Protein Modelling

Phospho-2-dehydro-3-deoxyheptonate aldolase (AroG) is an important enzyme for the phosphoenolpyruvate (PEP) catalytic reaction and has an important role in the metabolic pathways, i.e. tryptophan biosynthesis, in this project. Literature review shows that Phenylalanine binds to AroG to allosterically inhibit the condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate(E4P), thus subsequently lower the level of the product 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), the first step to synthesize chorismite which is the precursor of tryptophan. When the Ser at AroG 211 is mutated to Phe, allosteric inhibition produced by Phe is alleviated. To investigate the structural mechanism of allosteric inhibition on AroG by Phe and the alleviation in S211F mutant, it is proposed to be quantified and visualized using PyMOL, Gaussian16.0W, GaussView6.0, Swiss, AutoDockTools software.

1. Fundamental assumptions

1. The ligand receptor docking results predicted by AutoDockTools in a semi-flexible docking mode are bassically correct within the range that the conformation allows to change.
2. The mutant AroG tetrameric protein structure predicted by amino acid sequence on the Swiss website is bassically correct within the range that the conformation allows to change.
3. The relative position of the wild-type and point-mutation mutant AroG to the protein pocket bound to Phe and PEP does not change.
4. The catalytic activity of the AroG with PEP can be characterized by the binding energy of the two.

2. Acquisition of protein and small-molecule structures^[1]

Required structure	Method of obtaining
Wild-type AroG tetrameric protein structure	UniProt: P0AB91
AroG-S211F tetramer protein structure	Predicted by Swiss AutoDockTools
Phe and PEP small-molecule structures	Gaussian16.0W, GaussView6.0
Crystal structure of Phe, PEP binding to AroG	PDBe: 1kfl

3. AutoDock molecular docking^[2]

In order to quantify the allosteric inhibition of Phe on AroG and the mechanism of AroG-S211F alleviating the inhibition, this paper uses AutoDockTools software to get results. Firstly, dock Phe molecules in the wild type and mutant AroG respectively. Then, dock PEP molecules to the protein active center one by one. During this process, record the ligand-receptor binding energy , and visualized the corresponding protein structure with PyMol software to further explore the relationship between protein structure and the corresponding docking results.

The workflow is as follows:

4. Results and Discussion

4.1 Binding energy

1. Allosteric inhibition effect of Phe

The average value of the binding energy is obtained by repeating the docking several times. When the Phe ligand is not bound, the binding energy of aroG and PEP is -5.5 kcal/mol; and when the Phe is bound to aroG tetramer at the corresponding site, the binding energy becomes -5.2 kcal/mol.

Conclusively, the binding of Phe to AroG has an inhibitory effect of PEP binding to AroG.

2. Effect of point mutations on the catalytic activity of AroG

Number of Phe bound to AroG	Ligand receptor binding energy / kcal·mol-1
1	-5.9
2	-5.5
3	-5.7
4	-5.5
Combined energy sum	-22.6

Number of PEP bound to AroG-4Phe	Ligand receptor binding energy / kcal·mol-1
1	-5.2
2	-5.4
3	-5.3
4	-5.3
Combined energy sum	-21.2

Number of Phe bound to MutAroG	Ligand receptor binding energy / kcal·mol-1
1	-5.2
2	-5.2
3	-5.0
4	-4.6
Combined energy sum	-20.0

Number of PEP bound to MutAroG-4Phe	Ligand receptor binding energy / kcal·mol-1
1	-6.2
2	-6.2
3	-6.4
4	-6.3
Combined energy sum	-25.1

From the comparison of the table data, the binding ability of the mutant aroG and Phe is weaker than that of the wild-type aroG, and the binding ability of the mutant aroG to PEP, in the case that the corresponding site has been combined with Phe, is greatly improved compared to the wild-type aroG.

Therefore, without considering the software simulation docking error, it can be concluded that the Phe allosteric inhibitory effect of mutant aroG is weakened.

4.2 Protein pocket structure

1. The docking results of AroG with Phe and PEP

In the figure, the yellow is the experimentally measured conformation of Phe in the crystal protein bound by aroG and Phe, and the green is the docking site and the conformation of Phe predicted by AutoDockTools software. Obviously, the ligand receptor docking site predicted by the AutoDockTools software is completely consistent with the actual site, but there is a slight difference in the conformation of Phe. Therefore, the prediction of binding energy can be more credible.

The following figure shows the docking visualization results of PEP and AroG:

2. Comparison of the wild-type and mutant AroG

According to the docking results, the protein pocket conformation of wild-type and mutant AroG bound to PEP has not changed significantly. The Red is the experimentally measured conformation of Phe in the crystal protein bound by aroG and Phe, and the pink is the docking site and the conformation of Phe predicted by AutoDockTools software. In general, the changes in binding PEP sites are not very significant.

Phe site:

It can be seen from the docking results that the Ser at AroG 211 mutating to Phe changes the conformation of the protein pocket which originally binds to Phe, and the binding site of Phe changes accordingly. The binding energy calculated by AutoDockTools software shows that the binding ability of mutant aroG and Phe becomes weaker. Therefore, it can be concluded that the conformation of the mutant aroG and Phe binding protein pocket changes, so that the binding ability of Phe to it becomes smaller, and the allosteric inhibition effect of Phe is reduced, finally the catalytic efficiency of aroG on PEP increases.

5. Advantages and disadvantages of the model

5.1 Advantages of the model

1. The results of the experiments can be quickly obtained by analysis using available software tools
2. The accidental deviation caused by experiments is avoided
3. Lower cost, less time consuming and easier to study

5.2 Disadvantages of the model

The prediction results of existing software tools have limitations:

1. The Gauss software utilizes a semi-empirical molecular orbital theory algorithm when computing the steady-state conformation of the Phe and PEP small molecules, which reduces the accuracy of the calculation results
2. When the AutoDockTools calculates the docking of large-mass proteins and ligands, it is limited by the computing power; the receptor can not select too many flexible chains, and the accuracy of the flexible docking prediction algorithm is not very high; if the initial value of the docking is not set properly, the prediction results will fall in the local optimal solution to reach the global optimal solution.

---

Reference

[1] Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Gallo Cassarino T, Bertoni M, Bordoli L, Schwede T. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014 Jul; 42(Web Server issue):W252-8. doi: 10.1093/nar/gku340. Epub 2014 Apr 29. PMID: 24782522; PMCID: PMC4086089.

[2] Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010 Jan 30;31(2):455-61. doi: 10.1002/jcc.21334. PMID: 19499576; PMCID: PMC3041641.

---