Proof Of Concept
- For the respective proteins, We scavenge the RCSB Database for the ZAP1 protein structure and an online tool for getting the ITSrDNA Structure, picking the 1ZW8 protein structure which is a solution NMR structure.
- PyMOL Contribution: PyMOL is a molecular visualization tool used for editing and preparing protein and nucleic acid structures for computational studies. In this research, PyMOL was employed to: • Remove water molecules and heteroatoms (such as ligands, ions, and cofactors) from the downloaded Protein Data Bank (PDB) files. This step is crucial to eliminate non-essential molecules that could interfere with the docking process. Using PyMOL, these extraneous molecules were removed to avoid non-specific interactions during the docking process. Water molecules, in particular, can obscure important binding sites and lead to erroneous docking predictions. • Add polar hydrogens to the protein and DNA structures. This ensures that the hydrogen bonding, which plays a significant role in protein-DNA interactions, is accurately represented during the docking phase. This step is crucial as polar hydrogens contribute to hydrogen bonding, which is a major stabilizing force in protein-DNA interactions. Proper representation of hydrogen atoms ensures that electrostatic interactions and hydrogen bonding networks are accurately modelled during docking simulations. Without these, the docking algorithm may fail to recognize critical contact points between the protein and DNA.
- AutoDock Vina Contribution: AutoDock Vina is a widely-used software for molecular docking, which was utilized to assign Gasteiger charges to the protein structure. Gasteiger charges approximate partial charges on atoms, which are essential for modelling electrostatic interactions during docking Gasteiger charges were added to ensure proper electrostatic interactions during the docking process. Gasteiger charges are widely used in docking as they provide a quick and reliable approximation of partial charges on atoms, essential for accurate modelling of non-bonded interactions. By assigning these charges, the software prepares the protein for accurate prediction of binding affinities and docking orientations with DNA.
- Saving the processed file in the .pdb format
- Alpha fold 3: AlphaFold 3 is Google’s advanced protein structure prediction tool, which was utilized to explore protein-DNA docking. The FASTA sequence of the ZAP1 protein and the ITS rDNA were submitted, and AlphaFold generated multiple models in cif format. Five docked models were obtained, each representing possible protein-DNA complexes, which were analysed to explore different conformational possibilities. Alternative: HADDOCK (High Ambiguity Driven Docking)
- We select all residues for docking, and select the Centre of Mass restraint for docking, while keeping the default parameters.
- The results for each Protein-DNA Aptamer docking is obtained in the form of Clusters.
- Choosing the Cluster with lowest HADDOCK energy, and downloading all the structures In .pdb format associated with the cluster for MD runs.
- Using the Solution Builder tab on CHARMM-GUI web server, firstly inputting the docked Protein-Aptamer structure obtained from HADDOCK, and manipulating the residues in case of any problems.
- Then We in-silico generate a Rectangular WaterBox with edge distance of 10 Å, and add 0.15 M KCl in the solution, using the ion-placing method called Monte-Carlo, The Water Module used is TIP3
- We generate the Crystal type Box(Cubic), with the parameters, now automatically generating grid information for PME FFT.
- Afterwards, we confirm the overall structural integrity of the Protein-DNA complex in VMD (Visual Molecular Dynamics) after solvating it.
- MD simulations were carried out using GROMACS software, starting with energy minimization followed by NVT (constant Number of particles, Volume, and Temperature) and NPT (constant Number of particles, Pressure, and Temperature) equilibration for 100 ps.
- The production run was set for 10 ns, saving trajectory files every 1 ps for further analysis.
- Upon the completion of MD simulations, the obtained trajectory was evaluated using various quality assessment tools from the SAVES server to ensure the structural integrity of the Protein-DNA complex.
