Difference between revisions of "Structure-based mutation analysis Gaucher Disease"
(→OPLS-AA/L) |
(→OPLS-AA/L) |
||
| Line 524: | Line 524: | ||
user 3m25.940s |
user 3m25.940s |
||
sys 0m8.560s |
sys 0m8.560s |
||
| + | |||
| + | |||
| + | nstep=1100 |
||
| + | |||
| + | step=1100 |
||
| + | Reached the maximum number of steps before reaching Fmax < 1 |
||
| + | real 1m58.514s |
||
| + | user 3m46.010s |
||
| + | sys 0m9.730s |
||
Revision as of 16:08, 25 June 2012
The aim of this task was to carry out a thorough analysis of ten mutations and to classify them as disease-causing and non-disease causing. Technical details are reported in our protocol.
Contents
Cystral structure
<figtable id="tab:mutations">
| PDB | Res [Å] | R value | Coverage | pH |
|---|---|---|---|---|
| 2nt0 | 1.80 | 0.18 | 96% (40-536) | 4.5 |
| 3gxi | 1.84 | 0.19 | 96% (40-536) | 5.5 |
| 2v3f | 1.95 | 0.15 | 96% (40-536) | 6.5 |
| 2v3d | 1.96 | 0.16 | 96% (40-536) | 6.5 |
| 1ogs | 2.00 | 0.18 | 96% (40-536) | 4.6 |
The 5 crystral structures of glycosylceramidase with the highest resolution. The physiological lysosomal pH value is 4.5. 2nt0 was selected for the analysis. </figtable>
Mutations
<figtable id="tab:mutations">
| Nr | Pos P04062 |
Pos 2nt0_A |
From | To | Disease causing |
|---|---|---|---|---|---|
| 1 | 99 | 60 | H | R | No |
| 2 | 211 | 172 | V | I | No |
| 3 | 150 | 111 | E | K | Yes |
| 4 | 236 | 197 | L | P | Yes |
| 5 | 248 | 209 | W | R | Yes |
| 6 | 509 | 470 | L | P | No |
| 7 | 351 | 312 | W | C | Yes |
| 8 | 423 | 384 | A | D | Yes |
| 9 | 482 | 443 | D | N | No |
| 10 | 83 | 44 | R | S | No |
Mutations used for the structure-based mutation analysis. </figtable>
<figure id="fig:mutations">
</figure>
SCWRL
We employed SCWRL <ref name="scwrl">Qiang Wang, Adrian A. Canutescu, and Roland L. Dunbrack, Jr.(2008). SCWRL and MolIDE: Computer programs for side-chain conformation prediction and homology modeling. Nat Protoc.</ref> for substituting the wildtype residues listed in <xr id="tab:mutations"/> by the corresponding mutatant residues which are chosen from a rotamer library. <xr id="fig:scwrl"/> denotes the results.
<figure id="fig:scwrl">
1: H60R (2.88)
2: V172I (4.39)
3: E111K (8.14)
4: L197P (3.28)
5: W209R (9.23)
6: L470P (28.84)
7: W312C (0.18)
8: A384D (13.50)
9: D443N (2.36)
10: R44S (4.85)
Rotamers of SNPs from <xr id="tab:mutations"/>. Blue: wildtype; Red: rotamer SCWRL; In brackets: energy(mutant)-energy(wildtype). </figure>
None of rotamers chosen by SCWRL clashed with another side-chain or the backbone. The only mutation which led to a structural change was L470P. Here, the insertion of proline interrupted the beta-sheet. The hydrogen bonding network changed in case of mutation number 1, 5, 7, and 8 (cf. <xr id="tab:scwrl"/>). W209R introduces a hydrophilic arginine which forms a hydrogen bond to T180. Although not predicted by SCWRL, the arginine might impact the protein structure. W312C is located next to the active site (cf. <xr id="fig:mutations"/>) and there exists a hydrogen bond to E340. Substitution the hydrophobic tryptohphane by a hydrophlic cysteine in the vicinity of the active site might account for the disease-causing effect of this mutation.
As expected, all mutations increased the energy of the model (cf. the energy difference in brackets in <xr id="fig:scwrl"/>). The energy increased most in case of L470P due to the break of the beta-sheet. A384D and W209R also made the model less stable which is caused by substituting an unpolar residue by a charged residue. All four mutations which increased the model energy most are disease-causing.
<figtable id="tab:scwrl">
| Nr | Mutation | Wildtype | Mutatant | Clashes | Structural change | ||
|---|---|---|---|---|---|---|---|
| H-bonds | Hydrophobicity | H-bonds | Hydrophobicity | ||||
| 1 | H60R | T471 | Hydrophilic | G62 | Hydrophilic | No | No |
| 2 | V172I | Hydrophobic | Hydrophobic | No | No | ||
| 3 | E111K | Hydrophilic | Hydrophilic | No | No | ||
| 4 | L197P | Hydrophobic | Hydrophobic | No | No | ||
| 5 | W209R | Hydrophobic | T180 | Hydrophilic | No | No | |
| 6 | L470P | T482 | Hydrophobic | T482 | Hydrophobic | No | Yes |
| 7 | W312C | E340, C342, P316 | Hydrophobic | E340, C342 | Hydrophilic | No | No |
| 8 | A384D | Hydrophobic | V404 | Hydrophilic | No | No | |
| 9 | D443N | Hydrophilic | Hydrophilic | No | No | ||
| 10 | R44S | S13, Y487 | Hydrophilic | S13, Y487 | Hydrophilic | No | No |
Structure-based analysis of SNPs from <xr id="tab:mutations"/>. H-bonds: residues involved in forming hydrogen bonds (cut-off: 3.2 Å). </figtable>
We further noticed that SCRWL changed the backbone at some positions which led to different secondary structure assignments (<xr id="fig:scwrl_ss"/>). The positions at which the deviations could be observed were independent from the mutated sites.
<figure id="fig:scwrl_ss">
</figure>
FoldX
The superposition of the rotamer configurations predicted by FoldX and SCWRL are shown in <xr id="fig:foldx"/>. The predictions of both tools differed in case of four mutations. In case of H60R, the side-chain orientation of arginine predicted by FoldX forms two instead one hydrogen bonds to T741 and might therefore impact the protein structure more than the orientation of SCRWL. In case of A384D, the romater of FoldX might be more stable than the one of SCWRL since it has a higher distance to the surrounding residues. In case of D443N we prefer the prediction of SCWRL which is closer to the wildtype configuration. For the same reason we prefer the prediction of FoldX in case of R442. For the subsequent GROMACS analysis, we hence chose the FoldX model in case of mutation number 8 and 10 and the SCWRL models for all all other mutations.
<figure id="fig:foldx">
1: H60R (-0.39)
2: V172I (-0.80)
3: E111K (-0.36)
4: L197P (0.28)
5: W209R (2.86)
6: L470P (8.59)
7: W312C (1.88)
8: A384D (4.52)
9: D443N (-0.34)
10: R44S (0.60)
Rotamers of SNPs from <xr id="tab:mutations"/>. Blue: wildtype; Red: rotamer SCWRL; Orange: rotamer FoldX; In brackets: energy(mutant)-energy(wildtype). </figure>
A comprehensive list of the differences between the mutant and the wildtype models can be found here. The total energy increased in case of mutation number 4-8, and 10. Just as in case of SCWRL (cf. <xr id="fig:scwrl"/>), L470P, A384D, and W209R increased the energy of the model most. Since it is unlikely that mutations like V172I decrease the energy, we consider the energy calculations of SCWRL as more plausible.
Minimise
</figure> </figure>| <figure id="fig:minmise_scwrl_energies"> |
| <figure id="fig:minmise_foldx_energies"> |
<figure id="fig:minimise_scwrl_mutations">
1: H60R
2: V172I
3: E111K
4: L197P
5: W209R
6: L470P
7: W312C
8: A384D
9: D443N
10: R44S
Side-chain optimization of SCWRL models over five iterations minimise. Green: the input model. </figure>
<figure id="fig:minimise_foldx_mutations">
1: H60R
2: V172I
3: E111K
4: L197P
5: W209R
6: L470P
7: W312C
8: A384D
9: D443N
10: R44S
Side-chain optimization of FoldX models over five iterations minimise. Green: the input model. </figure>
Gromacs
Runtime analysis
To show the relationship between nsteps and runtime of 'mdrun', different nstep were chosen from 50 to 1000. Three different energy functions were selected:
- AMBER03 protein, nucleic AMBER94
- CHARMM27 all-atom force field (with CMAP)
- OPLS-AA/L all-atom force field
AMBER03
nstep=50
step=50 Reached the maximum number of steps before reaching Fmax < 1 real 0m7.446s user 0m13.230s sys 0m1.070s
nstep=100
step=100 Reached the maximum number of steps before reaching Fmax < 1
real 0m13.987s user 0m25.860s sys 0m1.530s
nstep=200
step=200 Reached the maximum number of steps before reaching Fmax < 1
real 0m27.186s user 0m51.340s sys 0m2.540s
nstep=300
Step=300 Reached the maximum number of steps before reaching Fmax < 1
real 0m40.664s user 1m16.690s sys 0m3.860s
nstep=400
step=360 Stepsize too small, or no change in energy.
real 0m48.479s user 1m32.195s sys 0m4.0230s
nstep=500
step=360 Stepsize too small, or no change in energy.
real 0m48.481s user 1m32.200s sys 0m4.190s
nstep=600
nstep=700
nstep=800
nstep=900
nstep=1000
step=360 Stepsize too small, or no change in energy.
real 0m48.475s user 1m31.650s sys 0m4.720s
nstep=1500
nstep=2000
step=360 Stepsize too small, or no change in energy. real 0m48.694s user 1m31.450s sys 0m4.990s
nstep=2500
nstep=3000
nstep=5000
step=360 Stepsize too small, or no change in energy. real 0m48.743s user 1m32.050s sys 0m4.550s
CHARMM27
nstep=50
step=50 Reached the maximum number of steps before reaching Fmax < 1
real 0m7.292s user 0m12.950s sys 0m1.050s
nstep=100
step=100 Reached the maximum number of steps before reaching Fmax < 1
real 0m7.292s real 0m13.785s user 0m25.850s sys 0m1.180s
nstep=200
step=200 Reached the maximum number of steps before reaching Fmax < real 0m26.843s user 0m50.240s sys 0m2.660s
nstep=300
Step=300 Reached the maximum number of steps before reaching Fmax < real 0m39.990s user 1m15.850s sys 0m3.470s
nstep=400
Step=348 Stepsize too small, or no change in energy. real 0m46.322s user 1m27.650s sys 0m4.150s
nstep=500
Step=348 Stepsize too small, or no change in energy. real 0m46.158s user 1m27.400s sys 0m4.280s
nstep=600
nstep=700
nstep=800
nstep=900
nstep=1000
Step=348 Stepsize too small, or no change in energy. real 0m46.121s user 1m27.680s sys 0m3.860s
nstep=1500
nstep=2000
Step=348 Stepsize too small, or no change in energy. real 0m45.345s user 1m28.090s sys 0m4.012s
nstep=2500
nstep=3000
nstep=5000
Step=348 Stepsize too small, or no change in energy. real 0m46.013s user 1m27.993s sys 0m4.432s
OPLS-AA/L
nstep=50
step=50 Reached the maximum number of steps before reaching Fmax < 1 real 0m6.063s user 0m10.750s sys 0m0.840s
nstep=100
step=100 Reached the maximum number of steps before reaching Fmax < 1 real 0m11.513s user 0m20.940s sys 0m1.340s
nstep=200
step=200 Reached the maximum number of steps before reaching Fmax < 1 real 0m22.378s user 0m41.760s sys 0m2.110s
nstep=300
Step=300 Reached the maximum number of steps before reaching Fmax < 1 real 0m32.959s user 1m1.960s sys 0m3.180s
nstep=400
Step=400 Reached the maximum number of steps before reaching Fmax < 1 real 0m43.790s user 1m22.880s sys 0m3.950s
nstep=500
step=500 Reached the maximum number of steps before reaching Fmax < 1 real 0m54.252s user 1m43.040s sys 0m4.720s
nstep=600
step=600 Reached the maximum number of steps before reaching Fmax < 1 real 1m5.020s user 2m3.660s sys 0m5.490s
nstep=700
step=700 Reached the maximum number of steps before reaching Fmax < 1 real 1m15.570s user 2m24.260s sys 0m6.150s
nstep=800
step=800 Reached the maximum number of steps before reaching Fmax < 1 real 1m26.511s user 2m44.730s sys 0m7.480s
nstep=900
step=900 Reached the maximum number of steps before reaching Fmax < 1 real 1m36.966s user 3m5.360s sys 0m7.810s
nstep=1000
step=1000 Reached the maximum number of steps before reaching Fmax < 1 real 1m47.718s user 3m25.940s sys 0m8.560s
nstep=1100
step=1100 Reached the maximum number of steps before reaching Fmax < 1 real 1m58.514s user 3m46.010s sys 0m9.730s
nstep=1500
step=1177 Stepsize too small, or no change in energy. real 2m6.700s user 4m2.190s sys 0m10.110s
nstep=2000
step=1177 Stepsize too small, or no change in energy. real 2m6.707s user 4m2.040s sys 0m10.470s
nstep=2500
nstep=3000
nstep=5000
step=1177 Stepsize too small, or no change in energy. real 2m6.734s user 4m2.126s sys 0m10.340s
Mutations
Mutation 1
Energy Average Err.Est. RMSD Tot-Drift ------------------------------------------------------------------------------- Bond 1624.88 820 5492.83 -5022.62 (kJ/mol) Angle 4289.66 76 402.822 -411.894 (kJ/mol) Potential -45289.6 2900 16896.6 -18817.6 (kJ/mol)
References
<references/>
