Investigation of molecular details of a bacterial cationic amino acid transporter (GkApcT) during arginine transportation using molecular dynamics simulation and umbrella sampling techniques

doi:10.21203/rs.3.rs-2881849/v1

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Research Article

Investigation of molecular details of a bacterial cationic amino acid transporter (GkApcT) during arginine transportation using molecular dynamics simulation and umbrella sampling techniques

https://doi.org/10.21203/rs.3.rs-2881849/v1

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published 21 Jul, 2023

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Context:

Cationic amino acid transporters (CATs) facilitate arginine transport across membranes and maintain its levels in various tissues and organs, but their overexpression has been associated with severe cancers. A recent study identified the alternating access mechanism and critical residues involved in arginine transportation in a cationic amino acid transporter from Geobacillus kaustophilus (GkApcT). Here, we used molecular dynamics (MD) simulation methods to investigate the transportation mechanism of arginine (Arg) through GkApcT. The results revealed that arginine strongly interacts with specific binding site residues (Thr43, Asp111, Glu115, Lys191, Phe231, Ile234, and Asp237). Based on the umbrella sampling, the main driving force for arginine transport is the polar interactions of the arginine with channel-lining residues. An in-depth description of the dissociation mechanism and binding energy analysis brings valuable insight into the interactions between arginine and transporter residues, facilitating the design of effective CAT inhibitors in cancer cells.

Methods

The membrane-protein system was constructed by uploading the prokaryotic CAT (PDB ID: 6F34) to the CHARMM-GUI web server. Molecular dynamics simulations were done using the GROMACS package, version 5.1.4 with the CHARMM36 force field and TIP3 water model. The MM-PBSA approach was performed for determining the arginine binding free energy. Further, the hotspot residues were identified through per-residue decomposition analysis. The characteristics of the channel such as bottleneck radius, and channel length were analyzed using the CaverWeb 1.1 webserver. The proton wire inside the transporter was investigated based on the classic Grotthuss mechanism. We also investigated the atomistic details of arginine transportation using the path-based free energy umbrella sampling technique (US).

Cationic amino acid transporters (CATs)

MM-PBSA

MD simulations

Umbrella sampling

Binding free energy

L-arginine is a semi-essential amino acid that serves as a precursor for many vital biological pathways, including the synthesis of nitric oxide, polyamines, creatine, agmatine, and urea [1]. Although the endogenous de novo production of arginine through the hepatic urea cycle by the activity of argininosuccinic acid synthetase (ASS) and argininosuccinic acid lyase (ASL) can supply the majority of arginine required for host metabolic in non-stressed states and healthy adults, under catabolic conditions and also pregnant dams, endogenously synthesis of arginine is insufficient and must be provided from the diet [2, 3]. Arginine transport across the biological membrane is mainly mediated by facilitative transporters called cationic amino acid transporters (CATs), which belong to the major solute carrier 7 families (SLC7) and amino acid polyamine cation (APC) superfamily [4]. The CAT transporters subfamily is classified into four transporters for cationic amino acids: CAT1 (SLC7A1), CAT2A (SLC7A2A), CAT2B (SLC7A2B), and CAT3 (SLC7A3) [5]. The CATs transporters designated as the y + system mediate pH insensitive and sodium ion independent transport of cationic amino acids through the trans-stimulation mechanism except for CAT2, which doesn’t exhibit a significant degree of trans-stimulation [6–8]. Having cytoplasmic N and C termini, CAT proteins comprise 14 transmembrane (TM) helices traversing through the membrane bilayer [9]. All CATs are N-glycosylated at two positions (N223 and N229) in the third extracellular loop [10]. CATs also contain three phosphorylation sites located in helix 5 and 6, and previous studies have shown that phosphorylation of these sites decreases the transportation activity of proteins [11]. While many protein transporters (approximately 70% of membrane proteins) function as oligomers in the membrane, the functional form of the CAT proteins is a monomer [12]. Among CAT isoforms, CAT1 is highly expressed in all tissue except the adult liver, and its ubiquitous expression is mainly regulated by cationic amino acids (arginine, lysine, ornithine) concentration, cytokines, and hormonal secretion [13].

There is no resolved structure available for the eukaryotic CAT1 transporter. However, the crystal structure of the homologous prokaryotic transporter of CAT1 from Geobacillus kaustophilus (GkApcT) has resolved; it has a 37% identity with the eukaryotic CAT1 at the sequence level [14]. Thus, GkApcT can be a suitable candidate for a comprehensive investigation of the arginine transportation mechanism through the CAT1 transporter. The transportation process in the GkApcT transporter operates via alternating access based on the formation of outward and inward barriers due to the structural rearrangement of the transporter [15]. The crystal structure of GkApcT has resolved in an inward occluded state where the intracellular gate is sealed via the interactions between Arg327 (in helix 8) and Thr241 (in helix 6), and Glu155 (in helix 3) and Asp237 (in helix 6), and the latter interaction, called the thin intracellular gate, is mediated with a water molecule (Fig. 1) [14]. Till physiological pH is below the given pKa = 8 for the Glu115 functional group, the protonated form of Glu115 is predominant in the structure. The opening of the cytoplasmic gate involves the deprotonation of Glu115 and a conformational shift of TM6 swing away from TM3 and TM8, allowing arginine to sink and release inward [14, 16]. In other words, deprotonation of Glu115 causes a repulsive force between Glu115 and Asp237 triggering the release of arginine into the cytosol. Through an insightful investigation of the arginine transport through the CAT family, Jungnickle and colleagues showed that the conserved aromatic residue, Phe231 in TM6 and Thr43 in TM1, serves as the thick extracellular gate element that separates the arginine binding site from the exoplasmic tunnel leading to the extracellular side of the membrane [14].

There are many studies indicating overexpression of CAT1 in different cancer cells, such as colorectal cancers, melanoma, breast cancers, chronic lymphocytic leukemia (CLL), and liver cancer [17–20]. Cancer cells are highly dependent on CAT1 transporters’ activity to maintain sufficient arginine required for rapid cell growth and proliferation [19]. The functional significance of CAT1 in tumor cell growth has been shown through genetic manipulation with the knockdown of CAT1 with small interfering RNA (siRNA), as well as employing monoclonal antibodies recognizing the extracellular domain of CAT1 in cancer cells exhibiting overexpression of CAT1. [19, 21, 22]. Thus, CAT1 can be a promising target for tumor-targeting therapy because CAT1 blockade is more selective and reduces the off-target effects [23]. CAT1 inhibitors act by depriving cationic amino acids such as arginine, thus, preventing protein synthesis and growth in the cancerous cells. Moreover, the amino acid arginine has been illustrated to facilitate protein synthesis and induce cell growth by regulating the mammalian target of rapamycin complex 1 (mTORC1) through mTORC1 kinase activation, and CAT1 inhibition is considered to suppress the mTORC1 pathway and subsequently cell growth [22].

At present, there is no experimental structural information for CAT1; however, there is an approximately 40% identity between CAT1 and its bacterial homolog at the sequence level. Therefore, in this study, we performed molecular dynamics simulation studies of GkApcT embedded in the lipid bilayer environment to investigate the critical amino acids involved in arginine binding. The umbrella sampling technique was conducted to gain insight into conformational changes and rearrangements in the transporter during arginine transportation. We were able to demonstrate the role of water molecules in the transportation of arginine across GkApcT. The results show that GkApcT is capable of transporting water molecules with low permeability compared to water channels such as aquaporin (AQP).

2.1. Preparing membrane-protein system

The three-dimensional structure of the GkApcT mutant (M321S), henceforth called pCAT, in complex with arginine (PDB ID: 6F34), was downloaded from the RCSB PDB repository. Since CAT1 is an integral protein, the CHARMM-GUI web tool was employed to construct a lipid bilayer membrane with a preference composition around protein [24]. The lipid bilayer consisted of 128 lipids per membrane leaflet maintaining the ratio of lipids in the K12 strain of E. coli membrane (see Table S1 for details) [25, 26]. CHARMM-GUI server generates input files required for GROMACS.

2.2. Molecular dynamics (MD) simulation production

MD simulation was carried out using GROMACS package, version 5.1.4 [27]. The force field chosen for the MD simulations was CHARMM36 [28]. Using CHARMM-GUI, the systems were solvated by using the TIP3P water model, and adequate counter ions were added to neutralize the system. The long-range electrostatic interactions were computed by using Particle-Mesh Ewald (PME) algorithm [29]. In order to remove any steric clashes and hindrances, the systems were energy minimized using the steepest descent algorithm [30]. The systems were maintained at melting temperature 303.15 K under 1 bar pressure, using a temperature coupling time constant of 1.0 ps and a pressure coupling time constant of 5.0 ps. The cutoff value for calculating short-range electrostatic interactions was set at 1.2 nm. The MD production was run under periodic boundary conditions (PBC) with a time step of 2 fs. The details of the MD simulation are provided in Table 1.

Table 1

Details of MD simulation systems
Simulation system	System shape	no. water molecules	no. K⁺ ions	no. Cl^- ions	Temp. (K)	Simulation time (ns)
Arg/pCAT/mem	rectangular	22916	124	61	303.15	600
pCAT/mem	rectangular	17615	111	47	303.15	300

2.3. MM-PBSA binding free energy

The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) approach was utilized to calculate the binding energy between pCAT and arginine [31]. The following equation was used in the total binding energy calculation:

\({\varDelta G}_{binding}={G}_{complex}- {G}_{protein}- {G}_{ligand}\) Eq. (1)

where G_complex is the total free energy of the complex (Arg/pCAT) and G_protein and G_ligand denote the free energy of the unbounded transporter (pCAT) and the Arg ligand, respectively. The above equation can also be alternatively expressed as:

\({\varDelta G}_{binding}={\varDelta E}_{MM}+ {\varDelta G}_{psolv}+ {\varDelta G}_{npsolv}-T\varDelta S\) Eq. (2)

where ΔE_MM indicates the molecular mechanics potential energy (Electrostatic and van der Waals contributions), and, ΔG_psolv and ΔG_npsolv are related to the polar solvation energy and non-polar solvation energy, respectively. The term TS is the entropic contribution to the binding energy.

The g_mmpbsa tool was used to calculate distinct factors of arginine and pCAT binding energy [32]. The grid spacing was set to 0.5 Å. Solvent and solute dielectric constants were set to 80 and 2, respectively. A total of 1500 frames were extracted from the last 200 ns (400–600 ns) of the resulting MD trajectory and implemented for the calculation of the average binding energy of the complex. Finally, the per-residue decomposition method was carried out to determine the role of residues of pCAT interacting with arginine.

2.4. Umbrella sampling simulation setup

To determine the potential binding locations of arginine along the transporter, we employed the Umbrella Sampling (US) method, which involved pulling the ligand through the transporter while generating potential location distributions [33]. This technique provides the free energy profile showing the energy boundaries along the transporter path, and we investigate the conformational alterations during the transportation process. The pulling method was carried out to create the initial configurations for performing umbrella sampling simulation. In pulling, a time-dependent external force was artificially applied to arginine along the z-axis (perpendicular to the membrane plane). As a result, arginine was pulled from the binding site toward the intracellular face of the transporter. The velocity and force constant used in pulling were 0.001 nm ps^− 1 and 1000 kJ mol^− 1 nm^− 2, respectively. We extracted a series of windows along the reaction coordinate with a spacing of 0.1 nm. The run time for each umbrella sampling window was 20 ns with a force constant of 1000 kJ mol^− 1 nm^− 2. Finally, the potential of mean force (PMF) was obtained using the weighted histogram analysis method (WHAM) implemented in GROMACS. All analyses were performed over the last 10 ns of the umbrella sampling trajectory.

The preparation of the structure and the images were carried out using VMD software, version 1.9.3 and Chimera, version 1.3 [34, 35].

2.5. Channel detection by CAVER Web

To identify static tunnels and channels within pCAT, CAVER web version 1.1 was used [36]. For this purpose, we extracted the pCAT coordinates from the last snapshot of the CMD trajectory of the Arg/pCAT/membrane system. Then, the structure was uploaded to the CAVER Web server and the tunnels were calculated using a probe radius of 1.2 Å, a shell radius of 3 Å, and shell depth 4 Å.

2.6. Identifying proton wires by Grotthuss wire analysis

In order to investigate the proton wire connected atoms with continuous hydrogen bond formation, we performed Grotthuss analysis by in-home script from GitHub (gitlab.com/KomBioMol/proton_wire) to find water networks connect source residue (here is arginine) or channel forming moieties (Glu115 and Asp237) and the target (periplasmic and cytoplasmic bulk solvent). Angle and distance criteria for hydrogen bonds were considered when the minimum angle between acceptor-hydrogen-donor is 100 degree and the maximum distance between the hydrogen bond acceptor and the donor is 0.35 nm.

3.1. Conformational Stability and Flexibility

To assess the stability of the Arg transporter in the simulated system, the backbone RMSD of protein was calculated during 600 ns of MD simulation (Fig. S1). According to the RMSD values, the system was equilibrated after 250 ns. Hence, we used the timeframe of 400 to 600 ns for the next analyses. To identify the flexible regions of the protein, the root-mean-square fluctuation (RMSF) of Cα atoms was compared for the pCAT and pCAT/Arg systems (Fig. 2). A comparison of the RMSF values between pCAT and pCAT/Arg systems showed that the residues located in loops and N/C termini of pCAT have greater flexibility. The residues located in loops and N/C termini of GkApcT are constructed of residues 1 to 35, 80 to 100, 130 to 160, 240 to 260, 335 to 355, and 400 to 420. The average value of RMSF was 0.05 nm. It may be concluded that arginine binding indirectly affects the flexibility of loops and N/C termini areas and shows that these residues have been involved in the new interactions.

3.2. Binding Energy Analysis

The MM-PBSA approach was applied to calculate arginine binding free energy. According to the RMSD graph (Fig. S1), the binding free energy of arginine was calculated based on the 1500 snapshots taken from the last 200 ns of trajectory. The results showed that the binding energy is about − 287 kJ.mol^− 1 mostly contributed by electrostatic interactions. The contribution of electrostatic interactions was − 625 kJ.mol^− 1, but most of these interactions are neutralized by the polar solvation energy (410 kJ.mol^− 1). To demonstrate the number of water molecules around the arginine ligand, the total number of water molecules was defined within the radius of 0.2 Å around arginine during the last 200 ns of trajectory (Fig. 3A). Having an average of three water molecules around arginine, the Arg binding site in pCAT has been well hydrated (Fig. 3B). The number of water molecules in the Arg binding site of pCAT may be low compared to other transporters. This is because the GkApcT structure has been resolved in an occluded conformational state, and it might have more capacity for water molecules permeation in its open conformational state. The water molecules form multiple hydrogen bonds with nearby polar residues, and they play a critical role in mediating the interactions between pCAT and arginine. A previous study has shown that the exclusion of water molecules in the ligand-receptor complex increases the ligand binding affinity [37]. Wang et al. illustrated that the interaction of ligands with residues in dry regions (non-solvated regions) is highly favorable regarding free energy [38]. These results are supported by Michel et al. who also demonstrated that in drug design, enhancing the affinity of lead molecules by displacing water molecules from a protein binding site can be a useful strategy [39]. Taken together, the water molecules inside the GkApcT transporter is to facilitate the transport of substrates across the channel by providing a medium for solvation, stabilizing the surrounding residues, and facilitating conformational changes during the transport process [14].

The contribution of each residue to the total energy of the arginine binding was calculated by the per residue energy decomposition (PRED) method. According to PRED analysis, the residues Thr43, Asp111, Glu115, Lys191, Phe231, Ile234, and Asp237 were the main contributors to the arginine binding toward pCAT (Table 2). The residue Thr43 (due to having a hydroxyl group in its side chain) and the negatively charged residues Asp111, Glu115, and Asp237 (because of owing the COO⁻ group in their side chains) form favorable salt bridge interactions with arginine. On the other hand, Lys191 (because of having an NH₃⁺ group in its side chain) makes a strong electrostatic repulsion with the guanidinium group of arginine (Fig. 4). Based on hydrogen bond analysis, Glu115, and Asp237 are each individually involved in 3 hydrogen bonds with the arginine ligand (Fig. S2). These results are in agreement with Jungnickle and colleague's results which revealed that residues Thr43, Glu115, Ala119, Lys191, Phe231, Ile234, Glu237, and Ser321 were crucial residues for binding and transport of arginine through pCAT [14]. In addition, it has been shown that Phe231, a conserved amino acid on the transmembrane (TM) helix 6, is crucial for ligand binding, and due to the interaction of Phe231 with Thr43 on the TM1, the extracellular side of the transporter is completely obstructed. However, the intracellular side is less sealed with a thin gate formed by the interactions between Met321, Ile234, and Glu115 [14]. The residue Lys191 displayed a negative contribution to the total binding energy of arginine, but, based on Jungnickle's work, it interacts with carbonyl groups in the unwound region of TM1 and has a role in stabilizing TM1 during the arginine transportation [14].

Table 2

Contribution of each amino acid in the arginine binding within p-CAT/Arg complex
Residues	van der Waals energy	Electrostatic energy	Polar solvation energy	SASA	Binding energy
Thr-43	3.1045	-20.8775	9.372	-1.1155	-9.5155
Asp-111	-0.0575	-37.6225	-4.744	0.0015	-42.2925
Glu-115	3.131	-110.2715	89.314	-1.0045	-18.8275
Lys-191	-0.276	28.3375	-2.487	-0.0725	25.507
Phe-230	-0.3	-3.758	0.038	-0.033	-4.0515
Phe-231	-4.220	-18.298	23.618	-1.631	-0.5305
Ala-232	-2.5115	-7.5945	16.1075	-0.9665	5.035
Ile-234	-1.494	-16.5875	13.89	-1.7905	-5.9925
Asp-237	5.8245	-119.5915	94.628	-1.0255	-20.1585
Tyr-268	-0.403	3.896	5.034	-0.258	8.269
Val-317	-0.95	-2.0245	-1.448	-0.4005	-4.8195
Ser-321	-0.2355	-2.525	-2.736	-0.002	-5.5
Notes: All Energy values are in kJ.mol^− 1

3.3. Protonation-dependent gating mechanism in the pCAT transporter

Previous studies have evidenced that in GkApcT, a water-mediated salt bridge between Asp237 in TM6B and Glu115 in TM3 results in the generation of the intracellular gate of this channel. [14, 16]. The residue Glu115 with a pK_a value of 8.22 is in a protonated state, and the importance of this residue is completely realized for the opening of the intracellular gate. When the proton is released from Glu115, owing to electrostatic repulsion between two acidic residues, TM6B moves away from TM3 and TM8. This movement may lead to a rupture of interaction between the α-amino group of arginine and the carbonyl groups of TM6 (Fig. S3) [14]. In the mutant protein structure (M321S) used in this study, the guanidinium group of arginine is close to both Glu115 and Asp237; therefore, unlike the alanine ligand in the native structure of GkApcT, the presence of arginine in the mutant protein, shifts the pK_a value to lower values through the inductive effect [40], resulting the deprotonation of Glu115. For this reason, the deprotonated state of Glu115 was selected for the simulation.

To measure the distance fluctuation between Asp237 and Glu115, the distance between the center of mass (COM) of Asp237 and the COM of Glu115 was computed in the Arg/pCAT complex during the last 200 ns of the MD trajectory (Fig. 5A). The average distance between the COM of these residues was around 1 nm during the simulation time. The positively charged guanidinium group of arginine keeps the distance between these two residues constant, which is well in agreement with previous studies. [14, 16]. During the arginine transportation, the interaction between these acidic residues is likely disrupted, and the formation of hydrogen bond interactions between carboxylate groups of these residues and the amine group of arginine facilitate the opening of the intracellular gate and thus achieving the entrance of arginine into the pathway. Once arginine passes the gate, the interaction between the acidic residues is re-formed. Can this gating mechanism be an intrinsic feature of the transporter and not related to the arginine ligand? To deal with this issue, the pCAT/memb system (pCAT without the arginine ligand) was MD simulated for 300 ns (Fig. S4). The average distance between COM of Asp237 and Glu115 fluctuated between 1 and 1.2 nm indicating the gating-like mechanism in the transporter without arginine (Fig. 5B). This further emphasizes that the gating mechanism of pCAT is intrinsic, meaning that it is dependent on conformational changes in protein itself rather than on the binding of other molecules, such as protons specifically; it has been proposed that the transporter undergoes an alternate access mechanism, where a large conformational changes occurs in the protein to switch the transporter between an outward-open and an inward-open conformation. The intrinsic gating mechanism exists in other transporters, such as glucose transporter GLUT1 mediating sugar transport and a prototype of the major facilitator superfamily (MFS) formate/nitrite transporters (FNTs) found in prokaryotes and lower eukaryotes. Deng et al. illustrated the foundation for the alternating-access mechanisms of GLUT1 transporter which undergoes a conformational change that alternates the orientation of the substrate-binding site between the extracellular and intracellular sides of the membrane, allowing for the transport of glucose across the membrane [41].

3.4. Characterization of potential channels and water distribution within pCAT

By definition, the channels are specified by two openings connecting different cellular environments. To detect the potential static channels within the pCAT transporter, the CAVER Web server [36] was used. To do this, the conformational structures of the pCAT protein were extracted from the MD trajectory of the related simulated system and uploaded to the CAVER Web server. We found no potential channels within the pCAT protein during 600 ns of MD simulation. It was not surprising because the original structure of the pCAT protein used in this study has been determined in the closed-state conformation (PDB ID 6F34). However, considering the optimal parameters for a tunnel, about 13 tunnels were identified within pCAT (Table S2). We chose a tunnel including the arginine binding site (See Table S2 and Fig. 6). As illustrated in Fig. 6, this tunnel is located in the center of pCAT, where the arginine is binding to the protein. Since pCAT is in the closed-state conformation, the tunnels identified by CAVER Web do not cover the whole arginine transporter channel. However, it seems that the selected tunnel is located in the main arginine pathway.

There are many membrane symporters that transport water molecules along their substrates [42]. The pCAT protein is no exception to this rule. Jungnickle and colleagues have shown that the extracellular and intracellular tunnels of pCAT are filled with water molecules, which are crucial in mediating the interaction between Asp237 and Glu115, and Thr241 and Arg327 [14]. Thermodynamically, the uptake of arginine disrupts the osmotic equilibrium of the cell with its surroundings, thus the water molecules enter the cell through the pCAT transporter to establish a new equilibrium. Water molecules also coordinate the interaction of the α-carboxylate group of the substrate with the unwound region of TM1 and the hydroxyl group of conserved Tyr268 on TM7 [14, 43].

To investigate the presence of water molecules chain within the pCAT transporter, a water wire (WW) or proton wire (PW) analysis was performed by the classical Grotthuss mechanism [44]. Indeed, a proton wire is a chain of atoms involved in hydrogen bonds in which a proton moves from one water molecule to the next. This results in achieving the net flux of water without moving oxygen atoms. We calculated the probability of the proton wire that connects the arginine (ligand) side chain to the bulk solvent in both intracellular and extracellular sides during the MD trajectory. In initial conformations, the residues surrounding arginine in the bottleneck of the channel (Leu48, Val118, Phe230, Phe231, and Ile234) create a hydrophobic barrier, and hence, arginine was entirely inaccessible to water molecules. In the final conformations, however, a PW composing around nine water molecules connects the carboxylate group of arginine to the intracellular bulk water (Fig. 7A). Totally, the probability of PW formation from arginine to the solvent is approximately 75% of all frames; indicating that arginine is accessible to water molecules and participate in the formation of proton wires. In addition, all atoms of the hydrophilic residues such as Thr43, Glu115, Asp237, Ser321, Ser91, Thr94, Ser89, Thr241, Glu15, Glu244, and Ser16 lining the channel can stabilize the PW and make the transporter’s internal space a host for water molecules.

The existence of proton wire was also assessed between Glu115 and Asp237 as the two crucial residues involved in the gating mechanism of pCAT (Fig. 7B). Our study revealed the participation of two water molecules in wire formation between carboxylate groups of Asp237 and Glu115. This PW continues to the barrel axis by five water molecules linking the key proton binding site. The probability of this PW was substantially high, around 90% of all frames during the MD trajectory. The PW analysis suggests that Glu115 can exchange protons freely with the intracellular side. This is due to the existence of arginine leading to a pKa lower for the glutamate residue. A previous computational study has revealed that deprotonation of Glu115 leads to the alternating access transition from the inward occluded state to the inward-open state [16]; indeed, during MD simulation, the initial closed state in the crystal structure evolves more open and expos to the cytoplasmic site leading to exposure of Glu115 to water molecules and the formation of continuous water wire. Indeed, protonation/deprotonation state of Glu115 yield conformational changes from occluded state to inward-open state that is consistent with previous studies on multidrug transporter EmrE in E. coli, which demonstrated that protonation and deprotonation of Glu14 in the binding site can change the orientation of amino acid side chains and alter the ligand [45, 46].

3.5. Force and potential of mean force profiles during the Arg Transportation

To investigate the ligand unbinding mechanism, arginine was pulled out of its binding site in the intracellular direction employing a pulling simulation method. In Fig. 8A, the average force profile as a function of the simulation time is depicted. At the beginning of the process (t = 0), arginine is involved with the mainly polar residues mentioned in PRED analysis; consequently, the pulling force was increased at the beginning of the arginine dissociation process. Based on the hydrogen bond analysis (Fig. 8A), it is shown that the arginine initially formed an average of 10 hydrogen bonds with surrounding amino acids within the first 0.9 nanoseconds. Afterward, an average of 7 hydrogen bonds were broken, resulting in a considerable drop in the average force profile. The arginine unbinding profile reached a peak value of nearly 1190 pN, and arginine was about to leave the binding site of the transporter and reach state (a), where hydrogen bonds formed between specific residues in the transporter and arginine (Fig. 8B). Specifically, the carboxylate groups of Asp237 and Glu115 interacted with the guanidinium group of arginine. Additionally, the α-amino group of arginine interacted with the carbonyl groups of both Phe231 and Ile234; the α-amino groups of both Ala232 and Thr43 interacted with the α-carboxyl group of arginine. These hydrogen bond interactions collectively obstructed the movement of arginine through the related pCAT channel. By pushing the side chains of Thr43, Ala232, Ile40, and Phe231 forward, arginine adopted a state (b), which was stabilized by the hydrogen bonds with Ile234, Ala39, Glu115, and Asp237. In this state, the unbinding profile reached a second peak of around 1235 pN required to pull arginine from this pose down to the channel. At point c, the hydrogen bonding interactions between the guanidinium group of arginine and the carboxylate group of Asp237, as well as between the amino group of arginine and the carboxyl group of Ala39, were still preserved. Subsequently, the pulling force decreased and fluctuated around zero, representing arginine is entirely out of the channel and quite solvent-exposed.

As depicted in Fig. 8A, there is only one minimum energy during the passage of the amino acid Arg through the pCAT transporter. Probably, this cannot be true for the passage of a permeant through a transporter. Many examples in biochemistry literature indicate numerous conformational changes and hence, the numerous minima and maxima during permeant transportation processes [47, 48, 49]. Thus, we performed an umbrella sampling approach to provide the free energy profile showing the energy boundaries along the transporter path, and to investigate the conformational alterations during the arginine transportation process. Several successive energy minima and maxima were obtained in the potential of mean force (PMF) graph (Fig. 9A), indicating conformational energy obtained after breaking and forming specific interactions between arginine and the pCAT protein’s residues. The first energy barrier (between states a and b in Fig. 9) is related to the rupture and re-establishment of hydrogen bonds between arginine and pCAT polar residues Asp237, Glu115, Phe231, and Thr43. These findings are consistent with PRED data, which identified these residues as the crucial interacting residues with arginine in the binding site. In the case of the b state, the hydrogen bonds between the α-amino group of Thr43 and the carboxylate group of Arg are broken, and Arg locates at a conformation that causes PMF to be a positive value. This barrier is related to the movement of arginine through the gate created by Asp237 and Glu115. The guanidinium group of arginine forms strong salt bridges with the carboxylate group of Asp237 and Glu115 residues. The stable interactions between arginine and two acidic residues of Asp237 and Glu115 have been observed in the pCAT crystallographic structure [14, 16]. At state c, a hydrogen bond is established between the hydroxyl group of Ser321 and the carboxyl group of arginine, as well as between the α-amino group of Gly235 and the carboxyl group of arginine. The acidic residues of Glu115 and Asp237 have still formed stable hydrogen bonds with arginine in this state. The next highest energy barrier (between states c and d in Fig. 9) corresponds to the disruption of H-bonds between arginine and the OH group of Ser321, as well as between arginine and Glu115. In addition, the carbonyl group of Tyr116 forms a hydrogen bond with the α-amino group of arginine and causes a sharp peak at around 0.6 nm in the PMF profile along the z-axis of the channel. (state d in Fig. 9). At state e, the critical interactions between arginine and the pCAT residue are: (i) H-bonds between the guanidinium group of arginine and the side chain’s carboxylate of Asp237; (ii) H-bond between the guanidinium group of arginine and the hydroxyl group of Ser91; (iii) H-bond between the guanidinium group of arginine and the hydroxyl group of Tyr116; and (iv) H-bond between the guanidinium group of arginine and the side chain amino group of Lys179. Surprisingly, the arginine permeant is undergone conformational change and 180^⸰-rotation during the d to e transition so that the carboxylate group of arginine is arrayed toward the intracellular side of the transporter. The PMF change from state e to f corresponds to the breakage of the H-bonds existing in the state E and the formation of new hydrogen bonds between the guanidinium group of arginine and the hydroxyl groups of Thr94 and Thr241. In addition, another new hydrogen bond is formed between the hydroxyl group of Ser89 with the carboxyl group of arginine. Finally, arginine leaves the transporter by breaking the hydrogen bonds between the carbonyl group of Glu15 and its guanidinium group and between the carbonyl group of Glu244 and its α-amino group. The PMF graph illustrated an energy barrier of about − 9.42 kJ.mol^− 1 related to polar interactions of arginine with Glu244, Ser89, Glu15, and Ser16, as well as the hydrophilic interaction of arginine with Gln10 at the exit region of the channel, where there may be more exposure to water molecules and PMF graph reached equilibration at around ~ 3 nm distance.

As illustrated in Fig. 9, the arginine rotation during the transportation process from d to e state causes the big PMF difference between these two states. Does this rotation require a conformational change to the pCAT transporter? Do the thin-gate residues (Asp237 and Glu115) experience a conformational change on the arginine rotation? To answer these questions, we measured the distance between the center of mass (COM) of Asp237 and Glu115 in each umbrella sampling window (Fig. S5). By measuring the distance between these two residues, we could monitor the changes in channel radius in each window. The average distance was around 1 nm, and the channel did not undergo significant changes during transportation. To support further our findings regarding the conformational changes during the arginine transportation mechanism, we conducted principal component analysis (PCA) on the trajectory data corresponding to each given window (Fig. S6). The 2D projections of trajectories on eigenvectors illustrate that the conformational changes in the channel during the arginine rotation were minor, and these conformations are very similar in terms of the dominant motions captured by the principal components. These findings suggest that the arginine transporter maintains its structural integrity during transportation, supporting its functional role as a transporter. Arginine passes through the pCAT channel without causing significant conformational changes in its structure. This rotation is influenced by the interaction of arginine with surrounding residues which makes arginine adopt a more favorable conformation.

Previous studies have shown that due to the high dielectric constant and the consequent shielding effects, water molecules can play a critical role in the strength of the interactions of the ligand with their neighbor residues; more water molecules, weaker hydrogen bonds, more fragile ionic bonds, and weak electrostatic bonds [50, 51]. For this reason, the number of water molecules around arginine in every specified conformation in the PMF profile was calculated (see inserted table in Fig. 9). It is shown that the average number of water molecules around arginine in states a and b is low and approximately equal to 3. As arginine moves from the binding site toward the intracellular side of the pCAT channel, the number of water molecules around the ligand increases gradually. The maximum number of water molecules related to state h, where arginine almost exits the transporter and is placed inside the solvent. The low number of water molecules in the binding pocket (states a and b in Fig. 9) is owing to the low bottleneck radius in pCAT and the proximity of arginine functional groups to channel lining residues. Horner and colleagues have shown that the mobility of the water molecules in transporters mainly depends on the number of hydrogen bond interactions they form with residues in the channel wall [52]. Thus, the formation of hydrogen bonds between water molecules and polar residues Asp237 (TM8), Glu155 (TM3), Glu115, Thr241 (TM6), Glu115 (TM3), and unwound region of TM1 lead to the movement of waters through the transporter channel. As a result, arginine moves toward the intracellular side of the cell along with many water molecules.

In the present study, we investigated the unbinding mechanism of arginine out of the corresponding binding pocket down toward the cell using molecular dynamics simulation and umbrella sampling techniques. PRED analysis showed that Thr43, Asp111, Glu115, Lys191, Phe230, Ile234, and Asp237 are the central interacting residues with arginine in the binding site. The potential of mean force obtained by umbrella sampling data illustrated that there are potential barriers along the channel, especially in the arginine-binding site, that hinder the movement of arginine. The transportation mechanism is primarily characterized by the sequential formation and breakage of hydrogen bonds between arginine and those residues lining the wall channel. Furthermore, MD studies unveiled the water permeation through the channel with low capacity revealing the implication of water molecules in mediating interaction between Asp237 and Glu115. Arginine binding promotes the deprotonation of Glu115 and the conformational transition to be more accessible to the water, which hints at the role of Glu115 as one of the main drivers of arginine transition through the channel. The obtaining results here facilitate a further explanation of the CAT transporters' activity which might be helpful in drug discovery and biochemical research to combat CAT-related cancers.

CAT Cationic Amino acid Transporter

SLC Solute Carrier

APC Amino Acid-Polyamine-OrganoCation

TM Transmembrane

PDB Protein Data Bank

MD Molecular Dynamics

MM-PBSA Molecular Mechanics Poisson-Boltzmann Surface Area

PMF Potential of Mean Force

PBC Periodic Boundary Conditions

VMD Visual Molecular Dynamics

RMSD Root-Mean-Square Deviation

RMSF Root-Mean-Square Fluctuation

PRED Per-Residue Energy Decomposition

PW Proton Wire

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Hamid Mahdiuni contributed to the study’s conception and design. Maral Afshinpour and Parinaz Parsi performed computation, data collection, and analysis. Maral Afshinpour wrote the original draft of the manuscript. Hamid Mahdiuni edited and reviewed the manuscript. All authors read and approved the final manuscript.

Data Availability

All data needed to evaluate the conclusions in the paper are present in the manuscript and the supplementary materials. MD simulations were performed adopting the GROMACS package. CHARMM-GUI web server was further adopted in the preparation of parameters and input for MD simulations. The preparation of the structure and the analysis of trajectories were performed by adopting the built-in tools in the GROMACS package, Chimera, and VMD software. The tunnels and channels were detected by the CAVER web server, version 1.1. Characteristics of the channels detected by the CAVER Web server are accessed with Job ID vqyyfr at the CAVER Web homepage: CAVER Web - tool for the analysis of tunnels and channels in protein structures (muni.cz)

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Investigation of molecular details of a bacterial cationic amino acid transporter (GkApcT) during arginine transportation using molecular dynamics simulation and umbrella sampling techniques

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Journal Publication

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Preparing membrane-protein system

2.2. Molecular dynamics (MD) simulation production

2.3. MM-PBSA binding free energy

2.4. Umbrella sampling simulation setup

2.5. Channel detection by CAVER Web

2.6. Identifying proton wires by Grotthuss wire analysis

3. Results and Discussion

3.1. Conformational Stability and Flexibility

3.2. Binding Energy Analysis

3.3. Protonation-dependent gating mechanism in the pCAT transporter

3.4. Characterization of potential channels and water distribution within pCAT

3.5. Force and potential of mean force profiles during the Arg Transportation

4. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

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