This year, our team has focused on complementary in silico and in vitro analysis of our selected proteins of interest for Methylene Blue (MB) degradation.
Previous studies on elucidating the molecular mechanism of biodegradation by ligninolytic enzymes have suggested the diversity of active binding sites for different common commercial dyes such as Congo Red and Methyl Orange [4]. In order to gain a deeper understanding of the molecular mechanisms of MB biodegradation by our proteins of interest, we performed in silico analysis with GROMACS molecular dynamics simulation.
First and foremost, we did protein structure preparation, which is the most important aspect of in silico analysis. High-resolution X-ray crystallography-resolved structures were selected from the RCSB Protein Database. For instance, the 0.93 Å structure of Phanerodontia chrysosporium Magnesium Peroxidase (PDB ID:3M5Q) was used in one of our analyses to ensure the validity of our GROMACS molecular dynamics simulation.
Apart from protein structures obtained from the RCSB Protein Database, the protein structure of a newly characterized Trametes versicolor lignin peroxidase isozyme (LPG3) was prepared with ColabFold, a multiple sequence alignment (MSA)-mediated AlphaFold protein structure prediction tool [3]. Cofactors were reconstituted into the protein structure by AlphaFill, a homology-based ligand transplantation algorithm [2].
MB-docked structures were prepared with SwissDock and analysed with UCSF Chimera prior to GROMACS Molecular Dynamics Simulation, for which the simulation results are shown below.
Our in silico analyses have demonstrated the MB degradation potential of all proteins of interest as suggested by our data on protein-ligand binding and interaction modes, supporting in vivo ability to form complexes with MB. This has prompted our plans to perform further analyses, such as with Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) to further decouple the energetics within the system, or co-incubation and validation with cryo-EM in wet lab.
Overall, the selection of all 5 proteins as potential candidates to proceed to in vitro analysis was based on literature review and our in-silico analysis workflow.
Wet Lab
The idea of building a Synechococcus elongatus shuttle vector first began with iGEM Team Marburg 2019’s shuttle vector BBa_K3228089. This plasmid backbone was further modified by Team HK_SSC 2021 to create BBa_K3776009. Our team has now designed our plasmid based on these two previous contributions in view of their modularity and compatibility with our selected chassis Synechococcus elongatus UTEX 2973.
After molecular cloning, further analysis on protein expression will be performed based on protocols contributed by iGEM Team HK_SSC 2021 and HK_SSC 2022.
Dry Lab
The interactions between Methylene Blue and Proteins of interest are analyzed by Discovery Studio [1], and visualized by PyMOL (Schrodinger LLC, 2020). Note that MB has different binding sites among all proteins of interest.
The above analysis serves as preliminary models to identify and visualize interactions between Methylene Blue and Proteins of interest.
van der Waals interactions contributed by T193,T196, T199, Q262, A299, T300, G301 and Q302 are present in the binding pocket. Hydrogen bonds are formed between MB and Q200, N260 and A304. Alkyl interactions are formed between MB and P303 and M305. π-anion interactions are present between MB and D198.
1KYA: Trametes versicolor Laccase (lac2)
van der Waals interactions contributed by L35, P123, and N 172 are present in the binding pocket. Hydrogen bonds are formed between MB and D142 and V145. π-alky interactions are formed between MB and P32.
van der Waals interaction contributed by I199 is present in the binding pocket. Hydrogen bonds are formed between MB and I295 and F301. π-alky interactions are formed between MB and I295 and L299. π-Sigma interactions are present between MB and I295.
Note that the Ligand Binding Mode with the lowest FullFitness Energetics in the cluster is utilized for subsequent GROMACS Molecular Dyamics Simulation Analysis.
In brief, FullFitness energetics takes into account the internal energy of the ligand and the enzyme, the interaction between the ligand and enzyme complex and the solvation energy by electrostatic ,and non-polar contributions proportional to the solvent accessible surface area (SASA) [1].
Reference:
[1] Grosdidier, A., Zoete, V., & Michielin, O. (2007). EADock: Docking of small molecules into protein active sites with a multiobjective evolutionary optimization. Proteins, 67(4), 1010–1025. https://doi.org/10.1002/prot.21367
[2] Hekkelman, M. L., De Vries, I., Joosten, R. P., & Perrakis, A. (2022). AlphaFill: enriching AlphaFold models with ligands and cofactors. Nature Methods, 20(2), 205–213. https://doi.org/10.1038/s41592-022-01685-y
[3] Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Овчинников, С. Г., & Steinegger, M. (2022). ColabFold: making protein folding accessible to all. Nature Methods, 19(6), 679–682. https://doi.org/10.1038/s41592-022-01488-1
[4] Sorhie, V., Chang, C., Bharali, P., & Bhaskar, M. (2022). UNDERSTANDING ENZYME-LINKED BIODEGRADATION BY MOLECULAR DOCKING OF SCHIZOPHYLLUM COMMUNE’S LACCASE, LIGNIN. . . ResearchGate. https://doi.org/10.14704/nq.2022.20.6.NQ22791
