iGEM 2021 — Team UChicago

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# Team:UChicago/Notebook

# UChicago's Lab Notebook

Our documentation of what was done on what days with some of our commentary.

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# Team:UChicago/Engineering

# Engineering Success

### The Process of Docking

One of the initial stages of our project was to computationally dock the substrate onto the active site of the enzyme. This was necessary so that the software we used for our MD simulations, Gromacs, could accurately predict the interactions between the two molecules. Normally, there are several ways to assess the validity of a docked state, involving energy values of the state, similarity to other docked molecules, and key active residues. We iterated docking over three stages using three different programs, testing the validity of our docking results from each program. The three programs we used were first Pymol, then Swiss Docking, then Autodock Vina through Chimera. With Pymol, we were easily able to see key active residues, as well as similarity to other docked molecules, but we were unable to asses the energy values of the state, and the docking was simply the manual single aligned state. We then moved onto Swiss Docking; for most of our complexes, this provided too many docked states, and even after biasing the simulation, we were unable to get results that were similar to other molecules docked in the active sight. Our final iteration was with AutoDock Vina, a module of chimera. This let us appropriately bias our simulations, giving us approximately 10 states with good free energy values that were comparable to varying substrates in the active sight we had found in literature. Below are docking examples with one of our laccases, CueO.

### Docking With Pymol

Pymol gave us a good place to start in terms of getting an idea of where the active site was on the molecule. However, because Pymol is unable to modify the position of the ligand to be in the lowest energy state within the active site, we were only able to use it as a baseline.

### Docking With Swiss Docking

Swiss Docking runs the molecule through different positions and finds a set of docked states with varying energy levels. Here we can see the lowest energy state produced from the Swiss Docking program for our ligand with the CueO laccase. However, this state did not match what we would expect based on our work from Pymol, or match with the active residues from literature.

![](https://static.igem.org/mediawiki/2021/7/77/T--UChicago--CueO_SwissDock.png)

### Docking With Autodock Vina

While the difference is subtle, this docking matched what we would expect based on the Pymol alignment from literature. Like the Swiss Docking program, Autodock Vina calculated the lowest state for this docking, as pictured here.

![](https://static.igem.org/mediawiki/2021/f/f3/T--UChicago--CueO_ADVina.png)

(blue is the ligand, tan is the CueO laccase)

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# Team:UChicago/Experiments

# Experiment

## Docking of ligand in active site

Docking was performed with AutoDock Vina through Chimera

 1. 1GYC, laccase from T. versicolor, complexed with triclosan

 2. 1GSK, laccase from B. subtilis (CotA), complexed with diclofenac

 3. 1KV7, laccase from E. coli (CueO), complexed with diclofenac

CotA and CueO were used for our collaboration with Team Aboa

Our protocol for using AutoDock Vina can be found [here](https://2021.igem.org/File%3AT--UChicago--ADVina.pdf)

**1GYC with triclosan**

Below is an image of triclosan docked in the active site of 1GYC. Here, we can see that the hydroxyl group on triclosan is pointing directly towards the histidine in 1GYC. We know that the electron transfer occurs along the his-cys pathway (Mot et al, 2012), so the close distance between the hydrogen and the histidine supports the validity of this structure.

![](https://static.igem.org/mediawiki/2021/d/dd/T--UChicago--1GYC_triclosan_AS.png)

**CueO with diclofenac**

Here, the magenta residues are Met and Asp amino acids that are expected to bind to the ligand (Wang et al). Indeed, diclofenac fits snugly within those bounds.

![](https://static.igem.org/mediawiki/2021/9/96/T--UChicago--CueO_diclo_AS.png)

**CotA with diclofenac**

![](https://static.igem.org/mediawiki/2021/1/19/T--UChicago--CotA_diclo.png)

Sources

[1] - Mot AC, Silaghi-Dumitrescu R. Laccases: complex architectures for one-electron oxidations. Biochemistry (Mosc). 2012 Dec;77(12):1395-407. doi: 10.1134/S0006297912120085. PMID: 23244736.

[2] - Wang H, Liu X, Zhao J, Yue Q, Yan Y, Gao Z, Dong Y, Zhang Z, Fan Y, Tian J, Wu N, Gong Y. Crystal structures of multicopper oxidase CueO G304K mutant: structural basis of the increased laccase activity. Sci Rep. 2018 Sep 24;8(1):14252. doi: 10.1038/s41598-018-32446-7. PMID: 30250139; PMCID: PMC6155172.

## Protonation

Because we want to compare two pH conditions in our simulations, this would affect the protonation state of each amino acid residue. To do this, we used the web server [H++](http://biophysics.cs.vt.edu/). Below, we have attached the outputs to our protonation:

[1gyc\_pH5](https://2021.igem.org/File%3AT--UChicago--Tver_pH5.zip)

[1gyc\_pH7.1](https://2021.igem.org/File%3AT--UChicago--Tver_pH7_1.zip)

>
To use these outputs in our simulation, we created and ran a python script that converted the AMBER coordinate and topology fies to GROMACS coordinate and topology files. The program we used is attached below.

topology\_converter.py

## MD Simulations

We ran our molecular dynamics simulations through GROMACS 2020.6. The basic steps of an MD simulation are solvation, minimization, equilibration, and then the final MD run. Solvation involves the creation of a box with your molecule of interest, adding solvents (typically water), and adding counterions such that the entire box has a neutral charge. Minimization calls a steep optimization algorithm to seek lowest energy conformation protein under linear restraints. Equilibration stabilizes the system at a constant volume (NVT equilibration) and at a constant pressure (NPT equilibration). Only once solvation, minimization, and equilibration are completed can the MD run be initiated.

Our protocols for MD simulations is attached below. Our MD runs were performed on UChicago’s Midway Cluster in the Research Computing Center.

md-protocol.docx

## MD Simulation Analysis

The two files we care about that are outputs to the MD run are the trajectory file (.xtc) and the run topology files (.tpr). From these files, we can view the conformational changes of the molecule over time, calculate the root mean square fluctuation of each residue, compute the radius of gyration, and complete a principal component analysis via eigenvalue decomposition.

Our protocols are attached below

md\_analysis.docx

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# Team:UChicago/Results

# Results

## Molecular Dynamics Simulations

We ran a 100ns simulation of 1gyc at 295K (summer temperatures) and pH 7. We did not include coppers or a ligand.

[

](https://static.igem.org/mediawiki/2021/4/4a/T--UChicago--1gyc_100ns_15s.mp4)

**Radius of Gyration**

Since protein is already globular in shape, the RG value is fairly consistent over time which is expected.

![](https://static.igem.org/mediawiki/2021/8/8c/T--UChicago--radius_of_gyration.png)

## Future Steps

Since we were unable to run a full simulation with embedded coppers and triclosan, next steps for this project would be to find correct parameters for the copper ions and complete simulations with laccase, triclosan, coppers, and specified temperature and pH combinations.

Once we have a simulation output containing both laccase and triclosan, we can find a proxy for the dissociation constant by inspecting the trajectory and seeing when triclosan leaves the active site. Comparing the dissociation constant between different conditions would tell us information about the reaction kinetics.

Since the scree plot of the entire protein showed no major conformational changes, we thought it would be revealing to analyze just the active site residues. Specifically, Mehra et al suggested that His-458 (green) and Asp-206 (yellow) residues form crucial interactions with laccase substrates, with the Nε H-atom of His-458 plausibly involved in the actual electron transfer [2]. It would be illuminating to track how the distance between triclosan and those residues vary over the course of the simulation.

![](https://static.igem.org/mediawiki/2021/b/b0/T--UChicago--mehra.png)

**References**

[1] - Kim and Nicell, 2006:

[2] - Mehra, R., Muschiol, J., Meyer, A.S. et al. A structural-chemical explanation of fungal laccase activity. Sci Rep 8, 17285 (2018). https://doi.org/10.1038/s41598-018-35633-8