Structure based mutation analysis of GBA

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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