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Molecular Interaction Mechanism of Tyramine, Phenethylamine Binding with Adrenergic Receptor [beta]2.

Byline: Ming Guo, Yan Wang, Xiaomeng Wang, Min Guo, Yanke Jiang and Yan Zhang

Summary: The interactions of [beta]-phenylethylamine ([beta]-PEA) and tyramine with adrenergic receptor [beta]2 (ADR[beta]2) were studied by molecular docking and molecular dynamics (MD) simulation methods. The binding free energies of the complexes were calculated and used to characterize the interactions of ADR[beta]2 with [beta]-PEA and tyramine, respectively. The driving force of ADR[beta]2 binding with [beta]-PEA includes Van der Waals interaction, electrostatic interaction and hydrophobic interaction, while only Van der Waals interaction exists between tyramine and ADR[beta]2. Tyramine exists in the flexible TM6 area of ADR[beta]2 which is unstable, so tyramine is hard to bind with ADR[beta]2. Compared with tyramine, [beta]-PEA firmly binds to the stable TM3 region through hydrogen bonds between [beta]-PEA and protonated Hie 65 of ADR[beta]2.

Keywords: Tyramine; [beta]-phenylethylamine; Adrenergic receptor [beta]2; molecular docking; Molecular dynamics simulation

Introduction

Tyramine is one kind of monoamine, also known as 2-(4-hydroxyphenyl) ethylamine which occurs naturally in cheese and other foods. A trace amount of tyamine can cause migraine. It can prompt the release of catecholamines, but cannot pass the blood-brain barrier [1-5]. Early in 1909 [6], Barger isolated tyramine from ergot, and pointed out that it had the similar function of the adrenaline towards blood pressure and uterine. Although the toxicity and activity of tyramine are relatively small, it can work for a long time. Barger subsequently separated the tyramine from rotten fish, and confirmed that tyramine can increase the blood pressure and excite the uterine [7]. In 1952, James and Kearns [8] reported that there was a toxin in American cockroach hemolymph which was poisoned and paralyzed by dual-chlorophenyl trichloroethane (DDT).

This toxin can lead the release of high frequency nerve impulses when disposed on the abdominal nervous systems of cockroaches, and high concentrations of it can block the nerve conduction after excitement. In 1984, Chang and his companion pointed out that [9] the insect neurotoxin may be tyramine, and its effect was not limited to insects' nerves. Therefore, the reasons for the tyramine can kill the insect may be the tyramine can restrain insects' nerves, blood circulation and digestive activity when the concentration of tyramine increased in DDT poisoned insects' hemolymph.

Phenethylamine (PEA), an aromatic amine, is a colorless liquid at room temperature. It is soluble in water, ethanol and diethyl ether. Similar to other low molecular weight amines, it smells fishy. It will react with carbon dioxide to form the corresponding carbonate solid when exposed to air. PEA is a strong salt base [10] which can form stable crystalline hydrochloride whose melting point is 217 degree Celsius. PEA may also be a skin irritant and a photosensitive agent.

[beta]-phenethylamine ([beta]-PEA) or 2-phenylethylamine, is an alkaloid and monoamine neurotransmitter. In the human brain, [beta]-PEA can act as the nerve regulating substances, neurotransmitters and trace amine [7, 11-14]. [beta]-PEA is a natural compound that is composed by the decarboxylation of the amino acid Phe which is treated by enzyme. It can also be found in many foods, such as chocolate, especially after microbial fermentation. Most people think that phenylethylamine in food is enough to work on the nerve. However, it is soon metabolized by the monoamine oxidase enzyme, preventing it concentrating on the brain effectively.

Adrenergic receptors (ADR) can be activated by catecholamines adrenaline and noradrenaline [15]. ADR receptors are divided into [alpha] and [beta] two types in the manner of react with pharmaceutical. [alpha] receptors are divided into [alpha]1 and [alpha]2 receptors; [beta] receptors are divided into [beta]1, [beta]2, [beta]3 receptors. [alpha]1 receptors [16] are mainly distributed throughout vascular smooth muscle, heart and liver. [alpha]2 receptors locate in the vascular smooth muscle, the presynaptic membrane, adipose cells, platelets, liver cells. [beta]1 receptors locate in the heart, adipose tissue and renal vascular, platelet, salivary glands, and gastrointestinal tract/mesenteric artery. [beta]2 receptors locate in the smooth muscle, skeletal muscle, liver and presynaptic membrane. [beta]3 receptors locate in fat muscle, liver and mast cells, fat cells, and kidney tissue, which indicate that it may have a moderating effect on lipolysis [17].

These ADRs in various organs are dominated by post-ganglionic sympathetic and can react with epinephrine and norepinephrine. Its chemical nature is still a mystery. Epinephrine has the effect on both [alpha] and [beta]-adrenergic receptors while norepinephrine mainly affects [alpha] receptors. [alpha] receptors can lead to vasoconstriction, pupil diffusion and can be inhibited by double-benzylamine, ergot poisoning and other [alpha] blockers [18]. [beta] receptors can cause bronchiectasis, vasodilation etc. and can be inhibited by 3, 4-dichloro-isopropyl (D-CI) and other blocking agents [19].

ADR is one of the biogenic amine receptor family members, the amino acid sequence of receptor protein can be divided into N-terminal, intracellular domain, C-terminal, conserved transmembrane domains and extracellular regions [20]. N-terminal is extracellular while C-terminal is intracellular. Seven conserved transmembrane domains' [alpha]-helixes across the phospholipid bilayer repeatedly (Fig. 1). Generally, the conservative of transmembrane region residues is relatively strong, but the residues of C, N-terminal and loop region are relatively variable, less conserved [20].

Molecular dynamic simulations of guest-host interactions are now being used as tools for understanding the complexation process, particularly the driving forces for complex formation as well as the optimal geometries of the resulting complexes [21, 22]. To investigate the binding models of ADR[beta]2 with PEA and tyramine, 1 ns MD simulation has been used on ADR[beta]2-PEA/tyramine complex. In this paper, the interactions of ADR[beta]2 with PEA and tyramine were analyzed and compared by MD simulations.

Experimental

Simulation and Template Processing

The molecular structures of tyramine and [beta]-PEA were shown in Fig. 2. The formula of tyramine is C8H11NO, molecular weight is 137 D, the surface charge is zero, and the number of potential hydrogen bond acceptor is one. The formula of [beta]-PEA is C8H11N, molecular weight is 121 D, the surface charge is zero, and the number of potential hydrogen bond acceptor is two. Tyramine is different from PEA whose H opposites to -CH2CH2NH2 is replaced by -OH. The 3D molecular structures of tyramine and [beta]-PEA were established by Chem3D Ultra 8.0 software, then the molecular structures of tyramine and [beta]-phenethylamine were carried out the MM2 energy minimization, The geometric parameter of histamine was optimized at the HF/6-31G* level with Gaussian09 package. Atomic charge parameters were obtained and then import the resulting parameter file into Amber for the subsequent calculations simulations.

The X-ray crystal structure of ADR[beta]2 (PDB code 2RH1) was used as the initial structure (Fig. 3). The crystal water and ligand dodecaethylene glycol, acetamide, 1,4-butanediol, cholesterol, maltose, palmitic acid and sulfate ion were removed from the crystal structure. The number of amino acid residues in the receptor was 230. There is a hollow cavity formed by seven sequentially connecting transmembrane [alpha]-helixs, and the hollow cavity also includes three extracellular loops (ECL1-ECL3) and another three intracellular loops (ICL1-ICL3). In three extracellular loops, ECL3 is relatively longer, ECL1 and ECL2 are short. In the three intracellular loops, ICL1 and ICL2 are very short, ICL3 is very long. There are about 88 special functional sites in adrenergic receptor, mainly in TM3, TM4, TM5, TM6 and TM7 five domains [23]. Four disulfide bonds are contained in the protein structure of the ADR[beta]2 and the interactions between them are basis of maintaining a high structure level of protein.

Docking

The docking studies were carried out on the workstation of Silicon Graphics O2 with AutoDock 4.2.3 program [24]. After correcting atom types and adding all polar hydrogen atoms, Kollman all-atom charges were assigned for ADR[beta]2. The geometry optimization of ligands tyramine, PEA was carried out using MM2 force field and Gasteiger-Huckle charge was supplied with the convergence criterion set at 0.05 kcal/(A mol) [25]. Tyramine took Leu115 as the center in the range of 10 A to optimize its configuration in the binding pocket and docking was continued for 20 Genetic Algorithm runs. The PEA was docked into the binding pocket in the same manner, whereas docking center is not Leu115 but Asp85.

Molecular dynamics simulation

Dynamics simulation was implemented in Amber 11.0 program package using the Amber FF03 and GAFF force field. Ligand charges and force field parameters of ADR[beta]2 file were transferred into Amber to generate the topology file and coordinate file. All simulations were carried out at neutral pH. These starting structures were then placed in a truncated octahedral periodic box of TIP3P water molecules. The distance between the edges of water box and the closest atom of solutes is at least 10 A. To maintain the electroneutrality of the system, counterions (Cl-) was added into complex. The system was minimized with the module SANDER in constant volume by 500 cycles of the steepest descent minimization followed by 500 cycles of conjugated gradient minimization. These procedures ensured that the initial experimental structure was maintained while the solvent was allowed to relax. The steps above all featured 2000 cycles of the steepest descent followed by conjugating gradient minimization.

After energy minimization, canonical ensemble (NVT)-MD was carried out for 100 ps, during which the system was heated from 0 K to 300 K [26, 27]. Finally, 1 ns isothermal isobaric ensemble (NPT)-MD simulation was applied without any restraints, the time-step for MD simulation was 2 fs, every 2 ps record track file at a time.

Calculations of free energy

The binding free energy was calculated in Amber 11.0 by MM-GBSA method. For each snapshot, the free energy of complex, protein and its ligand were calculated, separately. The binding free energy was obtained from the basic formula as follows:

IGbind = Gcomplex-(Greceptor+Gligand) = IGMM +IGsol -TIS (1)

where, Gcomplex, Greceptor and Gligand represent the mean conformation free energy of the complex, receptors and ligands; IGMM represents the free energy of molecular mechanics; IGsol is solvation free energy, TIS is entropy effect.

The energy of molecular mechanics was composed by electrostatic interactions and Van der Waals interactions.

IGMM = IGele + IGvdw (2)

Solvation free energy is divided into two parts: polar and non-polar:

IGsol = IG pol,sol + IGnonpol,sol (3)

IGpol,sol represents polar dissolution free energy, IGnonpol,sol represents non-polar dissolution free energy.

IGpol, sol can be acquired by solving the equations of Generalized-Bom, and IGnonpol, sol is obtained by the following formula:

IGnonpol, sol = I3 SASA (4)

Where, I3 represents the surface tension and is set to 0.0072 kcal/(mol A2), SASA is the solvent accessible surface area (A2). Conformational entropy's contribution (including translation, rotation and vibration) was assessed by N mode module using 40 snapshots. It is respectively conducted the structural optimization of complex, proteins and small molecules, in which the distance-dependent dielectric constant Iu = 4r, energy convergence criteria is the RMSD less than 10-5 kcal/(mol A2). In addition, the binding free energy was broken down into each residue by MM-GBSA method [28, 29]. This decomposition is only for the contribution of molecular mechanics and solvation energy, does not include the contribution of entropy.

Results and Discussions

Docking Results

The configuration of tyramine was optimized to find a spatial coordinates of molecular which made the potential energy function have a minimum value. The active pocket was selected as Leu115 centered 10 A range of docking tyramine into ADR[beta]2. PEA was docked into ADR[beta]2 in the same manner, whereas PEA binds to ADR[beta]2 in the pocket centered Asp85 not Leu115. As shown in Fig. 4, tyramine binds with residues Glu122, Val206 of ADR[beta]2 through hydrogen bonds and falls into the hydrophilic "pocket" that is formed by the Thr123, Cys125, Glu122, Ser161, Thr118 and Ser207. PEA forms hydrogen bonds with residues Asp85 and falls into the hydrophobic "pocket" that were formed by Met40, Ile43, Val44, Met82, Ala85, Val86, Val87, Pro88, Trp109, Ile112, and Val117. Obviously, the action sites and "micro-environment" of the two systems are different, this also lead to the difference of binding between the two ligands and ADR[beta]2.

Molecular dynamics simulations of two systems

The ADR[beta]2-tyramine and ADR[beta]2-PEA complex were further compared by 1 ns MD simulation in water solvents.

Throughout 1 ns MD simulation, the root mean squared deviations (RMSD) value curves of backbone atoms during the MD simulation have been obtained. As shown in Fig. 5(a) and Fig. 5 (b), the RMSD indicates that the conformation achieves equilibrium within 700 ps after the beginning of MD simulation. The corresponding RMSD of ADR[beta]2-tyramine and ADR[beta]2-PEA complex system fluctuates largely and experiences a long time before reach desired balance. The RMSD of tyramine rapidly increases over 2.5 A in the first 400 ps, and fluctuates sharply at a value of 2.3+-0.3 A in the next 600 ps. The RMSD of PEA is obviously lower than that of tyramine and it fluctuates around 1.3 A after 200 ps. The average RMSD values of 400 to 1000 ps are 1.8 and 1.2 A for tyramine and PEA, respectively. Generally, the bigger the molecular flexibility remains, the higher the RMSF value of protein backbone atoms is relative to the initial structures. RMSF of the two systems were monitored in the simulation.

As shown in Fig. 5(c), the protein structures of two systems share similar RMSF distribution and similar kinetics feature trend. ADR[beta]2 protein shows four main flexible regions, including TM7 (residues Thr28-Gln37), TM6 (residues Ile66-Thr72), ICL1 (residues Ser109-Lys121), and ECL3 (residues Ala148-Cys163). TM7 and TM6 are the specific functional regions of ADR[beta]2. The residues located in the active sites of ADR[beta]2-tyramine complex system: Ala63, Leu67, Met68, Val59 and Phe61 show greater conformation deviation than that in the ADR[beta]2--PEA complex system. ADR[beta]2-tyramine complex system and ADR[beta]2-PEA complex system exhibit similar RMSF distributions and kinetics trends, the RMSF value of ADR[beta]2--PEA complex system is slightly higher than the RMSF value of ADR[beta]2-tyramine complex system, indicating the amino acid residues of flexibility of ADR[beta]2--PEA complex system slightly higher than the amino acid residues of flexibility of ADR[beta]2-tyramine complex system.

The binding stability above will be further analyzed by hydrogen bonds and calculations of binding free energy.

Analysis of hydrogen bonds

Effects of hydrogen bonds on the organism are essential. Protein is made of amino acid combined each other by peptide bond (-NH-CO-). Different lengths of peptide chain connect to each other and fold into secondary, tertiary and quaternary structures through intermolecular interactions like hydrogen bonds. The hydrogen bond is an important factor in stabilizing [alpha]-helical structure of protein. The critical hydrogen bonding interaction at the binding sites of ADR[beta]2-tyramine system was monitored in the process of simulation and shown in Fig. 6.

In Fig. 6(a), tyramine and amino acid residues Glu122 and Val206 of ADR[beta]2 form hydrogen bonds located in the "micro environment" surrounded by Thr123, Cys125, Glu122, Ser161, Thr118 and Ser207. Fig. 6(b) shows the representative conformations selected from the dynamics simulation. The -NH2 of tyramine rotates 90Adeg in a parallel position with a benzene ring. The hydrogen bonds of tyramine with Glu122, Val206 were broke up and then it formed hydrogen bonds with the oxygen atoms of water molecules, Wat743 and Wat3399, and hydrogen atoms of water molecule Wat1219.

Table-1 lists the results of hydrogen bonds analysis in the 1 ns molecular dynamics simulation. The occupation of hydrogen bonds formed by tyramine with residues Glu122 and Val206 of ADR[beta]2 in the starting phase are 29.60%, 27.40%, respectively. But the occupation of the three hydrogen bonds formed by tyramine with three water molecules are 97.36%, 99.31% and 98.10% after 500 ps simulation, respectively.The bonds length is less than 3.5 A and the bonds angle is less than 60Adeg. This shows that hydrogen bonds between tyramine and residues Glu122, Val206 were broke up and formed hydrogen bonds with the surrounding water molecules because of solvent effects. This phenomenon may be due to the hydrophilic of tyramine and this can be verified by the analysis of binding free energy.

Residues Asp85 near the active site can form a hydrogen bond with residues Val89, and the duration of the hydrogen bond is 94.50%; residues Trp81 can form a stable hydrogen bond with Asp85, and the duration of the hydrogen bond is 92.60%; Val58 and Gly62, Val59 and Ala63, Pro60 Ala64 formed hydrogen bonds, and the occupation are all more than 90%, these formed hydrogen bonds can build a stable environment for the surrounding ligands. Meanwhile, these residues are belonging to the area of TM3 of ADR[beta]2 which has been confirmed [30] the focus area where the constitutive activity mutation occurs.

Table-1: Analysis of hydrogen bonds of ADR[beta]2-tyramine.

donor###receptor###Occupation###Distance###bond angle

###(%)###(A)a###(Adeg)b

Glu122 OE2###ligand N1###29.60###2.893 (0.007)###55.58 (0.03)

Val206 O###ligand N1###27.40###2.796 (0.002)###51.28 (0.02)

Glu122 O###ligand O1###18.55###3.016 (0.001)###50.08 (0.02)

Asp85 O###Val89 N###94.50###2.060 (0.002)###25.33 (0.06)

Trp81 O###Asp85 N###92.60###2.986 (0.002)###26.68 (0.01)

Val58 O###Gly62 N###98.70###2.199 (0.001)###34.89 (0.02)

Val59 O###Ala63 O###90.41###2.381 (0.003)###26.50 (0.03)

Pro60 O###Ala64 N###99.01###2.011 (0.001)###31.10 (0.09)

WAT O###ligand N1###97.36###2.196 (0.001)###34.64 (0.01)

WAT O###ligand O1###99.31###2.896 (0.003)###21.65 (0.02)

ligand O1###WAT H1###98.10###2.934 (0.002)###35.11 (0.01)

The critical hydrogen bonding interactions at the binding sites of ADR[beta]2-[beta]-PEA system in the initial structure and representative conformation of simulation were monitored (Fig. 7). Fig. 7(a), the N1 atom of [beta]-PEA forms hydrogen bonds with the OD1, OD2 atoms on Asp85 of protein ADR[beta]2 and PEA falls into the hydrophobic pocket composed by Met40, Ile43, Val44, Met82, Ala83, Val86, Val87, Pro88, Trp109, Ile112, and Val117. In Fig. 7 (b), N1 atom of PEA forms hydrogen bonds with Hie65, Gly62 by the receptors H1 and H2. Hie is the result of protonated N at the position of Iu of histidine. [beta]-PEA conformation changed during the simulation, the angle formed by N1-C1-C2 changed from 112.4 Adeg of the initial structure to 101.5 Adeg of the structure of simulation.

Table-2 shows that the analysis of hydrogen bonds of ADR[beta]2-[beta]-PEA system during the simulation. The occupation of hydrogen bonds between N1 atom and oxygen atom of Asp85 are 11.26%, 9.06% and 9.15%. The bond length is longer than 3.5 A and the bond angle does not meet the bonding conditions, which indicate that the hydrogen bonds between [beta]-PEA and residue Asp85 of ADR[beta]2 has been broken after simulation. The OD1, OD2 atoms of Asp85 competitively formed hydrogen bonds with the same hydrogen atom of amino of [beta]-PEA. Meanwhile, the space conformational changes of [beta]-PEA may be caused by the other reason that the hydrogen bond breaks up.

Table-2: Analysis of hydrogen bonds of ADR[beta]2-PEA

donor###receptor###occupation (%) distance(A)a###bond angle(Adeg)b

Gly62 O###ligand N1###84.06###2.343 (0.005)###41.99 (0.04)

Hie65 ND1###ligand N1###82.70###2.987 (0.003)###39.92 (0.03)

Asp85 OD1###ligand N1###11.26###3.890 (0.002)###82.29 (0.05)

Asp85 OD2###ligand N1###9.06###3.981 (0.003)###73.77 (0.07)

Asp85 OD2###ligand N1###9.15###3.852 (0.004)###68.27 (0.06)

Val59 O###Ala63 N###94.40###2.824 (0.004)###43.88 (0.05)

Ala64 O###Leu67 N###94.78###2.315 (0.003)###43.91 (0.03)

Ala64 O###Met68 N###90.08###2.623 (0.002)###40.21 (0.04)

Phe61 O###Hie65 N###97.74###2.648 (0.004)###37.59 (0.02)

Gly62 O###Ile66 N###94.07###2.536 (0.005)###39.18 (0.01)

After 1 ns dynamics simulation, [beta]-PEA formed hydrogen bonds with residues Gly62, Hie65. The occupations are 84.06%, 82.70% and bond lengths are 2.343 A, 2.987 A, respectively, which indicates that [beta]-PEA and Gly62, Hie65 formed stable hydrogen bond interactions. The contribution of hydrogen bond between ADR[beta]2 and [beta]-PEA was subsequently analyzed using calculation of free energy. Val59 and Ala63, Ala64 and Leu67, Phe61 and Hie65, Gly62 and Ile66, Ala64 and Met68 form important hydrogen bonds, which also make [beta]-PEA steadily, exist in TM3 area of ADR[beta]2.

The main contribution of residues

Residues Thr123, Cys125, Glu122, Ser161, Thr118 and Ser207 are the critical residues in tyramine-ADR[beta]2 complex system. Fig. 8 shows the spatial position change of the initial structure of tyramine and representative conformation from MD trajectories. As can be seen from Fig. 8, conformation of tyramine (green sticks model) changed, and hydrogen atom on the benzene ring and substituent group hydroxyl group (-OH) tends to be in the same plane, and offset from the position of the original structure, away from the direction of Glu122, Val206.

To verify this phenomenon, the position changes of tyramine relative to ADR[beta]2 were analyzed by distance between tyramine and its surrounding key residues Glu122, Val206. The results were shown in Table 3. The distance in the experiments is the distance between the two center atoms of molecules. The distance between tyramine and residue Glu122 is 5.08 A in the initial structure and increases to 7.91 A in the first 500 ps simulation. The distance continues to increase to 8.14 A at the balance stage, until the final distance 10.69 A at the end of the simulation. Similarly, the distance between tyramine and residue Val206 is also increasing from 5.04 A in the initial structure to 7.71 A in the end of the simulation during the 1000 ps simulation. These datas indicate that tyramine moves to the direction far from residues Glu122 and Val206 and are consistent with the observed results in Fig. 8.

The broken hydrogen bonds may be caused by the movement of tyramine in a solvent environment. Because of solvent effect, the hydroxyl of tyramine was surrounded by a mass of water molecules that make it away from the "naked" residues of the protein surface. So tyramine binds with ADR[beta]2 receptors weakly. The interaction forces between ligand and protein was mainly via electrostatic interactions, hydrogen bonding and Van der Waals interactions. The mechanism and energy contribution relationship of tyramine binding with ADR[beta]2 receptors will be discussed in the subsequent analysis of the binding free energy.

Table-3: Distances between tyramine and surrounding residues.

###initial value###warming###balance###dynamic simulation

Tyr-Glu###5.08###7.91+-0.06###8.14+-0.04###10.69+-0.15

Tyr-Val###5.04###5.63+-0.05###5.77+-0.09###7.71+-0.11

The spatial position change of PEA relative to ADR[beta]2 was shown in Fig. 9 from MD trajectories. The angle N1-C1-C2 of PEA changed from 112.4 Adeg in the initial structure to 101.5 Adeg in the representative structure.

The changes of average conformational distance between [beta]-PEA and its critical residues Gly62, Hie65 in the initial conformation, heating, balance and after simulation was monitored, showing in Table 4. The distance between [beta]-PEA and residue Gly62 is 4.61 A in the initial structure and increased to 4.74 A in the 500 ps simulation, the distance continues to increase 5.10 A at the balance stage, until the final distance 4.11 A at the end of the simulation. The distance between [beta]-PEA and residue Hie65 is fluctuating continually from 8.68 A in the initial structure to 7.15 A in the end of the simulation. The fluctuation is small and the distances getting smaller which indicates that [beta]-PEA gradually get close to these two residues and move to TM6 area. It is difficult for these two residues to get out of the active pocket, and Hie65 is a protonated histidine which is the key to activate ADR[beta]2 receptor.

[beta]-PEA can form stable hydrogen bonds with the acceptor protein probably form hydrogen bond with Hie65, thereby activating the adrenergic [beta]2 and affect the biological function of ADR[beta]2 receptor. The energy contribution relationship of [beta]-PEA binding with ADR[beta]2 receptors will be discussed in the subsequent analysis of the binding free energy.

Table-4: Distances between PEA and surrounding residues.

###initial value###warming###balance###dynamic simulation

PEA-Gly###4.61###4.74+-0.07###5.10+-0.07###4.11+-0.08

PEA-His###8.68###8.25+-0.08###8.64+-0.07###7.15+-0.09

The interaction analysis based on binding free energy calculation

The computational simulations of protein-ligand binding free energies play an important role in studying the active site. To elucidate the binding free energies of small molecules binding to protein, the binding free energies were usually calculated by free energy perturbation (FEP) [31, 32], thermodynamic integration (TI) [33], Molecular Mechanics Generalized Born/Poisson Boltzmann surface area (MM-GBSA/PBSA) methods. Though FEP and TI methods rigorous theoretical and calculation results are accurate, they need us to do a lot of samples and they are very time-consuming, limiting a large number of applications in analog design. MM-GBSA/PBSA calculation time is short, the high accuracy and relevance experiments, which are widely used in various systems.

The relative binding free energies of tyramine-ADR[beta]2 system were calculated using MM-GBSA /MM-PBSA methods. Table 5 shows the results of binding free energy of tyramine-ADR[beta]2 system. The calculated results of free energy by GB method and PB method are similar. The polar solvent free energy of tyramine-ADR[beta]2 complex calculated by GB method is -1947. 1 kcal/mol and by PB method is -2060.8 kcal/mol. The difference between the two methods isprominent, but the difference of IGpol, sol is only 0.1 kcal/mol. The binding free energy (IGbind) based on GB method was -7.1 kcal/mol, while the binding free energy (IGPB) based on PB method was -6.1 kcal/mol. According to the results, the two results of free energy methods are negative, indicating that tyramine and ADR[beta]2 binds each other stability.

The free energy analysis suggests that the Van der Waals interaction is the fundamental driving force of the binding process, and solvation free energy (IGsolv = 6.0 kcal/mol) has no contributions to binding energy. This confirms that tyramine is embedded in water molecules by forming hydrogen bonds with water molecules and then hindered the binding between tyramine and other residues. The electrostatic interaction has beneficial contribution to binding energy and its value is only -1.2 kcal/mol. This may because that the acid intensity of tyramine is weakened by the effect of decarboxylation of tyrosine and it tends to neutral in the solvent environment, the charging ability is also decreasing. Although the binding free energy is negative, the absolute value is only 6.1 kcal/mol, it describes the binding ability between tyramine and ADR[beta]2 protein is not strong. Solvent effect is probably the main reason that makes tyramine molecule free from protein molecules.

Table-5: Binding free energy calculations of tyramine-ADR[beta]2 (kcal/mol).

###Complex###Receptor###Ligand###Delta(I)

###Energy Component

###Mean###I###Mean###I###Mean###I###Mean###I

###Generalized Born

###Gvdw###-1553.3###0.79###-1541.5###0.77###-0.9###0.02###-10.9###0.04

###Gele###-13627.7###2.12###-13636.4###2.11###9.9###0.07###-1.2###0.08

###Gpol,sol###-1947.1###0.35###-1938.2###0.33###-15.6###0.03###6.8###0.09

###Gnonpol,,sol###99.6###0.06###99.2###0.05###2.1###0.01###-1.6###0.01

###GMM###-15180.9###2.27###-15177.8###2.25###9.1###0.07###-12.2###0.03

###Gsolv###-1847.4###1.35###-1838.9###1.33###-13.6###0.03###5.1###0.04

###G###-17028.4###1.87###-17016.8###1.88###-4.5###0.03###-7.1###0.09

###Poisson Boltzmann

###Gvdw###-1553.3###0.79###-1541.5###0.25###-0.8###0.01###-11.0###0.04

###Gele###-13627.7###0.47###-13636.4###0.33###9.9###0.07###-1.2###0.08

###Gpol,sol###-2060.8###0.13###-2050.5###0.62###-17.0###0.03###6.7###0.11

###Gnonpol,,sol###69.0###0.07###68.8###0.71###0.9###0.01###-0.7###0.01

###GMM###-15180.9###2.16###-15177.8###0.25###9.1###0.02###-12.2###0.04

###Gsolv###-1991.9###1.04###-1981.8###0.63###-16.2###0.03###6.0###0.11

###G###-17172.8###2.09###-17159.6###0.22###-7.1###0.01###-6.1###0.08

Table-6: Binding free energy calculations of PEA-ADR[beta]2 (kcal/mol).

###Complex###Receptor###Ligand###Delta(I)

###Energy Component

###Mean###I###Mean###I###Mean###I###Mean###I

###Generalized Born

###Gvdw###-1526.3###0.17###-1401.0###0.93###-13.2###0.02###-112.1###0.07

###Gele###-13660.2###2.91###-13561.8###2.92###-44.1###0.05###-54.3###0.14

###Gpol,sol###-1942.1###0.46###-1903.4###0.48###-76.6###0.02###37.9###0.07

###Gnonpol,,sol###102.9###0.06###103.1###0.06###92.6###0.01###-92.8###0.01

###GMM###-15186.5###0.37###-14962.8###0.39###-57.3###0.06###-166.4###0.16

###Gsolv###-1839.1###0.42###-1800.3###0.48###16###0.03###-54.8###0.08

###G###-17025.7###0.35###-16763.1###0.34###-41.3###0.16###-221.2###0.11

###Poisson Boltzmann

###Gvdw###-1526.3###0.17###-1401.0###0.99###-13.2###0.02###-112.1###0.07

###Gele###-13660.2###2.91###-13561.8###2.92###-44.1###0.05###-54.3###0.14

###Gpol,sol###-2056.0###0.35###-2016.1###0.38###-84.9###0.03###39.0###0.09

###Gnonpol,,sol###70.8###0.04###70.9###0.03###79.4###0.01###-79.5###0.01

###GMM###-15186.5###0.91###-14962.8###0.75###-57.3###0.06###-166.4###0.45

###Gsolv###-1985.2###0.35###-1945.2###0.37###-5.5###0.03###-40.5###0.09

###G###-17171.7###0.36###-16908.1###0.35###-62.8###0.16###-206.9###0.11

The relative binding free energy of [beta]-PEA-ADR[beta]2 complex is calculated using MM-GBSA/MM-PBSA methods. The energy contribution of each part can explain the interaction between protein-ligand systems. According to Table 6, the polar solvent free energy of [beta]-PEA-ADR[beta]2 complex calculated by GB method and PB method are -1942.1 and -2056.0 kcal/mol, respectively. The difference between two IGpol, sol (37.9 and 39.0 kcal/mol, respectively) is only 1.1 kcal/mol. Both the PB and GB methods can be used for the contribution of qualitative analysis. The standard deviation between two methods for [beta]-PEA-ADR[beta]2 complex is similar to that for tyramine-ADR[beta]2 complex.

The relative binding free energies calculated by MM-PBSA and MM-GBSA methods are -221.2 kcal/mol. (IGGB) and -206.9 kcal/mol (IGPB), respectively. The binding free energy is negative, indicated that [beta]-PEA could bind with ADR[beta]2 stably. Each energy analysis suggests that Van der Waals interaction, electrostatic interaction and hydrophobic interactions are the fundamental driving force of the binding process, and solution free energy IGsolv (37.9 kcal/mol) is not conducive to binding energy contribution.

The relative binding free energy of tyramine-ADR[beta]2 and [beta]-PEA-ADR[beta]2 complex are -7.1 and -221.2 kcal/mol respectively. According to Van't Hoff equation, the binding free energies of tyramine-ADR[beta]2 and [beta]-PEA-ADR[beta]2 complex are all negative, indicating the spontaneous binding process of tyramine and [beta]-PEA. [beta]-PEA has a smaller the binding free energy, indicating a more potent binding with ADR[beta]2 than tyramine. Therefore, [beta]-PEA-ADR[beta]2 complex is more stable than tyramine-ADR[beta]2 complex.

The binding energy difference is mainly due to the differences in electrostatic and hydrophobic interactions in energy term. Tyramine has a hydroxyl group, which is formed via tyrosine decarboxylation and this lead to the weak electrical and strong hydrophilic. Tyramine can be easily surrounded by water molecules in the solvent environment, and the hydrophobic interaction was small. But the benzene ring of [beta]-PEA has strong hydrophobic and electrical property. The positive amino of [beta]-PEA can bind with the carboxyl group of Gly62 of ADR[beta]2 in TM3 region to form salt bond, leading [beta]-PEA bonded to the adrenergic protein more firmly. The ability of [beta]-PEA binding to ADR[beta]2 is stronger than tyramine, and thus [beta]-PEA could have better effects on the ADR[beta]2 protein by comparing the two systems, and may have a greater impact on its biological functions.

Conclusions

In this paper, molecular docking, molecular dynamic simulations and binding free energy calculation had been used to investigate the interaction of tyramine-ADR[beta]2 and [beta]-PEA-ADR[beta]2. 1 ns MD simulation had been performed on ADR[beta]2-tyramine/[beta]-PEA complex to investigate their binding mode. MD simulation revealed that the structural domain TM7 (Thr28-Gln37), TM6 (Ile66-Thr72), ICL1 (Ser109-Lys121) and ECL3 (Ala148-Cys163) of protein ADR[beta]2 had higher flexibility and occur conformational changes during the progress of combing tyramine/[beta]-PEA.

The variation trend of system backbone atoms RMSD had been used to determine whether ADR[beta]2-tyramine/[beta]-PEA systems reached a steady state along within the time. It is significant to analyze the data only in stable system. By comparing the RMSD curves of the two systems, the fluctuation of tyramine residue is higher than that of [beta]-PEA, particularly the fluctuation of TM6 at the allosteric site is more significant (residues Ile66-Thr72). Residues Ala63, Leu67, Met68, Val59 and Phe61 at the active site of tyramine complex system show larger deviation.

Moreover, the complex systems of ADR[beta]2-tyramine and ADR[beta]2-[beta]-PEA exhibited similar RMSF distribution, which indicate that [beta]-PEA/tyramine combined with ADR[beta]2 sharing a similar mechanism. The calculate results of binding free energy show that the main driving forces of [beta]-PEA binding ADR[beta]2 receptor are Van der Waals, electrostatic interactions and hydrophobic interactions, while tyramine binding with [beta]2 adrenergic receptor mainly by Van der Waals interaction. In the process of simulation, the conformation of tyramine changed and a mass of water molecules formed a hydrogen bonds grid surrounded tyramine, solvated it and weakened its hydrophobic interactions due to its good hydrophilic nature. Further, tyramine, which is formed by the decarboxylation of tyrosine, has weak electric property and almost without static effect of ADR[beta]2.

In addition, tyramine is in TM6 region which is the flexibility region of ADR[beta]2 receptor, so the instability of the protein conformation also makes it difficult to combine stably with tyramine.

[beta]-PEA has stronger hydrophobic than tyramine, and the positively charged amino of [beta]-PEA can bind with carboxyl group of Gly62 of ADR[beta]2 which in TM3 region to form salt bond. Meanwhile, there are hydrogen bond interactions between Val59 and Ala63, Ala64 and Leu67, Phe61 and His65, Gly62 and Ile66, Ala64 and Met68 to keep the [beta]-PEA exist steadily in the TM3 region. The protonation is the key to activate the active site of ADR[beta]2. Through observing the conformation change of the active site in ADR[beta]2-PEA complex, we found that the nitrogen atom in the Iu position of Hie65, which formed hydrogen bonds with the [beta]-PEA had occurred protonation, and Hie65 lied in the specific function domain TM3 which is belonging to ADR[beta]2, declaring that the combination of [beta]-PEA makes ADR[beta]2 activated.

Through these multiple computational analysis, we compared the similarities and differences in ADR[beta]2 receptor protein combined with tyramine/[beta]-PEA, showing that [beta]-PEA can combine with ADR[beta]2 receptor preferably and activate it, thus its impact is greater. But tyramine can not bind to the residue of ADR[beta]2 receptor steadily. This difference depends on an oxygen atom on the structure of two small molecule ligands. The good hydrophilicity of tyramine make it surrounded by the water molecules, had solvent effects, and did not form a good combination effect with the ADR[beta]2 receptors, while hydrophobic and charge ability of [beta]-PEA are the main factor of it combining with ADR[beta]2 receptor, which finally protonated Hie65 and activated receptor. In summary, comparing the two biogenic amines, [beta]-PEA can activate ADR[beta]2 receptor, but tyramine can not make ADR[beta]2 receptor produce the biological effects.

Acknowledgments

This work was supported by the Cooperative Project of Zhejiang Province-Chinese Academy of Forest (2015SY11); the Zhejiang Provincial Natural Science Foundation (LY14E030016); The Pre-research Project of Research Center of Biomass Resource Utilization, Zhejiang A and F University (2013SWZ02-1), and the Key Laboratory of Chemical Utilization of Forestry Biomass of Zhejiang Province, Zhejiang A and F University.

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