Structure based mutation analysis of GBA

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Introduction

After the sequence based mutation analysis, as described in Task 6, a structure based mutation analysis of ten mutations (listed in the table below) was carried out. For the analysis, several different tools have been used. The different tools and the corresponding steps, applied in this analysis are described in more detail in this workflow for reasons of clarity.

Nr. SNP ID/Accession Number Database Position
including SP
Position
without SP
Amino Acid Change Codon Change
1 CM081634 HGMD 49 10 Gly - Ser cGGC-AGC
2 rs74953658, CM050263 dbSNP, HGMD 63 24 Asp - Asn tGAC-AAC
3 rs1141820 dbSNP 99 60 His - Arg CAC - CGC
4 CM880035 HGMD 159 120 Arg - Gln CGG-CAG
5 rs80205046, CM041347 dbSNP, HGMD 221 182 Pro - Leu CCC - CTC
6 rs74731340, CM970620 dbSNP, HGMD 310 271 Ser - Asn AGT - AAT
7 CM880036 HGMD 409 370 Asn - Ser AAC-AGC
8 CM993703 HGMD 350 311 His - Arg CAT-CGT
9 rs80020805, CM052245 dbSNP, HGMD 455 416 Met - Val cATG-GTG
10 rs113825752 dbSNP 509 470 Leu - Pro CTT - CCT



Structure Selection

To carry out a structure-based analysis of the mutations chosen in Task 6, a crystal structure had to be chosen. According to Uniprot there are 19 different crystal structures of glucocerebrosidase available. The table below shows the six different structures with a resolution of or better than 2 Angstrom. 2NT0 is chosen as template for the analysis carried out in this section, as no residues are missing, the R-value is quite low, and it has the best resolution among the structures without missing residues. Only incomplete structures have been resolved near the physiological pH (7.4), therefore a structure resolved at a more acid pH had to be chosen. The structure can either be downloaded from the PDB website or by using the script fetchpdb, which validates the ID and downloads the corresponding structure.


PDB ID Resolution [Å] R-factor Coverage pH # Missing Residues (A/B)
1OGS 2.00 0.195 4.6 0
2NT0 1.79 0.181 . 4.5 0
2V3D 1.96 0.157 6.5 9/8
2V3E 2.00 0.163 7.5 7/7
2V3F 1.95 0.154 6.5 8/14
3GXI 1.84 0.193 NULL 0


Mutation Mapping

Figure 1 shows the positions of the analyzed mutations in the original structure of 2NT0. As already mentioned in Task 5 and 6, one can clearly see that two mutations are next to the active site residues Glu235 and Glu340, namley the mutations at positions 120 and 311. The wildtype residues at these positions (Arg120 and His311) are known to form hydrogen bonds with the active sites and should therefore be quite important for function and structure. <ref>Kim et al., Crystal Structure of the Salmonella enterica Serovar Typhimurium Virulence Factor SrfJ, a Glycoside Hydrolase Family Enzyme. Journal of Bacteriology, 2009, p. 6550-6554, Vol. 191, No. 21 </ref> The other eight mutation positions are located all over the protein. The localization anf the amino acid properties of the different mutant and wildtype structures have already been analysed in Task 6.


Figure 1: 2NT0 with hilighted mutation positions (red) and active site residues (blue).
Figure 2: Close-up of active site of 2NT0 with hilighted mutation positions (red) and active site residues (blue).

SCWRL

SCRWL4 was applied ten times, once for each mutation. The resulting conformations of the mutants are visualized in Figure 3. For this representation the hydrogens of the SCWRL mutant structures have been removed to simplify the comparison with the other two structures. Figure 3 additionally shows the wildtype amino acids and the mutants created with the mutagenesis method of pymol. The conformations, created with SCWRL4 and pymol vary greatly. Only in mutation 9 they seem to be quite similar. Figure 4 shows a superposition of the wild type protein and the mutated proteins in cartoon representation. This shows that SCWRL did not only change the mutant residues, but also changed some beta sheets at the bottom of the structure (shown in green). This fact may be due to the different polar interactions of the mutants.

Figure 3: Wildtype amino acids (red) and mutations created with SCWRL (green) and pymol mutagenesis (orange) hilighted on the structure of 2NT0.
Figure 4: Cartoon representation of 2NT0, chain A (gray) superimposed with the resulting structures of SCWRL (green).




Minimise

Figure 5 shows the interesting positions with hilighted mutants and wild type residues of the pdb files obtained with Minimise after having applied the steps as indicated in the workflow. The hydrogens of the structures have been removed as well.

Figure 5: Wildtype amino acids (red) and mutations (green) created with Minimise. Polar interactions are shown with dotted lines.

Gromacs

Figure 6 shows the interesting positions with hilighted mutants and wild type residues of the pdb files obtained with Gromacs after having applied the steps indicated in the workflow. The hydrogens of the structures have been removed as well.


Figure 6: Wildtype amino acids (red) and mutations (cyan) created with Gromacs. Polar interactions are shown with dotted lines.

Polar Interactions

Polar interactions are crucial for the structue, and therefore function of a protein. A mutation of a single amino acid could therefore alter the features and appearance of a protein. Analyzing polar interactions may therefore help to determine whether a mutation is damaging or neutral. If the polar interactions, which are present in the wild type structure, are still formed after the mutation, the mutation may be tolerated, otherwise it should be damaging.

The table below shows the residues forming polar interactions with the mutant/wild type amino acid. The polar interactions can be seen in Figure 3, Figure 5 and Figure 6 as well, but one may not clearly distinguish, whether an interaction is only formed by e.g. the wild type or by wild type and mutant. To determine the interaction partners, the tool Pymol was used [actions -> find -> polar contacts -> to other atoms in object]. The table additionally indicates whether different polar interactions are present between the amino acids of interest in the wildtype and in the mutations. The Pymol Mutagenesis results were not taken into account, as no real simulations have taken place.


Mutation PDB
Wild type
Pymol
Mutagenesis
SCWRL Minimise Gromacs Differences
Wild Type Mutation Wild Type Mutation
1 S8 S8
F9
S8 S8 S8 S8 S8 no
2 K413
Y418


Y22
K413
Y418
K413
Y418
K413
Y418
K413
Y418
K413
Y418
no
3 T471
G62
T471
G62
T417 yes
4 G83
M85
S177
D282
N234
E340
G83
M85
G83
M85
S177



S339
G83
M85
S177
D282
N234
E340
G83
M85
S177



S339
G83
M85
S177
D282
N234
E340
G83
M85
S177
yes
5 S181
L185
S181
L185
S181
L185
S181
L185
S181
L185
S181
L185
S181
L185
no
6 L268
H273
H274
L268
H273
H274
L268

H274
L268
H273
H274
L268

H274
L268
H273
H274
L268

H274
T267
yes
7 S366
Y373
V375
S366
Y373
V375
S366
Y373
V375
S366
Y373
V375
S366
Y373
V375
S366
Y373
V375
S366
Y373
V375
no
8 D282
D283
R285
S339

D283
R285
D282

R285

Y313
E340
D282
D283
R285

D283
R285

Y313
E340
N234
E235
D282
D283
R285
S339

D283
R285

Y313
E340
yes
9 L420 L420 L420 L420 L420 L420 L420 no
10 T482 T482
V468
T482 T482 T482 T482 T482 no


Mutations 1, 2, 5, 7, 9 and 10 show in most cases (SCWRL, Minimise, Gromacs) the same polar interactions as the wild type and should therefore not have a big influence. The other mutations (3,4,6 and 8) form however additional bonds or miss some interactions which are present in the wild type. Interestingly, the most common mutation found in Gaucher Disease forms the same polar interactions as the wildtype.

Clashes or Holes

Furthermore it was analyzed whether the mutations cause clashes or holes in the protein structure. The following table shows whether the mutations created/minimized with Pymol, SCWRL, Minimise and Gromacs lead to structural inconsistency compared to the wildtype glucocerebrosidase (2NT0).


Mutation Pymol
Mutagenesis
SCWRL Minimise Gromacs
1 no
but different surface
no no no
but different surface
2 no no no no
3 no no
but different surface
no
but different surface
no
4 no no no no
but different surface
5 no no no no
6 no
but different surface
no
but different surface
no
but different surface
no
but different surface
7 no no no no
8 no no
but different surface
no no
9 no no
but different surface
no no
but different surface
10 no no
but different surface
no
but different surface
no



None of the mutations lead to clashes or holes in the protein structure, but some of the mutations cause a different surface. Mutation 6 leads to a different surface, no matter with which tool they were created or minimized. This is quite interesting as Serine and Asparagine are structurally and chemically very similar. Regarding the polar interactions, they are not identical.

Overall one would assume that the surface of the resulting protein is changed if a mutation leads to different polar interactions and if the mutations takes place inside a structural element. Mutation 1 leads to a different surface when build with Pymol Mutagenesis and Gromacs. The mutated residue is part of a secondary structural element, the mutated amino acid has a different polarity than the wildtype and the Pymol Mutagenesis build forms an additional hydrogen bond. These facts could explain the different surface. Mutation 2 does not lead to a different surface. This is quite interesting regarding the Pymol Mutagenesis Build, as this one forms completely different hydrogen bonds than the wildtype. The fact that the mutated residue is not part of a secondary structural element and that Asparagine and Aspartic Acid have a similar molar mass could be an explanation for the not altered surface. Mutation 3 leads to a different surface when minimised with SCRWL or Minimize. The polar interactions are always different to the ones formed by the wild type, no matter which tool was used. Furthermore the mutated amino acid is structurally very different to the wildtype amino acid. Mutation 4 results in a different surface when using the tool Gromacs. The mutated amino acid forms different polar interactions than the wild type, no matter which tool was used. Therefore it is quite interesting, that the surface is only altered when gromacs is used. Mutation 5 shows in all cases no different surface. This is quite interesting as the mutation is damaging. But as it also forms the same hydrogen bonds the structure should be the same. Mutation 6 has in all cases a different surface. There is also one missing hydrogen bond which might be responsible for that. Mutation 7 has no changes in the surface or any clashes or holes. The reason again may be the same hydrogen bonds as the wildtype. As the amino acids in wildtype and mutant also have the same properties it is what we expected. Mutation 8 shows a different surface with SCWRL and no changes in all other tools. That is quite interesting because the hydrogen bonds differ for all tools. So we would expect a different surface for all tools. Mutation 9 causes a different surface with SCWRL and Gromacs. As the hydrogen bonds are the same we would not expect that. But the amino acids in wildtype and mutant have different properties, so this may be a reason. Mutation 10 results in a different surface with SCWRL and Minimise. As the hydrogen bonds are the same, there has to be another reason. Interestingly Pymol shows one additional hydrogen bond but there is no change in the surface. The reason for the different surface with SCWRL and Minimise may again be because of the different amino acid properties.

Energy Comparison

The energy of a protein is an indication for its stability: the lower the more stable is the protein. Therefore a comparison of energy between the different mutations and wildtypes could be an indication whether a mutation could be damaging. If the energy of the mutated protein is much higher than the one of the wildtype protein it might be an indication that this mutation is damaging.

In this section, the energies of the structures, obtained with different minimization and modeling tools (SCWRL, FoldX, Minimise and Gromacs) are listed for each structure and for the wildtype (if available).

SCWRL

The table below shows the minimal energies for the different mutated structures obtained after the sidechain modelling with SCWRL. Most of the mutations are about the same energy value. Only Mutation 5 shows a much higher energy, which shows, that the protein is much less stable than if other resiudes are mutated. This indicates that Mutation 5 is a damaging mutation.

Mutation 1 2 3 4 5 6 7 8 9 10
Minimal Energy 351.329 348.659 350.017 355.416 473.454 364.148 352.615 362.604 354.98 375.976

FoldX

The total energies calculated with FoldX are shown in the table below. The differences between the wild type and the different mutant structures have been calculated and are listed in the table as well.


Mutation Total Energy Difference
WT -372.60 0
1 -225.82 -146.78
2 -228.18 -144.42
3 -226.97 -145.63
4 -226.38 -146.22
5 -196.84 -175.76
6 -224.01 -148.59
7 -228.48 -144.12
8 -217.29 -155.31
9 -221.71 -150.89
10 -218.65 -153.95

The mutated structures have a higher energy than the wildtype protein. Once again it is Mutation 5 having the highest energy and therefore being the least stable structure. The other mutations show similar energies.

Minimise

The total energies calculated with minimise are shown in the table below. The differences between the wild type and the different mutant structures have been calculated and are listed in the table as well.

Mutation Total Energy Difference
WT -12263.635255 0
1 -10405.258800 1858.37645
2 -10354.081889 1909.55337
3 -10313.900667 1949.73459
4 -10415.502839 1848.13242
5 -8558.228610 3705.40664
6 -10397.436817 1866.19844
7 -10427.599844 1836.03541
8 -3611.581531 8652.053719
9 -10030.430566 2233.20469
10 -10300.108046 1963.52721

The energy values of the mutated proteins are higher than the energy of the wildtype protein, which indicates that the mutated ones are less stable. This time Mutation 8 shows the highest energy and variance from the wildtype protein. Mutation 5 is also significantly higher than the average distance. Mutation 9 shows a slightly higher energy than most of the other mutations. This might be an indication, that Mutation 5, 8 and 9 are more damaging than the other mutations.

Gromacs

Wildtype

The wildtype glucocerebrosidase was simulated with three different force fields and different maximum number of steps to integrate or minimize (nsteps). The following force fields have been used:

  • AMBER03: Assisted Model Building and Energy Refinement
  • CHARMM27: Chemistry at HARvard Macromolecular Mechanics; all atom force field
  • OPLS/AA: Optimized Potential for Liquid Simulations; all atom force field
Correlation between nsteps and runtime of mdrun

The following table shows the measured time for mdrun for different nsteps and force fields. It also shows the steps it really took to calculate the lowest energy. Nsteps specify the maximum number of steps to integrate or minimize during the simulation. Therefore the computation time should be higher, the more steps have been performed. This fact can be observed in the table below. With the force fields AMBER03 and CHARMM27 it took less than 1000 steps to reach the minimum, so computation times for 1000 and 5000 are the same. Figure 7 shows the correlation between nsteps and the runtime (real time)of mdrun.

Figure 7: Correlation between nsteps and the runtime of mdrun for different force fields
nsteps AMBER03 CHARMM27 OPLS/AA
10 real: 0m4.615s
user: 0m2.910s
sys: 0m0.230s
Steps: 10
real: 0m10.760s
user: 0m2.760s
sys: 0m0.150s
Steps: 10
real: 0m3.801s
user: 0m2.280s
sys: 0m0.320s
Steps: 10
50 error occured real: 0m34.086s
user: 0m10.830s
sys: 0m0.260s
Steps: 50
real: 0m13.042s
user: 0m9.200s
sys: 0m0.620s
Steps: 50
100 real: 0m29.400s
user: 0m22.060s
sys: 0m0.640s
Steps: 100
real: 0m23.616s
user: 0m19.410s
sys: 0m0.590s
Steps: 100
real: 0m25.184s
user: 0m18.330s
sys: 0m0.740s
Steps: 100
500 real: 1m9.519s
user: 0m53.700s
sys: 0m1.010s
Steps: 242
real: 3m15.607s
user: 1m1.790s
sys: 0m1.070s
Steps: 307
real: 3m51.264s
user: 1m7.870s
sys: 0m1.590s
Steps: 500
1000 real: 2m9.160s
user: 0m52.990s
sys: 0m1.190s
Steps: 242
real: 3m22.812s
user: 1m2.120s
sys: 0m1.170s
Steps: 307
real: 5m9.851s
user: 1m38.390s
sys: 0m1.860s
Steps: 732
5000 real: 2m13.786s
user: 0m53.020s
sys: 0m1.030s
Steps: 242
real: 3m15.591s
user: 1m2.730s
sys: 0m1.560s
Steps: 307
real: 5m5.631s
user: 1m37.240s
sys: 0m1.830s
Steps: 732
Results of the different Force Fields

The results for the different force fields and different nsteps are listed in the following file for reasons of clarity: File:Glucocerebdosidase gromacs forcefields.pdf. The results are additionally visualized in the figures below.


AMBER03

Figure 8: AMBER03 with 10 nsteps.
Figure 9: AMBER03 with 100 nsteps.
Figure 10: AMBER03 with 500 nsteps.
Figure 11: AMBER03 with 1000 nsteps
Figure 12: AMBER03 with 5000 nsteps.

In Figure 8 you can see that with the AMBER03 forcefield bond, angle and potential come very soon near the optimum. Figure 9, 10, 11 and 12 show the lowest energy that can be reached with bond near 500, angle near zero and potential about -5000.

CHARMM27

Figure 13: CHARMM27 with 10 nsteps.
Figure 14: CHARMM27 with 50 nsteps.
Figure 15:CHARMM27 with 100 nsteps.
Figure 16: CHARMM27 with 500 nsteps.
Figure 17: CHARMM27 with 1000 nsteps
Figure 18: CHARMM27 with 5000 nsteps.

With the forcefield CHARMM27 many steps are needed to reach the optimum. In figure 13 you can see that it reaches a minimum for the potential about 0 and the minima for bond and angle (near 500 and zero). The potential does not go below zero until we use 5000 steps. There it comes to the minimum of about -5000, that was also reached with the AMBER03 forcefield, which is shown in figure 18.

OPLS/AA

Figure 19: OPLS/AA with 10 nsteps.
Figure 20: OPLS/AA with 50 nsteps.
Figure 21: OPLS/AA with 100 nsteps.
Figure 22: OPLS/AA with 500 nsteps.
Figure 23: OPLS/AA with 1000 nsteps.
Figure 24: OPLS/AA with 5000 nsteps.

With the forcefield OPLS/AA it is the same as with AMBER03. For ten steps (figure 19) you can see that bond, angle and potential fall and already with 50 steps (figure 20) they reache their minima. But here it is -10000 for the potential, which is much lower than with AMBER03. For bond and angle it is the same.

Mutations

For the ten mutated structures, only a simulation with the AMBER force field has been done. The table below shows the total bond, angle and potential energy of the mutations and the wildtype. Additionally the differences of the mutations in comparison to the wildtype are given. The results of the Gromacs calculations are shown in more detail in the following file: File:Glucocerebdosidase gromacs mutations.pdf. The plots of the Gromacs energies are shown in Figures 25 to 34.


Mutation Total Energy Bond Difference Bond Total Energy Angle Difference Angle Total Energy Potential Difference Potential
WT 1037.65 0 4355.86 0 -48425.9 0
1 1274.92 237.27 4255.25 -100.61 -47765.7 660.2
2 1456.65 419 4236.44 -119.42 -47077.9 1348
3 1231.56 193.91 4183.17 -172.69 -48600.7 -174.8
4 1338.99 301.34 4240.62 -115.24 -47096.1 1329.8
5 1478.43 440.78 4311.75 -44.11 77967 126392.9
6 1308.78 271.13 4244.41 -111.45 -47639.3 786.6
7 1201.46 163.81 4258.22 -97.64 -47883.1 542.8
8 1628.70 591.05 4182.29 -173.57 -40120 8305.9
9 1421.28 383.63 4230.01 -125.85 -47052.5 1373.4
10 1514.09 476.44 4283.65 -72.21 -46575.7 1850.2



Figure 25: GROMACS Energy for Mutation 1 (AMBER03, 500 nsteps)
Figure 26: GROMACS Energy for Mutation 2 (AMBER03, 500 nsteps)
Figure 27: GROMACS Energy for Mutation 3 (AMBER03, 500 nsteps)
Figure 28: GROMACS Energy for Mutation 4 (AMBER03, 500 nsteps)
Figure 29: GROMACS Energy for Mutation 5 (AMBER03, 500 nsteps)
Figure 30: GROMACS Energy for Mutation 6 (AMBER03, 500 nsteps)
Figure 31: GROMACS Energy for Mutation 7 (AMBER03, 500 nsteps)
Figure 32: GROMACS Energy for Mutation 8 (AMBER03, 500 nsteps)
Figure 33: GROMACS Energy for Mutation 9 (AMBER03, 500 nsteps)
Figure 34: GROMACS Energy for Mutation 10 (AMBER03, 500 nsteps)


The potential energy differences between mutation and wildtype are analysed to determine whether a mutation might be damaging. Mutation 8 and Mutation 5 have a significantly higher energy than all other mutations and might therefore be damaging. Mutation 3 has a lower potential energy than the wildtype, which indicates, that the mutated protein is more stable than the wildtype. As the energy is only slightly better than the one of the wildtype one can assume that this mutation is neutral. Otherwise, if the mutated protein would have been much more stable than the wildtype one, the mutation could also be damaging, as some flexibility of the protein could be necessary for the function. After Mutation 3 Mutation 7 shows the most similar energy to the energy of the wildtype. This might be an indication, that this mutation is harmless as well.


Discussion

In this section the results obtained with the different analysis steps applied in the structure based mutation analysis are combined to determine whether a mutation might be damaging or not.

Mutation SCWRL Minimise Gromacs FoldX
Energy
Polar
Interactions
Clashes
Holes
Energy Polar
Interactions
Clashes
Holes
Energy Polar
Interactions
Clashes
Holes
Energy
1 neutral neutral neutral neutral neutral neutral neutral non-neutral neutral neutral
2 neutral neutral neutral neutral neutral neutral neutral neutral neutral neutral
3 non-neutral non-neutral neutral non-neutral non-neutral neutral non-neutral neutral neutral neutral
4 non-neutral neutral neutral non-neutral neutral neutral non-neutral non-neutral non-neutral neutral
5 neutral neutral non-neutral neutral neutral non-neutral neutral neutral non-neutral non-neutral
6 non-neutral non-neutral neutral non-neutral non-neutral neutral non-neutral non-neutral non-neutral neutral
7 neutral neutral neutral neutral neutral neutral neutral neutral neutral neutral
8 non-neutral non-neutral neutral non-neutral neutral non-neutral non-neutral neutral non-neutral neutral
9 neutral non-neutral neutral neutral neutral non-neutral neutral non-neutral non-neutral neutral
10 neutral non-neutral neutral neutral non-neutral neutral neutral neutral non-neutral neutral

Mutation 1

The mutation at position 10 (not including the signal peptide) from Glycine to Serine does not lead to a change in present hydrogen bonds: Both in wildtype and in the different minimized mutated structures, a hydrogen bond to S8 is formed. The surface of the mutated protein, simulated with Gromacs, is however different from the wildtype surface. The energy of the mutation is higher than the one of the wildtype, no matter which simulation/minimization was used. But as this is true for each mutation in this analysis, this might not be an indication for a damaging effect: the energy is not significantly higher than the ones of the other mutations. As the surface is the only hint, that the mutation is damaging, and the rest is in favor of a neutral mutation, one would conclude in this case, that Mutation 1 is harmless. According to HGMD this classification is wrong: the mutation is damaging and causes Gaucher Disease.

Mutation 2

Mutation 2, located at position 24, describing a change from Arspatic Acid to Asparagine does neither lead to a change in polar interactions, nor does it lead to a different surface or clashes or holes. Additionally it does not have significantly high energy values in any of the different calculations. Therefore one would assume that this mutation is neutral. Once again, the prediction is wrong: the mutation is damaging.

Mutation 3

The mutated Histidine to Arginin at position 60 leads to either to the formation of a completly different hydrogen bond or to no binding at all. Furthermore the surface is different in both SCWRL and Minimize. Mutation 3 is the only mutation leading to a lower energy than the wildtype in the Gromacs calculations. It is not significantly lower and therefore very similar to the wildtype energy. It is hard to decide whether the mutation is damaging or not. The polar interactions and the surface indicate a damaging effect, whereas the energy comparison is in favor of a neutral effect. As the mutation is only listed in dbSNP, the mutation should be neutral.

Mutation 4

Mutation 4 seems to have a damaging effect: different or less hydrogens compared to the wildtype are formed, the surface is at least changed for one minimization tool and the energy is not significantly high or low. The prediction was made correclty: Mutation 4 is listed in HGMD.

Mutation 5

The mutation from Proline to Leucine at position 221 leads to an extremely high energy, which indicates, that the protein is very unstable. This is quite interesting, as the polar interactions and the surface remain the same compared to the wildtype glucocerebrosidase. But the fact that the energy is this high is a strong indicator, that this mutation is damaging. The prediction was made correclty: Mutation 5 is listed in both HGMD and dbSNP.

Mutation 6

Mutation 6 leads to a different surface with each minimization/simulation tool and to different hydrogen bonds compared to the wildtype glucocerebrosidase. As the energy comparison does not lead to contradicting indices, this mutation is classified as damaging. The prediction was made correclty: Mutation 6 is listed in both HGMD and dbSNP.

Mutation 7

The mutation at position 409 does neither change the polar interactions at this position nor the surface of the protein. Furthermore the energy comparison shows that it is closer to the wildtype energy than to an extreme high energy value. These facts indicate that the mutation is neutral. The prediction is not correct, as this mutation is the most common one found in patients with Gauchers Disease.

Mutation 8

Mutation 8 has a significantly higher energy than the other mutated structures and furthermore alters the surface of the protein and leads to different hydrogen bonds compared to the wildtype. These facts lead to the assumption that the mutation is damaging. The prediction was correct: Mutation 8 leads to Gaucher Disease.

Mutation 9

The mutation forms the same hydrogen bonds as the wildtype, but does lead to a different surface. As the energy comparison does not tell one otherwise, this mutation is thought to be harmless. The prediction is not correct, as this mutation is known to cause Gauchers Disease.

Mutation 10

Mutation 10 leads to the same results as mutation 9, it forms the same hydrogen bonds, causes in half of the minimization tools no different surface and the energy is not significantly higher. Therefore it is classified as harmless, which is true.


Summary

In this section we tried to predict the effects of mutations based on their polar contacts and energies compared to the wildtype. In half of the cases, the effect of the mutation was not predicted correctly. Even the most common mutation among people with Gauchers Disease was not classified correctly. This shows that the analyses applied in this sections are not suficient to classify the effect of mutations. Regarding the results for mutation 2 we would say, that it is not damaging, because all results indicated that the mutation has only marginal effect on the protein. But that was a wrong prediction. So there are more methods needed, to make a correct prediction. It is also hard to decide, which effect is the strongest. Has it a stronger effect that different hydrogen bonds are formed or that the energy is higher? If you look at mutation 3 you can see that it is not clear if we should predict the mutation damaging or not, because it shows different results. We think it depends on the position of the amino acid and its properties, which changes are more important or deleterious. The combination of structure and sequence based mutation analysis might lead to better results.



Additional analyses might help to better classify mutations. Therefore have a look at sequence-based mutation analysis and combined (sequence and structure) mutations analysis.

References

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