Rs121907968

General Information

SNP-id	rs121907968
Codon	485
Mutation Codon	Trp -> Arg
Mutation Triplet	gTGG -> CGG

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Sequence-based Mutation Analysis

Pysicochemical Properties

First of all, we explored the amino acid properties and compared them for the original and the mutated amino acid. Therefore we created the possible effect that the mutation could have on the protein.

Trp	Arg	consequences
aromatic, polar, hydrophobic, neutral	positive charged, polar, hydrophilic	Trp is very big, because of two aromatic rings in its structure. Furthermore, it is hydrophobic, whereas, Arg is a hydrophilic amino acid. Therefore, the changes in the 3D structure might be extreme and delete the function of the protein.

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Visualization of the Mutation

In the next step, we created the visualization of the mutation with PyMol. Therefore, we created a picture of the original amino acid (Figure 1), of the new mutated amino acid (Figure 2) and finally of both together in one picture whereas the mutation is white colored (Figure 3) . The following pictures display that the mutated amino acid Arginine looks very different to Tryptophan. Tryptophan has two huge aromatic rings. Contrary, Aspartate is also a long amino acid and forks at the end of the rest. Furthermore, Argenine is also orientated in a completely different direction. Because of the aromatic ring the differences between these two amino acids is really huge. Therefore, the amino acids will probably cause drastic effects on protein structure and function.

picture original amino acid	picture mutated amino acid	combined picture
Figure 1: Amino acid Tryptophan	Figure 2: Amino acid Arginine	Figure 3: Picture which visualize the mutation

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Substitution Matrices Values

Afterwards, we looked at the values of the substitution matrices PAM1, PAM250 and BLOSSUM62. Therefore, we looked detailed at the three values: the value for according amino acid substitution, the most frequent value for the substitution of the examined amino acid and the rarest substitution.

In this case, the substitution of Tryptophan to Arginine has very high values that agree with the most frequent value for PAM1 and PAM250. In contrast, BLOSOUM62 has a low value for this substitution which is near the value for the rarest substitution. The difference between the two PAMs and BLOSUM62 can be ascribed to the different preparations of these two kind of substitution matrices. The PAM-matrices is an evolutionary model whereas BLOSUM is based on protein families. Therefore probably this mutation is evolutionary not that unlikely whereas within a protein family it is very unusual. Therefore, according to PAM1 and PAM250 a mutation at this position will probably not cause structural changes which can affect functional changes whereas according to BLOSSUM62 a mutation at this position will probably cause structural and functional changes of the protein.

PAM 1			Pam 250			BLOSOUM 62
value amino acid	most frequent substitution	rarest substitution	value amino acid	most frequent substitution	rarest substitution	value amino acid	most frequent substitution	rarest substitution
2	2 (Arg)	0 (all, except Arg, Phe, Ser, Tyr)	2	2 (Arg)	0 (all, except Arg, His, Leu, Phe, Ser, Tyr)	-3	2 (Tyr)	-4 (Asn, Asp, Pro)

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PSSM Analysis

Besides, we looked additional at the position specific scoring matrix (PSSM) for our sequence. In contrast to PAM and BLOSOUM, the PSSM contains a specific substitution rate for each position in the sequence. Therefore, the PSSM is more position specific than PAM or BLOSOUM. We extracted the substitution value for the underlying mutation, the value for the most frequent substitution and the rarest substitution.

In this case the substitution rate for Tryptophan to Arginine acid at this position is very low and near the value for the rarest substitution. This means this substitution at this position is likely very uncommon which indicates that this substitution has bad effects as a consequence. Therefore, we conclude that this mutation will probably cause protein structure changes as well as functional changes.

PSSM
value amino acid	most frequent substitution	rarest substitution
-6	13	-7

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Conservation Analysis with Multiple Alignments

As a next step we created a multiple alignment which contains the HEXA sequence and 9 other mammalian homologous sequences from [UniProt]. Afterwards we looked at the position of the different mutations and looked at the conservation level at this position. The regarded mutation is presented by the colored column on Figure 4. Here we can see, that all the other mammalians have also at this position the amino acid Tryptophan. Therefore, the mutation at this position is highly conserved and a mutation there will cause probably huge structural and functional changes for the protein.

Figure 4: Mutation in the multiple alignment

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Secondary Structure Mutation Analysis

As a next step we compared the different results of the secondary structure prediction tools JPred and PsiPred. Afterwards we can examine in which secondary structure element and where therein the mutation takes place. This can give an overview of how drastic the mutation can be. In this case both tools agree and predict at the position of the mutation a coil. This has as a result, that the mutation at this position would not destroy or split a secondary structure element. It will probably only changes the coil between two secondary structure elements, but this can sometimes also cause a change of the the following secondary structure. We think that a drastic change of the protein structure and its function is unlikely because the mutation does not affect a secondary structure element. The change of the coil will probably only take places between two secondary structure elements which will probably not change the protein.

JPred:
...HHHHCCCCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHCCC...
PsiPred:
...HHHCCCCCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCC...

Comparison with the real Structure:

Afterwards we also visualize the position of the mutation (red) in the real 3D-structure of [PDB] and compare it with the predicted secondary structure. The visualization can therefore like above the predicted secondary structure display if the mutation is in a secondary structure element or in some other regions.

Here in this case the mutation position disagree with the position of the predicted secondary structure and is within a alpha-helices (Figure 5 and Figure 6). This means a mutation will probably destroy or split the alpha helix which affects drastic structural changes on the protein. We think that a structural change is very likely, because it is within a secondary structure element and will therefore cause extreme changes.

Figure 5: Mutation at position 485

Figure 6: Mutation at position 485 - detailed view

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SNAP Prediction

Next, we looked at the result of the SNAP prediction. For this prediction we took the amino acid of the certain position and checked every possible amino acid mutation. Afterwards we extract the result for Arginine which is the real mutation in this case. SNAP has as a result that the exchange from Tryptophan to Arginine at this position is non-neutral with a very high accuracy. This means that this certain mutation on this position cause very likely structural and functional changes of the protein.

Substitution	Prediction	Reliability Index	Expected Accuracy
R	Non-neutral	7	96%

A detailed list of all possible substitutions can be found [here]

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SIFT Prediction

Next, we used SIFT Prediction which displays if a mutation is neutral or not. Therefore, it first shows a row which contains a score for the particular mutation position to a certain amino acid. The amino acid which are not tolerated at this position are colored red. Besides, it also constructs a table which lists the amino acids that are predicted as tolerated and not-tolerated.

In this case, the only substitution that is tolerated is the one to Tryptophan itself. The substitution to Arginine is not-tolerated at this position (Figure 8). This means that this mutation at this position is probably not neutral and will cause probably structural and function changes of the protein. SIFT Matrix:
Each entry contains the score at a particular position (row) for an amino acid substitution (column). Substitutions predicted to be intolerant are highlighted in red.

Figure 7: Legend

Figure 8: SIFT Table Threshold for intolerance is 0.05. Amino acid color code: non-polar, uncharged polar, basic, acidic. Capital letters indicate amino acids appearing in the alignment, lower case letters result from prediction.

Predict Not Tolerated Position Seq Rep Predict Tolerated y v t s r q p n m l k i h g f e d c a 485W 0.97 W

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PolyPhen2 Prediction

Finally, we also regarded the PolyPhen2 prediction for this mutation. This prediction visualizes have strongly damaging the mutation probably will be. Therefore it gives the result for two possible cases: HumDiv and HumVar. HumDiv is the preferred model for evaluation rare alleles, dense mapping of regions identified by genome-wide association studies and analysis of neutral selection. In contrast, HumVar is the preferred model for diagnostic of Mendelian diseases which require distinguishing mutations with drastic effects from all remaining human variations including abundant mildly deleterious alleles. We decided to look at both possible models, which are agrees in the most cases.

In this case both models predict that the mutation is probably damaging (Figure 9 and Figure 10). This means that the mutation is not neutral and will probably destroy the structure and the function of the protein

Figure 9: HumDiv prediction

Figure 10: HumVar prediction

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Structure-based Mutation Analysis

Mapping onto Crystal Structure

Figure 11: Visualization of the mutation and important functional sites
Color declaration:
* red: position of mutation
* green: position of active side
* yellow: position of glycolysation
* cyan: position of Cysteine

First of all, we colored the important residues and also the mutated residue in the crystal structure, to see if the mutation is near of far away from the functional residues. As you can see on Figure 11, the mutation is located at the end of a helix. Therefore a change of the amino acid could change the structure of this helix, but probably this mutation does not destroy the complete helix. Furthermore, the functional residues of the protein are located far away from this mutation. So this mutation could not be explained by just looking at the mutation's location onto the structure.

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SCWRL Prediction

Because the mapping analysis does not give a good explanation why the mutation causes damages on the protein, we decided to analyse this mutation in more detail. Therefore, we looked for the structure of the original amino acid and the structure of the amino acid after the mutation event and compared them in size and orientation. For this purpose we used SCWRL.

picture original amino acid	picture mutated amino acid	combined picture
Figure 12: Amino acid Tryptophan	Figure 13: Amino acid Arginine	Figure 14: Picture which visualize the mutation

The structure of the different amino acids differ extremely (Figure 12, Figure 13). The size of the amino acids and the orientation of the amino acids is very different (Figure 14). Therefore this exchange could damage the protein.

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FoldX Energy Comparison

Next we use the FoldX energy tool to compare the energy values of the two different structures.

Original total energy	Total energy for the mutated protein	Strongest energy changes within the mutated protein
-154.17	-149.08	-

The energy of the mutated structure is a little bit higher than the value of the original structure. Therefore the mutated protein seems to be a little bit more unstable than the original one. Although the difference is not dramatically it could lead to a too rigid structure which means that the binding site is too rigid to bind on its ligands. To have the possibility to compare these energy values with the values of the other analysis tools, we calculated a ratio between these energy values.

Ratio of the original structure	Ratio of the mutated structure	Difference
100	96.80	3.20

The mutated structure has about 3% more energy than the original one.

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Minimise Energy Comparison

Next we use the minimise energy tool to compare the energy values of the two different structures.

Comparing Energy:

Original total energy	Total energy for the mutated protein
-9610.467157	-9608.663976

In this case the mutated structure has a little more energy than the original structure. To have the possibility to compare the energy values of the different tools we calculate the ratio.

Ratio of the original structure	Ratio of the mutated structure	Difference
100	99.98	0.02

In this case the difference between the two ratios is nearby 0.

Comparing Structure:

This tool also gives as output a [PDB] file with the position of the original and the mutated amino acid.

picture original amino acid	picture mutated amino acid	combined picture
Figure 15: Amino acid Tryptophan	Figure 16: Amino acid Arginine	Figure 17: Picture which visualize the mutation

These pictures (Figure 15, Figure 16, Figure 17) shows the difference of the structure of the two amino acids. The result is consistent with that from SCWRL (Figure 12, Figure 13, Figure 14). Both tools model the mutated amino acid with the same orientation.

Visualization of H-bonds and Clashse:

To get more insight in the effects of the mutated amino acid on the structure, we also analysed the H-bonds and clashes of the Serine residue.

H-bonds near the mutation	Clashes of the mutation
Figure 18: H-bonds of the original amino acid (colored in magenta)	Figure 19: H-bonds of the mutated amino acid (colored in red)	Figure 20: Possible clashes

Both pictures (Figure 18, Figure 19) shows a h-bond between the amino acid with another amino acid. Now we have to look on the picture in more detail to see, if they have a h-bond with the same amino acid. This is not the case here, so the wrong H-bond could be an explanation for a damage in the protein function. Furthermore, there do not exist any clashes between the mutated amino acid and the rest of the protein, which can be seen on Figure 20.

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Gromacs Energy Comparison

Comparing Energy:

To analyse the energy values calculated by Gromacs, we used the AMBER99SB-ILDN force field.

Here are the values of the original structure:

Energy	Average	Err.Est.	RMSD	Tot-Drift
Bond	1091.57	270	-nan	-1622.75
Angle	3326.81	62	-nan	404.076
Potential	-61304.1	960	-nan	-6402.44

Here you can see the values which gromacs calculated for the structure with the mutated amino acid:

Energy	Average	Err.Est.	RMSD	Tot-Drift
Bond	927.115	180	2166.81	-1050.91
Angle	3212.21	62	234.542	408.392
Potential	-49443.8	850	3236.86	-5516.44

One difference between gromacs and the other tools we used is, that gromacs also calculated the energy for the bonds and the angles. To compare the energies between the different tools we only consider the potential energy in our analysis, because the potential energy is the energy of the complete protein. Therefore, we calculated the ratio between the energies only for the potential energy.

Ratio of the original structure	Ratio of the mutated structure	Difference
100	80.65	19.35

The difference is with 20% high enough to explain a damage of the protein function.

Comparing Structure:

Gromacs also offers pictures of the mutated amino acids which can be seen in the following section.

picture original amino acid	picture mutated amino acid	combined picture
Figure 21: Amino acid Tryptophan	Figure 22: Amino acid Arginine	Figure 23: Picture which visualize the mutation

These pictures (Figure 21, Figure 22, Figure 23) are nearly equal to the pictures from SCWRL and minimise. Again we can see, that the structure of the amino acids is very different.

Visualization of H-bonds and Clashes:

To get more insight in the effects of the mutated amino acid on the structure, we also analysed the H-bonds and clashes of the Serine residue.

H-bonds of the original amino acid	H-bonds near the mutation	Clashes of the mutation
Figure 24: H-bonds of the original amino acid (colored in magenta)	Figure 25: H-bonds of the mutated amino acid (colored in red)	Figure 26: Possible clashes

Both amino acids have H-bonds with adjacent amino acids, but the mutated amino acid (Figure 25) binds to another residue than the original amino acid (Figure 24). Therefore, this could be an explanation if the protein function of structure is damaged. Furthermore, it was not possible to find any clashes between the mutated amino acid and the rest of the protein, which can be seen on Figure 26.

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