Difference between revisions of "Task 7: Structure-based mutation analysis"

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(Final results and discussion)
(Final results and discussion)
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=== I65T ===
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=== R71H ===
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=== R158Q ===
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=== R261Q ===
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=== T266A ===
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=== P275S ===
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=== T278N ===
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=== P281L ===
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=== G312D ===
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=== R408W ===

Revision as of 21:12, 2 July 2011

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):

1J8U experimental details.png

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:

Catalytic site and mutations.png


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.


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)

R158Q.png

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)

R261Q.png

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)

T266A task7.png

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)

P275S task7.png

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)

T278N task7.png

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)

P281L task7.png

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)

G312D.png

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)

R408W.png

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

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
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
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 PAH R158Q GROMACS Bond.png
Angle 2351.59 22 189.806 80.2662 PAH R158Q GROMACS Angle.png
Potential -36202.9 740 3182.59 -4873.47 PAH R158Q GROMACS Potential.png

R261Q

Category Average Err.Est. RMSD Tot-Drift Graph
Bond 705.135 210 2053.34 -1228.48 PAH R261Q GROMACS Bond.png
Angle 2356.01 21 179.696 95.0119 PAH R261Q GROMACS Angle.png
Potential -36270.8 690 3020.89 -4437.22 PAH R261Q GROMACS Potential.png

T266A

Category Average Err.Est. RMSD Tot-Drift Graph
Bond 740.335 240 2209.81 -1459.51 PAH T266A GROMACS Bond.png
Angle 2352.51 22 192.08 65.6116 PAH T266A GROMACS Angle.png
Potential -36283.8 750 3239.42 -4913.99 PAH T266A GROMACS Potential.png

P275S

Category Average Err.Est. RMSD Tot-Drift Graph
Bond 911.556 410 -nan -2575.37 PAH P275S GROMACS Bond.png
Angle 2338.07 33 -nan -61.3735 PAH P275S GROMACS Angle.png
Potential -36106.6 1000 -nan -6645.9 PAH P275S GROMACS Potential.png

T278N

Category Average Err.Est. RMSD Tot-Drift Graph
Bond 778.485 280 2383 -1740.86 PAH T278N GROMACS Bond.png
Angle 2344.25 25 207.028 26.8598 PAH T278N GROMACS Angle.png
Potential -36291.6 820 3493.71 -5504.87 PAH T278N GROMACS Potential.png

P281L

Category Average Err.Est. RMSD Tot-Drift Graph
Bond 728.439 220 2141.57 -1346.93 PAH P281L GROMACS Bond.png
Angle 2352.62 21 187.673 80.9581 PAH P281L GROMACS Angle.png
Potential -36549.7 740 3172.53 -4826.89 PAH P281L GROMACS Potential.png

G312D

Category Average Err.Est. RMSD Tot-Drift Graph
Bond 1074.93 550 3492.54 -3394.39 PAH G312D GROMACS Bond.png
Angle 2396.13 28 198.398 -68.8 PAH G312D GROMACS Angle.png
Potential -13546.2 23000 328802 -143251 PAH G312D GROMACS Potential.png


R408W

Category Average Err.Est. RMSD Tot-Drift Graph
Bond 1508.76 980 4742.06 -5893.55 PAH R408W GROMACS Bond.png
Angle 2482.65 99 386.434 -483.529 PAH R408W GROMACS Angle.png
Potential 4.48468e+07 4.3e+07 6.93881e+08 -2.71572e+08 PAH R408W GROMACS Potential.png

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. PAH GROMACS Timeplot.png


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
R261Q 13.58 75.18 61.6
T266A 13.58 74.01 60.43
P275S 13.58 78.76 65.18
T278N 13.58 79.9 66.32
P281L 13.58 77.32 63.74
G312D 13.58 93.54 79.96
R408W 13.58 139.7 126.12


I65T

R71H

R158Q

R261Q

T266A

P275S

T278N

P281L

G312D

R408W