Flexibility analysis of Native Pyridoxal Kinase and its complexes with ATP and ADP : A Molecular Dynamics Simulation Study

Received Jul 22 nd , 2015 Revised Aug 20 th , 2015 Accepted Aug 24 th , 2015 Pyridoxal Kinase (PLK) phosphorylates vitamin B6, a step required for the conversion of Vitamin B6 into pyridoxal 5-phosphate. The protein is cytoplasmic and is active as a dimer. Molecular dynamics (MD) simulation studies using a 25ns scale for PLK and its complex with ATP (Adenosine triphosphate) and ADP (Adenosine diphosphate) were carried out and the trajectory analysis revealed that the flexibility of the entire PLK molecule increases. In present study we have investigated the conformational changes in pyridoxal kinase (PLK) after binding of ligands (ATP/ADP). The stability of native and PLK in complex with ATP and ADP, was ascertained by MD simulations and mechanism of ligand binding was explored by essential dynamics. Simulation results also indicated that the van der Waals contribution was greater than the electrostatic interaction between the protein residues and the ligands. Further, the ligand (ATP/ADP) binding results into decrement and increment of fluctuations in certain regions of protein. Keyword:


INTRODUCTION
Pyridoxal kinase (PLK) is an enzyme belonging to ribokinase super-family that catalyzes the conversion of pyridoxal to pyridoxal 5'-phosphate (PLP).The two substrates of this enzyme are ATP and pyridoxal, whereas its two products are ADP and pyridoxal 5'-phosphate.PLP acts as an essential ubiquitous coenzyme in many aspects of amino acids and cellular metabolism such as transamination, decarboxylation, and synthesis pathways involving carbohydrates, sphingolipids, amino acids, heme and neurotransmitters [1].Humans are unable to synthesize PLP de novo and require its precursor in the form of Vitamin B6 (Pyridoxal (PL), Pyridoxine (PN), Pyridoxamine (PM) from the diet.
The objective of the current study is to perform a detailed examination of the structural flexibility and conformational changes in PLK after the binding of the ligands.The significant motions around the binding site in PLK+ (ATP/ADP) complex were analyzed with the help of essential dynamics.The findings thus obtained are useful for revealing the conformational changes in PLK after binding of the ligands.We demonstrate that the molecular basis of PLK function is largely determined by the mechanism in which, the ligand can modulate the conformational dynamics of PLK linked to the functional activities [8][9].Elucidation of the ligand binding mechanism is the necessary step to obtain more selective and potent drugs for this new potential target.

RESEARCH METHOD
The three dimensional structure coordinates of the native Pyridoxal kinase (PDB Id: 3H74) and its complexes with ATP (PDB Id: 3IBQ) and ADP (PDB Id: 3HYO) were obtained from Protein data bank [10].Missing residue in the respective PDBs were added by Modeller 9.14 [11], using native Pyridoxal kinase as the template.The molecular topology file and force field parameters that includes the charge for ligands, ATP and ADP were generated at the PRODRG [12] server.
MD simulations were performed using GROMACS 4.5.5 [13] with the GROMOS96 43a1force field [14].Native and the complex structures were solvated with SPC (simple point charge) explicit water model [15] embedded in 7.618 x7.618 x7.618 nm, 7.817 x 7.817 x7.817 nm or 7.592 x 7.592 x 7.592 nm boxes respectively.For neutralizing the system, 12 sodium ions were added to replace water molecules in the boxes.The simulation system was composed of 40149 (3H74), 46330 (3IBQ) and 40017 (3HYO) atoms respectively and was subjected to energy minimizationusing steepest descent method, until a tolerance of 10 kJ/mol was reached.The water molecules and ions, were energy minimized, keeping the protein and the ligand fixed followed by the minimization of the protein by fixing the main-chain and Ca atoms.Finally, the entire system was minimized.
Molecules were equilibrated with the fixed protein at 10K, 50K, 100K, 200K, 300 K each for 200 ps, taking the initial velocities from a Maxwellian distribution.It should be noted that the protein and the ligand were fixed during the process of heating up.So the solute (protein and ligand) was subsequently relaxed step by step, and heated up to 300 K by using 1 ns MD simulations.Interactions involving covalent bonds and shortrange non-bonded interactions were computed at every time step, and long-range electrostatic forces were computed at every two-time steps.All the bond lengths including hydrogen atoms were constrained by the LINCS algorithm [16].The electrostatic interactions were calculated using the Particle-mesh Ewald (PME) algorithm [17], with interpolation order of 4 and a grid spacing of 0.16 nm.The van der Waals interaction were treated using a cutoff 0.9 nm and the coordinates were saved every 2 ps.
Finally, the 25 ns molecular dynamics simulations were performed under normal temperature (300 K) and pressure (1 bar), using a temperature coupling time constant of 0.1 ps and a pressure coupling time constant of 1.0 ps [18].

RESULTS AND ANALYSIS
Molecular Dynamics simulations of the Pyridoxal Kinase complex with ATP (PLK+ATP) and the Pyridoxal kinase complexed with ADP (PLK+ADP) were performed using the explicit SPC water model by applying periodic boundary conditions.The total energies of both the simulation models versus simulation time are shown in Figure 1A.In general, the total energies of the system of the native and complex models remain stable as a function of simulation time.The length of the simulation is an important factor while considering the dynamics studies.The root mean square deviation (RMSD) values of all protein atoms of PLK, PLK+ATP and PLK+ADP are shown in Figure 1B, RMSD values become stable after ~15 ns in case of PLK+ADP and PLK whereas in PLK+ATP, it stabilizes after ~11 ns.Furthermore, as shown in Figure 1B, the RMSD for PLK+ADP is larger than that for PLK+ATP, which indicates that flexibility of PLK+ADP is comparatively more than PLK+ATP upon binding of ligand and the results are also confirmed by the larger fluctuation in radius of gyration in PLK+ADP to PLK+ATP shown in Figure 1C.It can be observed from the results that the binding of ligands (ATP/ADP) to protein increased the flexibility of protein and relatively more in PLK+ADP complex.To further investigate the significant motions, the root mean square fluctuations (RMSF) for all the Calpha atoms in PLK+ATP and PLK+ADP were computed (Figure 1D).In case of native Pyridoxal kinase, major fluctuations are present in residues 42-53, 150 -156, 193-197 and 251 -259.To support our results and further investigate the most significant collective modes of motion occurring during the simulations of the Native PLK, PLK+ATP and PLK+ADP, the covariance matrix corresponding to the C-alpha atoms coordinates was calculated and the essential dynamics was performed.The 3N eigenvalues (846 eigenvalues) of the covariance matrix were ranked in decreasing order of magnitude as shown in Figure 2

3.1Interaction between Pyridoxal Kinase and Ligand (ATP/ADP)
To explore the interaction between the ligands (ATP/ADP) and PLK, we calculated the energy contribution of the van der Waals interactions and short range electrostatic interactions.Distance between PLK and ligands (ATP/ADP) got varied as function of simulation time in compliance with Van der Waals interaction between them.This also strengthened that major contributing term in interaction between PLK and ligands (ATP/ADP) is van der Waals as shown in Figure 5. Average number of H-bonds between PLK-ATP is 8-12 and PLK-ADP is 6-9 which shows there is relatively stronger interaction in PLK and ATP to PLK and ADP which is also in accordance with nature of van der Waals interaction in Figure 4.
In  The two dimensional representatives for the interacting mode of (A) ATP with PLK in PLK+ATP complex at 7400ns (B) ADP with PLK in PLK+ADP complex at 8600 ps.

3.2Flexibility analysis in Pyridoxal kinase with ligands (ATP/ADP)
Binding of ATP decreases the fluctuations in the regions 111-124 (loop+sheet+helix), 151-156 (loop) and fluctuation increases in 46-48 (loop), 208-213 (catalytic site).Fluctuation increases in ATP -binding site in PLK+ATP complex.After ATP is bound to the pyridoxal kinase, whole protein structure becomes more compact than its normal state reported by Ming Hui et al [20], which is in agreement with our result.Whereas in case of PLK+ADP complex, fluctuation decreases after binding of ADP to protein in 43-50 (loop), 115-122 (loop + sheet), 193-195 and 253-260 (loop).Larger conformational change in binding site in PLK-ATP as compared to PLK-ADP because larger fluctuations exist in binding site.

CONCLUSION
From our findings we conclude that the flexibility of PLK-ADP complex is relatively more than its complex with ATP and flexibility is increased in both the cases of PLK-ATP, PLK-ADP after binding of ligands to protein (PLK).The atomic fluctuations, for the complexes are mainly localized on the loop part but a few are at the active site which is also in agreement with atomic fluctuation along the eigenvector 1. Interaction between the Pyridoxal kinase and ligands (ATP/ADP) is dominated by van der Waals interaction in preference to theelectrostatic contribution (protein-ligand distance curve).Interaction between Pyridoxal kinase and ATP is relatively stronger than Pyridoxal kinase and ADP and is revealed by the van der Waals energy and number of hydrogen bondspresent between them.ATP binding to PLK decreases the fluctuations in the regions 111-124 (loop+sheet+helix), 151-156 (loop) and increases the fluctuations in 46-48 (loop), 208-213 (catalytic site).On the other hand in PLK+ADP complex fluctuation decreases after binding of ADP to protein in 43-50 (loop), 115-122 (loop + sheet), 193-195 and 253-260 (loop) regions.Flexibility analysis results are quite consistent with the previous experimental results [20], and hence may be useful for in-depth understanding of the mechanism of phosphorylation and dephosphorylation of pyridoxal kinase.
or inhibition of PL Kinase may lead to interruption of the salvage

Figure 1 .
Figure 1.(A) Total energy of Native Pyridoxal kinase (Black), Pyridoxal kinase in complex with ATP (Red) and Pyridoxal kinase in complex with ADP (Blue) as afunction of simulation time for 25-ns MD simulation(B) Time dependence of RMS deviation (RMSD) for uncomplexed Pyridoxal kinase (Black), Pyridoxal kinase complexed with ATP (Red) and Pyridoxal kinase complexed with ADP (Blue) for all protein atoms over 25-ns MD simulation.(C) Radius of gyration for all protein atoms as a function of simulation time of Native Pyridoxal kinase (Black) Pyridoxal kinase+ADPcomplex (Red) and Pyridoxal kinase+ATP complex (black) over 25 -ns MD simulation (D) RMS fluctuation values for all the C-alpha atoms in Native Pyridoxal kinase (Black) Pyridoxal kinase+ATP complex (Red) and Pyridoxal kinase+ADP complex (Blue)

Figure 2 .
Figure 2. Eigenvalues of the covariance matrix resulting from the simulations of(A) NativePyridoxal kinase(B) Pyridoxal kinase+ATPcomplex and (C) Pyridoxal kinase+ADP complex.

Figure 3 .
Figure 3. Displacements of the components of the first eigenvectors for (A)Native Pyridoxal Kinase (B) Pyridoxal kinase+ATP complex (C) Pyridoxal kinase+ADP complex.
The van der Waals and ISSN: 2278-8115 IJCB Vol. 5, No. 1, August 2016, 13 -20 http://www.ijcb.inshort range electrostatic energies between PLK and ATP are shown in Figure 4A.Average van der Waals energy (black) and short range electrostatic energies (red ) for PLK+ATP is -319.395kJ/mol and -20.9556 kJ/mol respectively and for PLK+ADP is -189.813KJ/mol and -9.667KJ/mol respectively.For PLK with ligand (ATP/ADP) overall van der Waals contribution is more dominant than the electrostatic contribution shown in Figure 4. Interaction between Pyridoxal kinase and ligand (ATP/ADP) is mainly dominated by Van der Waals interaction in both the models.

Figure 4 .
Figure 4.(A) The van der Waals (Black) and short-range electrostatic energy (Red) between Pyridoxal kinase and ATP in PLK+ATP complex during the simulation.(B) The van der Waals (Black) and short-range electrostatic energy (Red) between Pyridoxal kinase and ligand ADP in PLK+ADP complex during the simulation.

Figure 5 :
Figure 5: Distance between (A) Pyridoxal kinase and ATP in PLK+ATP complex and (B) Pyridoxal kinase and ADP in PLK+ADP complex as a function of 25 ns simulation time.
pyridoxal kinase complexed with ATP; it forms H-bond with Asn239, Asn 243, Thr 179, Asn 141 and Cys 189 in original crystal structure.But Asn 243 is important residue to recognize ATP for binding because this residue is most of the time present during simulation H-bond formation between ATP and Protein.At different instant of simulation time, ligand ATP forms H-bond with different residue in binding region mainly Thr 179, Val 181, Cys 189, Leu 206, Pro 207, Gly 208, Tyr 210, Asn 211, Leu 242 and Asn 243 form Hbond with Pyridoxal kinase complexed with ATP.Residue in Loop region of binding site of ATP also shows hydrophobic interaction with ligand ATP.In case of Pyridoxal kinase complexed with ADP; it forms H-bond with Leu 116, Tyr 153, Gln 154 and Val 154.The interacting models derived by the LIGPLOT program[19] of the inhibitor ligands (ATP/ADP)with Pyridoxal kinase are illustrated in Figure 6A, 6B at 7400ps and 8600ps repectively, which were taken from the structures with the average electrostatic energy of each stage.IJCB Vol. 5, No. 1, August 2016, 13 -20 http://www.ijcb.in

Figure 6 :
Figure6: The two dimensional representatives for the interacting mode of (A) ATP with PLK in PLK+ATP complex at 7400ns (B) ADP with PLK in PLK+ADP complex at 8600 ps.