Part:BBa_K3461001
Mutated Phage P2 That Binds to S. pyogenes
This part is one of the 418 different "mutated" versions of Lactococcus Phage P2. We sequenced this part in order to identify specific properties of mutated Phage P2 that are able to bind to S. pyogenes.
First off, we'll give some background of our methodology. We utilized three different softwares: The Scripps Research Insitute’s Autodock Vina, BIOVIA Discovery Studio, and MODELLER. Autodock Vina is the main tool we used to predict the binding affinity between the phage and the bacteria. Therefore, the first step was to validate the software as well as having the binding affinity for the control group.
The Scripps Research Insitute’s Autodock Vina was validated as we docked Phage P2 with the receptor of its natural host, Lactococcus lactis. This also serves as our control group. However, the 3D model of the macromolecule on L. lactis in which Phage P2 binds onto wasn’t available. Therefore, utilizing previous research and 2D structures of the macromolecule from A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in L. lactis, we were able to model the receptor by utilizing BIOVIA Discovery Studio to predict possible bond angles. Hence, we were able to computationally model a three-dimensional structure of the L. lactis receptor.
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Using Autodock Vina, we then docked this modelled macromolecule with the Phage P2’s PDB structure, which can be found in RCSB Protein Data Bank. The binding affinity given by Autodock Vina was -11.38 kcal/mol. From our experience, Autodock Vina’s binding affinity score varies +/- 1.0 kcal/mol.
Thereafter, we used Autodock Vina to dock the PDB format of Phage P2 with the receptor of S. pyogenes, which is peptidoglycan, in which its three-dimensional structure can be found on PubChem. We found the binding affinity to be -4.50 kcal/mol, indicating that Phage P2 and S. pyogenes are unlikely to bind with one another as expected. This value of -4.50 kcal/mol will serve as our baseline.
Because the original Phage P2 is unlikely to bind to Peptidoglycan, receptor of S. pyogenes, we attempted to decrease the binding affinity value (meaning it binds with one another better) between Phage P2 and S. pyogenes by using MODELLER to model 450 different versions of Phage P2. MODELLER is used to predict a protein’s three-dimensional structures when the program is provided with an alignment of known sequences. The program then calculates many models that satisfies its restraints. MODELLER created 450 different probable models of the mutated Phage P2. Thereafter, we docked all 450 versions with the receptor of S. pyogenes via Autodock Vina. The average binding affinity score was -4.90 kcal/mol, the maximum was -3.51 kcal/mol, and the minimum was -6.60 kcal/mol. The minimum of -6.60 kcal/mol occurs at Phage P2 mutant #327, which is the part sequence we included here.
To explain the significance of our docking, we created another control group. We decided to dock Phage A25, a bacteriophage that naturally binds to S. pyogenes, to S. pyogenes. The binding affinity calculated by Autodock Vina was -7.54 kcal/mol.
Thus, this indicates that Phage P2 Mutant #327 may not bind to S. pyogenes as well as it would to L. lactis, nor does it bind to S. pyogenes as well as Phage A25. However, the value of -7.54 kcal/mol is close to -6.60 kcal/mol to suggest that it is probable that the Phage Mutant #327 will bind to S. pyogenes. Phage Mutant #327 also has significantly improved its binding affinity from the natural form of Phage P2, since the original Phage P2 with S. pyogenes resulted in -4.50 kcal/mol as its binding affinity.
Now, we have created a mutant of Phage P2 that can bind to S. pyogenes, which was initially outside its host range. However, we still have to make sure that this mutant Phage P2 can still bind to its original host, L. lactis.
Therefore, we tested this by docking the two together and found a binding affinity of -10.01 kcal/mol, which can be compared to the original Phage P2 binding with L. lactis’s binding affinity, which is -11.38 kcal/mol. This means that Phage P2 Mutant #327 is still most likely to be able to still bind to L. lactis.
In conclusion, our computational mutant Phage P2 has succeeded in binding to both its original host L. lactis and our targeted host S. pyogenes.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 520
Illegal PstI site found at 526 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 520
Illegal PstI site found at 526
Illegal NotI site found at 188 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 520
Illegal PstI site found at 526 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 520
Illegal PstI site found at 526 - 1000COMPATIBLE WITH RFC[1000]
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