Introduction

Blood Coagulation and Thrombus Formation.

The blood plays a vital role in the human body by transporting essential materials. Blood clotting, or thrombus formation, occurs to stop bleeding from wounds, with fibrin stabilizing the clot to aid healing. However, thrombosis, a condition involving excessive clotting, can lead to serious cardiovascular diseases (CVDs) like heart attacks and strokes. Given the high occurrence and treatment costs of CVDs, this research aims to develop affordable remedies using natural sources, specifically the fibrinolytic enzyme Dionain found in Dionaea muscipula (Venus flytrap).

Dionain and Its Previous Studies.

Dionain is a papain-like cysteine protease enzyme derived from Dionaea muscipula  (Venus flytrap), which is the dominant enzyme in Venus flytrap digestive juice. Further  findings about the enzyme itself are still limited, especially in terms of its fibrinolytic  activity. Enzymatic and structural characterization research by Risør, Michael W., et al.  show that further investigation on dionain may reveal its capabilities in acting as an  efficient, stable, and broad protease.

Experiment Methods

1. Structure Obtainment and Structural Validation

Risør et al [6] in his work has prevailed the crystal structure of the  Dionain enzyme from Dionea muscipula plant, and stored in the PDB  Database. That until Carter T. Butts and his team used this 3D structure  to validate their works on generating the 3D structure of Droserasin and  Nephentesin. Including in the work of Carter Butts, the team also  generate 3D structure of Dionain without plant specific insert (PSI) that  form a blockage on the catalytic pocket, where the catalytic residue  harbours. The model that’s been used in this project is the Dionain  without the mentioned PSI, with the catalytic residue of Cys26, Asp164,  His165, and Gln20 that form stability for the interactions. Ramachandran plot helps to  validate the quality of the  model used. Based on the  calculation, dionain shows a  great quality, with the score of  Ramachandran favoured as  95.05%, and Ramanchandran  Outliers of 0.45 (Also do not  consist of the mentioned  catalytic residues). This justify  the use of this model  throughout the project.

2. Protein-Protein Docking

Protein-protein docking part was performed  using ClusPro software, with additional  information of dionain’s catalytic residues as the  main interactor for the enzyme. The substrate  used was fibrin in it’s full chain form (PDB ID:  2HLO), and the rest of the parameters were left  default. The results showing a clear indication  that Dionain, as the cystein protease, being  predicted to interact with fibrin chain as shown  in the figure. Among all of the predicted  complexes, almost most of the complex that  shows interaction made a great potential of  dionain interacting with chain Gamma of the  fibrin. This occurrence, however, still dominated  by the Hydrophobic bond between the  interaction of the dionain’s catalytic residues  and gamma chain of the fibrin due to the size of  the fibrin used. With these findings, we then  proceed to check the interaction between  Dionain and the domains of the fibrin.

3. Protein-Peptide Docking

Protein-Peptide docking part was done by predicting the interaction between dionain enzyme and 6 of the fibrin domains (A to F).  The prediction was conducted using HADDOCK 2.4 protein-peptide parameters, while the prediction of the binding energy (ΔG)  and the visualization were done using PRODIGY [9], and PyMol & LigPlot+ respectively.

Result:

The prediction of this part resulting in 6 results (Dionain FibrinA,B,C,D,E, and F), where a clear indication of dionain’s  catalytic residues interacting with fibrin domain is seen at  dionain-fibrin F complex (Figure 4B). Out of the rest of the  complexes, Dionain-FibrinF showing the interaction of Cys26  (posses the role of nucleophilic attack,  thus degrading the substrate) using hydrogen  bond at Ser164 of the fibrin domain F, followed  up with the interaction of Asp164 and Gln20  (hydrogen bond) that’s been known to stabilitate  the interaction. This occurrence is a great indication  of the dionain fibrinolytic potential at domain F of  the fibrin, which is the composition of the fibrin  gamma chain. Alongside with the mentioned  interaction, this complex binding strength is  considered as moderate protein interaction, as the  predicted binding energy showing a value of-10.2 kcal.mol-1 and KD value of 3.1 x 10^-8.

4. Protein-Ligand Docking

The fragmentation of the substrate Fibrin using RPG were made to prepare the  ligands. The cleavage rule used to fragment this fibrin were constructed based  on modified Papain clevaging rules, with unique addition of Dionain  characteristics reported by Risør et al [6]. These fragments were then modeled  into ligands (consist of 6 amino acids) which serves and molecule 2 for the  Dionain to bind with (molecule 1). This process utilizes the Protein-ligand fully  flexible docking using HADDOCK 2.4 to examine the potential binding  interactions between the enzyme and the ligands.

Result:

The Protein-Ligand docking prediction showing the interaction of Dionain with the  ligand derived from Gamma chain using the catalytic residues. Cys26, His Asp  164, His 165, and Gln 20 was interacting with the same amino acid from the  ligand, indicating the potential of fibrinolytic activity. Dionain catalytic residues  interacted with Glu5 of the ligand, which was the fourth amino acid from the N  terminal of the ligand. Since the ligand were made from from the last three  previous amino acids and the first three from the next fragment, one would  expect that the predicted interaction will occur in the middle of the made ligand  (third amino acid). This occurrence was most likely due to the computational  limitation of the prediction parameters. Despite the limitation, the energy binding  of this interaction shows a -9.8 kcal.mol-1 and Kd value of 6.2 x 10-8, indicating  a intermediate strength.

Conclusion and Future Outlook

Based on the serial methods performed, Dionain has a  potential to not only interact, but digest the fibrin protein.  The predicted results from protein-protein, protein-peptide,  and protein ligand interaction revealed that dionain prefers  to interact with fibrin in the Gamma chain, with a clear  indication of digestion in the domain F of the chain,  followed by the results of the interaction between Dionain  and ligand derived from Gamma chain. The findings,  however, still contain some imperfections due to the  computational limitation of the parameter used. Elucidating  the behaviour of Dionain using different parameters in silico  will strengthen and refine the results, making it closer to  discovering new biological agent to combat CVDs.