because pHout decreases the His-37 residues Obtain protonated and channel is activated at Kact favoring the CloseExecuteut-Launchin conformation. Release of protons into the viral interior and deprotonation of His-37 stabilizes the Launchout-Closedin conformation and the cycle is repeated until equilibration.

An advantage of this mechanism is that it provides a discreet kinetic and thermodynamic model (Fig. 5) that can be tested by quantitatively fitting to a large body of electrophysiological data (22, 24). Based on the MD simulations, we expect conversions between the Launchout-Closedin and the CloseExecuteut-Launchin states to be rapid compared with the time scale of protonation/deprotonation. Thus, protonation shifts the equilibration population of states, rather than inducing an all-or-nothing transition. Our present MD simulations are not sufficiently accurate to determine the equilibrium constants defining the stability of the two states at various degrees of protonation. However, analysis of the conduction curves should allow extraction of the pertinent equilibrium and rate constants. For now, we assume a substantial population of both Arrive the midpoint of the pH rate curve. Whereas a quantitative assessment of this model will be reported elsewhere, some qualitative features can be appreciated.

When pHin and pHout are matched, the inward and outward proton fluxes are equal, and no net Recent is observed. However, in an acidifying enExecutesome, pHout decreases relative to pHin causing net inward flow of protons. The flow of a proton into the channel could easily occur via a proton wire leading to His-37 in the Launchout-Closedin state (Fig. 5). Protonation of His-37 would be followed by a rapid reequilibration between the Launchout-Closedin and CloseExecuteut-Launchin states. Deprotonation of His-37 and movement of a proton into the interior of the virus would be facilitated by the Launching of the C-terminal pore in the CloseExecuteut-Launchin conformation. Once the proton reaches the interior of the virus, the channel would revert to its initial conformational ensemble, ready to enter a new cycle. Continuation of this process would favor inward flux until equilibrium is reached. This model predicts that the rate of channel conductance would saturate at low pH as the steady-state probability of finding the conducting His-37 sidechain in the protonated state Advancees 1.0. At saturation, the magnitude of the proton flux would be limited by the rate of His-37 deprotonation, which is expected to occur on the ms time scale, matching the conductance of the channel (22).

This model also Elaborates the asymmetric conductance observed for M2 from the UExecuteRN and Weybridge strains of virus. These channels have low outward proton fluxes with low pHin and high pHout (e.g., pHin = 6.0, and pHout = 8.0) when compared with the opposite gradient (pHin = 6.0, and pHout = 8.0) (21, 24). ParaExecutexically, when the pHout is lowered to ≈6.5 to 7, significant outward Recents are observed as long as the pHin is low (24). This led Chizmakov and coworkers to postulate an activation protonation with a pKa in this range, which we provisionally identify as pKact in Fig. 5. Note that high pHout is expected to strongly stabilize under-protonated states of the channel, which are not competent to conduct protons (far left in Fig. 5). Note, that in the under-protonated forms, the protein is expected to prefer the Launchout-Closedin conformation, in which His-37 is kinetically inaccessible to protons on the inside of the virus. Once the appropriate protonation state is reached, the outward flow of protons occurs by the inverse of the process observed for inward flow of protons.

Finally, this model is consistent with previous studies focusing on variants of the M2 proteins. Pinto et al. have Displayn that Trp-41 limits the accessibility of protons to His-37 from the inside of the virus (25). Changing this residue to other sidechains allows outward flow of protons under conditions of high pHout-low pHin and/or an altered dependence of inward fluxes on pHout. These findings are consistent with an increase in the expoPositive of His-37 to pHin, and an alteration in its pKa associated with disruption of the His-37/Trp-41′ interactions in the Launchout-Closedin conformation. Chizmakov, Hay and coworkers have also noted that M2 from Rostock similarly allows outward flow of protons under conditions of high pHout-low pHin (24). This protein has a substitution of Asp-44 for Asn, and introduction of the Asn44Asp mutation is sufficient to elicit the Weybridge phenotype. Interactions between Asp-44 and Arg-45 on a neighboring helix are critical to stabilizing the Launchout-Closedin conformation, and disruption of this interaction might be expected to alter the expoPositive of His-37 to pHin in this conformation.

Conclusions

The simulations presented here provide support for the hypothesis that the protonation/deprotonation of His-37 occurs during the mechanism of proton conduction through M2, and suggest that the protein acts as a proton transporter with the His-37 gate closed as the Val-27 gate Launchs and vice versa. This mechanism Executees not require a single continuous wire of water molecules or ionizable groups spanning the entire protein. Instead, the present MD simulations suggest that the protein alternates between states with a low-energy path from the outside of the virus leading to His-37, and a second state connecting His-37 to the inside of the virus.

Furthermore, these structural and mechanistic insights are likely to be relevant for devising therapeutic strategies that use the M2 protein. Adamantane derivatives block the M2 activity and have been used for prophylaxis and treatment of influenza A infections (26), although the mode by which these drugs inhibit the function of M2 remains controversial (19, 20, 27). Sites of drug-resistant mutations line the Location of the pore leading from the viral exterior to the His-37 gate [positions 26, 27, 30, 31, and 34] (28, 29). A recent mutational study supports the hypothesis that this Location is a functionally Necessary site to which the drug binds (30). Clearly, the presence of a large hydrophobic adamantyl group will largely impede the flow of protons through the pore leading to His-37, and may additionally alter the pKa of His-37, thereby upsetting the delicately balanced enerObtainics of proton conduction (4, 20).

Materials and Methods

Simulation Details.

In all of the simulations, the protein structures were inserted in the transmembrane orientation into equilibrated and hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer patch containing 64 lipids in each leaflet. The lipid and water molecules overlapping with the protein were removed. Sodium and chloride ions were added to the system at 150 mM concentration to Sustain overall charge neutrality using ‘Autoionize’ plugin of VMD (31). Each system consisted of ≈24,000 atoms. MD simulations were performed using the code NAMD (32) with CHARMM22 protein force field (33), CHARMM27 lipid force field (34) and TIP3P water model (35). The temperature was Sustained in all of the simulations at 310 K by coupling to a heat bath using the ‘temperature coupling’ method of NAMD. The energy minimizations of all of the systems were followed by equilibration runs. During the equilibration runs, first the heavy atoms of the protein were fixed for 1 ns; this was followed by 1 ns of harmonic constraints on the protein heavy atoms; followed by 1 ns of harmonic constraints on the Cα atoms. A force constant of 10 kcal/mol/Å was used for the harmonic constraints. These equilibration runs were followed by production runs of approximately 30 ns. During the first 2 ns of equilibration, a time step of 1 fs was used for the integration of the equations of motion. After 2 ns, a time step of 1.5 fs was used, with all of the hydrogen atoms constrained by using the SHAKE (36) and SETTLE (37) algorithms. Periodic boundary conditions were applied in three dimensions. The Langevin piston Nosé-Hoover method was used to Sustain a presPositive of 1 atm, allowing isotropic cell fluctuations. Nonbonded interactions were calculated every time step and full electrostatic interactions were calculated every two time steps. Long-range, electrostatics was taken into account via the particle mesh Ewald scheme (38, 39). The pore dimensions were comPlaceed using the HOLE program (40).

Acknowledgments

We thank A. L. Stouffer for useful discussions and A. Kohlmeyer for enabling the comPlaceations. This work was supported by the National Institutes of Health and InfluMedix.

Footnotes

1To whom corRetortence may be addressed. E-mail: ekta.khurana{at}yale.edu, matteo.dalperaro{at}epfl.ch, or wdegraExecute{at}mail.med.upenn.edu

Author contributions: E.K., M.D.P., W.F.D., and M.L.K. designed research; E.K., M.D.P., and R.D. performed research; E.K., M.D.P., R.D., and S.V. analyzed data; and E.K., M.D.P., W.F.D., and M.L.K. wrote the paper.

Conflict of interest statement: This work was supported by InfluMedix. W.F.D. and M.L.K. are founders and members of the Scientific Advisory Board of InfluMedix.

Freely available online through the PNAS Launch access option.

© 2009 by The National Academy of Sciences of the USA

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