Substrate Recognition in the Escherichia coli Ammonia Channel AmtB

Aug 23, 2010 - Parc Científic de Barcelona. ... Although the Escherichia coli ammonia transporter B (AmtB) protein has been the focus of several rece...
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J. Phys. Chem. B 2010, 114, 11859–11865

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Substrate Recognition in the Escherichia coli Ammonia Channel AmtB: A QM/MM Investigation Thomas P. Nygaard,†,‡ Mercedes Alfonso-Prieto,§,| Gu¨nther H. Peters,† Morten Ø. Jensen,⊥,# and Carme Rovira*,§,|,∇ MEMPHYSsCenter for Biomembrane Physics, Department of Chemistry, Technical UniVersity of Denmark, DK-2800 Kgs. Lyngby, Denmark, Computer Simulation and Modeling Laboratory (CoSMoLab), Parc Cientı´fic de Barcelona, 08028 Barcelona, Spain, Institut de Quı´mica Teo`rica i Computacional (IQTCUB), Department of Life Sciences and Chemistry, Roskilde UniVersity, DK-4000 Roskilde, Denmark, and Institucio´ Catalana de Recerca i Estudis AVanc¸ats (ICREA), 08019 Barcelona, Spain ReceiVed: March 15, 2010; ReVised Manuscript ReceiVed: July 26, 2010

Although the Escherichia coli ammonia transporter B (AmtB) protein has been the focus of several recent studies, there are still many questions and controversies regarding substrate binding and recognition. Specifically, how and where AmtB differentiates between substrates is not yet fully understood. The present computational study addresses the importance of intermolecular interactions with respect to substrate recruitment and recognition by means of ab initio QM/MM simulations. On the basis of calculations with substrates NH3, NH4+, Na+, and K+ positioned at the periplasmic binding site (Am1) and NH3 and NH4+ at intraluminal binding sites (Am1a/b), we conclude that D160 is the single most important residue for substrate recruitment, whereas cation-π interactions to W148 and F107 are found to be less important. Regarding substrate recruitment and recognition, we find that only NH4+ and K+ reach the Am1 site. However, NH4+ has the largest affinity for this site due to its better dehydration compensation, while charge stabilization effects favor the binding of NH4+ over NH3 (i.e., if NH3 would enter the Am1 site, it is likely to be protonated). Therefore, we conclude that the Am1 site selects NH4+ over Na+, K+ and NH3. Our calculations also suggest that translocation of NH4+ from Am1 into the channel lumen is driven by rotation of the A162-G163 peptide bond, which coordinates NH4+ but not NH3 at both Am1 and Am1a/b sites. 1. Introduction Ammonia/ammonium (Amm) is an essential growth factor for some bacteria, yeasts, plants, and fungi, while for other organisms, it is a highly toxic metabolic waste product, for example, for mammalian cells.1,2 Proteins belonging to the ammonia/ammonium transporter (Amt) family have been identified in a wide range of organisms: (methyl)ammonia/ammonium permeases (Mep) in yeasts, Rhesus (Rh) blood group proteins in animals, and Amt proteins in bacteria and plants.3,4 Therefore, a detailed understanding of how these proteins work may have future pharmaceutical, agricultural, and biotechnological relevance.5-8 The first Amt protein for which high-resolution crystallographic structures were solved was Escherichia coli AmtB,9,10 followed by Archaeoglobus fulgidus Amt-1,11 and E. coli AmtB complexed with its physiological inhibitor GlnK.12,13 Three identical AmtB monomers form a trimer.9,10,14,15 Each monomer has 11 transmembrane-spanning R-helices arranged in a righthanded bundle. Depressions in both the periplasmic and the cytoplasmic surfaces (vestibules) lead into a hydrophobic pore lined by histidines H168 and H318 (Figure 1), which, by sharing a H-atom between their imidazole rings, are mutually fixed.9,10 * To whom correspondence should be addressed. E-mail: [email protected]. † Technical University of Denmark. ‡ Present address: Radiometer Medical, DK-2700 Roenshoej, Denmark. § Parc Cientı´fic de Barcelona. | Institut de Quı´mica Teo`rica i Computacional (IQTCUB). ⊥ Roskilde University. # Present address: D. E. Shaw Research, New York, New York 10036. ∇ Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA).

Figure 1. Simulated system. (A) Snapshot of the classical MD simulation43 showing a side view of one AmtB monomer. The arrow indicates the direction of physiological Amm transport. (B) Enlargement of the constriction region showing relevant substrate binding site residues and channel lining residues treated at the QM level in the simulations. NH4+ is shown at the Am1, Am1a (purple), and Am1b positions, while the Am2 position is only indicated. This structure is the starting one for the Am1-QM/MM simulations.

In the crystal structures of Khademi et al.,9 a periplasmic NH4+/ MeNH4+ molecule and three intraluminal gaseous NH3 molecules were resolved, and their positions were named Am1, Am2, Am3, and Am4, respectively.9 A subsequent molecular dynamics (MD) study of AmtB identified two intermediate positions, Am1a and Am1b between Am1 and Am2 (Figure 1B), which may accommodate NH4+.16 Even though NH3 recognition at the Am1 site has been proposed,17 the most

10.1021/jp102338h  2010 American Chemical Society Published on Web 08/23/2010

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TABLE 1: Proposed Functionality of Key Residues with Respect to Substrate Recognition at the Am1, Am1a, Am1b and Am2 Sites, Suggested from Crystallographic and Computational Studies residue(s) Q104 F107 and W148

D160

A162 S219

proposed function(s) Am1 NH4+ recognition due to steric exclusion effects16 stabilization and selection of charged substrates by cation-π interactions9,18,25,26,28 F107, a gate that can open and close spontaneously16,28 or in correlation with NH4+ contact16,25 electrostatic stabilization of positively charged substrate16,21,22 and/ or stabilization of protein structure9,10,16,20 proton acceptor in deprotonation pathway via a two-water wire23,25 stabilization of charged substrates,10,16,22,25 dynamically involved in translocating substrate across stack16 stabilization of charged substrates9,10,16,18,21,25 catalyzes the deprotonation of NH4+21

Am1a, Am1b, and Am2 proton acceptor in deprotonation pathway via the A162-G163 peptide bond16 A162, H168, W212, tetrahedral coordination of NH4+16,25 N216, and T273 W212 and F215 stabilization of charged substrates by cation-π interactions9,25 promotion of transfer and deprotonation (only F215)18 H2O and NH3 stabilization of NH4+16,24 D160

common view is that this site recognizes NH4+,9,10,18,19 whereas NH3 occupies the channel lumen. This is in accordance with experiments indicating a gradient driven NH3 uniport, that is, diffusion or passive transport,9,16,19 although an electrogenic transport mechanism (cotransport of H+ and NH3) still needs to be disproved.18 That NH4+ is the Amm species preferred at the Am1 site and that NH3 occupies the Am2-Am3 sites are also supported by computational studies.20-22 The most important residues located in the vicinity of the periplasmic substrate binding site Am1 are Q104, F107, W148, D160, A162, and S219 (Figure 1B). In the vicinity of the intraluminal positions (Am1a, Am1b, and Am2), the most important residues are D160, A162, H168, W212, N216, F215, and T273 (Figure 1B). The carbonyl oxygen of A162 is hereafter referred to as A162:CdO. The function(s) of these residues with respect to substrate recognition, suggested from crystallographic and computational studies, is summarized in Table 1. AmtB has been the focus of several recent computational studies,9,10,16,20-28 but still, many questions and controversies regarding the detailed mechanism of AmtB prevail. In particular, the functionality of key residues along the channel (Table 1) with respect to substrate binding, recognition, and translocation is controversial. Cation-π interactions have been proposed to be essential for binding NH4+ to the Am1 and/or Am2 site.9,26,28 However, this has been questioned in other studies.16,21,22 Because the function of cells crucially relies on differences in ion concentrations between the inside and the outside, Amt proteins need to differentiate between NH4+ and, for example, the naturally abundant ions Na+ and K+, which are of smaller and similar size, respectively. Obviously, any conductance of

Nygaard et al. Na+ and K+ across AmtB is prevented by the hydrophobic pore interior, where AmtB utilizes the titrability of NH4+ and conducts Amm as NH3. However, how and where AmtB differentiates between the substrates is not yet fully understood. On the basis of electrostatic calculations, it has been suggested that the hydrophobic constriction region functions as a filter selective against Na+ ions, while selectivity against K+ could not be explained.10 On the other hand, calculated relative free energy differences ∆G for binding of NH4+ vs monovalent alkali cations at the Am1 site have shown that all of the cations have some affinity for this site, but NH4+ is the favored species.22 Inhibition of AmtB uptake activity of MeAmm by NH4+,17,19 which is also observed in experiments on other Amt proteins,30-34 indicates that Amm is a better AmtB substrate than MeAmm. Moreover, only small or no inhibiton by alkali metal cations is observed in experiments on AmtB and other Amt proteins,18,30,31,33,34 proving the Am1 site to be highly NH4+ specific. The focus of the present study is to quantify the relative importance of intermolecular interactions (hydrogen bond and cation-π interactions) with respect to substrate recruitment and recognition in AmtB. For that purpose, we will perform QM/ MM Car-Parrinello simulations of substrate bound in the constriction region, in particular at the Am1, Am1a, and Am1b sites. The calculations will use snapshots of previous classical MD simulations,16 and the binding of four different substrates, NH4+, NH3, K+, and Na+, will be compared. It will be shown that NH4+ is the substrate with the highest affinity for the Am1 site, and only NH4+ and K+ can reach this position. 2. Materials and Methods 2.1. Initial Structures. The initial structures of AmtB channel were taken as snapshots from our previous classical MD simulations of the E. coli AmtB trimer (PDB 1U7G, at 1.35 Å resolution)9 in a fully hydrated lipid bilayer.16 Five MD simulations (each one 2-4 ns) were performed varying the protonation state of the intraluminal histidines and Amm. We identified, apart from the Am1 site found crystallographically (Figure 1), two intraluminal binding sites, Am1a and Am1b, both providing tetrahedral coordination of NH4+, indicating that NH4+ translocates from the substrate binding site (Am1) to intraluminal positions before its deprotonation.We selected one snapshot of the classical MD simulation in which the intraluminal histidines (His168 and His318) are protonated at Nδ and Nε, respectively. This was found to be the most likely protonation state in native AmtB in terms of the similarity with the crystal structure and the computed pKa.16 Single monomers with NH4+ positioned either at the periplasmic binding site (Am1) or at an intraluminal position (Am1a or Am1b) provided initial structures for the present QM/MM simulations (Figure 1 for Am1 and Figure S1B in the Supporting Information for Am1a/ b). The time evolution of the rmsd during the classical simulation from which the snapshot for the CPMD calculations was taken is shown in Figure S2 in the Supporting Information. An orthorhombic simulation box was constructed by stripping off the lipid bilayer and only including water molecules within the minimum and maximum x,y coordinates of the monomer (Figure 1A). Overall neutrality was achieved by replacing water molecules with chloride ions. A similar setup was used in a previous QM/MM study of proton exclusion in the GlpF aquaporin.35 2.2. QM/MM Calculations. To obtain optimum structures of substrate binding to the Am1, Am1a, and Am1b sites, we used the CPMD QM/MM method,36 which combines the first principles MD method of Car and Parrinello (CPMD)37,38 with

E. coli Ammonia Channel AmtB: A QM/MM Investigation a force-field MD methodology. General details of the CPMD QM/MM method are described in the Supporting Information. Valence orbitals of the QM atoms were expanded with a plane wave basis set up to 70 (calculations with NH3 and NH4+ as substrates) and 80 Ry (Na+ and K+). Ab initio pseudopotentials were used to describe the core electron-valence shell electron interactions.39 The PBE functional was used for exchange and correlation interactions.40,41 Even though this functional has a well-known tendency to overbind water systems, thus producing overstructured pair correlation functions,42,43 we selected it in view of its good performance in hydrogen bond interactions, including the NH3 dimer.44 Moreover, this is the same functional that we used in our previous study of the GlpF aquaglyceroporin.35 The simulations used a time step of 3 au and a fictitious electron mass of 500 au to solve the equations of motion. The deuterium mass was used for hydrogen. Previous work has demonstrated the reliability of the CPMD QM/MM method in the description of structural, energetic, and dynamic properties of systems of biological interest, including protein channels (see for instance refs 35, 45, and 46). The AMBER force field47 was used to account for interactions among the atoms of the MM region. The QM-MM partition was taken as follows. The QM region included the substrate and residues (amino acids + water molecules) either directly or potentially coordinated to it. In the case of the Am1 site, we included the side chains of Q104, F107, W148, S219, D160, the peptide bond of A162-G163, and five water molecules that were already present in the crystal structure.9 Even though D160 is further than 5 Å away, we included it in QM because previous work16 identified it as an important residue for NH4+ recruitment. Moreover, preliminary calculations of the binding energy using gas phase models (data not shown) confirmed that this residue contributes most to the binding energy at this site. F103 was excluded from QM since previous ab initio calculations showed that it has a minor role in substrate stabilization.26 Binding of all substrates at the Am1 site was analyzed. For the simulations of the Am1a and Am1b sites, the QM region included the side chains of D160, H168, W212, F215, N216, T273, H318, the peptide bond of A162-G163, and two NH3 molecules. Only NH4+ and NH3 were probed because it is believed that the alkali ions do not reach these sites. The following procedure was used to find the optimum position of the substrates in each site: After optimizing the initial structure, we performed a short QM/MM CPMD simulation (∼2 ps at 300 K) to equilibrate the local structure around the ion. In the case of the Am1a-NH4+ simulation, the substrate moved to the Am1b position during the simulation. In contrast, NH3 moved from the Am1b to the Am1a position. Afterward, the temperature was lowered progressively until a structure with small nuclear gradients was reached. Even though this procedure does not ensure completely that the substrate position that we reach is the global minimum, it is enough to capture trends among the different substrates. Noteworthy, the main changes observed in the position of the ions at the Am1 position (separation of the pair Na+/NH3 with respect to K+/NH4+) already occur during the initial structure optimization (i.e., prior to the QM/MM CPMD simulation, Figure S3 in the Supporting Information), and it is maintained during the simulation. The resulting structures were used for analysis. 2.3. Calculation of Interaction Energies. Preliminary values of interaction energies (∆Eint) between a specific substrate and the protein were obtained by means of single point gas phase density functional theory (DFT) calculations (using the CPMD code and the same DFT setup as in the QM/MM simulations

J. Phys. Chem. B, Vol. 114, No. 36, 2010 11861 described above) including only the residues/water molecules of the QM region. The interaction energy of each substrate (∆Eint) was computed via the supermolecular approach, as a difference between the energy of the complex (e.g., NH3 complexed with Q104, F107, W148, S219, D160, and A162G163 + 5 water molecules in the case of the Am1-NH3 simulation) and those of the isolated fragments (e.g., NH3 and the rest of the complex, following the above example). As a first approximation, and with the purpose of just ranking the binding energies for different substrates, the structures of the fragments were not optimized. To estimate the contribution to the binding energy of a given residue surrounding the substrate, we repeated the calculations in the absence of this particular residue from the complex. The binding energy change can be attributed to the effect of this particular residue. Even though the values obtained are not expected to be additive, this analysis gives an idea of the contribution of a given residue to the total binding energy, relative to another residue. Similar analyses are commonly used to evaluate fragment contributions in molecular systems.48 Binding energies between each substrate and the protein at each binding site were also computed at the QM/MM level, as the difference between the energy of the complex (e.g., NH3 · · · protein) and those of the fragments (e.g., NH3, protein):

∆Eint ) Ecomplex - (Eprotein + Eligand) The contribution of a given protein residue (e.g., D160) to the binding energy was estimated as:

∆∆Eint ) ∆Eint - ∆Eintmutant where the corresponding mutant (i.e., D160G) was generated in silico by moving this particular residue to the MM region and turning off its partial charges and van der Waals parameters. It should be noted that although GGA functionals perform well for interactions dominated by electrostatics, such as ion · · · water interactions, the energies are likely to be affected by an error bar of the order of 1-2 kcal/mol.45,49 For this reason, only trends among different substrates are meaningful. Interaction energies computed using QM/MM turned out to be qualitatively similar to the ones obtained using gas phase models (Table S1 in the Supporting Information), indicating that long-range interactions do not affect the strength of the interactions between the neighboring residues and the substrate. 2.4. Analysis of Substrate Coordination. Coordination numbers were defined as the number of interactions between substrate atoms and any atom of neighboring residues and water molecules. An interaction was identified when the distance was shorter than a cutoff value, obtained from previous theoretical studies of the charged substrates in bulk water.50,51 In the case of cation-π interactions, the cutoff distances were adopted from previous gas phase DFT calculations reported for complexes between cations and aromatic amino acid motifs (F, W).52 Cutoff distances and the corresponding bulk water coordination numbers are listed in Table S2 in the Supporting Information. 3. Results and Discussion 3.1. Substrates at the Am1 Site. The final optimized structures of the substrate · · · protein complexes (initially) at the Am1 z-position are shown in Figure 2. All substrates exhibit favorable intermolecular interactions with their neighboring residues and water molecules (the coordination number is 4 for

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Nygaard et al. TABLE 2: Total Interaction Energies (∆Eint) between Substrates and the Protein Obtained from QM/MM Calculations at the Final Optimized Structuresa Am1

Am1b

substrate

∆Eint

∆Gbind

∆Eint

NH3 NH4+ Na+ K+

-30.4 -156.9 -164.5 -135.5

-23.5 -68.1 -56.4 -47.9

-7.8 -162.0

a All energies are in kcal/mol. Also listed is the estimated binding free energy ∆Gbind (see the text for more details).

Figure 2. Substrates at the Am1 position. (A-D) QM/MM optimized structures with the four different substrates (ball and stick): NH3, NH4+, Na+ (orange), and K+ (green), initially at the Am1 position. Only QM atoms are shown. Residues within coordination distances (see Table 4) are connected to the substrates by dashed lines. (E) Change in the z-positions of substrate heavy atoms relative to the Am1 position during the QM/MM simulations.

Figure 3. Substrates at the Am1a and Am1b positions. QM/MM optimized structures with NH4+ (A) and NH3 (B) initially at the Am1b position. Only QM atoms are shown. Residues within coordination distances (see Table 4) are connected to the substrates by dashed lines. The intraluminal NH3 coordinating to the substrate is shown in licorice.

the four substrates NH3, NH4+, Na+, and K+). Most of the interactions involve water molecules, and NH4+ and K+ are the only substrates that interact with A162. Close inspection of Figure 2A-D reveals that NH4+ and K+ are slightly displaced toward the channel lumen relative to the other substrates. This is confirmed by analysis of the change in the z-position during the optimizations, shown in Figure 2E. NH4+ and K+ are the only substrates that remain close to the Am1 site, whereas NH3 and Na+ move up toward the periplasmic side. Comparison of the degree of coordination of the substrates in the protein relative to bulk water (Table S2 in the Supporting Information) shows that Na+ and K+ lose coordination upon binding to the protein, whereas NH4+ maintains its optimum 4-fold coordination. This suggests that NH4+ has the largest affinity for the Am1 site. The energetic analysis provides further insight into the differences among substrates. According to the solvation free energies (see Table S3 in the Supporting Information for literature values of ∆Gsol), dehydration is more costly for Na+ than for K+ and NH4+ and, consequently, demands a larger compensation from substrate-protein interactions. The neutral substrate, NH3, shows the lowest dehydration cost. Ranking of the substrates according to their AmtB binding affinities requires knowledge about the binding free energies ∆Gbind, which can be calculated as ∆Gbind ) ∆Gcom - ∆Gsol, where ∆Gcom is the complexation free energy associated with bringing the substrate from the gas phase into the solvated substrate · · · protein complex. According to Table S3 in the Supporting Information, the entropic contribution T∆Ssol is small as compared to ∆Hsol and approximately of the same size (-5.1 to -7.7 kcal/mol) for all charged substrates. Thus, we can apply the approximation ∆Gbind ∼ ∆Hcom - ∆Hsol and use for ∆Hcom the calculated interaction energy (∆Eint, listed in Table 2). The values obtained for ∆Gbind are given in Table 2. It should be noted that the computed values of ∆Eint (and, therefore, ∆Gbind) should be taken as an upper bound to the real interacting energy, as they

have been obtained using the frozen fragment approximation (i.e., the structures of the isolated substrates and the protein were taken as that of the substrate · · · protein complex; see the Materials and Methods section).53 For this reason, only trends among different substrates are meaningful. The results of Table 2 show that NH4+ has the largest affinity, followed by Na+ and K+. They also give an answer to the question asked above; NH4+ is preferred over other monovalent cations such as Na+ and K+ due to a larger compensation of the dehydration cost from substrate-protein interactions. The estimated binding free energy for NH3 is much smaller than for the charged substrates (Table 2). This implies that differentiation between charged and neutral species is due to the larger stabilization of charged substrates relative to the neutral ones at the Am1 site. Therefore, if NH3 would enter the Am1 site, it is likely to be protonated, since NH4+ has a larger affinity for the site. In conclusion, by means of a fine-tuned balance between charge stabilization and dehydration cost, the Am1 site in AmtB has high NH4+ specificity with respect to neutral molecules like NH3 and other monovalent ions such as Na+ and K+. This explains the experimental observation that monovalent alkali cations do not inhibit MeAmm conduction in AmtB and other Amt proteins.9,18,22,25,29-31,33,34 3.2. Substrates at the Am1a/Am1b Sites. Because NH4+ is expected to deprotonate in between the Am1 and the Am2 positions,7,27,31,35 it is of interest to examine the structure and energetics of NH4+ and NH3 at the Am1a/Am1b intermediate positions. In the simulation with NH4+ initially at the Am1a position (Figure S1B in the Supporting Information), it was found that the substrate moves further down in the pore lumen toward the Am1b position (Figure 3A), losing coordination to N216:Oδ and T273:Oγ and gaining one interaction with H168: Nε. This is the same configuration found in the simulation with NH4+ initially at Am1b. Both the coordination pattern (four

E. coli Ammonia Channel AmtB: A QM/MM Investigation TABLE 3: Contributions to the Total Interaction Energy from Residues and Water Molecules Surrounding the Substratesa position

fragment

NH3

NH4+

Na+

K+

Am1

Q104 F107 W148 D160 A162:CdO S219 w1 w2 w3 w4 w5 A162:CdO H168 W212 F215 N216 T273 NH3

-2.6 +0.6 -1.7 -2.6 -0.3 0.0 -1.5 +0.8 -18.0 +3.0 -2.5 +3.7 -3.6 +0.5 -0.9 0.0 +0.5 +0.2

-7.2 -4.0 +3.1 -39.5 -7.1 -11.2 -16.5 -2.8 +2.4 -11.1 -6.4 -33.9 -23.8 -5.1 -3.0 -2.9 -5.1 -18.3

+1.1 -4.1 +3.1 -38.9 +3.5 -6.0 -21.9 -13.5 -15.6 -16.6 -7.7

-4.2 -4.3 +2.5 -42.4 -8.5 -9.7 -13.6 +0.1 +2.7 -10.1 -6.0

Am1b

a

All energies are in kcal/mol.

intermolecular contacts, which is its optimum coordination number in solution; Table S2) and interaction energy, ∆Eint, of NH4+ at the Am1b position are similar to the ones at Am1 (Table 2). Instead, NH3 moves from Am1b to the Am1a position (Figure 3B) during the simulation. Intraluminal NH3 turns out to be weakly bonded (Table 2), forming only one interaction with the protein (His168-Nε · · · H-NH2). This is in agreement with the view that NH3 is in a “gaseous” state when occupying the pore lumen.9 3.3. Main Intermolecular Interactions. 3.3.1. Interactions with D160. Table 3 lists the interaction energies between the individual substrates and its neighboring residues at the Am1 site. Even though D160 is distant (>6 Å) from the substrates, its carboxylate group provides the largest interaction energy for any of the charged substrates; specifically, it contributes more than twice the size of any other interaction energy. Most likely, this is due to the combined effect of polarization of the two-water wire connecting D160 to the charged substrates (w4 and w5 in Figure 2; note that this hydrogen-bonding network is broken for the neutral NH3) and the through-space electrostatic interaction between the two charged moieties (NH4+ and D160). In fact, gas phase MP2 calculations by Lin et al.27 demonstrated that this electrostatic interaction is significant. Hence, D160 is the single most important interaction for stabilizing positively charged substrates in the binding pocket, which is in accordance with related observations.21,22 The contribution of Q104, F107, W148, A162:CdO, and S219 is comparatively low. Interestingly, a recent mutagenesis study has reported increasing MeAmm conduction rates with respect to wild type AmtB in the following order: F107A < W148A < S219A.18 This was ascribed to a decreasing substrate affinity for the Am1 site and consequently a lowering of the rate-limiting barrier for translocation across the F107/F215 stack. Hence, by providing a higher substrate affinity at the expense of a lower conduction rate, the presence of F107, W148, and S219 is presumably crucial for the enhanced scavenging efficiency of AmtB at very low substrate concentrations. 3.3.2. Interactions with A162. At the Am1 position, the carbonyl group of A162 interacts with the substrate only in the case of NH4+ and K+. As a consequence of this interaction, these substrates move slightly below the Am1 position and

J. Phys. Chem. B, Vol. 114, No. 36, 2010 11863 concurrently displace one water molecule (w3 in Figure 2B,D). The fact that A162:CdO drives a partial dehydration of NH4+ is in agreement with previous observations.25 On the contrary, A162:CdO does not coordinate to Na+ nor to NH3. Seemingly, the nature of the Am1 site only allows cations of similar size to interact strongly with A162:CdO. However, recognition of NH4+ and MeNH3+ from monovalent alkali metal cations before translocation of substrates into the channel lumen is probably achieved by better compensating the dehydration cost of NH4+ and MeNH3+. Comparison of the initial structures for the Am1a and Am1b simulations (Figure S1B in the Supporting Information) with the optimized structures (Figure 3) shows that the initial orientation of A162:CdO, directed into the pore lumen (Figure S1B in the Supporting Information), is only stable for NH4+ (Figure 3A), whereas A162:CdO reorients toward the Am1 site for NH3 (Figure 3B). Apparently, A162:CdO has rotational freedom to coordinate NH4+ at both extra- and intraluminal positions, inferring that it can guide the substrate into the channel lumen. The ability of A162:CdO to coordinate NH4+ at the Am1 position, as well as to reorient from intra- to extraluminal positions, supports its previously proposed roles in NH4+ stabilization10,16,22,25 and in guiding NH4+ across the F107/F215 stack.16 3.3.3. Interactions with Q104. As shown in Figure 2, there are water molecules hydrogen bonded to Q104 (w1 in Figure 2A-D and w2 in Figure 2C) that provide a stabilizing effect. Thus, in addition to its possible role in substrate differentiation by steric exclusion effects,16 Q104 may also indirectly play a role in stabilizing NH4+ along the first part of the translocation pathway by polarizing water molecules. The conformation of Q104 in the Am1 site (Figure 2) resembles the one encountered in the crystal structures of wild type AmtB10 but not of engineered AmtB.9 Upon superposition of the crystal structures, it is obvious that the wild type conformation of Q104 is sterically possible only when NH4+ and not MeNH3+ occupies the Am1 site. Hence, substrate stabilization due to water polarization by Q104 can be expected to have less effect for MeNH3+ than for NH4+, although polarization and steric exclusion effects are not mutually exclusive. This provides an explanation to the fact that NH4+ is a better AmtB substrate than MeNH3+.17,19 3.3.4. Cation-π Interactions. Analysis of the distances between the substrates and its neighboring residues (Table 4) reveals that none of the charged substrates come within cation-π interaction distances to aromatic residue F107 and W148 in the Am1 position, nor to F215 and W212 in the Am1b position (Figures 2 and 3 and Table 4). For all charged substrates at the Am1 site, the interaction energies to F107 or W148 are |∆Eint| e 4.3 kcal/mol (Table 3), and their contribution is much lower than some of the water molecules. Correspondingly, the magnitude of the interaction energies with F215 or W212 at the Am1b site is only |∆Eint| e 5.1 kcal/mol, and they contribute much less than A162 and H168 (Table 3). Therefore, as compared with other interaction energies at the Am1 and Am1b sites (Table 3), cation-π interactions appear to be not significantly large. Our results for the Am1 site contrast with a previous ab initio study by Liu and Hu,26 which concluded that cation-π interactions provide a large stabilization of NH4+ at this position. In particular, the interaction energies determined for W148 and F107 with NH4+, +3.1 and -4.0 kcal/mol, respectively (Table 3), differ considerably from the values -18.4 and -10.9 kcal/ mol reported by Liu and Hu.26 This discrepancy could be due to differences in the simulated system (native vs mutated

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TABLE 4: Coordination Distances (in Å) of the Different Substrates at the Am1 (Left) and NH3/NH4+ at the Am1a/b Positions (Right), Respectively Am1

Am1a/b

residue

NH3

NH4+

Na+

K+

residue

NH3

NH4+

Q104:Oε Q104:Nε W148:Nε D160:Oδ A162:CdO S219:Oγ w1 w2 w3 w4 w5 F107 W148

3.57 4.40 5.29 6.82 4.07 2.11 2.20 1.58 3.54 3.05 2.07 5.10 5.67

3.82 5.31 3.74 6.27 2.10 1.98 1.65 6.40 2.61 1.94 4.08 4.03 5.01

4.27 3.34 4.23 6.99 4.30 3.97 2.27 2.47 2.42 2.29 4.65 5.48 5.01

4.70 5.69 4.98 6.55 2.72 2.69 2.71 4.82 3.77 2.87 4.80 5.12 5.24

A162:CdO G163:N-H H168:Nε W212:Nδ N216:Oδ N216:Nδ T273:Oγ NH3 W212 F215

3.88 3.66 2.15 5.51 6.22 7.26 5.20 4.57 7.84 3.69

2.00 6.14 1.69 2.33 4.18 6.23 3.80 1.87 4.86 4.38

structure), the setup of the system (crystal structure vs MDequilibrated one), or in the method to calculate interaction energies. The calculations of Liu and Hu were performed on an engineered Amtb (F68S, S127P, and K265L mutants). Because of the F68S mutation, Q104 (a residue interacting with the substrate at Am1) adopts a different configuration than in the wild-type structure, as discussed previously.16 In our calculations, the mutations of the crystal structure were reverted to the appropriate wild-type residues and the orientation of Q104 changed to the wild-type one during the initial long time scale MD simulation.35 In contrast, the simulations of Liu and Hu were performed directly in the crystal structure, without optimization or previous classical MD simulation. Concerning binding energies, the calculations by Liu and Hu26 are based on simplified gas phase models; therefore, polarization effects from surrounding residues are not explicitly taken into account. In particular, only F103, F107, W148, and S219 were included in their model, which thereby neglect the important contributions from Q104, A162:CdO, and, most importantly, D160. On the basis of our results, we find that cation-π interactions provide stabilization of charged substrates at the Am1 site, but in contrast to previous suggestions, they do not provide the major contribution. 4. Conclusions In this work, we have conducted QM/MM simulations of E. coli AmtB with four different substrates: NH3, NH4+, Na+, and K+. Our main motivation was to clarify the relative importance of different interactions with respect to substrate binding and selectivity. We find that D160 contributes the most in stabilizing and recruiting charged substrates at the Am1 site; it accounts for 30-40% of the total interaction energy between the substrates and the surrounding residues. Previously proposed cation-π interactions to W148 and F107 only account for ∼6% of the total interaction energy. Therefore, these do not provide the main contribution for binding charged substrates at the Am1 position. We also find that Q104 can provide stabilization of substrates through water polarization. The effect is expected to have less effect for MeNH4+ than for NH4+ and may provide some explanation for the experimentally observed differences in affinities.17,19 Among the four different substrates, we find that NH4+ has the largest affinity for the Am1 site. The smaller affinity observed for K+ and Na+ toward the Am1 site is mainly due to the reduced compensation of their dehydration cost via substrate-protein interactions, while the smaller affinity observed for NH3 is mainly due to a larger stabilization of charged substrates relative to neutral substrates at the Am1 site. The

fact that A162:CdO coordinates NH4+ but not NH3 at the Am1 and Am1a/b sites, together with the observed rotation of the A162-G163 peptide bond, suggests that A162 can guide NH4+ into the channel lumen. Acknowledgment. We thank Flemming Y. Hansen for valuable discussions. We acknowledge financial support by the Spanish Ministerio de Educacio´n y Ciencia (Grant FIS210803845) and the Generalitat de Catalunya (GENCAT) (Grant 2109SGR-1309). M.A.-P. thanks the F.I. fellowship program of the GENCAT. We acknowledge the computer support, technical expertise, and assistance provided by the Barcelona Supercomputing Center-Centro Nacional de Supercomputacio´n (BSC-CNS). T.P.N. acknowledges financial support from the HPC-EUROPA Program of the European Commission for a visit to BSC-CNS (Barcelona, Spain) and funding from the Center for Biomembrane Physics (MEMPHYS), which is supported by the Danish National Research Foundation. The molecular images in this paper were created with Visual Molecular Dynamics.54 Supporting Information Available: General details of the QM/MM calculations. Figure S1 of the QM fragment chosen for the Am1 and Am1a/b simulations. Table S1 of the gas phase interaction energies. Substrate coordination in water. Table S2 of coordination numbers. Table S3 of thermodynamic data. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Schneider, M.; Marison, I. W.; von Stockar, U. J. Biotechnol. 1996, 46, 161–185. (2) Solomon, E.; Berg, L.; Martin, D. Biology, 5th ed.; Saunders College Publishing: Orlando, 1999. (3) Murzin, A. G.; Brenner, S. E.; Hubbard, T.; Chothia, C. J. Mol. Biol. 1995, 247, 536–540. (4) Transport Classification Database TCDB, 2005; http://www.tcdb. org/. (5) Kumar, A.; Kaiser, B. N.; Siddiqi, M. Y.; Glass, A. D. M. Funct. Plant Biol. 2006, 33, 339–346. (6) Loque, D.; Von Wiren, N. J. Exp. Bot. 2004, 55, 1293–1305. (7) Madhani, H. D.; Fink, G. R. Trends Cell. Biol. 1998, 8, 348–353. (8) Smith, D. G.; Garcia-Pedrajas, M. D.; Gold, S. E.; Perlin, M. H. Mol. Microbiol. 2003, 50, 259–275. (9) Khademi, S.; O’Connell, J.; Remis, J.; Robles-Colmenares, Y.; Miercke, L. J. W.; Stroud, R. M. Science 2004, 305, 1587–1594. (10) Zheng, L.; Kostrewa, D.; Berneche, S.; Winkler, F. K.; Li, X. D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17090–17095. (11) Andrade, S. L. A.; Dickmanns, A.; Ficner, R.; Einsle, O. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 14994–14999.

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