Structural Comparison of the Wild-Type and Drug-Resistant Mutants

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Structural Comparison of the Wild-Type and Drug-Resistant Mutants of the Influenza A M2 Proton Channel by Molecular Dynamics Simulations Ruo-Xu Gu, Limin Angela Liu, Yong-Hua Wang, Qin Xu, and Dongqing Huang Wei J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp312396q • Publication Date (Web): 17 Apr 2013 Downloaded from http://pubs.acs.org on April 24, 2013

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The Journal of Physical Chemistry

Structural Comparison of the Wild-Type and DrugResistant Mutants of the Influenza A M2 Proton Channel by Molecular Dynamics Simulations Ruo-Xu Gu†, Limin Angela Liu‡, Yong-Hua Wang*§, Qin Xu†, Dong-Qing Wei*† †

State Key Laboratory of Microbial Metabolism and Shanghai Jiao Tong University, Shanghai Minhang District, 200240, China ‡

§

Fred Hutchinson Cancer Research Center, Seattle WA, 98109, United States

College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, 510640, China

ACS Paragon Plus Environment

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ABSTRACT: The influenza A M2 channel in the viral envelope is a pH-regulated proton channel that is crucial for viral infection and replication. Amantadine and rimantadine are two M2 inhibitors that have been widely used as anti-influenza drugs. However, due to naturallyoccurring drug-resistant mutations, their inhibition ability has gradually decreased. These drugresistant mutations are found at various positions on the transmembrane domain of the M2 protein and could be categorized to three types: mutations close to the drug-binding site located at the pore-facing positions (V27A, A30T, S31N, and G34E); mutations at the interhelical interfaces at the N-terminal half of the channel (L26F); and mutations outside the drug-binding site lying at the interhelical interfaces (L38F, D44A). Investigating the structures and the M2inhibitor interactions of these mutants would illuminate drug inhibition and drug resistance mechanisms and guide the design of novel anti-influenza drugs targeting these drug-resistant mutants. In this study, we chose four mutations at different positions (V27A, S31N, L26F, L38F) and conducted molecular dynamics simulations on both the apo-form and the drug-bound forms. The protein structures as well as the water structure in the channel pore were analyzed. Stable water clusters facilitating drug binding were found. Both the protein pore radii profiles and the structure of the water clusters were sensitive to the mutations. Based on our simulations, we compared the structures of the mutated proteins and proposed possible mechanisms for drug resistance of these mutations.

Keywords: proton conduction; drug inhibition; water cluster; protein-drug binding; pore radius profile


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INTRODUCTION

The influenza matrix protein 2 (the M2 protein) in the viral envelop is a selective proton channel sensitive to the pH environments1. Because of its essential role in the process of viral infection and replication, inhibiting the M2 channel by small ligands has been used as a strategy for treating the influenza pandemic2-5. Amantadine (AMA) and rimantadine (RIM) are M2 inhibitors that have been used as anti-influenza drugs for several decades, but they have lost their efficacy gradually because of various naturally-occurring drug-resistant mutations, as shown in Fig. 1. The binding cavity of these inhibitors is identified in the channel pore6,7, constituted by several pore-facing residues including Val27, Ala30, Ser31, and Gly348-10 (i.e., the pore-binding site, Fig. 1).

Figure 1. Mutations at different positions of the M2 channel. Mutations of residues at the pore-facing positions (V27A, A30T, S31N, G34E), mutations at the interhelical interfaces of the N-terminal side of the channel (L26F), and mutations at the C-terminal side (L38F, D44A) of the channel are shown in green, red, and blue, respectively. Only three subunits of the channel are shown for clarity. The pore-binding site is also labeled.


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The channel pore of the M2 protein is a homo-tetramer and is assembled by four transmembrane (TM) peptides9,11. Therefore, one mutation in the monomer unit would introduce four different residues in the channel protein. The drug-resistant mutations could be discriminated by their positions on the TM helices. Mutations of pore-facing residues (V27A, A30T, S31N, and G34E) are all located at the N-terminal half of the TM helices and are close to the pore-binding site (Fig. 1)5,12. Mutations of pore-facing residues (e.g., His37 and Trp41) at the C-terminal half of the channel have deleterious effects on the biological function of the channel as these residues are crucial for proton conduction and channel gating. Mutations have also been found at the interhelical interfaces of the channel. Some of these mutations are located at the Nterminal half of the channel and are close to the pore-binding site (e.g., L26F), whereas others lie at the C-terminal side (L38F13,14 and D44A15) and are outside the pore-binding site and therefore may induce drug resistance allosterically. We will refer to these three types of mutations as: pore-facing mutations, N-terminal interhelical mutations, and C-terminal interhelical mutations, respectively (Fig. 1).

Based on the 3D structures of the M2 channel10, one may infer the drug resistance mechanisms for the different mutations. For example, mutations at the helical interfaces may disturb helixhelix packing, whereas pore-facing mutations may influence the properties of the channel pore directly. The drug resistance mechanisms for the pore-facing mutations have been proposed and generally agreed upon based on the existing 3D structures of the channel. However, there are still some debates regarding the drug resistance mechanisms of the interhelical mutations (such as L38F and D44A)16.


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Extensive experiments and computations have been carried out to study the aforementioned mutations. Wang et al.4 conducted molecular dynamics (MD) simulations to explore interactions between different inhibitors and the M2 channel (including the wild-type (WT) M2 and two drug-resistant mutations, L26F and V27A). They found enlarged pore radii for the V27A and L26F mutants, which may lead to weaker drug binding and consequently drug resistance. Balannik et al.17 compared mutant M2 structures with pore-facing mutations and discussed the effects of these mutations on proton conduction. Rosenberg et al.7 found that the V27A and S31N mutants did not bind with the inhibitors in the pore-binding site by surface plasmon resonance experiments. The structures of these two mutants have been solved and enlarged pore radius were found in both mutants15,18, which may explain the weakened binding and resultant drug resistance.

A special note about the mutation S31N is that the existing 3D structures of the M2 channel proteins place this residue at either the pore-facing position or an interhelical position. Due to this difference, two different drug resistance mechanisms have been proposed for this mutation. When this residue is at the interhelical position, the S31N mutation destabilizes the assembly of helices at the helical interfaces and increases the pore radius11, leading to weakened drug binding. When this residue is facing the pore, the S31N mutation reduces the pore radius9,19, causing drug molecules to fail to bind to the channel pore.

Our previous study found significant differences between the M2 structures in the protein data bank due to the protein length used in the experiment (i.e., whether the structure contains the C-


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terminal amphipathic helices or not, see Fig. 2)10. For instance, the four TM helices packed loosely in several early structures with wide channel pores, whereas more tightly-packed channel structures were found when the C-terminal amphipathic helices were included and/or when different experimental conditions were used20-22. However, most of the previous computational studies used only the TM domain of the M2 channel that is a rather flexible structure. Therefore, novel studies using longer proteins and more tightly-packed structures are necessary in order to investigate the effects of the C-terminal amphipathic helices on the structure and the stability of the M2 mutants.

Molecular modeling and molecular dynamics (MD) calculations are powerful techniques for investigating the ligand-protein interactions23,24. In our work, we carried out MD simulations of the wild-type (WT) M2 and four mutants (V27A, S31N, L26F, and L38F) in order to explore the effects of these mutations on the channel structure and ligand binding. We used the ssNMR structure (PDB ID: 2L0J) in our simulations in order to consider the effects of the C-terminal amphipathic helices on the mutated structure19. Our simulations found that all of the mutations changed the pore radius and affected the highly-ordered water structure in the channel pore, which may be responsible for the reduced efficacy of the inhibitors in these mutants4,25. Our work is a systematic comparison of M2 structures with mutations at different positions. We obtained several novel findings that provide possible explanations for the drug resistance mechanisms of these four mutations. The understanding of these mechanisms would be helpful for designing novel inhibitors targeting the drug-resistant channels.

METHODS


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1. Simulation System Construction. We conducted MD simulations of the WT M2 channel protein as well as four mutant channels (V27A, S31N, L26F, and L38F) in bilayer environment. For each M2 structure (WT or the mutants), the apo-form, the AMA-bound form and the RIMbound form were simulated. Fifteen simulation systems were built in total, as shown in Table 1.

Table 1. Molecular dynamics (MD) simulations carried out in this study.

Ligand-bound forms System

Apo-form AMA

RIM

WT (2L0J) L26F

5 ns MD simulation with protein backbone constrained, followed by 15 ns MD simulation with no constraints

V27A S31N

ligand insertion into the apo-form followed by 5 ns MD simulations with protein backbone constrained, followed by 15 ns MD simulation with no constraints

L38F

The initial conformation used in these simulations was constructed by inserting the M2 channel

(PDB

ID:

2L0J)19

into

a

pre-equilibrated

bilayer

containing

200

dipalmitoylphosphatidylcholine (DPPC) molecules. 7 lipid molecules were removed from each leaflet to avoid bad contacts between the lipids and the M2 protein. The system was then solvated by TIP3P water molecules with a width of 1.5 nm at each side of the bilayer. Chloride ions were added as counter ions to neutralize the simulation system. The final system contained one M2 channel, 186 DPPC molecules, ~6470 TIP3P water molecules, as well as ~10 chloride ions. The dimension of the simulation box was 7.5 nm × 7.5 nm × 7.5 nm. The channel axis was parallel to the Z-axis of the simulation system.


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For this wild-type apo-form structure, we carried out 15 ns MD simulation with all protein atoms constrained to pack the lipids around the M2 channel properly. After that, we built ligandbound forms for the WT channel and generated the four different mutants (both apo-form and ligand-bound forms). For these structures, 5 ns MD simulation was conducted first with the protein backbone atoms constrained allowing the protein side chains to move freely. Then, 15 ns simulation without any constraints was carried out. For the simulations of the ligand-bound forms, the inhibitor molecule was positioned in the pore-binding site in the vicinity of the Ser31 residues according to the ssNMR structure (PDB ID: 2KQT)8 with the positively-charged nitrogen atom pointing towards the His37 residues. Simulation procedures for all of the fifteen systems are listed in Table 1.

2. Simulation Protocol. All simulations were carried out using the GROMACS package26,27. The integration time step was 2 fs. The NPT ensemble was used and the system was maintained at 300 K and 1 atm by the Parrinello-Donadio-Bussi V-rescale thermostat28 and Berendsen algorithms, respectively. Semi-isotropic pressure was applied with the pressure along the Z-axis being independent from that in the X-Y plane. The van der Waals interactions were treated with a cut off of 1.4 nm, whereas the long-range electrostatic interactions were computed by the PME method. All bond lengths were constrained by the LINCS algorithm29,30. The proteins and the ligands were described by the OPLS all-atom force field30 whereas the Berger’s united-atom force field31 was used to describe the lipid molecules. Similar force fields were used in Chakrabarti et al.’s publications32,33 and our previous simulations10. The topology files for the ligands

were

generated

by

the


Topolbuild

package

8

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(http://www.gromacs.org/Downloads/User_contributions/Other_software,

access

date:

07/09/2012). The trajectories were saved every 10 ps.

3. Data Analyses. Secondary structures of the proteins during the simulations were calculated using the DSSP package34,35. Pore radius profiles of the M2 channels were calculated using the HOLE2 package36. The pore radius profiles were calculated based on 10 snapshots extracted randomly from the last 10 ns trajectory and the average was reported. Water density in the channel pore was defined as the number density of the water oxygen atoms and was calculated by the g_densmap tool of the GROMACS package. Protein structure figures were generated using the VMD package37.

RESULTS AND DISCUSSION

We studied the drug-resistant mutations of the M2 channel according to their physical location in the channel. Pore-facing mutations may modify the pore radius and the pore electrostatic properties by varying the size and charge of the residue side chain, whereas interhelical interfacial mutations may influence the M2 channel structure by affecting tetramer packing. However, interhelical mutations at the C-terminal half of the channel apparently have different effects on ligand binding compared with those at the N-terminal half because they do not participate in binding of the drug molecules.

The V27A (pore-facing mutation), L26F (N-terminal interhelical mutation), and L38F (Cterminal interhelical mutation) mutants were simulated as examples of these three types of


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mutations. The S31N mutant was also investigated because it is the most widely found drugresistant mutation but its drug resistance mechanism has not been fully understood. In Stouffer et al.’s crystal structure (PDB ID: 3C9J)9, Ser31 residues are pore-facing and mutation of them to the large asparagine decreases the channel pore radii. However, in the sNMR structures (PDB ID: 2RLF and 2KIH)11,15, the S31 residues lie at the helical interfaces and the S31N mutation increases the channel pore size by destabilizing helical packing. In the initial structure used in our MD simulations (PDB ID: 2L0J), the Ser31 residues are pore-facing. We will next present our MD simulations that compared the structures of these mutants to explore their possible drugresistant mechanisms.

1. Protein Structural Stability During the MD Simulations. MD simulations were conducted for the WT M2 channel and the four drug-resistant mutants (V27A, S31N, L26F, and L38F). Because of the C-terminal domain’s important role for the stability of the TM domain and the proton conduction rate21,38, we used an initial structure (PDB: 2L0J) including the C-terminal amphipathic helices to explore their effects on the structures of the M2 mutants.

For each structure, the apo-form, the AMA-bound form, as well as the RIM-bound form were simulated, as shown in Table 1. The backbone RMSD value of each simulation leveled off around 3 Å after ~5 ns of the production simulations for the entire channel (Fig. S1 in the Supporting Information). More specifically, the backbone RMSD values of the TM domain in all the simulations fluctuated around 1.5 Å (Fig. S2), which is similar to the results of Wang et al.’s simulations4 where only the TM domain was used (PDB ID: 3LBW). In contrast, the RMSD


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values of the C-terminal amphipathic domain stabilized around 3.2 Å (Fig. S2), implying that the C-terminal amphipathic helices were more flexible during our simulations.

Protein secondary structures in all MD simulations are similar and the results for the apo-form of the WT M2 are shown in Fig. 2 as an example. The four subunits constituting the M2 channel all maintained the α-helix structure (in blue in Fig. 2) except for several terminal residues and the residues connecting the TM helices and the C-terminal amphipathic helices. The terminal residues didn’t show α-helix structure mainly because of weak backbone hydrogen bonds at the protein endings. In the initial structure (PDB ID: 2L0J), a single residue (residue 47) forms a rigid turn between the TM helix and the C-terminal amphipathic helix19, whereas in our simulations, three flexible residues (residues 46 to 48) (in yellow in Fig. 2) were found at this region between the TM helix and the C-terminal amphipathic helix.

Our MD simulations were too short to investigate extensive conformational changes of the channel and could not determine the conformational distribution of the C-terminal amphipathic helices. However, our calculations showed the stability of the C-terminal amphipathic helical conformation in 2L0J in short MD simulations, as shown by the secondary structure in Fig. 2.


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Figure 2. Secondary structure of the four subunits (chains A-D) of the M2 channel during the MD simulations. The result of the apo-form of the WT M2 is shown as an example. The structure of the M2 channel is shown with the TM helices and the C-terminal helices in blue and the turn connecting these two helices in yellow. The X-axis represents the simulation time, whereas the Y-axis lists the residue numbers of the four subunits of the channel.

The four TM helices (numbered as A-D) are arranged in a symmetrical tetramer conformation in the initial structure (PDB ID: 2L0J). Although some NMR studies39 and most M2 structures in the protein data bank support this symmetrical tetramer conformation, several MD simulations40 and NMR experiments have revealed a different structure referred to as a dimer of dimers conformation41,42. As proposed by Forrest et al.40, the channel may be in the tetramer conformation if the distances between the opposing TM helices are similar (i.e., the distance between chains A and C, and that between chains B and D), whereas the channel would be in the dimer of dimers conformation otherwise. The distances between the mass centers of adjacent TM helices and those between the opposing ones were stabilized around 1.0 nm and 1.4 nm during the simulations, respectively, as shown in Fig. 3. This result is consistent with Chen et al.’s


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calculations43 and indicates that the M2 channel maintains the symmetrical tetramer conformation for the apo-form and the ligand-bound forms of different mutants in short MD simulations.

The stability of the secondary and the quaternary structure of the M2 channel in all simulations indicates that these mutations do not cause significant changes to the overall structure of the M2 channel. Therefore, these mutations probably induce drug resistance by disturbing the structure of the binding cavity, which is explored below.

Figure 3. Distances between the mass centers of the M2 protein backbones of the TM helices (chains A-D). Only the result for the apo-form of WT M2 is shown. The results of the other simulations are highly similar. These results imply a symmetrical tetramer conformation of the M2 channel.

2. Structural Comparison of the WT M2 and the Four Mutant Channels. The protein structure and the pore radius at the N-terminal entrance of the channel were affected by all of the four mutations studied here (V27A, S31N, L26F, and L38F). The average


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structures of the WT M2 and the four mutants (the apo-forms) during the last 10 ns simulations were calculated. The averaged structures of the wild-type and the four mutant channels and their channel pore radius profiles are shown in Fig. 4 with the mutations labeled. This figure summarizes how the mutations affect the channel pore, which will be discussed in detail in this section.

Figure 4. The average structures of the wild-type and the four mutant channels during the last 10 ns simulations and their corresponding pore radius profiles. The protein backbones are shown in ribbon model in white. The side chains of key residues are represented in ball-andstick model with the carbon atoms in cyan and orange. Nitrogen and oxygen atoms are shown in blue and red, respectively. The channel pores are shown in different colors according to the pore radii (red if the radius is 2.8 Å).

The average pore radius profile of the apo-form of the WT M2 and the mutants are also shown in Fig. 5A. The standard deviations of the M2 pore radius profiles are shown in Fig. S3. The shape of the radius profile of the WT M2 is consistent with that of the experimental


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structures11,19,25. From the profile (Figs. 4A and 5A), we can see that there is a channel gate at the C-terminal exit around His37 and Trp41 and a secondary gate at the N-terminal entrance in the vicinity of Val27. The pore radius reaches its maximum value and the channel forms a large cavity in the vicinity of Gly34 (Figs. 4A and 5A).

Mutation of Val27 to residues with smaller side chains such as alanine (V27A, Fig. 4B) destroyed the hydrophobic gate formed by this residue and increased the pore radius by ~2 Å at the N-terminal end (Figs. 4B and 5A). This result is consistent with Wang et al.’s MD simulations4 and Pielak et al.’s NMR structure18. In the apo-form of the V27A mutant channel, the C-terminal end of the channel was also widened by ~1 Å around His37, possibly due to weakened interhelical packing propagated from an enlarged pore at the N-terminal end of the channel.


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Figure 5. Pore radius profiles of the 15 WT and mutant apo and drug-bound M2 channels. Panels A, B and C show the pore radius profiles for the apo-form, the AMA-bound form and the RIM-bound form of the WT and the four mutant channels, respectively. The pore radii are shown as a function of the position along the Z-axis of the channel. The positions of the pore-facing residues are labeled by boxes in green (Val27 and Ser31), red (Gly34), and blue (His37 and Trp41). The standard deviations of the pore radii are shown in Fig. S3.


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Leu26 and Leu38 are residues at the N-terminal and C-terminal interhelical interfaces of the channel, respectively. Mutation of them to residues with larger side chains such as phenylalanine (Figs. 4D and E) destabilizes the tetramer assembly and helical packing and increases the pore radii in the vicinity of Val27 by ~0.5 Å (Figs. 4D, 4E and 5A).

The pore radii for the L26F mutant are slightly different from Wang et al.’s results4. In the latter work, the pore radius of the N-terminal entrance was wider than our results by ~1 Å. We propose that this difference may be due to the different initial structures used in our calculations. The TM-only construct used in Wang et al.’s study packed slightly more loosely and hence may be more sensitive to this mutation, whereas the C-terminal amphipathic helices included in our simulation stabilized the structure of this mutant.

The channel pore radius of the L38F mutant was enlarged by ~0.5 Å in the vicinity of Val27 and His37 compared to the WT. Because Leu38 is located at the C-terminal half of the channel, this result suggests that destabilization of the helical assembly at the C-terminal half of the channel affects the protein structure at the N-terminal side, agreeing with the allosteric drug resistance mechanism proposed in earlier work11,13. We propose that the large side chains of Phe38 weakened the helical assembly and induced conformational changes of the pore-facing residues (e.g., Val27) at the N-terminal half of the channel, so that the channel pore radii were increased. Close examinations of the Val27 side chain conformations and interhelical distances among the Val27 side chains (shown in Fig. S4 and Table S1 in the Supporting Information) support this allosteric mechanism.


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In the ssNMR M2 channel structure (PDB: 2L0J, the starting structure of our simulations), the Ser31 residue is facing the channel pore. Mutation of this residue to asparagine introduces large side chains into the channel pore (Fig. 4C) and affects the protein assembly at the N-terminus. The pore radius profile for S31N shows a slightly enlarged pore in the vicinity of Val27 by ~0.5 Å. However, the pore radii around Asn31 decreased significantly by ~1.5 Å (Fig. 4C and Fig. 5A). This simulation result agrees with Stuffer et al.’s hypothesis that the S31N mutation decreases the channel pore radius9, but disagrees with our previous MD simulations. Our previous simulations used a solution NMR structure with a different conformation for the Cterminal amphipathic helices (PDB ID: 2RLF) from 2L0J where longer and more flexible turn (or loop) connected the TM helix and the C-terminal amphipathic helix9,15,18. In this sNMR structure, the S31 residue is positioned at the interhelical region resulting in the S31N mutation increasing the channel pore radius10.

Figures 5B and 5C show the effects of drug-binding on the M2 channel pore radius profiles in the WT and the four mutants. These results will be discussed in more detail in Section 4.

3. Comparisons of the Water Structures Inside the WT M2 and the Four Mutants Channels. Channel pore hydration is crucial for channel function because the protons exist in the form of hydronium ions and are transferred via water molecules from the N-terminal entrance to the vicinity of His37 during proton conduction19,44,45. Previous NMR11 and X-ray structures25 have found water molecules positioned immediately below Val27 and ordered water clusters


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stabilized by hydrogen bonds with Gly34 and His37. The crystal structure of the TM domain of the M2 channel (PDB: 3LBW) revealed highly-ordered water molecules inside the channel pore. In the crystal structure, bulk water was found immediately below the Val27 residues, whereas a stable water cluster forming hydrogen bonds with the Gly34 backbone oxygen atoms were found above the His37 residues. This water cluster contained six water molecules with two of them arranged on the top of other four forming a pyramidal structure (Please refer to Fig. 1 of Ref. 25). Molecular dynamics simulations of the TM domain of the channel also found large water densities indicating ordered water molecules around these regions in the channel pore, agreeing with the observations by crystallography25.

In this work, we focused on addressing three questions. The first is whether the inclusion of the C-terminal amphipathic helices would affect the water structure inside the pore. The second is whether the four mutations would affect the water structure. And the third is how drug binding affects the water structure inside the pore. As our main goal of the study is to find the effects of mutations on drug-channel binding, we will focus on the water structure at the N-terminal side of the His37 residues in the drug-binding pocket. We will examine the water structure in the WT channels first and then move on to discuss the effects of mutations on the water structure.

3.1. Highly-ordered Water Structure in the Channel Pore of the WT M2. In order to investigate the structure and dynamics of water molecules in the channel pore, we first computed the 3D water densities in the channel pore of the WT M2 (both the apo-form and the ligand-bound form). The results revealed large water densities at specific positions in the channel pore, as shown in Fig. 6. In the apo-form of the WT M2 channel, the water structure extended for ~2-3 helical


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turns at the N-terminal half of the channel, and three density maxima were found in the vicinity of the Ser31 side chains, and the Ala30 and Gly34 backbone oxygen atoms (Fig. 6), implying ordered water structures around these three residues.

Next, we counted the number of hydrogen bonds between the water molecules and these three residues (Ser31, Ala30, and Gly34) of each M2 subunit. The results are shown in Table 2. The number of hydrogen bonds between water molecules and these three residues were found to be ~0.84, 0.84, and 0.66, respectively (Table 2), for the apo-form of the WT M2. These values indicated extensive hydrogen bond interactions between these residues and the water molecules in the channel pore, and the water molecules were likely stabilized around these positions by these hydrogen bonds.

Thirdly, we calculated the 2D water density maps in the channel pore to evaluate the water distributions in the channel pore and to compare the water structures of the different mutants. The results are shown in Fig. 7. The water density map in the channel pore of the WT M2 (Fig. 7A) shows large water densities at positions near Ser31, Ala30 and Gly34, consistent with Fig. 6 and the hydrogen bond analysis results in Table 2.

The above analysis revealed a highly-ordered water structure inside the channel pore in the drug-binding pocket in the apo-form and ligand-bound forms of the WT channel. These water molecules were organized into three layers in the apo-form (Fig. 6). Each of these layers contained four water molecules. The top layer was near the Ser31 side chain oxygen atoms, the middle layer was below the Ala30 backbone oxygen atoms, and the bottom layer was below the


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Gly34 backbone oxygen atoms. These layers of water molecules formed extensive hydrogen bonds with residues Ala30, Ser31, and Gly34 (Fig. S5A and Table 2). In the ligand-bound forms, only two layers of water were found in the drug-binding site, as the top layer near Ser31 was now occupied by the drug molecule (Figs. S5B-C and Figs. 6B-C). In our simulations, there were also one or two additional water molecules at the spaces between these water layers (the dashed red circles in Fig. 7A) forming pyramidal structure with the four water molecules in a layer. This pyramidal structure is highly consistent with that found in the most recent X-ray crystal structure (PDB ID: 3LBW) and in Wang et al.’ MD simulations4,25.

The above described channel water structure was quite dynamic during the simulation. There were frequent exchanges among the water molecules between different layers, as shown in Fig. S6. These water molecules also exchanged with water molecules in the bulk at the frequency of about once in tens of picoseconds.


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Table 2. Numbers of hydrogen bonds between the water (Wat) molecules in the channel pore and the Ser31 side chains or the Ala30 and Gly34 backbone oxygen atoms for each subunit (chains A-D) and their averages in the wild-type channel simulations. Subunit

Apo-form

AMA-bound form

RIM-bound form

A

B

C

D

Average

Wat-Ser31

0.78

0.85

0.85

0.87

0.84 ± 0.03

Wat-Ala30

0.87

0.85

0.82

0.82

0.84 ± 0.02

Wat-Gly34

0.45

0.84

0.58

0.77

0.7 ± 0.2

Wat-Ser31

0

0

0

0

0

Wat-Ala30

0.85

0.85

0.88

0.75

0.83 ± 0.05

Wat-Gly34

0.60

0.74

0.70

0.52

0.64 ± 0.09

Wat-Ser31

0

0

0

0

0

Wat-Ala30

0.09

0.92

0.93

0.05

0.5 ± 0.4

Wat-Gly34

0.41

0.13

0.60

1.00

0.5 ± 0.3


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Figure 6. Water densities in the channel pore of the WT M2 for the apo-form, the AMAbound form, and the RIM-bound form show three layers of structured water molecules. Water densities are represented by the surface model in light blue. The ligands (AMA or RIM) are shown in green, whereas the residues at the pore-binding site are shown in cyan. Only three subunits of the channel are shown for clarity.


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Figure 7. The three layers of structured water molecules and water density maps in the channel pore of the 15 WT and mutant apo and drug-bound M2 channels. Panels A, B and C show the results of the apo-forms, the AMA-bound forms, and the RIM-bound forms,


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respectively. Pore-facing residues and the water molecules in the three layers are shown in balland-stick model on the left, with the carbon, nitrogen, and oxygen atoms and water spheres in green, blue, red, and magenta, respectively. The water molecules positioned in between these water layers are represented by red dashed circles. Corresponding positions of the water layers in the density map are labeled. The water densities are shown in gray in the density maps, whereas the densities of ligands are shown in blue in Panels B and C. The vertical axis (Y-axis) of the density maps is along the channel pore from the N-terminus to the C-terminus, whereas the horizontal axis (X-axis) is perpendicular to the channel axis.

Different from the crystal structure, we found more structured water molecules in the drugbinding site inside the channel pore. For instance, the crystal structure (PDB ID: 3LBW)25 revealed only one layer of water molecules in this region (corresponding to layer 3 in our simulations, Fig. 6). These water molecules were stabilized by the Gly34 backbone oxygen atoms. The crystal structure also contained two water molecules above this water layer that formed a pyramidal structure with the water layer (see Fig. 1 in Ref. 25). The water molecules immediately below the Val27 residues (close to Ser31) were unstructured in 3LBW. These differences may be attributed to the smaller pore radius near Val27 in our simulations (Fig. 4A and Fig. 5A) that may restrain the water molecules in more ordered conformations. Moreover, the G34A mutation in the 3LBW structure may affect the pore hydration by the hydrophobic side chains of Ala34, as MD simulations by Acharya et al.25 found much higher water density below residue Ala30 in the WT channel than in the G34A mutant (Figure 4 in Ref. 25).


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3.2. Changes of Water Densities in the Channel Pore Induced by Mutations. Fig. 7A compares the water densities in the channel pore of the WT M2 and the four mutants. Water densities for the WT M2 are very low in the vicinity of Val27 and His37, consistent with the small pore radii (Fig. 5A) at these positions. However, water densities increased significantly around Ala27 in the V27A mutant. This is likely because mutation of valine to alanine destroyed the hydrophobic gate and increased the channel pore radius (Fig. 4B and Fig. 5A) so that passage of water molecules through the N-terminal end became much easier. Water densities around Ser31 and Gly34 in the V27A mutant dispersed to a wider region and became mostly unstructured, implying destabilization of the highly-ordered water structure due to the wider channel pore.

Similar results were found for the L26F and L38F mutants, as these two mutants share similar pore radius profiles (Fig. 5A). The water densities around Val27 were also increased but to a less extent compared with V27A, a result consistent with the smaller increase of the pore radii at this position (Fig. 4D, E, and Fig. 5A). The water densities in the channel pore also became dispersed in these two mutants (particularly for L38F, Fig. 7A). Destabilization of the water structure in the channel is probably because of the weakened or destroyed secondary hydrophobic gate at the Nterminus. In the WT M2, passage of water molecules through this hydrophobic gate is more energetically costly and the entered water molecules became somewhat trapped in the channel pore, whereas in the above discussed three mutants (V27A, L26F, and L38F), the water molecules enter and exit the channel pore more freely because of the enlarged pore radii around residues 27 and the water structure in the channel pore is hence less stable. We would also like to note that, in the V27A and L38F mutants, the water structures were much less stable than the L26F mutant channel. In these two mutants, the water densities in the channel pore were spread


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out throughout the channel pore, implying the lack of stable water molecules (shown in Fig. 7A). For the V27A mutant, the absence of structured water is probably because of the very large Nterminal pore radius that allows water motion with little energy barrier. However, for the L38F mutant, the lack of structured water may be likely a result of the destabilization of the whole TM domain (as the pore size was increased throughout the entire TM domain).

For the S31N mutant, the pore radius around Asn31 was decreased significantly (Fig. 4B and Fig. 5A), therefore, the water molecules interacting with the Ser31 residues disappeared. The water densities for the other two water layers were more structured compared with the other three mutants (Fig. 7A).

The above results suggest that the mutations (V27A, S31N, L26F, and L38F) affected the channel helical assembly and pore radii, causing changes to the water density and water structure in the drug-binding site. In addition, drug binding also strongly affected the water structure inside the channel pore. We will discuss this issue further in the next section.

4. Ligand Binding in the Channel Pore and Possible Drug Resistance Mechanism. 4.1. Ligand Binding Closes the N-Terminal Hydrophobic Gate. The pore radius around Gly34 was enlarged by ligand binding in the simulations for the WT M2. In the apo-form, the pore radius reached its maximum value (~3 Å) at position of Z = ~13 Å (around residue Ser31) and then the radius value reduced slowly to ~2.5 Å at position of Z = ~18 Å (around residue Gly34). In the ligand-bound forms, the radii reached a plateau (~3 Å) from Z = ~13 Å to Z = ~18 Å (compare the black lines in Figs. 5A-C). This result is consistent with our previous simulations10


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where ligand binding in the pore-binding site increased the radii in the pore region of the channel.

Pore radius around the N-terminal hydrophobic gate (Val27) was decreased slightly by ~0.3 Å in the ligand bound form compared with the apo-form. The decreased radius around Val27 is consistent with Yi et al.’s simulation results46, where ligand binding helped close the secondary hydrophobic gate formed by Val27. Similar decrease of the pore radius around the hydrophobic gate was also found in the simulations of the ligand-bound form for the V27A, L26F, and L38F mutants (Figs. 5B and C). These results could be explained by the following two reasons: (1) The hydrophobic groups of ligands formed hydrophobic interactions with the Val27 side chains and increased helical packing in this region; (2) The hydrophobic groups of the ligands displaced water molecules around Val27 (or Ala27 for V27A) so that the pore radius decreased.

The N-terminal pore radius didn’t change significantly in the S31N mutant of the ligand-bound forms compared with the apo-form, as the large side chains of Asn31 decreased the pore radii.

Our results indicated that ligand binding in the WT and the mutant channels increased channel helical packing at the N-terminal side of the channel and decreased the channel pore radii around the N-terminal hydrophobic gate formed by Val27.

4.2. Hydrophobic Interactions and Water-Mediated Hydrogen Bonds are Responsible for Ligand Binding. The ligand (AMA or RIM) was found to bind in the cavity immediately below the Val27 residues, in the vicinity of the Ser31 residues, with the positively-charged nitrogen


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atom pointing toward the His37 residues throughout the simulation. This result is similar to Cady et al.’s ssNMR structure8 (PDB ID: 2KQT) and our previous MD simulations10. The binding conformation of the ligand in the channel pore was found to be quite stable.

Hydrophobic interactions and water-mediated hydrogen bonds were responsible for ligand binding. The adamantane cage of the ligand formed hydrophobic interactions with the Val27 side chains and helped close this hydrophobic gate of the channel (Figs. 5A-C), whereas the positively-charged nitrogen atom formed water-mediated hydrogen bonds with the Ala30 and Gly34 backbone atoms (Figs. 7B, 7C and Fig. S5). The hydrophobic and electrostatic interaction energies between the ligand and the channel were summarized in Table S2.

As in the apo-form of the WT M2 channel, highly-ordered water molecules were found in the ligand-bound structures. Figures 7B and C show the two layers of water molecules between the ligand and the His37 residues, corresponding to layer 2 and layer 3 in the apo-form (Figs. 6 and 7A). These two layers were constituted by four water molecules stabilized by Ala30 and Gly34 backbone oxygen atoms, respectively (Figs. 7B, 7C, and Fig. S5). The average numbers of hydrogen bonds between the water molecules and the Ala30 and Gly34 backbone oxygen atoms were ~0.83 and 0.64, respectively, for each chain in the WT AMA-bound form, as shown in Table 2. There are additional water molecules (red dashed circles in Figs. 7B and C) between these two water layers and between layer 3 and the His37 residues, arranged in a pyramidal structure as in the apo-form. The positively-charged nitrogen atom of the ligand and the water molecules in layer 2 were also arranged in a pyramidal structure (Fig. S5). These ordered water molecules mediated the extensive hydrogen bond networks between the ligand and the M2


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protein (Figs. 7B, 7C and Fig. S5), facilitating ligand binding in the channel pore. These results are consistent with Wang et al.’s simulation results4.

Water density maps of the ligand-bound forms in Fig. 7B show high water densities at the corresponding positions of the water layers, indicating highly-ordered water structures. The water density map in Fig. 7C and the average hydrogen bond numbers in Table 2 indicate that the water structures were more dispersed in the RIM-bound form. This could be attributed to the additional methyl group in RIM that is connected with the positively-charged nitrogen atom. This hydrophobic methyl group may perturb the water structure.

4.3. Less Stable Ligand Binding in the Mutants and Possible Drug Resistance Mechanisms. AMA/RIM densities in Figs. 7B and 7C were distributed in a wider range in the radial direction (along the X-axis) in the V27A, L26F, and L38F mutants compared with the WT, implying less stable ligand binding in these three mutants. We calculated the hydrophobic and electrostatic interaction energies between the ligands and the M2 channel (Table S2) and we found that all four mutants had less stable interaction energies than the WT, with the weakest drug-binding mutant being the S31N mutant channel. Although such interaction energies between the drug and the channel are only crude estimates of the true binding affinity, they do provide us a handle on the favorable interactions that may form between the drug molecule and the channel and give us a sense on the stability of the ligand-bound forms.

The two layers of water molecules in the WT ligand-bound forms (Figs. 6B and 6C) were also found in the AMA-bound form for the V27A, L26F, and L38F mutants, but the corresponding


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water densities were decreased by ~30% compared with those of the WT AMA-bound form (Figs. 7B and C). The water densities for the RIM-bound form of these mutants were dispersed widely in the channel, indicating unstructured water molecules.

These results suggest that the water clusters and ligand binding in the drug-resistant mutants are less stable than in the WT, which could be attributed to the enlarged channel pore radii in these three mutants. Based on these results, we propose that, in the V27A, L26F, and L38F mutants, the hydrophobic gate formed by residue 27 is disturbed and the pore radii of the Nterminal half of the channel are increased, therefore, the channel becomes more flexible. As a result, the water structures facilitating ligand binding become less stable and the inhibitors become more flexible in the enlarged channel pore. Weakened binding and drug resistance are, therefore, resulted. Wang et al.4 found that the enlarged channel pores of the V27A and L26F mutants could be occluded by molecules with larger and more extended hydrophobic groups. Their results are consistent with the drug resistance mechanism we propose here.

Drug resistance mechanism for the S31N mutant is somewhat different. The AMA/RIM molecules were bound in a deeper position, closer to the His37 residues in the S31N mutant, as shown in the density maps in Figs. 7B and C. The distances between the mass center of inhibitors and that of the His37 side chains were ~1.2 nm for the WT M2 as well as for the V27A, L26F, and L38F mutants, whereas the values were reduced to ~0.7 nm for the S31N mutant. It seemed that the binding site in the channel pore of the WT M2 was partly occupied by the large side chains of Asn31 so that the inhibitors were pushed into deeper positions by the Asn31 residues. The decreased channel pore radius around Asn31 may also increase the energy


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barrier for the ligand to enter the channel. Therefore, we postulate that the S31N mutant is drugresistant because of the increased energy barrier for ligand to enter and bind in the pore-binding site due to the large Asn side chain and smaller channel pore. Our result is similar to Stouffer et al.’s results9, where the reduced channel pore radius is considered as the reason for drug resistance. Given this drug resistance mechanism, designing drugs that are small in size that may enter the channel pore more easily may yield promising results.

CONCLUSIONS

In the present study, we carried out molecular dynamics simulations to study the structures of the WT and four drug-resistant mutants (V27A, S31N, L26F, and L38F) of the influenza A M2 proton channel. We compared the pore radius profiles of the apo, AMA-bound, and RIM-bound forms and analyzed the water structure and ligand-binding conformations of these channels. From these structural differences, we proposed drug resistance mechanisms for these four mutations at different positions in the channel.

We found that, in the WT channel, highly-ordered water molecules existed in three layers in the drug-binding site inside the channel and were stabilized by hydrogen bond interactions with residues Ser31, Ala30, and Gly34. This structured water cluster contributed to stable ligand binding via extensive hydrogen bonding networks. However, in the V27A, L26F, and L38F mutants, the mutations increases the channel pore radii at the N-terminal end of the channel, causing the pore water molecules to become less stable. The enlarged pore radii and disordered pore water molecules led to weakened ligand binding and hence drug resistance. In the S31N


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mutant, the pore-binding site is partly occupied by the Asn31 side chains so that the pore-binding site is not easily accessible for the inhibitors to bind, hence drug resistance occurs.

Compared with previous experiments and MD simulations, our work revealed the following new findings: (1) The C-terminal amphipathic helices strongly affect the channel structure. The presence of these helices increased the structural stability of the TM domain and caused the channel pore to be narrower, leading to more tightly-packed channel structures and more stable structured water molecules in the channel pore than previous found25. (2) The highly-ordered water structures were destabilized in the V27A, L26F, and L38F mutants mainly due to the increase in pore radii at the N-terminal end of the channel. (3) The S31N mutant led to drug resistance by partially occluding the pore-binding site, supporting Stouffer et al.’s theory9. (4) The weakening of helical packing at the C-terminal side of the channel in the L38F mutant caused a widening of pore radii at the N-terminal end of the channel, suggesting an allosteric drug-resistance mechanism.

In the present study, the channel structure and ligand binding in neutral pH environment are examined. How channel structures and ligand binding change in low pH environment when the channel is open is not yet fully understood. In addition, we only compared the structural characteristics of ligand binding in different mutants and estimated the drug-channel binding affinity using simple calculations of interaction energies, without computing the actual binding energies for the ligands. More accurate binding energy calculations between drug molecules and the M2 wild-type and mutant channels are underway in our lab and will be reported in a separate study.


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Corresponding Author [email protected]; [email protected]

ACKNOWLEDGMENT The authors thank Jacob Ulmschneider for insightful discussions. This work is supported by grants from the National High-Tech R&D Program (863 Program Contract No. 2012AA020307), the National Basic Research Program of China (973 Program) (Contract No. 2012CB721000), the Key Project of Shanghai Science and Technology Commission (Contract No. 11JC1406400), and Ph.D. Programs Foundation of Ministry of Education of China (Contract No., 20120073110057), which were awarded to D.Q. Wei.

Supporting Information Available: Interaction energies between the ligands and the M2 channels, RMSD values of the M2 channels, standard deviations of the channel pore radii, Val27 side chain dihedral angles and the pairwise distances among the Val27 residues, hydrogen bond networks among the inhibitor, the water molecules, and the M2 channels, and the dynamics of water molecules in the M2 channel during the MD simulations. This material is available free of charge via the Internet at http://pubs.acs.org.


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Structural Comparison of the Wild-Type and Drug-Resistant Mutants

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