Hello everyone

I wanted to dock NQO1 and p53 (protein-protein docking).

NQO1 (a homodimer) is a quinone reductases which has FAD bound to both of its active sites. In order to catalyse the reduction of quinones (its substrate), the FAD within the active site has to be reduced to FADH2 by the oxidation of NADPH which then makes way for the incoming quinone (ping-pong mechanism).

NQO1 is also known to protect p53 from Ubiquitin independent 20S proteasomal degradations and so mutations affecting NQO1 could alter p53 stability (this is my study interest). In pursuit of investigating this association, I want to dock p53 with NQO1 homodimer (having both FAD and NADPH within the active sites).

Now here's the problem

In the absence of a crystallographic structure bearing both the ligands together (FAD and NADPH), I want to dock NADPH first to the NQO1 protein (protein-ligand docking). Being new to docking and after reading multiple threads, I realized that validating my docking protocol is essential.

To validate my docking protocol (done using AutoDock Vina), I considered a crystallographic complex protein (1kbq, https://www.rcsb.org/structure/1KBQ) with two ligands in it – namely FAD and 5-methoxy-1,2-dimethyl-3-(4-nitrophenoxymethyl)indole-4,7-dione and performed the docking of the same complex (I removed 5-methoxy-1,2-dimethyl-3-(4-nitrophenoxymethyl)indole-4,7-dione from the structure and used it as a ligand). Ligand and protein preparation was carried out using AutoDock Tools.

Please note – I tried docking using ligand obtained from two sources

1. Ligand from PubChem (didn’t yield appropriate pose even after energy minimization or reduction of active torsions, Structure labelled B in the figure attached)

2. Ligand derived from 1kbq itself (yielded appropriate pose after reduction of active torsions without energy minimization) (structure A and its derivatives)

As I have learned, to validate my docking, I need to compare RMSD between the crystallographic structure and my docked pose which should be below 2A (pardon me if I am wrong). Although my docking seems perfect (as evident from the last figure, after reducing 4 out of 5 active torsions for the ligand) I have failed to calculate the RMSD. I have tried online servers like COMPARE (https://webs.iiitd.edu.in/raghava/ pldbench/compare.php) and LS-align (https://zhanglab.ccmb.med.umich.edu/LS-align/) but no results. I have also tried comparing the RMSD using Discovery studio visualizer wherein I have used the original crystallographic structure bearing both the ligands as reference by selecting my target ligand in the structure (Structure>RMSD> Set reference) and compared it to my docked pose (out_ligand_1.pdbqt Structure>RMSD>All atoms/ Heavy atoms). The message prompted is

“The following molecule(s) failed due to the number of atoms not matching the reference ligand”

1. Please help me calculate the RMSD between the two structures.

2. Further, by the looks of it (as the docked pose superimposes the crystallographic structure) can I assume that my docking protocol is validated?

If yes,

3. Should I dock NADPH to both the active site one at a time (as there are two active sites within the homodimer) or both the active sites can be docked simultaneously (if yes, kindly guide me)?

4. If my docked NADPH exhibits interactions similar to other ligands (interactions mapped from reported NQO1 crystallographic structures), can I use this structure for protein-protein docking using online servers?

Thanking you for your patient reading.