Molecular Dynamics Simulations on the ... - ACS Publications

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Molecular Dynamics Simulations on the Mechanism of Transporting Methylamine and Ammonia by Ammonium Transporter AmtB Jinan Wang,† Huaiyu Yang,† Zhili Zuo,‡ Xiuhua Yan,† Yong Wang,† Xiaomin Luo,† Hualiang Jiang,† Kaixian Chen,† and Weiliang Zhu*,†,§ Drug DiscoVery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China, School of Biomedical Sciences, Curtin UniVersity of Technology, Perth WA 6485, Australia, and School of Science, East China UniVersity of Science and Technology, Shanghai, 200237, China ReceiVed: May 18, 2010; ReVised Manuscript ReceiVed: September 23, 2010

AmtB is one of the ammonium transporter proteins facilitating the ammonium transport across the cellular membranes. Experimentally, the substrate used in in vitro studies is the radio labeled [14C]methylammonium, rather than ammonium itself. To explore the similarity and difference of the conduction mechanism of methylamine and ammonia molecules through AmtB, molecular dynamics simulations on 22 carefully designed systems were performed, which demonstrated that methylamine could be automatically transported in a very similar way to ammonia. The driving force for the conduction is mainly the hydrogen bond network comprising His168, His318, and Tyr32, working in coordination with NH-π interaction with residue Trp212. Then, Ser263 translocated the substrates from the exit gate into the cytoplasm by hydrogen bond interaction. The aromatic ring of Trp212 acted like a springboard to facilitate the translocation of the substrates from site Am2 to Am4 via NH-π interaction. Without the mediation of Trp212, further movement of substrate in the channel would be hampered by the strong hydrogen bonding from His168. In agreement with experimental results, the substrates could be transported by W212F mutant but not by W212A within the simulation time as long as 20 ns. In addition, we predicted that the mutants S263D and S263C remain the function of the transporter but S263A does not. The difference of transporting the two substrates is that methylamine involves more hydrophobic interactions than ammonia. In conclusion, methylamine molecule is a good mimic for investigating the translocation mechanism of ammonium transporter AmtB. Introduction Ammonium is the preferred source of nitrogen for many bacteria, fungi, and plants; its transport across the cellular membranes is certainly a fundamental process throughout all domains of life.1,2 Ammonium transporters are membrane proteins facilitating the ammonium transport in plants,3 bacteria,4 yeast,5 and animals.6 Recently, many reviews have been published on their functions and conducting mechanism.7-14 AmtB is one of the ammonium transporters present in the bacterial inner membrane between the cytoplasmic and periplasmic spaces, which is inhibited at high extracellular ammonium concentrations by the formation of the complex with the regulation GlnK protein, a member of PII protein family.2,15,16 Sufficient NH3 could diffuse into cell through hydrophobic membranes at medium to high concentration of external ammonium to satisfy cellular requirements. While at low ammonium concentration, the GlnK protein does not interact with AmtB, then the function of AmtB for ammonia/ammonium conduction is activated.2 As the extracellular ammonia should exist predominantly in the positively charged form under physiological conditions, there have been long-lasting controversies regarding the transport mechanism and the identity of the transported species. There are at least three suggested * To whom correspondence should be addressed. Phone: +86-2150805020. Fax: +86-21-50807088. E-mail: [email protected]. † Chinese Academy of Sciences. ‡ Curtin University of Technology. § East China University of Science and Technology.

conducting mechanisms, namely, the electronetural NH3 transport,17-22 NH3/H+ symport,22 and NH4+ transport.23 The crystal structures of AmtB from Escherichia coli,17,18 the homologous protein Amt-1 from Archaeoglobus fulgidus,24 and RH50 from Nitrosomonas europaea25 have shown that the channel is narrow and highly hydrophobic thus is favorable to conduct electronetural species (NH3) rather than ammonium ion. Computational simulations26-36 also supported the postulation that the ammonium ion is recruited in the periplasmic vestibule, but it is the ammonia that is transported into cytoplasm by AmtB. We noticed that the substrate used in in vitro transport assays was the radio-labeled ammonium analogue [14C]methylammonium, rather ammonium/ammonia itself.20-23,37 Subsequently, a fundamental question is raised that whether methylamine is an efficacious and precise mimic of ammonia as a probe molecule to explore the function of AmtB. Moreover, many site-directed mutagenesis experiments have been carried out, for instance, the replacement of the highly conserved residue Trp212, which locates below the residue Phe215 and opposite the residue His168, with alanine-disabled the function of AmtB, while the mutant of Trp212Phe retained most of the activity.22 Although this mutagenesis experiment firmly established the central role of residue Trp212 in the function of AmtB, an immediate question is whether this conclusion is true in the case of the endogenous substrate ammonium/ammonia and what is the exact role of the residue at atomic level. To the best of our knowledge, very limited theoretical study has been carried out for investigating whether methylamine is a good mimic of

10.1021/jp104508k  2010 American Chemical Society Published on Web 10/26/2010

Transporting Methylamine and Ammonia by AmtB

J. Phys. Chem. B, Vol. 114, No. 46, 2010 15173 also endorsed the critical role of the hydrogen bond formed by the residue 263, and the NH-π interaction formed by the residue 212 in transporting the substrate through the channel. Experimental Methods

Figure 1. (a) Structure of AmtB (1U7G.pdb). Residues 214-272 are not shown for clarity. Some important residues are shown in stick representation. CH3NH2 and NH3 are variably put at the four sites (Am1-Am4, red spheres) in simulations. (b) Side view of the simulation system.27 The AmtB is shown as a green ribbon, phosphate atoms are drawn as cyan spheres, the other atoms of the lipid are represented as magenta lines, and water molecules are displayed as red and white sticks. The front half of the bilayer is not shown for clarity.

ammonia in both cases of wild type and mutants of AmtB. Hu et al35 compared the intermolecular interaction energies for the different substrate on the site Am1 (Figure 1a) by quantum mechanics/molecule mechanics calculations and found that the interaction energies for the ammonium-AmtB complex and the methylammonium-AmtB complex were at substantial magnitude, -43.19 and -35.61 kcal/mol, respectively. However, the overall similarity and difference between the methylamine and ammonia conduction mechanisms by AmtB are still unknown at the atomic level. To answer these questions, we performed MD simulations on the 22 carefully designed simulation systems (Table 1), which showed that both methylamine molecule and ammonia molecule could pass through the wild type, Trp212Phe, and some other mutants of AmtB by very similar mechanism, while failed to be transported by Trp212Ala or Ser263Ala mutant. These studies

The computational approach for the MD simulations was similar to our previous work.27 In brief, the X-ray structure of AmtB (PDB entry 1U7G, Figure 1a) determined by Khademi et al17 was used. The three mutated residues (F68S, S126P, and K255L) and the engineered Met residues (S atoms were replaced by Se) were modified back to their native states. The protein was fitted into the dipalmitoylphosphatidylcholine (DPPC) bilayer with 288 lipids (14 400 atoms) to generate a suitable membrane system. Then the protein/DPPC systems were solvated in a bath of 14 375 SPC water molecules. The substrate molecular CH3NH2 or NH3 were manually added to proper sites (Am2, Am3, and Am4, Figure 1a). Na+/Cl- ions were then added to neutralize the modeling system to perform the modeling under simulated physiological conditions (Figure 1b27). The first protonation state of His168-His318 discussed in our previous study,27 which the hydrogen atoms were added to Nε of His168 and Nδ of His318, was used in all simulations. In total, 22 simulation systems were designed as shown in Table 1. The simulations A1, A2, and A3 were performed on the wild type AmtB but with different substrate to explore the similarity and difference of the conducting process between methylamine and ammonia; the simulations B1, B2, C1, and C2 were designed to study the role of the residue Trp212 in conducting the substrates; the simulations from D1 to D6 were designed for studying the function of Ser263 in substrate conduction, and the simulations from E1 to E4 and F1 to F6 were designed for investigating the role of the residues Phe31 and Tyr32 in substrate translocation. All MD simulations were performed using the GROMACS package version 3.2.138-40 with the GROMOS87 force field for protein, ions, CH3NH2 and NH3, and the parameters for lipid were the same as those used in previous MD studies of lipid bilayers.41-44 The charges of CH3NH2 and NH3 were obtained by a restrained ESP-fit method using the ChelpG approach.45 During MD simulation, all the bonds with hydrogen atoms were constrained with the linear constraint solver (LNCS) algorithm46 and the integration step of 2 fs was used. Electrostatic interactions were calculated using the particle-mesh Ewald method.47 The cutoff for Lennard-Jones interactions was set as 9 Å. The temperature was kept constant at 323 K by coupling the water, ions, lipids, CH3NH2 or NH3, and protein separately to a thermal bath using the Berendsen thermostat48 method with a coupling time of 0.1 ps. A constant pressure of 1.0 bar was applied independently in X, Y, and Z directions of the system with a coupling constant of 1.0 ps. First, the systems were subjected to energy minimizations using the steepest-descents algorithm to remove unfavorable contacts. After minimization of the whole system, the first 100 ps MD simulation was carried out to heat the solvent molecules, ions, and lipids to 323 K with the protein, lipid, and the substrate fixed. Following that, the second 100 ps MD simulations were performed to heat all the atoms in the system to 323 K with the protein main chain, the phosphorus atoms of the lipid, and substrates fixed. Third, with the whole system relaxed except for protein CR atoms and substrates the equilibration was completed after 1 ns MD simulation. Then the conventional molecular dynamics were performed and stopped when the substrate left the channel. The program LIGPLOT version 4.4.249 was used to calculate the hydrogen bond and hydrophobic interaction between the sub-

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TABLE 1: The Information for 24 MD Simulations on AmtB Transportera

a

simulation

protein

Am2

Am3

Am4

time (ns)

conductivity

A1 A2 A3 A′ B1 B2 C1 C2 D1 D2 D3 D4 D5 D6 E1 E2 E3 E4 F1 F2 F3 F4 F5 F6

wild type wild type wild type wild type W212F W212F W212A W212A S263A S263A S263D S263D S263C S263C F31A F31A F31V F31V Y32A Y32A Y32F Y32F Y32V Y32V

CH3NH2 CH3NH2 NH3 NH3 CH3NH2 NH3 CH3NH2 NH3 CH3NH2 NH3 NH3 CH3NH2 NH3 CH3NH2 CH3NH2 NH3 CH3NH2 NH3 CH3NH2 NH3 CH3NH2 NH3 CH3NH2 NH3

CH3NH2

CH3NH2

NH3

NH3

NH3

NH3

4.5 2.5 9 8 4 3 20 20 20 20 14 15 14 16 8 1 2 14 10 14 13 11 11 4

conductive conductive conductive conductive conductive conductive nonconductive nonconductive nonconductive nonconductive conductive conductive conductive conductive conductive conductive conductive conductive conductive conductive conductive conductive conductive conductive

A′ and D2 were from ref 27.

strate and protein. An interaction was counted as hydrophobic interaction if the distance of two atoms between the substrate and the transporter is less than 3.9 Å, and counted as a hydrogen bond if (i) it is between a listed donor and acceptor and (ii) the angles and distances formed by the atoms surrounding the hydrogen bond lie within the default criteria (90°).50 Result and Discussion CH3NH2 Conduction through the Wild Type AmtB. Two simulations, namely A1 and A2, were carried out for studying the transportation of methylamine (Table 1). The difference between A1 and A2 is that there are three CH3NH2 molecules located at Am2, Am3, and Am4 sites (referred to as M1, M2, and M3 hereafter), respectively, in A1, while there is only one CH3NH2 molecule at Am2 site (referred to as M1′ hereafter) in A2. A1 showed that M3 quickly exited the channel (∼102 ps, Figure 2), followed by M1 (∼1822 ps), and the last one, M2, left the channel at ∼2678 ps, while A2 demonstrated that the CH3NH2 exited the channel at ∼920 ps (Figure S1 in Supporting Information). Although the conduction rate of the substrates through the channel could not be estimated based on the trajectories, the two simulations suggested that the number of the methylamine molecules in the chamber does not essentially affect the conductivity of the transporter. For closely inspecting the whole transport mechanism of M1 (trajectory A1), both the hydrogen bond and hydrophobic interactions between the substrate and the protein were plotted along the simulation time in Figure 3. Five residues in the chamber were found forming hydrogen bonds frequently in the transport process, viz., His168, His318, Tyr32, Ser263, and Ile110 (Figure 3a), and 11 residues were involved in the hydrophobic interaction (Figure 3b, and Figure S2 in Supporting Information). Therefore, these interactions paved the way for transporting the substrate. Trajectories A3 and A′ showed that the same residues as observed in A1, viz., His168, His318, Tyr32, and Ser263, were involved in the hydrogen bond network, providing the major force to transport NH3. The critical role of His168 and His318 in substrate conductance was investigated by Javelle et al,37

Figure 2. Process of the three methylamine molecules leaving the channel in trajectory A1. Side views of the starting and five snapshot structures (102, 1820, 1822, 2676, and 2678 ps). CH3NH2 molecules are drawn as white and blue stick-balls, and water molecules are displayed as red and white sticks. F31, F107, H168, F215, S263, H318, and V314 are displayed as colored sticks. Hydroxyl of Ser-263 interacting with CH3NH2 molecule via a water bridge (1820 and 2676 ps).

through analyzing 14 engineered polar and nonpolar variants of these histidines in AmtB, which showed that both histidines were absolutely required for optimum substrate conductance.

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Figure 4. Process of M1 moving from Am2 down to Am4 with the help of Trp212 by NH-π interaction. Side views of four snapshots (445, 451, 456, and 457 ps) are shown. The hydrogen bond and NH-π interactions are shown as red dashed lines. The relevant distance are also shown as red character.

Figure 3. The residues involved in hydrogen bonds and hydrophobic interactions with CH3NH2 (M1) versus simulation time in the trajectory A1. (a) Time-dependent hydrogen bonds formed between M1 and the residue of the channel. (b) Time-dependent hydrophobic interaction between M1 and the residue of the channel; different colors are used for the sake of clarity.

We have investigated the role of the Ser263 in transporting NH3 by simulating the Ser263Ala mutant AmtB (D2 in Table 1) and found that the mutant could not transport ammonia molecules within the simulation time as long as 20 ns.27 Recently, the crystal structure of the GlnK-AmtB complex showed that Ser263 could also form hydrogen bond with the charged guanidinium moiety of GlnK-R47, demonstrating that this residue is directly involved in the binding of GlnK.51,52 The Moving of M1 from Site Am2 to Am4 was Facilitated by Hydrogen Bond, Hydrophobic, and NH-π Interactions. As the simulation started, M1 at Am2 with a hydrogen bond to His168 at the beginning (start, Figure 2) fluctuated down toward Trp212 to form the obvious NH-π interaction,53-57 one special kind of cation-π interaction with an interaction distance around 3.2 Å (445 ps, Figure 4). As the cation-π interaction with a six-membered ring is stronger than a five-membered ring,58 the substrate spontaneously slipped to form stronger NH-π interaction with the six-membered ring moiety (451 ps, Figure 4). As the simulation underwent, M1 gradually moved down to form a hydrogen bond with His318, and then one more hydrogen bond was formed with Tyr32 when M1 eventually arrived the site around Am4 (456 and 457 ps, Figure 4). During this process, the important residues involving hydrophobic interaction with CH3NH2 were M23, I28, I110, I114, I208, W212, and F215 (Figure 3b), which could obviously facilitate the translocation of CH3NH2 down to Am4. During the simulation, we found

that M2 could move up toward the site Am2, and could also move from the site Am2 down to Am4 by the same manner as M1 as we discussed before (Figure S3, Supporting Information). The Conducting of M1 from Site Am4 to Exit Gate was Facilitated by Hydrogen Bond, Hydrophobic Interaction, and Water Molecule. As the simulation further underwent, the hydrogen bond network among M1, His318, and Tyr32 was observed (457 ps, Figure 4), which drove the substrate to move further down. Very interestingly, a water molecule was discovered around the gate site, which might form a hydrogen bond with M1 at 1820 ps (Figure 2). At ∼1822 ps, the hydrogen bond between M1 and Ser263 was found, and this hydrogen bond finally pulled M1 out of the exit gate formed by Phe31 and Val314. The exit process of M2 in the channel was also similar to that of M1. At ∼2676 ps, the hydroxyl of Ser263 interacted with M2 via a water bridge followed by the hydrogen bond between M2 and Ser263 observed at ∼2678 ps (Figure 2), pulling M2 out of the exit gate into the cytoplasm. Therefore, Ser263 is the last key residue in the whole transport process, and we postulate that Ser263Ala mutation would disable the function of the transporter as no hydrogen bond would be formed between Ala263 and the substrate. Indeed, the mutant Ser263Ala could not translocate the substrates (trajectories D1 and D2, Table 1).27 During the simulations, water could move into the channel from the bottom but stop at around the site Am4 and also could move out of the chamber spontaneously (Figure S4, in Supporting Information), but no water wire through the channel was observed; the residue Tyr32 prevents the water molecule from moving further into the center of the channel by forming a hydrogen bond. This is consistent with the crystal structure obtained by Zheng et al.18 Information from the Simulation A2 and A3. A2 was carried out with only one methylamine molecule (M1′ hereinafter) at the site Am2. The MD simulation showed that the transport mechanism of M1′ from simulation A2 is very similar to that of M1 from simulation A1. M1′ could move down to the bottom of the channel via the hydrogen bond network

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Figure 6. Distance between Phe31 and Val314 versus time in the processes of CH3NH2 molecules leaving in the trajectory A1 (line is colored by black) and NH3 leaving in trajectory A′ (line is colored by red). The black shadow highlight the time when CH3NH2 passed through the exit gate.

Figure 5. (a) The φ-torsion of Gly163 versus time in trajectory A1. Curve is obtained by 5 ps averaged. (b) The superposition of the upconformation (snapshot at 1425 ps, carbon atoms colored by yellow) and down-conformation (snapshot at 1758 ps, carbon atoms colored by green) of Gly163, together with Ala162, Phe107, Phe215, and His168.

working in coordination with NH-π and hydrophobic interactions. The residues His168, His318, Tyr32, Ser263, and Trp212 are the key residues for driving M1′ passing through the chamber to the exit gate. With the help of water molecules, Ser263 could drag M1′ out of the exit gate into cytoplasm via hydrogen bond (Figure S1 in Supporting Information). A3 was carried out with only one ammonia molecule in the channel. Further analysis found that the exit process of NH3 is also very similar to that of M1′, and the NH3 was conveyed to the bottom region of the channel via the hydrogen bond network involving His168, His318, Tyr32, and NH-π interaction formed with Trp212, then finally exited the channel mediated by Ser263 and water molecules around the gate (Figure S5 in Supporting Information). Entering Mechanism. Ala162 is buried and packed against the edge of the pore-blocking aromatic ring of Phe107 and Phe215 at the entrance of AmtB channel. Zheng et al18 speculated that the carbonyl oxygen could serve as a transient hydrogen bond acceptor for conducting substrate molecules in a transient open state of the periplasmic pore entry. Indeed, our MD simulations revealed that Ala162 could form a hydrogen bond with methylamine by its carbonyl oxygen (Figure 5b), meanwhile, the φ-torsion of Gly163 could shift from ∼140 to

∼80° in trajectory A1 (Figure 5a), leading to the switch of the carbonyl group between down-conformation and up-conformation (Figure 5b). It is worth noticing that the conformation shift was frequently observed in all the simulation trajectories during almost all the simulation time, indicating that the shift is an intrinsic vibration mode. When Ala162 took its up-conformation, a strong hydrogen bond might be formed with a substrate around site Am1 (Figure 1a). As Ala162 changed its up-conformation to down-conformation, the substrates could be dragged into the inner chamber through the entrance lid composed of Phe107 and Phe215. Therefore, our simulations supported the speculation that the function of Ala162 is to actively guide substrate into the inner area of the channel via direct hydrogen bonding interaction. Nagaard et al33 also found this conformation change in MD simulation, although the conformation change in their simulation shifted only when an ammonium ion occupied in the site Am2 with only the protonation state that the Nδ atom of His168 and the N
atom of His318 were protonated. Distance between the Gating Residues. Phe31 and Val314 are two residues forming the exit gate, therefore, the distance between the residues, RF-V, characterizes the gate state of the protein. There are two different crystal structures (PDB: 1XQF and 1XQE) of AmtB available, belonging to different space groups P63 and R3, with different RF-V distances of 8.0 and 10.5 Å, respectively.18 Accordingly, an exit gating mechanism was proposed that the transporter requires remarkable structural changes during NH3 translocation.18,24 Figure 6 is the distance evolution along simulation time for AmtB conducting methylamine or ammine, taken from trajectory A1 and A′. Impressively, RF-V could reach as large as 10.2 Å during the simulation A1, almost the same as the distance determined in the structure 1XQE. However, limited distance fluctuation was found during the methylamine passing through the gate (at 0-100 ps, 1800-1900 ps, and 2600-2700 ps, Figure 6). The same limited distance fluctuation was also found in the trajectory A2. So it is reasonable to conclude that the AmtB has the structural flexibility to undergo such large fluctuation as determined by X-ray crystallography but that remarkable conformational change is not essential for the passage of both CH3NH2 and NH3 through the exit gate. This observation is in good agreement with our previous simulation for the passage of ammonia through the channel.27

Transporting Methylamine and Ammonia by AmtB

Figure 7. Three snapshots from the mutant W212A are shown. (a) The 20 ns from trajectory C2. (b) The 20 ns from trajectory C1. (c) The 6.5 ns from trajectory C1. Ammonia molecule and methylamine molecule are shown as colored sticks and balls, H168, H318, V314, F107, F215, A212, I208, and F31 are shown as colored sticks. The hydrogen bonds are shown as red dashed lines.

The Role of Trp212 in Conducting Substrate via NH-π Interaction. Javelle et al22 experimentally demonstrated that Trp212Ala mutant is inactive while the Trp212Phe mutant retains the most activity of the wild type. To illustrate the importance of Trp212 in transporting both CH3NH2 and NH3, simulations B1, B2, C1, and C2 were performed on mutants of AmtB in which Trp212 was mutated to phenylalanine and alanine (Table 1), respectively. Noteworthy, both CH3NH2 and NH3 could be fluently transported by both the wild type (A1, A2, and A3) and the Trp212Phe mutant (B1 and B2) within 7.66 ns, which is in agreement with Javelle’s result,22 while neither CH3NH2 nor NH3 was found passing through the chamber of Trp212Ala mutant during the simulation time as long as 20 ns. In details, CH3NH2 was observed to leave the channel at ∼924 ps (trajectory A2, and Figure S1 in Supporting Information) and ∼3215 ps (trajectory B1, and Figure S6 in Supporting Information), and NH3 left the channel at ∼7660 ps (trajectory A3, and Figure S5 in Supporting Information) and ∼2460 ps (trajectory B2, and Figure S7 in Supporting Information). However, the trajectory C1 showed that CH3NH2 was always fluttering around the site Am2 (Figure 1), and trajectory C2 revealed that NH3 was almost stuck at Am2 all the time during the simulation as long as 20 ns. To interpret the disability of Trp212Ala mutant in conducting CH3NH2 and NH3, trajectories C1 and C2 were carefully checked. It was found that both substrates could form hydrogen bond with His168, firmly astricting the molecules around the site Am2, indicating that the hydrogen bond is strong enough to prevent them from moving down the channel (Figure 7a,b), resulting in the disability of Trp212Ala mutant. In the wild type, with the help of Trp212 via NH-π interaction the substrates were easy to be translocated as discussed above (Figure 3). It was also noticed that CH3NH2 was vibrating a little fiercer than that of the ammonia molecule in Trp212Ala mutant, occasionally thrusting in between His168 and His318 to form two hydrogen bonds with the His168 and His318, respectively (Figure 7c). This interaction was also found in trajectory A1, where the substrate could be dragged down to the bottom of the channel by Trp212 and His 318. To interpret why Trp212Phe retained the most activity of the wild type,22 the distance between the nitrogen atom of the substrates and the aromatic ring of the mutated Phe212 was monitored (Figure 8). As expected, NH-π interactions, which played an essential role in translocating the substrates from Am2 to Am4 as discussed above, were observed with an average interaction distance of ∼5.3 and ∼4.0 Å in trajectory B1 and B2, respectively (Figure 8). Therefore, Trp212Phe mutant should have similar function to the wild type. As shown in Figure 8,

J. Phys. Chem. B, Vol. 114, No. 46, 2010 15177 we found that the interaction distance when transporting CH3NH2 is larger than that when transporting NH3, reflecting that the NH-π interaction between CH3NH2 is weaker than NH3. Meanwhile, we also noticed that the interaction distance in B1 was larger than that in trajectories A2 by about 0.5 Å, showing that the NH-π interaction between CH3NH2 and Phe212 in Trp212Phe mutant should be weaker than that in the wild type. This might be the reason why Trp212Phe mutant is still functional. However, the difference of the NH-π interactions is very little among trajectories A′, A3, and B2, thus we postulated that the activity of Trp212Phe mutant in conducting ammonia might be almost the same as wild-type. As the methyl group of CH3NH2 could also interact with the aromatic ring of Trp212, the relative fraction of the time spending making interactions of -CH3 or -NH2 with Trp212 was calculated (Figure S8, Supporting Information), which demonstrated that -NH2 forms more frequently interaction with Trp212 than -CH3, suggesting that HN-π interaction is more important than HC-π interaction in the conduction. The Role of Ser263 at the Gating Stage. As discussed above, Ser263 translocated the substrates from the exit gate into the cytoplasm by hydrogen bond formed with the substrates. To validate this observation, the mutants S263A, S263D, and S263C were designed for MD simulations. The first mutation (S263A) would disable the function of the mutant protein as a transporter while the other two should remain the function as both Asp and Cys have the capability to form hydrogen bond with the substrate. Indeed, the simulation results (D1 to D6 in Table 1, and Figure S9 in Supporting Information) are in good agreement with our postulation. Furthermore, we performed a sequence alignment of different ammonia channel protein among 7 different species (Figure S10, Supporting Information) and found that polar residue at this position is conserved in the bacterial, fungal and other species, demonstrating again that the polar residue 263 is important to the channel’s function. The Roles of Phe31 and Tyr32. As pointed out above, the residues Phe31 and Tyr32 were frequently interacting with the substrates during the conduction process. Accordingly, 10 MD simulations were carried out to study how important the two residues are to the transporter function. All the 10 simulations showed that both NH3 and CH3NH2 could be conducted by the mutants F31A, F31V, Y32A, Y32F, and Y32V within the simulation time of 20 ns (Figure S11 in Supporting Information), demonstrating that Phe31 and Tyr32 are important residues but not essential for substrate conduction. The Mechanics of Conducting Methylamine is Very Similar to Ammonia in AmtB. On the basis of the information from trajectories A1, A′, A2, and A3, we found that the key residues involved in hydrogen-bonding interaction for conducting both CH3NH2 and NH3 are the same, namely, His168, His318, Tyr32, and Ser263. We also observed that Trp212 is equally essential for conducting the two substrates and discovered that the translocation mechanism from the sites Am2 to Am4 and from Am4 to the cytoplasm as well as the gating mechanism for conducting CH3NH2 are very similar to that for NH3. Therefore, we concluded that the methylamine is a good mimic for investigating the conduction mechanism of ammonium transporter AmtB. However, there were also some differences between the two species in transporting. The methyl group of methylamine could form hydrophobic interaction with the residues in the channel, especially the residues Trp212, Ile28, Phe31, Tyr32, ILe266, Leu208, ILe110, and Leu114, to facilitate its transport (Figure 3b, and Figure S2 in Supporting Information). During our

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Figure 8. Distance between the nitrogen atom of the substrate molecules and the ring of the residue 212 versus time. (a1) The substrate is methylamine (M2) from trajectory A1. (a′) The substrate is ammonia (nha1) from the trajectory A1 (ref 27). (a2) The substrate is methylamine from trajectory A2. (a3) The substrate is ammonia from trajectory A3. (b1) The substrate is methylamine from trajectory B1. (b2) The substrate is ammonia from trajectory B2.

simulations, we found that methylamine could induce larger fluctuation of the residues of the channel, especially the His168 and His318, and larger fluctuation of the exit gate. But in general, the hydrogen bond network formed by the substrate and the residues in the channel is the major driving force to transport the substrates, thus, there is no obvious difference between transporting the ammonia and methylamine.

role in other proteins. This result provides molecular and atomic level insights into mechanisms underpinning the experimental observation that the Trp212Phe mutant retained the most activity of the wild type AmtB and that the Trp212Ala mutant disabled the function of the AmtB. The simulations predicted that Ser263Asp and Ser263Cys mutants remain the function of the protein as a transporter but not Ser263Ala.

Conclusion On the basis of the analyses of the 24 simulation trajectories (Table 1), the similar transporting mechanism of NH3 and CH3NH2 is acquired that the substrates passed through the channel facilitating by the NH-π interaction with residue 212 and hydrogen bond network formed with His168, His318, Tyr32, and Ser263. Therefore, the methylamine is a good mimic for investigating the conduction mechanism of AmtB. Besides that, our simulations also reinforced the essential role of Trp212 in the conducting of CH3NH2 or NH3 via NH-π interaction, and this kind of NH-π interaction may also play an important

Acknowledgment. This work was supported by grants from the National Natural Science Foundation (20721003), CAS foundation (KSCX2-YW-R-208 & KSCX2-YW-R-168), Shanghai S&T Foundation (09540703900), and National Science & Technology Major Project (2009ZX09301-001). Supporting Information Available: The initial coordinates of the simulated systems are available upon request. Figures S1-S11 are located in SI. This material is available free of charge via the Internet at http://pubs.acs.org.

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