Rs121907974

General Information

SNP-id	rs121907974
Codon	211
Mutation Codon	Phe -> Ser
Mutation Triplet	TTC -> TCC

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.

Phe	Ser	consequences
polar, tiny, hydrophilic, neutral	aliphatic, hydrophobic, neutral	Ile is much bigger than Ser and also is branched, because it is an aliphatic amino acid. Therefore the structure of both amino acids is really different and Ile is too big for the position where Ser was. Therefore, there has to be a big change in the 3D structure of the protein and the protein probably will loose its function.

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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 for both together in one picture whereas the mutation is white colored (Figure 3). The following pictures display that the mutated amino acid Serine looks very different to Phenylalanine. Phenylalanine has a huge aromatic ring. Contrary, Serine is very smaller and differs a little bit in the orientation. This shows that the amino acids have huge structural differences which will probably cause dramatic effects on protein structure and function.

picture original amino acid	picture mutated amino acid	combined picture
Figure 1: Amino acid Phenylalanine	Figure 2: Amino acid Serine	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 the 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 Phenylalanine to Serine has low values that are nearer to the values for the rarest substitution for all three matrices. Therefore, an exchange at this position is very unlikely and a mutation will almost certainly cause structural changes which can affect functional changes.

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	28 (Tyr)	0 (Asp, Cys, Glu, Lys, Pro, Val)	2	20 (Tyr)	1 (Arg, Asp, Cys, Gln, Glu, Gly, Lys, Pro)	-2	3 (Tyr)	-4 (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 Phenylalanine to Serine 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 concluded that this mutation will probably cause protein structure changes as well as functional changes.

PSSM
value amino acid	most frequent substitution	rarest substitution
-5	11	-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 on this position. The regarded mutation is presented by the first colored column (Figure 4). Here we can see, that all the other mammalians have the amino acid Phenylalanine at this position. 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 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. Furthermore, HEXA_HUMAN does not posses any disordered regions and therefore, a mutation in a coiled region do not change a functional important region. 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.

JPred:
...EEEECCCCCEEEEEECCCCCCCHHHHHHHHHHHHHHCCCEEEEEEECCCCCCCCC...
PsiPred:
...EEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHCCCCEEEEEECCCCCCCEEC...

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 (Figure 5 and Figure 6). The visualization can display if the mutation is in a secondary structure element or in some other regions.

Here in this case the mutation position almost agree with the position of the predicted secondary structure and is within a coil. Like explained above this means a mutation will probably not destroy a secondary structure element which affects no drastic structural change. Otherwise it can cause a change at the position of the two nearest secondary structure element which can has a functional loose as a consequence. We think that a structural change is unlikely, because it is not within a secondary structure element and will therefore not cause extreme changes.

Figure 5: Mutation at position 211

Figure 6: Mutation at position 211 - 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 Serine which is the real mutation in this case. SNAP has as a result that the exchange from Phenylalanine to Serine at this position is non-neutral with a high accuracy. This means that this certain mutation at this position cause very likely structural and functional changes of the protein.

Substitution	Prediction	Reliability Index	Expected Accuracy
S	Non-neutral	5	87%

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 of 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 Phenylalanine itself (Figure 8). The substitution to Serine is not-tolerated at this position. 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 w v t s r q p n m l k i h g e d c a 211F 1.00 F

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PolyPhen2 Prediction

Finally, we also regarded the PolyPhen2 prediction for this mutation. This prediction visualizes how 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 (Figure 9 and Figure 10), which agreed in the most cases.

In this case both models predict that the mutation is probably damaging. 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 in Figure 11, to see if the mutation is near of far away from the functional residues. As you can see on the picture, the mutation is located within a loop and far away from the functional residues. Therefore, we do not know in which way this mutation affects the global structure of the protein.

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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 Phenylalanine	Figure 13: Amino acid Serine	Figure 14: Picture which visualize the mutation

As you can see on Figure 12, the original amino acid (Phenylalanine) has a ring structure and is therefore, an aromatic amino acid. The mutated amino acid, visualized in Figure 13, is a Serine, which is smaller than Phenylalanine. Therefore there is no problem with the space for the amino acid. Serine is smaller than Phenylalanine and has enough space which means there should not be any clashes with other amino acid residues or with the backbone (Figure 14).

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FoldX Energy Comparison

One important point in the analysis of mutations is to look at the energy of the protein with the original and with the mutated amino acid. Often the energy increases dramatically with the mutated amino acid. This means, that the protein becomes very unstable and therefore, it is often possible that the protein can not bind its ligands any longer. Otherwise, it is also possible, that the protein with the mutated amino acid has a lower energy than the original protein. This means, that the protein is too rigid and loses its flexibility. Than it is also possible, that the protein can not bind the ligands any longer.

Therefore, we compared the energy of our protein with different methods. Here we want to present the result of FoldX.

Original total energy	Total energy for the mutated protein	Strongest energy changes within the mutated protein
-154.17	-144.25	-

In this case, the energy of the mutated protein is higher, than the energy of the original protein. Therefore, this means, that the mutated protein is not that stable than the original protein. So it is possible, that the mutated protein loses its function, because it is too unstable to bind the ligand.

We also will compare the energy values of these two structure with other methods. Because of the different calculation methods, it is not possible to compare the energy values directly. Therefore we decided to calculate the ratio between the energy values of the two structures. Our original mutation has the value 100, with this value we calculate the value of the mutated structure.

Ratio Original	Ratio mutated protein	Difference
100	93.66	6.34

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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	-9594.637506

The total energy of the mutated structure is a little bit higher than the energy of the original protein structure. 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 protein	Ratio of the mutated protein	Difference
100	99.84	0.16

Therefore, we can see, that the mutated structure has only 0.16% less energy than the original structure.

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 Phenylalanine	Figure 16: Amino acid Serine	Figure 17: Picture which visualize the mutation

If we have a look at the pictures (Figure 15, Figure 16), we can see that the location of the residue of the two amino acids is very similar (Figure 17).

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 of the mutated amino acid	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

On the pictures you can see, that neither the original amino acid (Figure 18) nor the mutated amino acid (Figure 19) has any H-Bonds with other residues or the backbone of the protein. Therefore, it is not possible to explain the damage of the protein by missing H-bonds. Furthermore, we can see, that there are no clashes between Serine and the rest of the protein (Figure 20), which means that the protein do not have to fold in another way because of clashing residues.

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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	1166.24	390	3425.52	-2317.42
Angle	3275.2	50	185.491	324.728
Potential	-46177.4	3600	49139.5	-22414.8

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 original amino acid	Ratio mutated amino acid	Difference
100	75.32	24.68

The difference between the energies calculated by Gromacs is much higher than the difference of the energy values calculated by the other tools. But otherwise, Gromacs use a real physical force field and therefore, it should be the most accurate method to analyse the energy of different structures. In this case the energy of the mutated structure is much higher than the energy of the original structure. Therefore it is possible, that the protein become unstable with the mutation and therefore does not work any more.

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 Phenylalanine	Figure 22: Amino acid Serine	Figure 23: Picture which visualize the mutation

These pictures (Figure 21, Figure 22, Figure 23) is very similar to the pictures created by SCWRL (Figure Figure 15, Figure 16, Figure 17). The mutated amino acid is much more smaller than the Phenylalanine and therefore does not need that much space. Otherwise it is possible, that because of the smaller amino acid, there are missing H-Bonds in the protein.

Visualization of H-bonds and Clashes:

To check if this is the case, we analysed the H-Bonds and clashes between the mutated amino acid and the rest of the protein.

H-Bonds of the original amino acid	H-bonds of the mutated amino acid	Clashes of the mutation
Figure 24: H-bonds of the original amino acid	Figure 25: H-bonds of the mutated amino acid	Figure 26: Possible clashes

Both amino acids do not have any H-Bonds with the rest of the protein (Figure 24, Figure 25). Therefore a missing H-Bond does not cause the damage on the protein. Furthermore, it is not possible to find any clashes (Figure 26) in between the protein.

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