Protein-DNA docking plays a critical role in understanding the molecular interactions governing regulatory functions in cells. This study presents a computational docking experiment between the ZAP1 protein and the Internal Transcribed Spacer (ITS) rDNA. The ZAP1 protein, which plays a significant role in zinc homeostasis, was sourced from the RCSB Protein Data Bank (PDB ID: 1ZW8). The ITS rDNA sequence was also retrieved and prepared for the docking study. Prior to docking, structural refinements such as the removal of water molecules, heteroatoms, and the addition of polar hydrogens were performed using PyMOL to enhance the accuracy of the docking results. This research demonstrates the critical importance of proper protein and DNA preparation in bioinformatics docking procedures.

Protein-DNA interactions are essential for many cellular processes such as transcriptional regulation, replication, and repair. Understanding these interactions at a molecular level provides insights into the mechanisms of gene expression regulation and genome stability. In this study, we explore the interaction between the ZAP1 protein, a transcriptional regulator involved in zinc homeostasis, and the Internal Transcribed Spacer (ITS) rDNA, a non-coding region involved in the maturation of ribosomal RNA. The docking experiment was carried out using molecular modeling techniques to provide a preliminary understanding of their binding interface.

Protein and DNA Selection
The ZAP1 protein was selected for this study due to its regulatory role in zinc metabolism and its potential interaction with ITS rDNA. The ZAP1 protein structure (PDB ID: 1ZW8) was downloaded from the RCSB Protein Data Bank. The ITS rDNA sequence, representing the internal transcribed spacer region, was also retrieved for docking analysis.

Preprocessing Procedures
Before the docking process, the protein and DNA structures require several preprocessing steps to ensure high-quality results.

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 to avoid non-specific interactions during the docking process.
• Add polar hydrogens to the protein and DNA structures to ensure accurate representation of hydrogen bonding during the docking phase.

Contribution:
AutoDock Vina was utilized to assign Gasteiger charges to the protein structure, essential for modeling electrostatic interactions during docking. By assigning these charges, the software ensures accurate prediction of binding affinities and docking orientations with DNA.

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.

An alternative approach:
HADDOCK is a flexible docking software that integrates experimental data, such as NMR and mutagenesis results, to predict protein-protein, protein-DNA, or protein-ligand interactions. In this study, HADDOCK was chosen for its ability to:

• Use active and passive residues to guide the docking process, incorporating prior knowledge of the protein-DNA binding interface.
• Perform rigid-body docking followed by semi-flexible refinement and explicit water refinement, increasing the accuracy of the predicted protein-DNA complexes.

The SAVES server integrates multiple validation tools to assess the quality of the protein structure before proceeding to molecular dynamics simulations. In this study, the following tools were used:

• ERRAT: This tool evaluates the quality of non-bonded atomic interactions within the protein structure. It assigns a quality factor indicating the extent of reliability. ERRAT helps identify regions in the model where the geometry might be distorted, which could affect subsequent docking or simulations.
• Verify3D: This tool checks the compatibility of the 3D structure with its 1D amino acid sequence. A high score in Verify3D suggests that the protein's 3D conformation is consistent with its sequence.
• WHATCHECK: This tool performs an in-depth check of the stereochemistry of the protein, including bond angles, bond lengths, and torsion angles.
• PROCHECK: This tool analyses the protein structure's backbone conformation by generating a Ramachandran plot.

Following the molecular docking procedure, five docked structures were successfully obtained, and they are now ready to undergo molecular dynamics simulations. Due to the high computational demand, we utilized IIT Roorkee's supercomputer, PARAM GANGA, to efficiently initiate the simulations, ensuring faster and more accurate results.