Atomic coordinates and crystallized structures of ten selected scorpion neurotoxins, 1AHO, 1B7D, 1DJT, 1DQ7, 1PTX, 1SN4, 1ZYW, 2A7T, 2I61, and 2SN3 [16–25] were obtained from the PDB at the Brookhaven National Laboratory [26]. All the structures were solved with <2.5Å resolutions, and the sequence identity among the majority of proteins in the dataset was less than 40%. Also these neurotoxins were selected so as all of them had 60–65 residues.

We obtained the sequence version of the human D1 dopamine receptor from Swiss-Prot (http://expasy.org/sprot). Then a BLAST (http://www.ebi.ac.uk/blast2/index.html) sequence analysis was performed against the whole PDB, to select the correct template that could be used to generate a model of the D1 dopamine receptor. From the selected template, the three-dimensional (3D) structural model for the D1 dopamine receptor was generated by the biopolymer homology modeling software of Swiss-model/Deep view 3.7. The generated D1 dopamine receptor model was then checked with known agonists and antagonists.

We computed the conservation of amino acid residues in each scorpion neurotoxins using the Clustal-W multiple alignment program [27].

CHARMM force field [28] (license No. 070815-29) was used to minimize the structures of scorpion neurotoxins and of the D1 dopamine receptor before docking. Molecular docking was performed using protein–protein docking software GRAMM v. 1.03 [29]. The GRAMM v. 1.03 is the best protein–protein docking software certified in the CAPRI (Critical Assessment of PRediction of Interactions) test and the full results of CAPRI are made available on its website (http://capri.ebi.ac.uk). The algorithm used by GRAMM v. 1.03 performs Fast Fourier transformation methodology by employing smoothed potentials, refinement stage and knowledge-based scoring. We used a fine-grid projection of a softened Lennard–Jones potential function [30] and then the top 4000 grid-based predictions are subjected to a conjugate minimization in continuous 6D rigid body space with the same soft potential. One representative prediction for each minimum is stored and the number of initial predictions falling into this minimum is marked as the volume of the minimum. The average radius of such minima on our smoothed landscape is 5 Å.

Interaction free energies are crucial for analyzing binding propensities in proteins. We computed binding affinity, desolvation free energy, electrostatic and contact free energies of docked structures using the program FastContact [31]. FastContact provides a fast estimate of the interaction free energy between two proteins. The interaction between two proteins is estimated as ΔGbind, where:

Solvent accessibility is the ratio between the solvent accessible surface area of a residue in three-dimensional structure and that in an extended tripeptide conformation. We have analyzed the solvent accessibility of binding amino acid residues in the toxins as well as in the D1 dopamine receptor before and after the docking simulations. A majority of binding residues showed a change from an exposed state before binding to a buried state after binding. We obtained the solvent accessibility information using NetASA program [34].

Stabilization center residues are defined by considering the contact map of a protein. Two residues are in contact if there is at least one pair of heavy atoms with a distance less than the sum of the van der Waals radii of the two atoms plus 1.0 Å. A contact is considered long range if it is between residues that are separated by at least 10 residues in the amino acid sequence. Two residues are stabilization center elements if they are involved in long-range contacts and if at least one supporting residue can be found in each of the flanking tetrapeptides of these residues, in such a way that at least seven out of the possible nine interactions are formed between the two triplets [35,36]. We used the server for identifying the stabilization centers at http://www.enzim.hu/scide.

Stabilizing residues play an important role in the stabilization of proteins. We have computed the stabilizing residues in all 10-scorpion neurotoxins and for the D1 dopamine receptor using parameters such as surrounding hydrophobicity, long-range order, high conservation score, and if it belongs to a stabilization center. We used the public server available at http://sride.enzim.hu for this purpose [37].

Molecular modeling of the D1 dopamine receptor was achieved on the basis of the crystal structure of the human beta2 adrenergic G-protein-coupled receptor [7] solved at 3.4 0 Å (PDB ID 2R4R). The sequence identity between the D1 dopamine receptor and the Beta2 adrenergic G-protein coupled receptor is 37%. Detailed information about the human beta2 adrenergic G-protein-coupled receptor and the D1 dopamine receptor is depicted in Table 1. BLAST output showed that the human beta2 adrenergic G-protein-coupled receptor exhibited an excellent E-value, 2×10−61, and a bit score of 232. The alignment between the D1 dopamine receptor and the human beta2 adrenergic G-protein coupled receptor is shown in Fig. 1. The three-dimensional (3D) structural model for the D1 dopamine receptor was generated based on the human beta2 adrenergic G-protein-coupled receptor template by using the biopolymer homology modeling software of Swiss-model/Deep view 3.7.

The modeled D1 dopamine receptor structure was checked with known agonists and antagonists. The agonists are A77636, A68930, dihydrexidine (DHX), dinapsoline, dinoxyline and dopamine. The antagonists are LE300, SCH23390. We obtain the 2D structures of agonists and antagonists from Pubchem and the 2D structure was converted to 3D structures by using CORINA software. All of them were bound with modeled D1 dopamine receptor at their specific regions. We showed dopamine binds with the predicted structure of D1 dopamine receptor in Fig. 2. The geometric qualities of the models were assessed by PROCHECK. This provides a good validation of the predicted structure for the D1 dopamine receptor and of the predicted binding site of dopamine.

We computed the conservation of residues using ClustalW multiple alignment program [27]. It was observed that highly conserved residues in scorpion neurotoxins are tyrosine, cysteine, glycine, leucine, phenylalanine and partially conserved residues are aspartic acid, asparagine, serine, lysine, and isoleucine. It is notable observation that these highly conserved residues were involved in the docking sites with the D1 dopamine receptor. Hence it can be thought that the conserved residues play a major role as docking sites for the toxin molecule. Among the major conserved residues in the toxin molecule, aspartic acid, lysine residues were involved in binding sites more frequently than others. The multiple alignment for all the scorpion neurotoxin is shown in Fig. 3.

CHARMM force field [28] was used to minimize the structures of scorpion neurotoxins and of the D1 dopamine receptor before docking. All the ten scorpion neurotoxin selected for this study showed a good docking behaviour with the D1 dopamine receptor. The interface residues of the D1 dopamine receptor and the scorpion neurotoxins involved in binding are shown in Table 2. The docked complexes of the scorpion neurotoxin with the D1 dopamine receptor are shown in Fig. 4. It can be seen from Fig. 4 that there is a high degree of uniformity in the docking regions and also with the docking residues. We calculate the overall binding free energy for each of these ten complexes; the results are shown in Fig. 5. It was observed that the complex formed with neurotoxin (PDB ID 2A7T) shows the highest overall binding affinity. From the X-ray structure report of 2A7T neurotoxin [23], residues Glu(2), Asp(50), Arg(64) that protrude outwardly in this neurotoxin are the main residues for interaction with the receptor. This experimental result is well supported by our computational method for 2A7T. Also, this is the main reason for the highest binding affinity of this neurotoxin towards the D1 dopamine receptor. The complex formed with (PDB ID 2SN3) had the lowest binding affinity with the D1 dopamine receptor. Further, it could be seen from Fig. 5 that the binding free energies are almost similar for all other scorpion neurotoxins with the D1 dopamine receptor.

Table 3 shows the minimum and maximum binding energies for individual residues after the formation of the complex. It can be seen from Table 3 that the minimum energy for binding ranges from 1.988 to 10.600-kcal/mol, while the maximum binding energy ranges from 0.481 to 1.903-kcal/mol. It can be observed that, except for 2SN3, all other binding energies are almost similar. Among the selected neurotoxins, 2A7T shows the highest binding affinity towards the D1 dopamine receptor. Our results on the binding effect of scorpion toxins with the D1 dopamine receptor and other GPCRs are correlated with the following experimental reports. Ismail et al. reported that scorpion Leiurus quinquestriatus venom blocked the dopaminergic receptors during envenomation [38]. Teixeira et al. reported a good experimental evidence for interaction between scorpion toxins with GPCR-like adrenergic and cholinergic receptors [39]. Scorpion venoms mediate their autonomic effects through direct agonist actions on post-junctional muscarinic M₃ cholinoceptors and α-adrenoceptors that were reported by Matthew et al. [40]. Fernandes et al. proved the effects of scorpion toxin, tityustoxin on the release of [3H] dopamine of rat brain prefrontal cortical slices [41]. Scorpion toxins active in pancreatic secretion include those that act on ion channels (Na⁺, K⁺, and Ca²⁺), G Protein coupled receptors (GPCR), and signalling kinases related to protein kinase C [42].

Solvent accessibility of all the residues in both toxins (Table 4) and the D1 dopamine receptor molecule (Table 5) before and after binding were computed with NetASA [34]. It can be seen from both tables that the solvent accessibility change is mainly from the exposed state before binding to the buried state after binding. Moreover, such changes are comparatively much more for toxin molecules than for the receptor molecule. We also computed the desolvation energies before and after the binding by using the formula:

We computed the stabilization center for the complex using web server Scide [35]. The results of the stabilization center are shown in Table 6. One or more residues act as stabilization centre(s) that were also involved in the binding with the receptor shown in bold in Table 6. This can be seen in all the scorpion neurotoxins, except 1PTX and 2SN3. Among the selected neurotoxins, 2A7T has five residues, acts as stabilization center, and is also involved in binding with the D1 dopamine receptor. On the other hand, the neurotoxin 2SN3 has no residues that can act as stabilization center and are involved in binding. From this result, we concluded that stabilizing centers may enhance the stability of the complex and also may support the docking process.

We computed the stabilizing residues in the scorpion neurotoxin molecules and also in the complex using web server Sride [37]. The results of the stabilizing residues are shown in Table 7. Cys(48) in 1AHO, Leu(51) in 1PTX and Tyr(7) in 1ZYW were found as both stabilizing residues in the toxins and were also involved in binding. Ile(6) in 1DJT and 2A7T and Val(45) in 2A7T were found to be stabilizing residues in both toxins and also in the complex. This behaviour could not be generalized for other toxin molecules in the sense that certain residues that act as stabilizing residues in the toxin were not seen as the stabilizing residue when they formed the complex with the D1 dopamine receptor molecule. However, the complex formed could be considered to be quite stable as the results from the stabilizing residue were very favourable.