Difference between revisions of "Structure-based mutation analysis Gaucher Disease"

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Stepsize too small, or no change in energy.
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Stepsize too small, or no change in energy.
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=== Mutations ===
 
=== Mutations ===

Revision as of 14:28, 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.

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">

2nt0_A with the selected mutations used for the structure-based analysis. Blue: wildtype residues; Red: mutant residues; Orange: active site residues E235 and E340.

</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">

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">

Seconary structure elements of 2nt0_A (grey) compared to secondary structure elements of models built by SCRWL.

</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">

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">
Energy of the SCWRL models vs. the number of minimise iterations.
<figure id="fig:minmise_foldx_energies">
Energy of the FoldX models vs. the number of minimise iterations.

<figure id="fig:minimise_scwrl_mutations">

Side-chain optimization of SCWRL models over five iterations minimise. Green: the input model. </figure>

<figure id="fig:minimise_foldx_mutations">

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=40
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

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/>