Structural optimization and docking studies of anatoxin-a : A potent neurotoxin

In this investigation, our aim was to get more insight on the geometry optimization, structural properties and molecular interaction of anatoxin-a, a naturally occurring potent neurotoxin. The geometry of the anatoxin-a was fully optimized in terms of density functional theory Gaussian 09. Calculations for structural parameters viz. total energy, dipole moment, electro-negativity, chemical hardness, chemical softness, electronic chemical potential and electrophilic index of optimized geometries has been carried out. The energy difference between HOMO-LUMO was found to be -4.89 eV. Furthermore, the molecular docking of anatoxin-a with nicotinic acetylcholine receptor (nAChR) was performed in order to find the molecular interaction involved in the inhibition process of nicotinic acetylcholine receptor by anatoxin-a by using Glide 5.9. Our results clearly indicates that anatoxin-a bind to the A-chain of nAChR with hydrophobic interactions between anatoxin-a and Phe214, Tyr277 and Thr281 residues of protein with bond length 1.97, 1.96 and 2.04 Å, respectively. The glide energy and docking score were found to be -18.242 and -4.567, respectively.


INTRODUCTION
The presence of toxic cyanobacterial blooms in eutropic lakes, rivers and reservoirs has been reported during the last four decades all over the world.The continuous increment in the number of cases of animal poisoning and human illness have altered worldwide attention on one of the most potential cyanotoxin anatoxin-a, generated by some strains of Anabaena (particularly Anabaena flos-aquae), Aphanizomenon, Microcystis, Planktothrix and Oscillatoria (Fawell et al., 1999;Viaggiu et al., 2004).Chemical synthesis of anatoxin-a hydrochloride and its conformational study with the crystal structure description has been done in past (Koskinen and Rapoport, 1985).Anatoxin-a has a semi -rigid bicyclic secondary amine structure and two enantiomeric forms, from which only (+)-anatoxin-a is produced in nature.The investigations on the characterization of anatoxin-a was effectively initiated in 1961 following after the death of cows that had ingested water from a lake containing an algal bloom in Saskatchewan, Canada (Carmichael et al., 1975;Carmichael and Gorham, 1978;Devlin et al., 1977;Moore, 1977).Anatoxin-a was implicated in the death of a 17-year-old boy who died 2 days after swallowing water while swimming in a pond containing an algal bloom (Behm, 2003).The toxin was termed very fast death factor (VFDF) because intraperitoneal (i.p.) injection of toxin-producing cells or cell culture filtrates into mice induced paralysis, tremors, mild convulsions and death within 2 to 7 min.Anatoxin-a has also been implicated in cases of animal poisoning following consumption of water containing blooms of Anabaena flos-aquae although no quantitative exposure data are available (Carmichael and Gorham, 1978;Edwards et al., 1992;Gunn et al., 1992;Pybus et al., 1986).The signs of anatoxin-a contact are failure of coordination, muscular fasciculations, convulsions and finally death of a person by respiratory paralysis.From the two modes of action of anatoxin-a, firstly it binds with the receptor and acts as an agonist of acetylcholine by stimulating the nerve.Upon binding of anatoxin-a with the receptor, it is not expelled due to which the nerve impulse does not fade.In its second mode of action, anatoxin-a inactivates the acetylcholine which is released by normal impulses and thus it inhibits the acetylcholinesterase enzyme.Cholinesterase, takes part in the hydrolysis of acetylcholine (a neurotransmitter), and is not capable of degrading the anatoxin-a due to which it causes permanent stimulation of the muscle cells which leads to paralysis.This result is an over stimulation of the muscle that leads to limp paralysis and death, when the muscles of the chest responsible for breathing are affected.Anatoxin-a has become a very useful agent in investigating nicotinic acetylcholine receptors (nAChRs) because it is resistant to enzymatic hydrolysis by acetylcholinesterase and 100-fold more selective for nicotinic acetylcholine receptors than for muscarinic acetylcholine receptors (Aronstam and Witkop, 1981).When acetylcholine is released at the neuromuscular junction of motor neurons, it binds to muscle cell receptor molecules consisting of a neuromuscular binding site and an ion channel.This triggers ionic currents that induce muscle cell contraction.The extracellular acetylcholinesterase acts on acetylcholine by degrading the neurotransmitter to prevent overstimulation of the muscle.After the contamination with anatoxin-a, the muscle cells keep on stimulated and causing muscle twitching, fatigue and finally, paralysis takes place.There are animal studies reports on the acute lethality of anatoxin-a resulted in the overstimulation of respiratory muscles may result in respiratory arrest and rapid death (Carmichael et al., 1975;1977;Stevens and Krieger, 1991).The main known toxic effect of anatoxin-a is acute neurotoxicity that is manifested as progressive clinical signs.

Quantum chemical calculations
The geometry optimization of anatoxin-a was done by means of density functional theory with a hybrid function B3LYP and because of its excellent compromise between computational time and description of electronic correlation.All the calculations were performed by Gaussian 09 suite of program at 6-311G** basis set (Becke, 1993;Frisch et al., 2009;Goel and Singh, 2013;Goel and Kumar 2014;Lee et al., 1988).The input file for the simulation of geometry optimization was a Z-matrix, generated with the help of Gaussview 5.0 that was also used in the visualization of output files.The elec-tronic absorption spectra for the optimized structure of Anatoxin-a was calculated at TD-DFT/B3LYP/6-311G** basis set both in gas and solvent phase (water).Energy calculations for HOMO, LUMO, electronegativity (χ), chemical hardness (η), soft-ness (S) and electrophilic index (ω) were also calculated by using Koopmans theorem for closed shell molecule (Koopmans, 1934) and defined as: Chemical hardness determines the resistance to change in the electron distribution.It is associated with the stability and reactivity of a chemical system and calculated by using Equation 1.
The softness is the property of a molecule which helps in the calculation of chemical reactivity.It is the reciprocal of hardness and calculated by using the Equation 2.
Electronic chemical potential is the negative of electronegativity of a molecule and determined by using the Equation 3.
Electronegativity describes the tendency of an atom or a functional group to attract electrons towards it.Higher the associated electronegativity, more an element or compound attracts electrons.
Electronegativity is determined according to Equation 5.
Electrophilic index (capacity of a species to accept electrons) is calculated by using the electronic chemical potential and chemical hardness as given away in Equation 4. It is the measure of stabilization in energy after a system accepts extra amount of electronic charge from environment (Koskinen and Rapoport, 1985;Lee et al., 1988).

Molecular docking studies
The protein-ligand docking is a well established computational technique widely applied in structure-based drug design, constantly used to predict the correct 3D conformation of a protein-ligand complex (Sergio et al., 2006).The molecular docking of anatoxin-a with nAChR (PDB ID: 2BG9) was performed using Glide version 5.9 by Maestro 9.4 software interface in Schrodinger 2013 (Friesner et al., 2006).The protein was prepared using Maestro's protein preparation wizard in which the water molecules were deleted, hydrogens were added, bond orders were assigned, and the overall structure was minimized to RMSD of 0.30 Å using OPLS2005 force field.The five active sites within the protein were predicted using SiteMap module version 2.7 (SiteMap, version 2.7, Schrodinger, LLC, New York, NY, 2013) in Maestro 9.4, which uses a novel algorithm for rapid binding site identification, from which the active site with the best site score was selected for preparing the grid.The centroids of the selected amino acid residues in the active site were taken into consideration for grid preparation.The ligand was prepared using Maestro's LigPrep module version 2.6.Further, the docking of anatoxin-a with nAChR was done by Glide using Extra Precision (XP).The best pose was selected on the basis of docking score, Glide energy and rank.The most energetically favourable conformation was selected and visualized using Maestro's XP viewer and PyMol (DeLano, 2002).

Molecular geometry and ultraviolet spectra analysis
The geometry of anatoxin-a has been optimized at B3LYP/6-311G** basis set which has been shown in Figure 1.The optimized geometry showed positive vibrational frequencies indicating the global minimum on the potential energy surface was attained during optimization.The single-point energy calculations were performed, and zero-point corrected total energies recorded.In addition to the characterization of anatoxin-a, the gas phase geometries, harmonic vibrational frequencies and binding energies were also computed.Further, the structural parameters like bond angle (Table 1) and bond length (Table 2) between different atoms of anatoxin-a from optimized geometry were also calculated.The ultraviolet spectra analyses of anatoxin-a was carried out both in water and gas phase at TD-DFT/B3LYP/6-311G**.The theoretical electronic excitation energies, oscillator strengths and absorption wavelength of anatoxin-a in gas and solvent phase were computed (Table 3) which show that the calculated absorption maxima are at 423, 349 and 243 nm in gas phase, while it was observed at 392, 362 and 258 nm in water.The molecular orbital geometry illustrates that the absorption maxima of Anatoxin-a corresponds to the electron transition between frontier orbitals (HOMO and LUMO).

Frontier molecular orbitals (FMOs)
The FMO plays an important role in the computation of quantum chemistry, optical and electric properties of a molecule (Goel and Kumar, 2014).The HOMO and LUMO are known as FMOs in which, HOMO represents the ability to donate an electron and LUMO acts as an electron acceptor.The gap between the energies of HOMO and LUMO helps in the determination of chemical reactivity, kinetic stability, chemical hardness and softness and optical polarizability of a molecule.The hard molecules are not more polarizable than the soft ones because they need big excitation energy.For evaluating the energetic activities of anatoxin-a, we did the calculations in water and gas phase at B3LYP/6-311G** level.The energies of the highest and second highest occupied molecular orbital, lowest and the second lowest unoccupied molecular orbital were calculated (Table 4).The molecular orbital surfaces and energies (eV) of HOMO, HOMO-1, LUMO and LUMO+1 in gas phase for anatoxina are shown in Figure 2. The energy differences between the HOMO and LUMO in anatoxin-a are -5.150,-4.945 eV in water and gas phase, respectively, that shows the chemical stability of the molecule.The HOMO lies on whole molecule except the acetyl group while LUMO lies on the acetyl group and some atoms of seven membered ring within the anatoxin-a molecule.

Docking analysis
Protein-Ligand docking is a well established computational technique widely applied in structure-based drug design (Badry et al., 2003).This methodology evolved constantly to predict the correct three-dimensional

Bond type Bond length(Å) Bond type Bond length(Å)
conformation of a protein-ligand bound complex.Anatoxin-a is a severe neurotoxin that act as mimic of acetylcholine (irreversibly binds with the nAChR) used for docking experiment.Binding of Anatoxin-a causes overstimulation of mussels, results in fatigue and then para-lysis (Wonnacott et al., 1991).Docking study is planned to know the mode of binding and forces responsible for the stabilization of Anatoxin-a at the nAChR surface.The anatoxin-a was docked with nAChR using Glide in order to study the binding mode of the anatoxin-a with NAR.The docking score and glide energy were found to be -4.567 and -18.242, respectively.The present study indicates that the anatoxin-a binds mainly to the hydrophobic pocket of chain A of the nAChR protein (Figures 3 and 4).The residues involved in polar interactions with anatoxina are Phe214, Val218, Ile219, Ile264, Leu273, Tyr277, Met278, Phe280, Thr281 and Phe284 (Figure 3).The complex of anatoxin-a and nAChR is stabilized by hydrophobic interactions between the ligand and Phe214, Tyr277 and Thr281 amino acid residues of the protein of bond lengths 1.97, 1.96 and 2.04 Å, respectively (Figure 3b).This study shows that anatoxin-a binds to the nAchR and acts as an agonist of acetylcholine and also inactivates it as reported previously.

Conclusions
The structure of anatoxin-a has been optimized by the Gaussian 09 at B3LYP/6-311G** level.At this level, the total energy, dipole moment, degrees of freedom and the energy difference between HOMO and LUMO were found to be -520.107a.u., 2.643 Debye, 84 and -4.945 eV, respectively.As anatoxin-a is a very fast neurotoxin, it is not degraded by cholinesterase and thus causes permanent stimulation of muscle cells leading to paralysis.The present study highlighted the structural parameters of anatoxin-a, which will be a useful base for rational drug designing as well as for experimental studies.The docking studies suggest that the anatoxin-a bound to the nAChR with hydrophobic interactions and inactivates the acetylcholine.The structure and binding related scatter data will be very useful in the cure of the neurodegenerative disorder, such as Alzheimer's disease by using the modified analogues of Anatoxin to further elucidate the receptor sub-types (Wonnacott et al., 1991;Araoz et al., 2010).This may lead to the development of new drugs which do not have the toxicity like anatoxin-a, but which act merely as acetylcholine replacement candidates.

Figure 2 .
Figure 2. Molecular orbital surfaces with their corresponding energy (eV) for HOMO, HOMO-1, LUMO and LUMO+1 in gas phase in anatoxin-a computed by TD-DFT.

Figure 3 .
Figure 3. Anatoxin-a docked in the binding pocket of nAChR using Glide 5.9.Different views of nAChR and anatoxin-a conformation.a) overview in cartoon model.b) Residues around the 4 Å region of binding site of anatoxin-a, the anatoxin is shown in ball and stick and hydrophobic interactions are depicted in pink dotted lines.Images are generated using PyMol and Maestro's XP viewer.

Figure 4 .
Figure 4. Surface view of anatoxin-a binding pocket.a) protein surface (grey colour) and ligand surface (blue colour) are shown separately which shows that ligand is embedded in the binding pocket of protein.b) Hydrophobic pocket of nAChR and anatoxin-a, the anatoxin-a represents in ball and stick model and the binding pockets in cyanocolor, in which the green color represents the binding pocket residues.Images are generated using PyMol and Maestro's XP viewer.

Table 1 .
Bond Angles in degree (˚) for optimized structure of anatoxin-a.

Table 2 .
Bond distances in Å for optimized structure of anatoxin-a.