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

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(Minimise)
(Runtime analysis)
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#OPLS-AA/L all-atom force field
 
#OPLS-AA/L all-atom force field
   
  +
<figure id="fig:runtim">
==== AMBER03 ====
 
  +
[[File:runtime.png|thumb|400px|left|<caption> Runtime of minimization with Gromacs for different nsteps by using the OPLS-AA/L force field and the wildtype structure 2nt0_A.</caption>]]
  +
</figure>
   
  +
<br style="clear:both;">
nstep=50
 
 
step=50
 
Reached the maximum number of steps before reaching Fmax < 1
 
real 0m7.446s
 
user 0m13.230s
 
sys 0m1.070s
 
   
  +
In <xr id="fig:runtim"/> we showed the runtime plot for different setting of nstep(from 50 to 5000).
 
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 ===
 
=== Mutations ===

Revision as of 16:32, 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

SCWRL models

1: H60R

<figure id="fig:minimise_scwrl_e">

Energy of the SCWRL mutant models compared to the SCWRL wildtype models over five iterations minimise. </figure>

<figure id="fig:minimise_scwrl_m">

Side-chain conformation of the SCWRL mutant models compared to the SCWRL wildtype models over five iterations Minimise. </figure>

FoldX models

<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:

  1. AMBER03 protein, nucleic AMBER94
  2. CHARMM27 all-atom force field (with CMAP)
  3. OPLS-AA/L all-atom force field

<figure id="fig:runtim">

Runtime of minimization with Gromacs for different nsteps by using the OPLS-AA/L force field and the wildtype structure 2nt0_A.

</figure>


In <xr id="fig:runtim"/> we showed the runtime plot for different setting of nstep(from 50 to 5000).

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/>