1. Introduction
G protein-coupled receptors (GPCRs) are one of the largest class of transmembrane proteins used as a therapeutic target. GPCRs are cell surface proteins involved in mediating and regulating wide range of biological processes including immune system, odor, vision, homeostasis, etc. [
1,
2]. They have also been associated with many disease conditions, such as Alzheimer’s disease, depression, pancreatic cancer, type 2 diabetes mellitus, obesity, cardiovascular diseases, Parkinson’s disease, schizophrenia, and neurological diseases [
3,
4]. As of 28 October 2022, nearly two-third responses of human hormones and one-third of FDA approved drugs directly involve targeting GPCRs (
https://gpcrdb.org), while approximately 500 novel drug candidates targeting GPCRs are in clinical trials [
5,
6,
7]. Human GPCRs can be classified into five different classes—class A (rhodopsin family), class B1 (secretin family), class B2 (adhesion family), class C (glutamate family), class F (frizzled or taste 2) [
3]. As of 28 October 2022, out of 826 human GPCRs identified, 165 are validated drug targets and more than 350 have been regarded as druggable (
https://gpcrdb.org/structure/statistics) [
2]. The function and physiological effect of GPCRs is obtained through their ligand recognition and receptor activation. The activation of GPCRs depends on their endogenous ligands and signals such as amines, peptides, lipids, proteins, small molecules, hormones, neurotransmitters, photons, odors, chemokines, etc. and a variety of intracellular transduction cascades (involving different G-proteins and second messengers) [
3,
8]. Owing to the pharmacological significance of GPCRs, investigating and exploring the ligands that interact with GPCRs and activates them is of immense importance.
Interestingly, the G-Protein Coupled Receptor (GPCR)—GPR109A, a class A GPCR, (also known as hydroxycarboxylic acid receptor 2—HCAR2 or HM74A in humans and PUMA g in mice) is expressed in variety of cells and tissue types, more robustly in osteoclastic precursor macrophages [
9,
10]. GPR109A is now recognized as an important target of niacin (the essential nutrient, vitamin B3 or nicotinic acid) and subsequent interaction of these two molecules led to widespread clinical examinations for the treatment of dyslipidemia and to increase HDL cholesterol [
11,
12]. Niacin has also been reported to limit lipolysis and hepatic acid accumulation independent of GPR109A without significant metabolic disturbances while fasting [
13]. Hippuric acid (HA) which is structurally similar to niacin, and one of the naturally occurring compound belonging to Phenolic Acids (PA) available in blueberry diet has been shown to interact with GPCR—GPR109A inhibiting the process of bone resorption and thereby increasing the bone mass [
14]. We therefore discuss the interaction of HA with GPCRs in relation to bone resorption and bone formation.
Although, importance in bone formation and resorption has mostly been given to micronutrients such as calcium, vitamin D, phosphate and macronutrients comprising fats and proteins [
15], recent studies by Chen et al., have shown significantly increased bone formation in rapidly growing male and female rodents when supplemented with a blueberry diet [
14]. Specifically, hippuric acid (HA), 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), and PA mixture comprising of all the seven metabolites were found to be potentially bioactive on stimulating osteoblast differentiation and proliferation in cell cultures [
14,
16]. Gene deletion studies (GPR109A
−/
−) in mice, conducted by Chen et al., have revealed significantly higher bone mass and strength in tibia and spine of mice (weaned 4-week-old and 6-month-old) using densitometric, bone histologic, and molecular signaling analytic methods [
17]. It was also observed that there is a significant decrease in the several bone resorption markers in serum and bone marrow plasma of GPR109A
−/
− mice. Additionally, in GPR109A
−/
− mice compared with their respective untreated control mice, HA considerably inhibited bone resorption and increased bone mass in wild type mice but had no additional effects on GPR109A
−/
− mice [
17].
Studies on the structural determinants of GPR109A receptor binding to niacin have been investigated using site-directed mutagenesis by Tunaru et al., revealed putative ligand binding residues in GPR109A receptor, and did not show any interaction of niacin to its close homolog with 95% amino acid sequence identity, GPR109B (also known as hydroxycarboxylic acid receptor 3—HCAR3 and HM74 in humans) [
18]. Based on the results of (GPR109A
−/
−) knockout studies, site-directed mutagenesis and structural similarity, it can be predicted that the actions of PAs on bone cells are mediated through a mechanism involving specific activation of GPR109A receptor. Other studies involving ligand binding assay (
35S-GTPγS) using membrane prepared from mouse fetal calvarial cells have shown that HA stimulates
35S-GTPγS binding to membranes transfected with GPR109A but not to GPR109B. It has been hypothesized that HA mediates it action on bone cell through binding to GPR109A [
19]. Further, results have also confirmed that HA binds to GPR109A similar to niacin but has a weak interaction with GPR109B [
19].
In the present study, we implemented a computational pipeline involving molecular docking studies, molecular dynamics simulations and Molecular Mechanics Poisson Boltzmann Surface Area (MM/PBSA) calculations [
20] combined with in silico mutational analysis and time-resolved circular dichroism spectroscopy to reveal the structural determinants of HA interaction with GPR109A. Taken together, our descriptive and predictive models accord with published findings and support the idea that HA interacts with a higher affinity towards GPR109A than GPR109B, and the residues involved in binding HA to GPR109A are very similar to the ones that mediate binding of niacin molecule.
2. Results and Discussion
In the present work, we have performed two separate MD simulation runs on each GPR109A-HA and GPR109B-HA complexes. For both the complexes, the first run (
pose-0) is the AutoDock predicted top ranked pose with largest population size for both GPR109A and GPR109B structures, whereas the other run (
pose-1) is the conformation from second largest AutoDock scored cluster. For easy interpretation in the naming pattern,
pose-A0 and
pose-A1 refers to GPR109A-HA complex whereas
pose-B0 and
pose-B1 refers to GPR109B-HA complex. All these complexes were subjected to 200 ns MD simulation followed by trajectory analysis; RMSD based structural clustering of GPR109A/B-HA interaction (
Supplementary File S1), and MM/PBSA calculations, which predicts comparative binding energy (not the absolute binding free energy) to check the energetic stability of all the complexes. RMSD plot (
Figure 1) of the protein-ligand complex for all four simulations shows the stability of the complex during the simulation.
2.1. Interaction of Hippuric Acid with GPR109A
The docked structure of HA with GPR109A in
pose-A0 (
Figure 2A) shows HA at a H-bond distance of R111 (a TMH3 residue). During MD simulations, it is observed that HA makes H-bond interaction with TMH4 residue K166, as well as with the ECL2 residues S178 and S179 (
Table 1). These interactions of HA with GPR109A residues also can be seen in the LigPlot schematic representation of the representative structure of the most dominant cluster from MD simulation (
Figure 3A). Visualization of automated docking through VMD shows that HA aromatic ring is stacked between the aromatic sidechains of F255 (TMH6) and F276 (TMH7) and similar arrangement is also observed in cluster representative structure from MD simulation (
Figure 2B). Stacked in between the aromatic rings of F255 (TMH6) and F276 (TMH7), HA makes stable contacts in the binding pocket by forming H-bonds with K166-S178-S179 triad as well as hydrophobic interactions with F255-F276 throughout the MD simulation as detailed in ligplot diagram (
Figure 3A).
In
pose-A1, the conformation from second largest AutoDock scored cluster, HA is docked in the pocket formed by TMH4, TMH5 and TMH6, near to ECL2 without any H-bond interaction with residues in ECL2 region (
Figure 2A). During the MD simulations, HA interacts via H-bonds with R111, K166 and R251 (
Table 1). However, in the cluster representative structure, HA occupies the similar binding confirmation as
pose-A0 (
Figure 2B) making interactions with S178, S179 and K166 (
Figure 3B).
The simulation results have also shown that HA occupies a binding site similar to that of niacin in GPR109A. Comparing HA binding with the results from our earlier studies of niacin [
21] and acifran [
22] with GPR109A, it is evident that the residues in ECL2 region (S178 and S179) and TMH4 region (K166) along with F255 (TMH6) and F276 (TMH7) are mainly involved in ligand binding. The ligand binding site of GPR109A comprising the residues in ECL2 region (S178, S179) was also experimentally reported in a mutagenesis study by Tunaru et al. [
18]. HA being a hydroxyl carboxylic compound and structurally similar to niacin, it is expected to interact in a similar manner and occupy the similar binding site to niacin.
2.2. Interaction of Hippuric Acid with GPR109B
In the first complex of GPR109B-HA, i.e., top scored AutoDock predicted model
pose-B0 (
Figure 4A), HA is present at the H-bond distance of R111 (TMH3), and the aromatic ring is surrounded by hydrophobic residues of TMH2; V83, Y86, and Y87, ECL1; W93, and TMH3; V103 and L104. However, in the cluster representative structure from MD simulation (
Figure 4B), HA makes two H-bonds; one with Y86 (TMH2) through carboxyl group and another with C177 (ECL2) (
Figure 3C) and the HA aromatic ring is surrounded by the hydrophobic side chain of residues from TMH2; V83, Y86, ECL1; W93 (ECL1), and TMH3; V103, L104, and F107. H-bond analysis have further shown that, HA makes H-bonds with Y86, Y87 (TMH2), C177 (ECL2) and S91 (ECL1) (
Table 2) during MD simulation.
In
pose-B1, the conformation from second largest AutoDock scored cluster (
Figure 4A), HA carboxyl group makes H-bond with Y87 (TMH2), and the aromatic ring of HA is near to the residues L76 (TMH2) and F107 (TMH3). After MD simulation, in the cluster representative structure from the most dominant cluster, HA occupies similar conformation as that of
pose-B0 (
Figure 4B) and forms a similar H-bond profile as that of
pose-B0 (
Table 2). Our simulation results have also shown that GPR109B harbors a different binding site for HA than that of GPR109A (
Supplementary Files S2 and S3). In GPR109B, HA interacts with the residues from ECL1 (Y86, Y87, S91) and TMH4 (C177) region occupying a binding site in the crevice formed by ECL2, TMH3 and TMH4.
2.3. MM/PBSA Calculation
GPR109A-HA complexes: The calculated binding energies of GPR109A-HA complexes;
pose-A0,
pose-A1 are −22.77 (±4.67), and −14.96 (±4.87) kcal/mol, respectively (numbers in the parenthesis are standard deviations) (
Table 3). The negative energy values suggest that all the complexes are energetically stable. In
pose-A0, HA is stabilized by forming H-bonds with ECL2 residues K166, S178 and S179, a salt bridge with K166 (TMH4), and hydrophobic interactions with F255 (TMH6) and F276 (TMH7) during MD simulation. However, in
pose-A1, HA is stabilized by the H-bond formation with R111, R251 and K166. Compared to
pose-A0, the H-bond occupancy for HA is less in pose-A1 and has weaker binding energy as demonstrated by MM/PBSA energy.
GPR109B-HA complexes: For GPR109B-HA complexes;
pose-B0 and
pose-B1 the calculated binding energies are −13.52 (±7.07) and −1.48 (±5.78) kcal/mol, respectively (numbers in the parenthesis are standard deviations) (
Table 3). In both these complexes, HA is stabilized by the formation of H-bonds with Y87, S91, C177 (ECL2) and Y86 (TMH2). In addition to H-bond, HA is also stabilized by the formation of a salt bridge with R111 (TMH3), and R251 (TMH6). MM/PBSA binding energy values shows that
pose-B1 is energetically less favorable compared to
pose-B0.
According to calculated binding energy values, all the complexes of GPR109B-HA are stable. Compared to GPR109A-HA complexes, GPR109B-HA complexes are energetically much less favorable. This is well supported by the finding of the weak binding of HA with GRP109B as observed by Chen et al., [
19]. Comparing the MM/PBSA binding energy of GPR109A-HA complex with GPR109A-Niacin (−6.2 ± 5.1 kcal/mol) study from our previous study, GPR109A has higher affinity for HA [
21].
2.4. Chimeric Structure Analysis
The results from H-bond analysis and MM/PBSA calculation have shown that HA has higher binding affinity with GPR109A than that of GPR109B. The major residues that are responsible for binding of HA to GPR109A are present on TMH4 and ECL2—
K166 (TMH4), S178 (ECL2) and S179 (ECL2) while residues in
TMH2 (Y86, Y87) and ECL1 (S91) play a major role in binding HA to GPR109B. To understand the importance of the specific residues of GPR109A and GPR109B for binding of HA, we generated two in silico chimeras of GPR109A and GPR109B similar to Tunaru et al., study [
18]. Chimera 3A4B consists of first three TM helices including the junction—TMH2/ECL1 from GPR109A and remaining four helices from GPR109B, while in chimera 3B4A, first three TM helices including the junction TMH2/ECL1 are taken from GPR109B and remaining four helices from GPR109A [
21].
The binding affinity of HA with chimera 3A4B and chimera 3B4A estimated from MM/PBSA calculation is −7.53 kcal/mol (±5.72) and −23.54 kcal/mol (±7.17), respectively (
Table 3). MM/PBSA analysis from MD simulations have shown that the affinity of HA decreases for chimera 3A4B than that for GPR109A. This decrease in interaction can be attributed to the loss of one of the major interacting residues S178 to I178 in chimera 3A4B. Additionally, HA has smaller H-bond occupancy with chimera 3A4B as compared to GPR109A (
Table 1 and
Table 4). The weak interaction is due to the absence of the polar residue S178 and loss of interaction with R251 in chimera 3A4B. In contrast to
pose-A0, carboxylic group of HA has H-bond interaction with R111 in chimera 3A4B. Absence of residue—S178 in the ECL2 region of chimera 3A4B might have paved a path for HA to move deep inside down the transmembrane helix to interact with R111. The cluster representative structure from MD simulation shows carboxyl group of HA interacting with the side chain of basic residue K166 (
Figure 3E).
The main residues of GPR109B interacting with HA via H-bonds and hydrophobic interaction are in ECL2, THM2 and TMH3 region. For the second chimera 3B4A, which contains first three helices including ECL2 from GPR109B and remaining from GPR109A, MM/PBSA analysis shows that HA has stronger affinity for chimera 3B4A as compared to GPR109B but has comparable binding energy to GPR109A
pose-A0. Representative structure forms the most dominant cluster for chimera 3B4A shows that HA occupies a binding site surrounded by TMH3 and TMH4 with hydrophobic interaction mainly from F255, H189, F186 and L258 (
Figure 3F). Furthermore, HA is stabilized in the binding site of chimera 3B4A by H-bond interaction with basic residues R111, H189 and R251 (
Table 5). The presence of 4 helices (TMH4, TMH5, TMH6 and TMH7) along with ECL2 and ECL3 from GPR109A in chimera 3B4A shifted the binding site of HA away from the location in GPR109B. Even in the presence of all the major interacting residues from GPR109B (Y87, C177, Y86, S91, L104) in chimera 3B4A, HA was found to move towards the binding site of GPR109A. This strengthens the finding that HA has stronger binding affinity to GPR109A. The major difference in the amino acid sequence near binding site of GPR109B and chimera 3B4A is the presence of S178 in the later. S178 has been a crucial residue in the binding of HA to GPR109A and similar other small carboxylic acid ligands like niacin and acifran [
21,
22]. The abrogation of binding for niacin and acifran with GPR109A mutant S178I also explains the importance of S178 in carboxylic acid ligands binding.
In a mutagenesis study by Tunaru et al., for similar chimeric structures, it was shown that chimera 3A4B was inactive with niacin and had very low binding of EC
50 (half maximal effective concentration) value greater than 100 µM for acifran [
18]. However, chimera 3B4A had stronger interaction with acifran (EC
50 value ~2 µM) and weak binding with niacin (EC50 value > 100 µM). HA, which is quite larger than niacin in size, but similar to acifran (in terms of size and structure), shows strong interaction with chimera 3B4A similar to that of acifran. This indicates that residues in ECL2 (S178, S179) and TMH3/TMH4/TMH6 (R111, K166, R251) regions are the major contributors in strong binding of HA to GPR109A.
2.5. Free Energy Landscape
Free energy landscape (FEL) analysis was performed using cpptraj module of AMBER16 [
23] to explore the lowest energy conformation obtained during the simulation. A stable protein-ligand system must possess a well-defined energy minimum. In this study, results of FEL for a 200 ns simulation were obtained by mapping Gibbs free energies to the first two principal components—PC1 and PC2 (
Figure 5). Principal Component Analysis (PCA) is a method of accessing most significant dynamics by transforming fast local atomic motions from MD trajectories into dominant functional motions. PC1 and PC2 capture the most dominant functional motions from MD trajectories [
24]. The energy minimum and energetically favored protein-ligand conformations are represented by dark purple spots in FEL whereas, yellow area represents unfavorable conformations. GPR109A-HA complex, pose-A0 (
Figure 5A) shows a distinct energy minimum displaying a stronger binding as compared to all other complexes. Chimeric structures (
Figure 5E,F) have a less pronounced energy minima in the FEL representation showing a weak binding of HA.
2.6. Circular Dichroism
Time resolved circular dichroism spectra revealed that HA interaction with GPR109A and GPR109B led to small conformational changes in the secondary structure content (α-helix, β-sheet and turns) of the protein (
Table 6). A small decrease in the alpha helical content of GPR109A (5.3%) and GPR109B (9.6%) was observed on interaction with HA. Marginal increase in the turns were observed in GPR109A (5.7%) after its interaction with HA, whereas for GPR109B, interaction with HA led to the slight decrease (4.8%) in turns. This slight change in secondary structure by HA binding might be responsible for the activation of GPR109A, as small conformational changes in the secondary structure of GPCRs are associated with its activation [
25].
2.7. Alanine Mutation Analysis
Since GPR109A-HA complexes are energetically more stable over GPR109B-HA, we considered GPR109A-HA complexes for further in silico mutation analysis to compare the importance of the residues of GPR109A that are interacting with HA for stable binding. Through interaction analysis of GPR109A with HA in MD simulations, we concluded that residues K166 (TMH4), S179 (ECL2), S178 (ECL2) and positively charged residues R111 (TMH3) and R251 (TMH6) are important for HA binding to GPR109A. Additionally, aromatic residues F255 (TMH6) and F276 (TMH7) are also concluded as important residues as they are found to be in the surrounding of HA during MD simulations. Based on these observations, we took the representative structures of the most dominant cluster from MD trajectory of complexes pose-A0 and pose-A1 and then subjected the identified critical residues to Alanine mutation. The mutated complex (GPR109A-HA) was energy minimized followed by 50 ns MD simulations for further analysis.
K166-S178-S179 Alanine triple mutant: According to H-bond analysis of
pose-A0 complex, residues; K166 (TMH4), ECL2 residues; S178 and S179 are involved in H-bonding with HA during MD simulation. So based on these results, we generated Ala triple mutants for these residues taking representative structure form the most dominant cluster as the starting structure. Visualization of 50 ns MD trajectory and H-bond analysis both show that due to the Ala mutation HA moved away from TMH4 residue K166A, and ECL2 residues: S178A and S179A during MD simulation. Instead, HA’s carboxyl group made a H-bond with N-terminal residue N17 with occupancy of 20% of the whole MD trajectory. Aromatic ring was surrounded by TMH6 hydrophobic residues; I254, F255, and L258, while in the wild type it is stacked between residues; F255 (TMH6) and F276 (TMH7) (
Supplementary File S4). Thus, according to this triple mutant, K166 (TMH4), ECL2 residues S178 and S179 are important for anchoring HA into the binding site similar to our calculations performed on our previous work on niacin binding to GPR109A [
21].
R111-R251 Alanine double mutant: As HA is energetically stable and makes H-bonds with R111 and R251 in
pose-A1, we generated a double Ala mutant. In the alanine double mutant, HA moved towards ECL2 residues; S178 and S179 during MD simulation and made H-bonds with these residues with an occupancy time of 12% and 19 %, respectively. The HA aromatic ring was surrounded by TMH6 hydrophobic residues: I254, F255, L258, and TMH7 residue F276. Compared to K166A-S178A-S179A triple mutant where HA was unable to reach the arginine binding site, in this specific double mutant, HA was unable to stay near the R111A (TMH3) and R251A (TMH6) due to the absence of any salt bridges. As a result, HA moved towards the ECL2 residues; S178 and S179 (
Supplementary File S5).
F255-F276 Alanine double mutant: In complexes
pose-A0 and
pose-A1, it has been observed that residues F255 (TMH6) and F276 (TMH7) were present near the HA aromatic ring during MD simulations, thus, we considered analyzing the importance of these residues in HA binding to GPR109A. We took the cluster representative structure of two complexes,
pose-A0 and
pose-A1, to generate the alanine mutant complexes: F255A-F276A-A0 and F255A-F276A-A1 In
pose-A0, HA makes H-bonds with K166 and ECL2 residues in the wildtype receptor; S178, and S179, and the aromatic ring is stacked between F255 (TMH6) and F276 (TMH7). The H-bond calculation for mutant complex, F255A-F276A-A0, shows that H-bonds of HA with K166, S178 and S179 that were present in wild type complex are also present in the mutant complex with an occupancy time of 17%, 62% and 47%, respectively. Due to alanine mutation of residues F255 (TMH6) and F276 (TMH7), the HA aromatic ring no longer remains in this position during MD simulation and instead shifts to a new site surrounded with ECL2 residues; F186, W188, and H189 (
Supplementary File S6). In complex
pose-A1 in WT receptor, the HA carboxyl group interacts with R111 (TMH3), R251 (TMH6), and the aromatic ring is surrounded by hydrophobic residues F255 (TMH6) and F276 (TMH7). In contrast, according to H-bond analysis, HA makes interactions with R111 (TMH3), K166 (TMH4), and R251 (TMH6) with an occupancy time of 57%, 24%, and 52%, respectively, in the F255A-F276A-A1 mutant complex. The HA aromatic ring is surrounded with residues; I254, L258, Y269 throughout the MD simulation. Thus, according to both the mutant complexes (F255-F276-A0 and F255-F276-A1), the hydrophobic residues F255 (TMH6) and F276 (TMH7) in wild type GPR109A are essential to strengthen HA binding (
Supplementary File S7). However, as these aromatic side chains are not present in the mutants, HA makes alternate interactions with other residues because of the presence of relatively stable H-bonds and salt bridges despite the absence of hydrophobic interactions with the aromatic residues.
K166-S178-S179-R111-R251-F255-F276 Alanine combined mutant: Finally, we also analyzed the combined alanine mutant of all the above seven residues. To generate the alanine mutant, we considered a frame from MD simulation with
pose-A0 in which the HA carboxyl group made interactions with S178, S179 (ECL2 residue), and the aromatic ring is surrounded by F255 (TMH6) and F276 (TMH7). Visualization of MD trajectory shows that HA moves away from the initially bound position after 4 ns of the MD simulation run, and by the time the simulation reaches the time of 6 ns, HA completely moved away from the initial binding site and exited out from the GRP109A protein (
Supplementary File S8). This specific combined mutant confirms the loss of interaction of HA with protein pointing out the importance of these residues (K166-S178-S179-R111-R251-F255-F276) in HA binding.
4. Conclusions
Structural basis of HA binding to GPR109A based on computational analysis (automated docking, MD simulation, H-bond analysis, and MM/PBSA analysis) clearly revealed that HA has stronger binding with GPR109A, while the binding of HA with GPR109B is comparatively weaker. Our computational results are in agreement with our experimental findings on HA interaction with GPR109A and GPR109B [
19]. Furthermore, MD simulations of WT and ALA mutant receptors highlighted important residues of GPR109A for HA binding. Chimeric constructs of GPR109A/GPR109B study strengthens the importance of residues S178, R111 and R251 for HA binding. Drastic reduction in binding energy with chimera 3A4B shows that ECL2 residue S178 is a marker for binding HA. Similar residues have already been found essential for binding of niacin and acifran to GPR109A [
18,
22]. Additionally, time-resolved circular dichroism spectra showed small conformational changes in the secondary structure content (α-helix, β-sheet and turns) of GPR109A and GPR109B on HA binding. Overall, the results presented here indicate that GPR109A serves as an important potential target for HA binding. Moreover, in the future, experimental techniques such as, bioluminescence resonance energy transfer (BRET) [
33], atomic force microscopy based force spectroscopy (AFM-FS) [
34], molecular recognition imaging (MRI) [
35], isothermal titration calorimetry (ITC) and fluorescence cross-correlation spectroscopy (FCCS) [
36], surface plasmon resonance (SPR) spectroscopy [
37] can be helpful to study and validate GPR109A/B interactions with HA.