Team:SNU India/Molecular Modelling
Molecular Modelling
We also wanted to check structure-function correlation in a bacterial laccase, for this purpose we chose Bacillus pumilus laccase which was characterised part (BBa_k863000) available in the iGEM kit. A model was generated using the SWISS-MODEL tool on EXPASY (Waterhouse et al, 2018) using the Bacillus subtilis CotA laccase structure as a template (PDB ID: 1GSK), having 70.12% sequence similarity with bpul. The modelled protein was then validated through Ramachandran plot generated through RAMPAGE tool (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) which is visualized from UCSF Chimera (Pettersen et al, 2004) as shown in the image below suggesting that greater than 99% of residues in the modelled structure are in favoured and allowed regions.
Number of residues in favoured region (~98.0% expected): 483 ( 95.6%) Number of residues in allowed region ( ~2.0% expected) : 20 ( 4.0%) Number of residues in outlier region : 2 ( 0.4%)
Next, the protein structure model thus generated was prepared (as mentioned in Mehra et al 2018) and docked using AutoDock Vina (Trott and Olson, 2010). In the docked model it was observed that both p-coumaric acid and Estradiol were docking at a similar ligand binding site near the T1 copper center with binding affinities (Table 1) We were also able to identify the protein residues involved in polar contacts with the docked molecules. Glutamine at position 144 and 427 being involved in interaction with p- coumaric acid, whereas glutamine at position 144 and argininine and valine at position 480 and 482 respectively were involved in interaction with estradiol.
Modelling binding of p-coumaric acid and estradiol binding to fungal laccase.
In order to identify the binding pockets of p-coumaric acid and estradiol and residues involved in their interaction with fungal laccase we performed molecular in-silico docking. The protein crystallographic data for the fungal laccase was taken from RCSB’s protein database (PDB ID: 1GYC). This laccase was prepped for docking using UCSF Chimera molecular modeling system (http://www.rbvi.ucsf.edu/chimera/). The protein structure was first prepared for docking using the inbuilt DOCK PREP plugin where proper charges were assigned to the copper centers and Gasteiger values were calculated. The parameters used for our analysis were based on published literature for docking substrates on different laccase (Mehra R et al 2018). We used pub-chem structures of both the two ligands that is for estradiol (Pub-chem ID- 5757) and for p-coumaric acid (Pub-chem ID- 637542) which were minimized using the inbuilt plugins and brought to the lowest possible energy conformation. The prepped protein and ligands were then docked using Autodock vina (http://vina.scripps.edu/) plugin in UCSF chimera using standard values. In the docked model we got a number of different conformations of estradiol and p-coumaric acid where it was interacting with the laccase. The best structures are those which have more no. of hydrogen bonds and low scores. In our model we can see that p-coumaric acid and estradiol have both docked near the T1 copper site having low scores, making 2 and 1 hydrogen bonds respectively. These results suggest both p-coumaric acid and estradiol can bind to the same active site on fungal laccase and hence p-coumaric acid could be used as a substrate for the fungal laccase.
Figure 2: Left panel images are for docking estradiol with Tvl laccase and right panel images shows docking results for p-coumaric acid. Upper panel shows the binding pockets of p-coumaric acid and estradiol. Both bind at the same pocket near the T1 copper site and have hydrogen bonds with the adjacent amino acids improving the stability of the docked structure. Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
References
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