Task 7: Structure-based mutation analysis
Contents
Task description
A detailed task description can be found here.
Selection of protein structure
We had the following choice of reference structures for PAH:
| Entry | Method | Resolution (A) | Chain | Positions |
|---|---|---|---|---|
| 1DMW | X-Ray | 2.00 | A | 118-424 |
| 1J8T | X-Ray | 1.70 | A | 103-427 |
| 1J8U | X-Ray | 1.50 | A | 103-427 |
| 1KW0 | X-Ray | 2.50 | A | 103-427 |
| 1LRM | X-Ray | 2.10 | A | 103-427 |
| 1MMK | X-Ray | 2.00 | A | 103-427 |
| 1MMT | X-Ray | 2.00 | A | 103-427 |
| 1PAH | X-Ray | 2.00 | A | 117-424 |
| 1TDW | X-Ray | 2.10 | A | 117-424 |
| 1TG2 | X-Ray | 2.20 | A | 117-424 |
| 2PAH | X-Ray | 3.10 | A/B | 118-452 |
| 3PAH | X-Ray | 2.00 | A | 117-424 |
| 4PAH | X-Ray | 2.00 | A | 117-424 |
| 5PAH | X-Ray | 2.10 | A | 117-424 |
| 6PAH | X-Ray | 2.15 | A | 117-424 |
All these structures have in common that they did not solve the structure of the whole PAH protein. They only solve the catalytic domain of PAH, the missing parts are the tetramerisation domain and the regulatory domain which are located at the N- and C- terminal ends. In addition, there is no complete true apo structure available either. All structures have at least a Fe2+ atom bound. Because of this we thought it might be better if we select a structure which has all reaction components or at least most of them bound in the catalytic site in order to get a good picture of the binding site configuration. Though, only 1KW0 and 1MMK fulfilled the constrains that all reaction components are bound.
In the end we did not select 1KW0 or 1MMK, we decided us for the structure 1J8U which is complexed with Fe2+ and BH4 (5,6,7,8-TETRAHYDROBIOPTERIN). Only. This has simple reasons. First of all it has the lowest resolution (1.5 Angstrom) and secondly we already used this structure in previous task as our reference structure for PAH. So we think to keep our experiments more consistent we should stay with this structure. Furthermore, we identified this structure to have no gaps and it solves the complete catalytic domain (as all available structures). Also, the R-Value looked good to us which is 0.157.
To sum it up our selected structure 1J8U has the following experimental metrics (taken from PDBe):
Mapping mutations to the structure
We identifier the following functional residues and catalytic sites with the help of UniProt entry P00439 and Catalytic Site Atlas. We looked for catalytic sites in the structure of 1J8U.
We identified the following functional residues and catalytic sites:
- HIS 285, functional part: side chain (from CSA)
- HIS 290 (from UniProt)
- GLU 330 (from UniProt)
- SER 349, functional part: side chain (from CSA)
In the following picture we can see the Fe+2 atom as a brown sphere, BH4 as a cloud of green blue and red spheres, the location of the mutated residues in orange (mutation I65T and R71H are not included) and the four identified catalytic sites in yellow:
I65T
This mutation is not part of our structure but we would say probably no effect on catalytic site because it is too far away.
R71H
This mutation is not part of our structure but we would say probably no effect on catalytic site because it is too far away.
R158Q
Probably no effect on catalytic site because it is too far away.
R261Q
Probably no effect on catalytic site because it is too far away.
T266A
No direct influence on catalytic site residue. However, this residue is located what we would define as the catalytic center.
P275S
Probably no effect on catalytic site because it is too far away.
T278N
No direct influence on catalytic site residue. However, this residue is located what we would define as the catalytic center. ´
P281L
Probably direct influence on catalytic site residue HIS 285. In addition, this residue is located what we would define as the catalytic center.
G312D
Probably no effect on catalytic site because it is too far away.
R408W
Probably no effect on catalytic site because it is too far away.
Binding Site
To get the binding sites of the two compounds (red) we selected the residues (orange) within 6 Angstrom range of the compounds.
Binding residues:
| 245 | 247-251 | 254-255 | 263-266 | 281 | 285-286 | 290 | 307 | 321-322 | 325-326 | 330 | 345-346 |
In this case the residues in range of the compounds are orange, the mutated residues out of range are blue and the mutated residues in range are white. The residues 266 and 281 are affected by our mutations and in range of the compounds.
Introducing mutations to 1J8U
Introducing mutations to 1J8U with SCWRL
We had to employ several steps to introduce our mutated residues to our structure with SCWRL:
1. extract amino acid sequence from PDB file
/apps/scripts/repairPDB 1J8U.pdb -seq > 1J8U_seq.txt
2. convert all upper case residues to lower case
vim 1J8U_seq.txt
:%s/.*/\L&/g
3. create one sequence file for each mutation and put the residue to mutate as an uppercase letter
4. execute SCWRL for each mutation
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R158Q.txt -o 1J8U_R158Q.pdb | tee scwrl_r158q.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R261Q.txt -o 1J8U_R261Q.pdb | tee scwrl_r261q.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_T266A.txt -o 1J8U_T266A.pdb | tee scwrl_t266a.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_P275S.txt -o 1J8U_P275S.pdb | tee scwrl_p275s.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_T278N.txt -o 1J8U_T278N.pdb | tee scwrl_t278n.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_P281L.txt -o 1J8U_P281L.pdb | tee scwrl_p281l.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_G312D.txt -o 1J8U_G312D.pdb | tee scwrl_g312d.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R408W.txt -o 1J8U_R408W.pdb | tee scwrl_r408w.out
SCWRL versus PyMol: Comparison of the rotation of the side chains
I65T
Could not compare the side chain of this mutation since this position is not included in 1J8U.
R71H
Could not compare the side chain of this mutation since this position is not included in 1J8U.
R158Q
Orange: side chain of the WT Yellow: side chain of mutated residue (PyMol) Pink: side chain of mutated residue (SCWRL)
As seen in the picture the mutated side chain of SCWRL (in pink) points now in the same direction as the lower part of the WT side chain (seen in orange). In contrast to that the calculated side chain rotation of pymol points towards the adjacent alpha-helix.
R261Q
Orange: side chain of the WT Yellow: side chain of mutated residue (PyMol) Pink: side chain of mutated residue (SCWRL)
All three side chains point into the same direction. However, we could observe that the side chain calculated by SCWRL is rotated around the Y-axis.
T266A
Orange: side chain of the WT Yellow: side chain of mutated residue (PyMol) Pink: side chain of mutated residue (SCWRL)
All three side chains point into the same direction. the rotation of pymol and SCWRL is the same. However, this is not surprising since alanine has no side chain.
P275S
Orange: side chain of the WT Yellow: side chain of mutated residue (PyMol) Pink: side chain of mutated residue (SCWRL)
All three side chains point into the same direction. Also the rotation of the mutatant side chain of pymol and SCWRL is the same.
T278N
Orange: side chain of the WT Yellow: side chain of mutated residue (PyMol) Pink: side chain of mutated residue (SCWRL)
The distinction between the mutated side chain position of pymol and SCWRL is that the mutated side chain of SCWRL is flipped to the empty C branch of the WT and pymols side chain is flipped to the CO branch of the WT. Hence, we may assume that they form different polar interactions.
P281L
Orange: side chain of the WT Yellow: side chain of mutated residue (PyMol) Pink: side chain of mutated residue (SCWRL)
The two mutated side chains of pymol and SCWRL point into the same direction. However, they are differently rotated. The side chain produced by poymol points to the FE atom and the side chain produced by SCWRL points to the BH4 molecule.
G312D
Orange: side chain of the WT Yellow: side chain of mutated residue (PyMol) Pink: side chain of mutated residue (SCWRL)
The mutated side chains of pymol and SCWRL are rotated into different directions by approximately 190° around the Y-axis. This leads to a different orientation of the COO ends in pymol and SCWRL.
R408W
Orange: side chain of the WT Yellow: side chain of mutated residue (PyMol) Pink: side chain of mutated residue (SCWRL)
Also in our last mutation the mutated residue is rotated differently in pymol and SCWRL. The mutated residue of SCWRL points into the same direction as the WT whereas the the side chain produced by pymol is somehow horizontal to that.
Hydrogen bond network of the WT and mutants
The given H-bond distances in the tables below are calculated from N-O. However, normally an H-bond is defined to be from NH-O. Hence, this explains the great values for H-bonds we obtained during our measurements. So we assume that the distance from N-H is 1 Angstrom. In order to get the "real" H-bond length now we have to substract this 1 Angstrom from our N-O distance. For example if our measured distance was 3 Angstrom then our corrected H-bond distance would be 3-1 = 2 Angstrom.
Furthermore, it seems like SCWRL confused the atom indices during its calculation. This has the effect that they may differ from the original structure. This leads to the problem that the atom index can not be taken directly to compare whether an H-bond in the original structure and in the mutated structure go from and to the same atoms. In order to solve this problem we set an remark whether this is the same h-bond in the WT or mutant structure.
I65T
We could not analyze the hydrogen bonding network of this position since it is not included in the J18U PDB structure.
R71H
We could not analyze the hydrogen bonding network of this position since it is not included in the J18U PDB structure.
R158Q
| R158 | Q158 |
|---|---|
|
|
WT (R158) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | From Atom Index | To Atom | To Atom Index | Distance (Ansgstrom) | Remark |
| R158 | SC | E280 | SC | N | 344 | O | 1360 | 2.9 | - |
| R158 | SC | E280 | SC | N | 343 | O | 1365 | 2.8 | - |
| R158 | SC | E141 | SC | N | 344 | O | 201 | 3.6 | - |
| R158 | SC | Y154 | SC | N | 343 | O | 306 | 3 | - |
| R158 | BB | Y154 | BB | N | 334 | O | 298 | 2.9 | - |
| R158 | BB | F161 | BB | O | 337 | N | 363 | 3.2 | - |
| R158 | BB | A162 | BB | O | 337 | N | 374 | 2.9 | - |
Mutant (Q158) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | From Atom Index | To Atom | To Atom Index | Distance (Ansgstrom) | Remark |
| Q158 | SC | Y154 | SC | N | 341 | O | 306 | 3.1 | - |
| Q158 | SC | I269 | SC | N | 341 | O | 1264 | 3.5 | - |
| Q158 | BB | Y154 | BB | N | 334 | O | 298 | 2.9 | - |
| Q158 | BB | F161 | BB | O | 337 | N | 363 | 3.2 | - |
| Q158 | BB | A162 | BB | O | 337 | N | 374 | 2.9 | - |
R261Q
| R261 | Q261 |
|---|---|
|
|
WT (R261) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | To Atom | Distance (Ansgstrom) |
| R261 | SC | THR238 | BB | NH2 | O | 2.3 |
| R261 | BB | LEU258 | BB | N | O | 2.9 |
| R261 | BB | ARG241 | BB | O | N | 2.9 |
Mutant (Q261) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | To Atom | Distance (Ansgstrom) |
| Q261 | BB | LEU258 | BB | N | O | 2.9 |
| Q261 | BB | ARG241 | BB | O | N | 2.9 |
T266A
| T266 | A266 |
|---|---|
|
|
WT (T266) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | From Atom Index | To Atom | To Atom Index | Distance (Ansgstrom) | Remark |
| T266 | SC | E286 | SC | O | 1247 | O | 1413 | 3.1 | - |
| T266 | BB | E286 | SC | N | 1241 | O | 1413 | 2.8 | Same H-Bond as A266 to E286 |
Mutant (A266) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | From Atom Index | To Atom | To Atom Index | Distance (Ansgstrom) | Remark |
| A266 | BB | E286 | SC | N | 1507 | O | 1710 | 2.8 | Same H-Bond as T266 to E286 |
P275S
| P275 | S275 |
|---|---|
|
|
WT (P275) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | From Atom Index | To Atom | To Atom Index | Distance (Ansgstrom) | Remark |
| P275 | BB | R270 | SC | O | 1320 | N | 1286 | 2.9 | Same H-Bond as S275 to R270 |
Mutant (S275) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | From Atom Index | To Atom | To Atom Index | Distance (Ansgstrom) | Remark |
| S275 | BB | R270 | SC | O | 1609 | N | 1281 | 3.1 | Same H-Bond as P275 to R270 |
T278N
| T278 | N278 |
|---|---|
|
|
WT (T278) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | From Atom Index | To Atom | To Atom Index | Distance (Ansgstrom) | Remark |
| T278 | SC | E280 | BB | O | 1350 | O | 1361 | 2.7 | - |
| T278 | SC | E280 | BB | O | 1350 | N | 1358 | 3.3 | Same H-Bond as N278 to E280 |
Mutant (N278) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | From Atom Index | To Atom | To Atom Index | Distance (Ansgstrom) | Remark |
| N278 | SC | E280 | BB | O | 1345 | N | 1353 | 3.4 | Same H-Bond as T278 to E280 |
P281L
| P281 | L281 |
|---|---|
|
|
WT (P281) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | To Atom | Distance (Ansgstrom) |
| P281 | BB | TYR268 | SC | O | OH | 2.3 |
Mutant (Q261) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | To Atom | Distance (Ansgstrom) |
| L281 | BB | TYR268 | SC | O | OH | 2.3 |
G312D
| G312 | D312 |
|---|---|
|
|
WT (G312) H-Bonds
There are no hydrogen bonds to residue 312.
Mutant (Q261) H-Bonds
There are no hydrogen bonds to residue 312.
R408W
| R408 | W408 |
|---|---|
|
|
WT (R408) H-Bonds
| From AA | From SC/BB? | To AA | To SC/BB? | From Atom | To Atom | Distance (Ansgstrom) |
| R408 | SC | LEU311 | BB | NH2 | O | 2.8 |
| R408 | SC | ALA309 | BB | NH2 | O | 3.0 |
| R408 | SC | LEU308 | BB | NH2 | O | 2.9 |
Mutant (W408) H-Bonds
Clashes with PHE410 and VAL412.
FoldX
Execution
We run foldX in the runfile mode and used the following runfile:
<TITLE>FOLDX_runscript;
<JOBSTART>#;
<PDBS>#;
<BATCH>list_stability.txt;
<COMMANDS>FOLDX_commandfile;
<Stability>Stability.txt;
<END>#;
<OPTIONS>FOLDX_optionfile;
<Temperature>298;
<R>#;
<pH>7;
<IonStrength>0.050;
<water>-CRYSTAL;
<metal>-CRYSTAL;
<VdWDesign>2;
<OutPDB>false;
<pdb_hydrogens>false;
<END>#;
<JOBEND>#;
<ENDFILE>#;
This says we are calculating the stability (energy) of the PDB structures defined in the list_stability.txt with the following content:
1J8U.pdb
_1J8U_G312D.pdb
_1J8U_P275S.pdb
_1J8U_P281L.pdb
_1J8U_R158Q.pdb
_1J8U_R261Q.pdb
_1J8U_R408W.pdb
_1J8U_T266A.pdb
_1J8U_T278N.pdb
The other lines define some options like that the calculation runs at 298K and with a physiological PH of 7. Then we executed this runfile with the following command:
/apps/FoldX_30b5/foldx -runfile runfile_stability.txt | tee stability_stdout.txt
Results
| total energy | Backbone Hbond | Sidechain Hbond | Van der Waals | Electrostatics | Solvation Polar | Solvation Hydrophobic | Van der Waals clashes | entropy sidechain | entropy mainchain | sloop_entropy | mloop_entropy | cis_bond | torsional clash | backbone clash | helix dipole | water bridge | disulfide | electrostatic kon | partial covalent bonds | energy Ionisation | Entropy Complex | Number of Residues | |
| 1J8U.pdb | 13.58 | -196.05 | -55.77 | -379.28 | -19.47 | 492.46 | -495.68 | 34.69 | 194.54 | 454.27 | 0 | 0 | 0 | 11.69 | 227.66 | -15.13 | -14.15 | 0 | 0 | 0 | 1.45 | 0 | 307 |
| _1J8U_G312D.pdb | 93.54 | -193.28 | -49.89 | -380.58 | -22.09 | 497.08 | -497.31 | 90.61 | 191.44 | 452.73 | 0 | 0 | 0 | 18.93 | 227.32 | -15.85 | 0 | 0 | 0 | 0 | 1.77 | 0 | 307 |
| _1J8U_P275S.pdb | 78.76 | -193.22 | -49.86 | -378.73 | -19.74 | 493.76 | -494.26 | 72.12 | 190.73 | 453.21 | 0 | 0 | 0 | 18.92 | 228.52 | -15.95 | 0 | 0 | 0 | 0 | 1.77 | 0 | 307 |
| _1J8U_P281L.pdb | 77.32 | -193.26 | -49.88 | -379.66 | -20.05 | 494.58 | -496.29 | 72.83 | 191.19 | 452.95 | 0 | 0 | 0 | 18.99 | 227.31 | -15.95 | 0 | 0 | 0 | 0 | 1.87 | 0 | 307 |
| _1J8U_R158Q.pdb | 77.89 | -193.3 | -47.69 | -377.81 | -17.08 | 490.87 | -494.58 | 71.81 | 189.62 | 451.69 | 0 | 0 | 0 | 18.72 | 227.12 | -16.12 | 0 | 0 | 0 | 0 | 1.76 | 0 | 307 |
| _1J8U_R261Q.pdb | 75.18 | -193.14 | -48.65 | -378.39 | -19.89 | 492.32 | -494.84 | 71.79 | 189.89 | 451.43 | 0 | 0 | 0 | 18.85 | 227.08 | -15.95 | 0 | 0 | 0 | 0 | 1.77 | 0 | 307 |
| _1J8U_R408W.pdb | 139.7 | -191.5 | -48.31 | -379.92 | -19.75 | 491.96 | -498.23 | 137.59 | 189.86 | 452.27 | 0 | 0 | 0 | 18.81 | 226.94 | -14.86 | 0 | 0 | 0 | 0 | 1.77 | 0 | 307 |
| _1J8U_T266A.pdb | 74.01 | -193.48 | -49.47 | -378.06 | -19.73 | 491.38 | -494.45 | 71.98 | 190.15 | 450.98 | 0 | 0 | 0 | 18.87 | 227.18 | -15.95 | 0 | 0 | 0 | 0 | 1.77 | 0 | 307 |
| _1J8U_T278N.pdb | 79.9 | -192.73 | -49.32 | -379.04 | -19.77 | 493.36 | -495.18 | 74.69 | 190.87 | 451.81 | 0 | 0 | 0 | 19.38 | 227.34 | -15.95 | 0 | 0 | 0 | 0 | 1.79 | 0 | 307 |
As expected our WT structure has the lowest total energy with a value of 13.58 kcal/mol. Interestingly, the mutations R408W and G312D cause the most increase of energy to the protein with values of 139.7 kcal/mol and 93.54 kcal/mol. The other mutations are also harmful in terms of stability to the protein. However, these mutations only introduce a total energy of 75-79 kcal/mol.
Minimise
| Mutation | Energy |
|---|---|
| WT | -7383.985291 |
| I65T | - |
| R71H | - |
| R158Q | -7400.825142 |
| R261Q | -7456.793410 |
| T266A | -7392.572699 |
| P275S | -7418.432874 |
| T278N | -7379.215571 |
| P281L | -7401.621858 |
| G312D | -5643.645312 |
| R408W | -5438.301688 |
Gromacs
Mutations
| Mutation | Steps | Potential Energy | Maximum Force | Norm of Force |
|---|---|---|---|---|
| WT | 36 | -3.7828219e+04 | 2.0795129e+02 | 2.1377096e+01 |
| R158Q | 328 | -3.7326676e+04 | 5.1247034e+02 | 1.7521566e+01 |
| R261Q | 363 | -3.7302664e+04 | 1.4321185e+02 | 1.3723365e+01 |
| T266A | 315 | -3.7422707e+04 | 3.7855130e+02 | 1.6683250e+01 |
| P275S | 256 | -3.7569789e+04 | 3.6591925e+02 | 1.9855389e+01 |
| T278N | 272 | -3.7567461e+04 | 6.8363385e+02 | 2.0818382e+01 |
| P281L | 336 | -3.7656289e+04 | 1.6796155e+02 | 1.4324168e+01 |
| G312D | 334 | -3.7583254e+04 | 8.6771161e+02 | 2.3178673e+01 |
| R408W | 320 | -3.6683172e+04 | 6.0292523e+02 | 2.2048130e+01 |
I65T
Could not compare the side chain of this mutation since this position is not included in 1J8U.
R71H
Could not compare the side chain of this mutation since this position is not included in 1J8U.
R158Q
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 726.495 | 230 | 2165.47 | -1392.61 | 
|
| Angle | 2351.59 | 22 | 189.806 | 80.2662 | 
|
| Potential | -36202.9 | 740 | 3182.59 | -4873.47 | 
|
R261Q
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 705.135 | 210 | 2053.34 | -1228.48 | 
|
| Angle | 2356.01 | 21 | 179.696 | 95.0119 | 
|
| Potential | -36270.8 | 690 | 3020.89 | -4437.22 | 
|
T266A
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 740.335 | 240 | 2209.81 | -1459.51 | 
|
| Angle | 2352.51 | 22 | 192.08 | 65.6116 | 
|
| Potential | -36283.8 | 750 | 3239.42 | -4913.99 | 
|
P275S
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 911.556 | 410 | -nan | -2575.37 | 
|
| Angle | 2338.07 | 33 | -nan | -61.3735 | 
|
| Potential | -36106.6 | 1000 | -nan | -6645.9 | 
|
T278N
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 778.485 | 280 | 2383 | -1740.86 | 
|
| Angle | 2344.25 | 25 | 207.028 | 26.8598 | 
|
| Potential | -36291.6 | 820 | 3493.71 | -5504.87 | 
|
P281L
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 728.439 | 220 | 2141.57 | -1346.93 | 
|
| Angle | 2352.62 | 21 | 187.673 | 80.9581 | 
|
| Potential | -36549.7 | 740 | 3172.53 | -4826.89 | 
|
G312D
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 1074.93 | 550 | 3492.54 | -3394.39 | 
|
| Angle | 2396.13 | 28 | 198.398 | -68.8 | 
|
| Potential | -13546.2 | 23000 | 328802 | -143251 | 
|
R408W
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 1508.76 | 980 | 4742.06 | -5893.55 | 
|
| Angle | 2482.65 | 99 | 386.434 | -483.529 | 
|
| Potential | 4.48468e+07 | 4.3e+07 | 6.93881e+08 | -2.71572e+08 | 
|
Timerun
The calculation of the time runs could not be done by a script, therefore we ran gromacs for just AMBER03, AMBERGS and CHARMM each with the nsteps of 125, 250, 500 and 1000.
Final results and discussion
Comparison of the delta energies of the different methods and mutations
| Mutation | FoldX – Total WT Energy | FoldX – Total Mutant Energy | FoldX – DELTA E | Minimise – Total WT Energy | Minimise– Total Mutant Energy | Minimise – DELTA E | Gromacs – Total WT Energy | Gromacs – Total Mutant Energy | Gromacs – DELTA E |
| R158Q | 13.58 | 77.89 | 64.31 | -7383.985291 | -7400.825142 | -16.839851 | -37828.219 | -37326.676 | 501.543 |
| R261Q | 13.58 | 75.18 | 61.6 | -7383.985291 | -7456.793410 | -72.808119 | -37828.219 | -37302.664 | 525.555 |
| T266A | 13.58 | 74.01 | 60.43 | -7383.985291 | -7392.572699 | -8.587408 | -37828.219 | -37422.707 | 405.512 |
| P275S | 13.58 | 78.76 | 65.18 | -7383.985291 | -7418.432874 | -34.447583 | -37828.219 | -37569.789 | 258.43 |
| T278N | 13.58 | 79.9 | 66.32 | -7383.985291 | -7379.215571 | 4.76972 | -37828.219 | -37567.461 | 260.758 |
| P281L | 13.58 | 77.32 | 63.74 | -7383.985291 | -7401.621858 | -17.636567 | -37828.219 | -37656.289 | 171.93 |
| G312D | 13.58 | 93.54 | 79.96 | -7383.985291 | -5643.645312 | 1740.339979 | -37828.219 | -37583.254 | 244.965 |
| R408W | 13.58 | 139.7 | 126.12 | -7383.985291 | -5438.301688 | 1945.683603 | -37828.219 | -36683.172 | 1145.047 |
I65T
We could not perform a structure based mutation analysis for this mutation since it was not solved in our structure.
R71H
We could not perform a structure based mutation analysis for this mutation since it was not solved in our structure.
R158Q
The mutation from amino acid arginine to glutamine on position 158 is not really closely located to any functional or catalytic residue. However, it is located in the catalytic domain of PAH (as all our mutations, though with exception of I65T and R71H which are located on the regulatory domain). Hence, we do not assume that this mutation has any direct influence on these sites.
When we compared the rotation of the mutant generated by SCWRL with the rotation of the mutant generated by pymol with the rotation of the WT side chain we found out that the mutated side chain of SCWRL now in the same direction as the lower part of the WT side chain. In contrast to that the calculated side chain rotation of pymol points towards the adjacent alpha-helix.
During our H-bond network analysis we found out that the WT, R158 is strongly interconnected with other residues by h-bonds. To be more precise we found 7 h-bonds, 4 h-bonds are side chain to side chain h-bonds and the other three are backbone to backbone h-bonds. When mutations are incorporated side chain to side chain and side chain to backbone interactions run in danger to get lost, hence the protein stability might be affected negatively. Whereas h-bonds between backbone to backbone and backbone to side chain are not affected by mutations since they start from a backbone which always stays as it is.
So we found that three side chain to side chain interactions got lost. In our mutant Q158 we now find the three backbone to backbone h-bonds as in our WT and two side chain to side chain h-bonds. One of these h-bonds is the old one Q158 -> Y154 which got preserved and we also found a new h-bond which does not exists in our WT, which is Q158 -> I269. However, the distance of this H-bond is 3.5 angstrom (real h-bond length is 2.5) which is kind of far apart for an h-bond. We assume that the threshold for an H-bond is 2.5 angstrom so we guess this h-bond interaction here is very weak.
In our energy calculations with the methods foldX, minimise and gromacs we found indeed an increase of the energy which is visible from a positive delta value. However, this positive delta value occurred only for the calculations of gromacs and foldx. Minimise calculated a slighly negative delta value which means that our mutant is more stable than the WT. This seems to be a little awkward since we found out that several H-bonds get lost due to the mutation.
R261Q
The illustration shows the residue 261 (red), the residue 238 (yellow), the iron (orange) and the BH4 molecule (blue).
The mutation seems to disrupt the H-bond to Thr238. This H-bond is probably necessary for the correct positioning of the helix next to R261 in the catalytic site. This helix contains the BH4 binding site. A shift of the helix would result in a change in the distance of the iron atom and the BH4 molecule. These two molecules need to interact in the reaction. Therefore it is likely, that the catalytic activity of this mutant is at least lowered. The illustration above shows the key players.
FoldX predicts an increase in energy, mostly caused by Van-der-Waals-clashes. Minimise predict the mutant to be more stable than the WT. Gromacs indicates an increase in potential energy, whereas the maximum Force decreased and the norm of force also decreased. Probably this is caused by a loss of interactions, one of them is probably the H-bond between residue 238 and 261.
T266A
The mutation from amino acid threonine to alanine on position 266 is not really closely located to any functional or catalytic residue. However, it is located in the catalytic domain of PAH (as all our mutations, though with exception of I65T and R71H which are located on the regulatory domain). Hence, we do not assume that this mutation has any direct influence on these sites.
When we compared the rotation of the mutant generated by SCWRL with the rotation of the mutant generated by pymol with the rotation of the WT side chain we found out that all three side chains point into the same direction. The rotation of pymol and SCWRL is the same. However, this is not surprising since alanine has no side chain.
During our H-bond network analysis we found out that the WT, T266 is interconnected with other residues by h-bonds. To be more precise we found 2 h-bonds, 1 h-bonds is a side chain to side chain h-bond and the other one is a backbone to side chain h-bond. When mutations are incorporated side chain to side chain and side chain to backbone interactions run in danger to get lost, hence the protein stability might be affected negatively. Whereas h-bonds between backbone to backbone and backbone to side chain are not affected by mutations since they start from a backbone which always stays as it is.
So we found that one side chain to side chain interaction got lost. As expected our mutant A266 preserved the backbone to side chain h-bond from A266 to E286.
In our energy calculations with the methods foldX, minimise and gromacs we found indeed an increase of the energy which is visible from a positive delta value. However, this positive delta value occurred only for the calculations of gromacs and foldx. Minimise calculated a slighly negative delta value which means that our mutant is more stable than the WT. This seems to be a little awkward since we found out that several H-bonds get lost due to the mutation.
P275S
The mutation from amino acid proline to serine on position 275 is not really closely located to any functional or catalytic residue. However, it is located in the catalytic domain of PAH (as all our mutations, though with exception of I65T and R71H which are located on the regulatory domain). Hence, we do not assume that this mutation has any direct influence on these sites.
When we compared the rotation of the mutant generated by SCWRL with the rotation of the mutant generated by pymol with the rotation of the WT side chain we found out that all three side chains point into the same direction. Also the rotation of the mutatant side chain of pymol and SCWRL is the same.
During our H-bond network analysis we found out that the WT, P158 is interconnected with one other residue by an h-bond. This is an backbone to side chain h-bond. When mutations are incorporated side chain to side chain and side chain to backbone interactions run in danger to get lost, hence the protein stability might be affected negatively. Whereas h-bonds between backbone to backbone and backbone to side chain are not affected by mutations since they start from a backbone which always stays as it is.
As expected this mutation is preserved in our mutant S275. However the distance of this h-bond increased from 2.9 angstrom in the WT to 3.1 angstrom in the mutant. So we guess that this might have a minor influence on the protein stability.
In our energy calculations with the methods foldX, minimise and gromacs we found indeed an increase of the energy which is visible from a positive delta value. However, this positive delta value occurred only for the calculations of gromacs and foldx. Minimise calculated a slighly negative delta value which means that our mutant is more stable than the WT.
T278N
The mutation from amino acid threonine to asparagine on position 278 is not really closely located to any functional or catalytic residue. However, it is located in the catalytic domain of PAH (as all our mutations, though with exception of I65T and R71H which are located on the regulatory domain). Hence, we do not assume that this mutation has any direct influence on these sites.
When we compared the rotation of the mutant generated by SCWRL with the rotation of the mutant generated by pymol with the rotation of the WT side chain we found out that the distinction between the mutated side chain position of pymol and SCWRL is that the mutated side chain of SCWRL is flipped to the empty C branch of the WT and pymols side chain is flipped to the CO branch of the WT. Hence, we may assume that they form different polar interactions.
During our H-bond network analysis we found out that the WT,T278, is interconnected with other residues by h-bonds. To be more precise we found 2 h-bonds, which are side chain to back bone h-bonds. When mutations are incorporated side chain to side chain and side chain to backbone interactions run in danger to get lost, hence the protein stability might be affected negatively. Whereas h-bonds between backbone to backbone and backbone to side chain are not affected by mutations since they start from a backbone which always stays as it is.
So we found that one side chain to backbone interaction got lost. In our mutant N278 we now find only one H-bond preserved which is the N278 -> E280 H -bond. So we may assume that we have a slightly decreased protein stability. Furthermore the hydrophobic threonine got interchanged with a non hydrophobic residue which also might have an influence.
In our energy calculations with the methods foldX, minimise and gromacs we found indeed an increase of the energy which is visible from a positive delta value.
P281L
The illustration above shows residue 281 (red), residue 268 (yellow), the iron atom (orange) and the bh4 molecule (blue).
This mutation removes the helix-breaker P from the end of a helix. In the prediction of SCWRL the effect of such a mutation will not be seen. It is possible, that the broken helix extends and causes changes in the backbone conformation. Residue 281 is in the near of the bound iron atom. The extension of the helix could cause major changes in the surface of the binding site of the iron, which is essential for the reaction. Therefore the mutation is probably limiting the catalytic activity of PAH. The illustration above shows the key players.
G312D
The illustration above shows residue 312 (red), the iron atom (orange) and the BH4 molecule (blue).
The residue 312 has no interactions. The mutation does not seem to influence any parts of the catalytic site or the binding sites. This mutation is probably not influencing the enzyme activity.
R408W
The illustration above shows the residue 408 (red), the residues 311, 309, 308 (yellow), the oligomerization domain (blue) and the H-bonds between the oligomerization domain and the catalytic domain (yellow lines).
The mutation R408W is in the flexible loop identified in the B-factor analysis during the discussion of the disorder prediction (task 3). This is a "hinge" loop between the oligomerization and the catalytic domain of the protein. As one can see in the illustration above, there are only five H-bonds between these two domains and three of them are defined by R408. The mutation causes several clashes in the prediction of SCWRL and probably these three h-bonds are broken. Therefore the flexibility between the two domains is somehow changed. PAH exists in the cell in an equilibrium between its homodimer and its homotetramer form. The dimer is less active than the tetramer form. It is supposed there is a mechanism, which changes the positions of the two domains relative to each other in order to realize this equilibrium. Through the dramatic change of the h-bonds between these two domains, this mechanism is probably broken, perhaps oligomerization itself is at least to some part broken. Therefore R408W is probably a dramatic change, which results in a dramatic decrease of PAH activity.
