Structure-based mutation analysis Gaucher Disease

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Revision as of 19:52, 25 June 2012 by Zhangg (talk | contribs) (Mutations)

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>


<figtable id="tab:mutations">

Nr Pos
From To Disease
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.



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
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.



FoldX <ref>Schymkowitz J., Borg J., Stricher F., Nys R., Rousseau F., Serrano L. (2005) The FoldX web server: an online force field. Nucleic Acids Research.</ref> is a force field for assessing the impact of point mutations. We called FoldX to determine the optimal side-chain conformation of the mutated site and compared the energy of the mutant model with the wildtype model in order to assess the severity of each mutation.

<xr id="fig:foldx"/> depict the side-chain conformation predicted by FoldX in comparison to SCWRL and the wildtype. The predictions of FoldX and SCWRL differed in four cases. 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 which suggests that these mutations are disease-causing. The fact that some mutations even increase the stability of the model indicates a mild effect on the protein function.


MINIMISE is a program that applies the CHARMM22 force-field and a rotamer library for minimizing the energy of models. We employed five iterations MINIMISE to refine the models generated by SCWRL and FoldX and compared the energy of the most stable mutant model to the most stable wildtype model in order to assess the effect of each point mutation.

SCWRL models

The energy of all SCWRL models could be reduced up to the second iteration and than gradually increased again. We therefore considered the models after two iterations MINIMISE as the most reliable ones which were used to assess the impact of the mutations on the phenotype. For this, we compared the MINIMISE energy of the mutant model to energy of the wildtype model (cf. the values in brackets in <xr id="fig:minimise_scwrl_e"/>). As already observed when employing SCWRL (cf. <xr id="fig:foldx"/>) and FoldX (cf. <xr id="fig:foldx"/>), L470P, D443N, and W209R significantly increase the the energy of the model. However, H60R and D443N now also lead to unstable models which would suggest a effect on the phenotype.

<figure id="fig:minimise_scwrl_e">

Energy of the SCWRL mutant models compared to the SCWRL wildtype models over five iterations MINIMISE. In brackets: energy(mutant 2nd iteration)-energy(wildtype 2nd iteration). </figure>

<xr id="fig:minimise_scwrl_m"/> show to which extent the MINIMISE optimization alters the side-chain conformations of the mutated residue compared to the wildtype residue. Altogether, the side-chain orientation of the mutated residues is changed only slightly and less than the side-chain of the wildtype model. Note that not only the side-chain of the mutated residue changes but also the side-chains of neighbouring residues.

<figure id="fig:minimise_scwrl_m">

Side-chain conformation of the SCWRL mutant models compared to the SCWRL wildtype models over five iterations MINIMISE. In brackets: energy(mutant 2nd iteration)-energy(wildtype 2nd iteration). </figure>

FoldX models

<figure id="fig:minimise_foldx_e">

Energy of the FoldX mutant models compared to the FoldX wildtype models over five iterations MINIMISE. In brackets: energy(mutant 2nd iteration)-energy(wildtype 2nd iteration). </figure>

<figure id="fig:minimise_foldx_m">

Side-chain conformation of the SCWRL mutant models compared to the FoldX wildtype models over five iterations MINIMISE. In brackets: energy(mutant 1nd iteration)-energy(wildtype 1nd iteration) </figure>


Runtime analysis

To show the relationship between nsteps and runtime of 'mdrun', different nstep were chosen from 50 to 5000. 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

<figtable id="runtime_table">

Forcefield nstep=50 nstep=100 nstep=200 nstep=300 nstep=400 nstep=500 nstep=1000 nstep=2000 nstep=5000
(360 steps to reach the minimum)
7.446 13.987 27.186 40.664 48.479 48.481 48.475 48.694 48.743
(348 steps to reach the minimum)
7.292 13.785 26.843 39.990 46.322 46.158 46.121 45.345 46.013
(1177 steps to reach the minimum)
6.063 11.513 22.378 32.959 43.790 54.252 107.718 126.707 126.734

Runtime of minimization with Gromacs for different nsteps by using AMBER03, CHARMM27 and OPLS-AA/L forcefield. Only the 'real time' returned from linux 'time' command is shown here. The unit of time is second. </figtable>

<xr id="runtime_table"/> presented the running time of Gromacs minimization for different setting of nsteps by using different forcefield. Nsteps told the program the maximum number of steps to minimize during the simulation. The computation time went higher when the Nsteps was higher. The table showed also the the steps the program needed to reach the minimum. By using AMBER03 and CHARMM27, only 360 and 348 steps were needed. By using OPLS-AA/L, it was 1177 steps, therefore it ran slower than the other two.

<figure id="fig:runtim">

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


In <xr id="fig:runtim"/> we showed the runtime plot for different setting of nsteps(from 50 to 5000). By using the OPLS-AA/L force field, the program needed 1177 steps to reach the minimum. From nsteps=50 to nsteps=1177, the runtime increased lineraly because it would run exactly nsteps time. After nsteps=1177, since the minimum has reached, the program would always stopped at 1177 steps no matter what the setting of nsteps was, therefore the running time stayed in the same.


<figtable id="mutation_gromacs">

WT H60R V172I E111K L197P W209R L470P W312C A384D D443N R44S
Average Energy Bond 1699.12 1624.88 1581.54 1973.46 1470.86 1598.3 1694.03 1579.31 1451.23 1621.83 1685.66
Difference Energy Bond 0 -74.24 -117.58 274.34 -228.26 -100.82 -5.09 -119.81 -247.89 -77.29 -13.46
Average Energy Angle 4375.68 4289.66 4371.18 4382.41 4381.32 4387.08 4425.35 4361.97 4361.9 4367.22 4368.95
Difference Energy Angle 0 -86.02 -4.5 6.73 5.64 11.4 49.67 -13.71 -13.78 -8.46 -6.73
Average Energy Potential -44445.9 -45289.6 -45025.6 -43553.6 -45475.1 -45265.6 -44266.1 -45024.9 -45805.7 -44803.9 -43924.1
Difference Energy Potential 0 -843.7 -579.7 892.3 -1029.2 -819.7 179.8 -579 -1359.8 -358 521.8

The average bond, angle and potential energy of the mutations and the wildtype, and the corresponding differences. </figtable>