The significant increase in resolution of the AdiC structure was achieved by adapting and adding three steps to our previously published protocol [16]. First, we introduced a size exclusion chromatography (SEC) step before crystallization. Then, crystals were flash-cooled in liquid ethane. Finally, we merged data sets from multiple crystals and applied anisotropic correction to the data (see the “Methods” section).
Overall structure
The crystal structure of AdiC from E. coli was solved at the unprecedented resolution of 1.7 Å by X-ray crystallography (Additional file 1: Table S1). AdiC is homodimeric and each monomer consists of twelve transmembrane α-helices (TMs) with intracellular N- and C-termini (Fig. 1). The high quality of the obtained electron density map can be assessed in Additional file 1: Fig. S2. The structure described here was obtained in the outward-open state. TM1–TM10 form a barrel-like structure surrounding a relatively large solvent filled cavity, which is exposed to the periplasmic space (Fig. 1B). Ten TMs constitute the core of the protein and adopt the 5+5 inverted repeats topology, which is typical of the APC superfamily [12, 13]. TM1 and TM6 are discontinuous, each forming two short α-helices: TM1a and TM1b, and TM6a and TM6b, respectively (Fig. 1 and Additional file 1: Fig. S3A). These short α-helices are connected by loops, which are known to be involved in substrate-binding [14,15,16]. The interface of the dimer was calculated with PISA [20] and the surface is equal to 2629 Å2. Most of the AdiC homodimer interface is formed by non-polar amino acids from TM11 and TM12, where residues of TM11 from one monomer interdigitate with residues of TM12 from the other monomer (Additional file 1: Fig. S3B). Further interactions between the two monomers are mediated by the loops between TM2 and TM3, the cytoplasmic ends of TM2 and TM3, the cytoplasmic halves of TM10, and the C-termini. The latter embrace neighboring monomers (Fig. 1A, bottom).
The unprecedented high resolution of the AdiC structure allowed for the identification of numerous water molecules and networks. Such structural information is very rare for membrane proteins and offers a unique opportunity to dissect these crystallographic water molecules and networks and directly characterize their dynamics and stability with molecular dynamics (MD) simulations. Therefore, a set of four independent all atom MD simulations was performed under physiological conditions. Each simulation was carried out up to 500 ns. The structure of AdiC remained stable throughout all the simulations (average RMSD: 2.1 ± 0.1 Å). The analyses focused on three important regions of the AdiC transport protein: (i) the substrate-binding site, (ii) the W202 and W293 gates, and (iii) the cavity at the dimeric interface.
Water shapes and stabilizes the substrate-binding site
The substrate-binding site of AdiC has previously been identified and described [14,15,16]. For comparison purposes, we define the solvent accessible volume between the indole nitrogen atom of W293 and the oxygen atom of the S26 side chain as the substrate-binding site of AdiC in outward-open crystal structures. In the 2.2 Å AdiC structure, seven water molecules were identified in this volume for each protomer [16] (Additional file 1: Fig. S4A). The significant increase in resolution in the presented high-resolution structure, allowed us to identify twenty-one and fifteen water molecules in monomers A and B, respectively (Additional file 1: Fig. S4B). This comparison reflects the important enhancement in water information obtained from the 1.7 Å resolution crystal structure. The water molecules in the substrate-binding site form a water cluster network (Fig. 2). We defined hydration water as water molecules that interact directly with the protein (Fig. 2, orange spheres), and bulk water as water molecules that do not interact with the protein, but only with other water molecules (Fig. 2, gray spheres). This allowed us to discover water molecules that are involved in the shaping and stabilization of the substrate-binding site by linking different TM domains (Fig. S5A and Table S2 in Additional file 1).
Seven water molecules act as placeholders for substrates atoms
Previously, we showed that two water molecules serve as placeholders for specific agmatine nitrogen atoms in the absence of ligand in the substrate-binding site of AdiC [16]. These water molecules, labeled H2O3 and H2O13 in Fig. 3A, occupy the nitrogen atoms positions of the primary amino and Nη1 guanidinium groups of agmatine. Due to the achieved high resolution, additional water molecules could be identified in the substrate-binding pocket of the new AdiC crystal structure (Fig. 3A). Based on the modeled L-arginine, the new water molecules H2O5 and H2O7 are located near the positions expected to be occupied by the two oxygen atoms of the L-arginine carboxyl group in the outward-open, substrate-bound state (Fig. 3B). This finding suggests that H2O5 and H2O7 replace the oxygen atoms of the L-arginine substrate in its absence. We then compared the modeled L-arginine (Fig. 3B) bound to the outward-open conformation (Additional file 1: Fig. S1, state b) with the L-arginine bound in the outward-facing, occluded AdiC-N22A structure (Additional file 1: Fig. S1, state c) [14]. This comparison revealed that in the outward-facing, occluded conformation, one oxygen of the L-arginine carboxyl group takes the position of H2O5, while the second oxygen moves away from the H2O7 position (Fig. 3C). In addition, conformational change from the outward-open to the outward-facing, occluded state positions the oxygen atom of the S26 hydroxyl side chain near the position of H2O7 (Fig. 3C). H2O5 and H2O7 are in hydrogen bonding distance to H2O6, which would be replaced by the Cα-atom of L-arginine (Fig. 3B) or by the corresponding carbon atom in agmatine. Thus, H2O5 and H2O7 stabilize the agmatine by weak C–H hydrogen bonds [21]. This observation would explain how the binding site of AdiC can accommodate both, L-arginine and agmatine. H2O19 forms a lone pair-π-interaction [22] with the side chain of W293 in the outward-open conformation (Fig. 3A). This interaction is disrupted and H2O19 is replaced by Nη2 from the L-arginine guanidinium group in the outward-facing, occluded conformation. Interestingly, this direct interaction with the middle gate residue W293 could represent a potential trigger in the transport mechanism of AdiC. Finally, the water network within the substrate-binding site is completed by the newly identified bulk water molecule H2O17, which is in hydrogen bond distance to H2O19 and H2O6 (Fig. 3A). With the exception of H2O13, the MD simulations support the presence of all water molecules acting as substrate atoms placeholders: H2O3: 74 ± 9%, H2O5: 65 ± 2%, H2O6: 33 ± 11%, H2O7: 17 ± 2%, H2O17: 28 ± 3%, and H2O19: 31 ± 12%. In summary, we suggest that binding of L-arginine to AdiC in the outward-open conformation replaces water molecules H2O3, H2O5, H2O6, H2O7, H2O13, H2O17, and H2O19 (Fig. 3A). The interchange between these seven structural water molecules and L-arginine has the advantage to preserve the geometry of the binding site in the substrate-free and substrate-bound outward-open states (Additional file 1: Fig. S1, states a and b), thus keeping the energetics in the protein minimal. It is noteworthy that in the L-arginine bound outward-facing, occluded AdiC-N22A structure [14], the position of H2O7 is taken by the S26 hydroxyl group, indicating a potential collective behavior between the water molecules, L-arginine, and S26 during the binding event and the transport dependent conformational change.
Crystallographic water molecules and networks constraining the spatial orientation of the gates W202 and W293 in the outward-open conformation and analyses of their stability with MD simulations
Conformational changes of AdiC upon binding of L-arginine were suggested [14, 18], which result among other things in the occlusion of the bound substrate from the periplasm by the movement of W202 (transition from states b to c in Additional file 1: Fig. S1). We thus investigated the possible implication of water molecules for the orientation of W202 in the conformation open to the periplasm (state a of the transport cycle in Additional file 1: Fig. S1). The side chain of W202 (TM6a) in the outward-open conformation forms hydrogen bonds with two water molecules: H2Oα and H2Oβ (Fig. 4A, B). These water molecules interact with N198(O) (TM6a) and G404(O) (TM11) and A401(O) and G404(O) (TM11), respectively. A third water molecule (H2Oγ) bridges the amide nitrogen atom of Q194 (TM6a) and W400(O) (TM11). Together, these three water molecules form a string of water at the interface between TM6a and TM11. Importantly, the indole nitrogen atom of the functionally relevant W202 residue does not form direct hydrogen bonds with the protein. Instead, it interacts indirectly with water molecules H2Oα and H2Oβ, thus adopting a specific spatial orientation. In addition, hydrophobic interactions are observed between W202 and hydrophobic residues in TM6 (I205) and TM10 (V358, I359 and L362) (Fig. 4B). As a consequence, the spatial orientation of the thin gate W202 in the outward-open conformation might be controlled by a water network and hydrophobic side chain interactions. However, in the molecular dynamics (MD) simulations, the observed water network (H2Oα-H2Oγ) interacting with W202 in the AdiC crystal structure (Fig. 4B) was disrupted, allowing W202 to sample a large range of conformations (Fig. 4D and Additional file 1: Fig. S6). We hypothesize that this water network is most likely only formed during crystallization.
The middle gate residue W293 [14] is located in the substrate-binding pocket of AdiC. In contrast to W202, the spatial orientation of W293 is stabilized by a hydrogen bond to H2O9 (Fig. 4C). This crucial water molecule is in turn stabilized through H-bond interactions with N22(O) and S289 (Fig. 4C and Additional file 1: Fig. S5D). W293 is also interacting with the N22 side chain (amide-π interaction) and neighboring hydrophobic residue. The H2O9-mediated interaction could help orient W293 in space, which is important since L-arginine and agmatine have indicated cation-π interactions between this tryptophan side chain and the guanidinium groups of the substrates [14,15,16]. This constrained orientation of W293 is supported by the MD simulations (Fig. 4E and Additional file 1: Fig. S6). The hydrogen bond interactions between H2O9 and N22(O), and the nitrogen atom and hydroxyl group of the W293 and S298 side chains is present in about 26 ± 6% of the conformations sampled during MD simulations. The amide-π interaction with N22, either directly or mediated through a water molecule, was more persistent during MD (43 ± 18%). This finding is supported by the N22A mutation, which locks AdiC in the outward-facing occluded state [14].
Potential mechanistic importance of gate flexibility in the transport cycle
Based on the MD simulations performed using the here presented high-resolution crystal structure of AdiC in the outward-open state, we found that the gate W202 is mobile adopting different conformations (Fig. 4D). In contrast, the mobility of gate W293 is constrained in this state (Fig. 4E). In a previous study [18], a transient cation-π interaction of L-arginine with W202 was proposed, before binding to W293 in the substrate-binding site. Together with our finding on the mobility of W202 in the outward-open state, we propose that W202 might screen for substrate. Once L-arginine is transiently bound to W202, the substrate would slide into the substrate-binding site, where it interacts via cation-π interaction with W293 and hydrogen bonds with other amino acids. This mechanism might be extrapolated to the inward-open conformation, i.e., W293 would be mobile looking for substrate, in this case agmatine, while W202’s mobility would be restrained—see Fig. 5 for a summary and a refined transport cycle. This mechanistic insight from the AdiC gate residues W202 and W293 might be expanded to AdiC homologs of enteropathogens as well as the amino acid-diamine transporters PotE [23] and CadB [24] from E. coli. In such members of the amino acid/polyamine/organocation (APC) transporter superfamily [7, 8], tryptophan residues corresponding to the W202 and W293 gates in AdiC are found at corresponding positions in their amino acid sequences (Additional file 1: Fig. S7).
Water containing cavity at the dimer interface
We identified a heart-shaped cavity at the interface of the AdiC dimer that is lined by mostly hydrophobic amino acids, F84, L85, V363, L366, F414, L417, and M418, and a few polar groups, Q88, Y367, and T421 (Additional file 1: Fig. S8). The high-resolution structure allowed us to detect a residual positive Fo-Fc density in this cavity during protein model refinement (Additional file 1: Fig. S9A). Solutes from the protein buffer and mother liquor, i.e., Tris(hydroxymethyl)aminomethan (Tris), Polyethylenglycol 400 (PEG400), and n-nonyl-β-D-glucopyranoside (see the “Methods” section), were too large to fit into the relatively small positive Fo-Fc density. Therefore, we hypothesized the presence of a dynamic interfacial water network and placed water molecules into these Fo-Fc densities during refinement (Additional file 1: Fig. S9A, B). This resulted in the identification of eleven water molecules in the interdimeric cavity (Additional file 1: Fig. S9C). The distances between the water molecules ranged from 1.9 to 3.8 Å. The lower limit is too short to be within hydrogen bond distance, and therefore, not all positions can be occupied simultaneously, supporting our hypothesis of a dynamic water network (Additional file 1: Fig. S9D). Since the data set used for the high-resolution structure determination was derived from twenty-two individual data sets, the observed water network would represent an ensemble of possible positions of water molecules in the cavity. We investigated the hydration of the cavity using the set of MD simulations and identified the positions with the highest probabilities of occupancy. All simulations started with an unsolvated cavity and, within the first 100 ns, water molecules started diffusing into the cavity, mostly from the ligand-binding pocket. The cavity remained hydrated throughout the simulation. On average, the cavity was occupied by approximately 2 water molecules, with the following frequencies for the main configurations: one (20%), two (38%), three (29%), and four (9%) water molecules. We observed that water molecules preferably occupied positions a-d and only rarely positions e–k (Additional file 1: Fig. S9E, F). This was further supported by the better-defined electron densities observed during refinement for positions a–d (Additional file 1: Fig. S9A, B). Since the distances between water molecules a-d are between 1.9 and 2.2 Å, they cannot exist simultaneously. During the MD simulations, water molecules were observed at different positions and frequencies (Additional file 1: Fig. S10). The interfacial water networks composed of three molecules were mainly localized in the continuous density defined by water molecules a–d (Additional file 1: Fig. S9E). However, they occupied intermediate positions in order to accommodate the hydrogen-bonding distance between them.
Noteworthy is that the water molecules at positions a–d formed hydrogen bonds with the hydrophilic side chains in the cavity (Q88, Y367 and T421), thus bridging monomers A and B (Fig. 6). This suggests that water molecules are involved in the stabilization of the AdiC dimer. To test this hypothesis, thermostability assays were performed to compare melting temperatures (Tm) between wild-type AdiC (AdiC-wt) and three mutants that would disrupt the water network (Q88E, Y367F and T421V) (Fig. 6B). All mutants eluted at similar volumes as wild-type AdiC during SEC indicating retention of the dimeric state after mutagenesis (Additional file 1: Fig. S11). The mutation Q88E replaces the nitrogen of the amide group by an oxygen atom, which precludes the side chain to serve as a proton donor. With pKa values of 3.8 and 4.2 calculated using ROSIE [25] and DelPhiPKa [26], the carboxyl group of the introduced glutamate side chain is deprotonated at physiological pH. The mutations Y367F and T421V replace the side chain hydroxyl groups by a proton or a methyl group, which impedes the formation of hydrogen bonds. In all cases, the thermostability of AdiC was significantly reduced, supporting the importance of water for dimer stabilization. The most drastic change was observed for AdiC-Y367F with a remarkable difference of almost 10 °C (Fig. 6B). For this mutant, the missing interaction of the hydroxyl group of Y367 with the amide group of Q88 (Fig. 6A) in AdiC-Y367F probably also contributed to the dimer destabilization (Fig. 6B).