Rs61731240

General Information

SNP-id	rs61731240
Codon	179
Mutation Codon	His -> Asp
Mutation Triplet	CAT -> GAT

Back to [Sequence-based mutation analysis]

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 concluded the possible effect that the mutation could have on the protein.

His	Asp	consequences
aromatic, positive charged, polar, hydrophilic	negative charged, small, polar, hydrophilic	On the one side, both amino acids are polar, but on the other side, His is positively charged, while Asp is negatively charged, which is an essential difference between these both amino acids. Therefore, it is very likely, that this change causes big changes in the structure of the protein and the protein therefore will probably not work any longer. Furthermore, the structure of the two amino acids is very different, because of the aromatic ring of His.

Back to [Sequence-based mutation analysis]

---

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 Aspartate looks very different to Histidine. Histidine has an aromatic ring. Contrary, Aspartate is smaller and forks at the end of the rest. Furthermore, it is also orientated in a completely different direction. This shows that the amino acids have huge structural differences which will probably cause dramatical effects on protein structure and function.

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

Back to [Sequence-based mutation analysis]

---

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 value.

In this case, the substitution of Histidine to Aspartic acid has very low values that are nearer to the values for the rarest substitution for PAM1 and PAM250. Contrary, for BLOSOUM62 the value for the amino acid substitution Histidine to Aspartic acid is average. This means the most frequent substitution value is almost as far away as the rarest substitution. The difference between the two PAMs can be ascribed to the different preparations of these two kind of substitution matrices. The difference between the two PAMs and BLOSUM62 can be ascribed to the different preparations of these two kinds of substitution matrices. The PAM-matrices are evolutionary models whereas BLOSUM is based on protein families. Therefore probably this mutation is evolutionary not that unlikely whereas within a protein family it is more unusual. Therefore, according to PAM1 and PAM250 a mutation at this position will almost certainly cause structural changes which can affect functional changes. The value from BLOSSUM62 is not really significant and therefore we are not able to determine effects on 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
3	20 (Gln)	0 (Ile, Met)	4	7 (Gln)	2 (Ala, Cys, Gly, Ile, Leu, Met, Phe, Thr, Trp, Val)	-1	2 (Tyr)	-3 (Cys, Ile, Leu, Val)

Back to [Sequence-based mutation analysis]

---

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 Histidine to Aspartic acid at this position is very low and near the value for the rarest substitution. This means that this substitution at this position is likely very uncommon which indicates that this substitution has bad effects as 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
-3	9	-5

Back to [Sequence-based mutation analysis]

---

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, which can be seen in figure 4. The regarded mutation is presented by the colored column. Here we can see, that all the other mammalians have the amino acid Histidine at this position. Therefore, the mutation at this position is highly conserved and a mutation will cause probably huge structural and functional changes in the protein.

Figure 4: Mutation in the multiple alignment

Back to [Sequence-based mutation analysis]

---

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 and the protein does not possess any disordered regions. The change of the coil will probably only take places between two secondary structure elements which will 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. The visualization can display if the mutation is in a secondary structure element or in some other regions, which can be seen in Figure 5 and Figure 6.

Here in this case the mutation position agreed 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 loss 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 179

Figure 6: Mutation at position 179 - detailed view

Back to [Sequence-based mutation analysis]

---

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 Aspartic acid which is the real mutation in this case. SNAP has as a result that the exchange from Histidine to Aspartate acid at this position is non-neutral with a very 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
D	Non-neutral	6	93%

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

Back to [Sequence-based mutation analysis]

---

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 (compare Figure 8).

In this case, the only substitution that is tolerated is the one to Histidine itself. The substitution to Aspartic acid 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 m w f c i y v l t a s p e r n d k g Q 179H 0.99 H

Back to [Sequence-based mutation analysis]

---

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, which agreed 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

Back to [Sequence-based mutation analysis]

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 (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.

Back to [Structure-based mutation analysis]

---

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 at 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 Histidine	Figure 13: Amino acid Aspartate	Figure 14: Picture which visualize the mutation

As you can see in the picture above (Figure 12, Figure 13, Figure 14), the structure of the two amino acids are very different. Normally, there is a Histidine on the structure, which has a ring structure and needs a lot of space around it. Now the new amino acid (Aspartate) is very small. Therefore the binding in the protein can change because of the smaller amino acid.

Back to [Structure-based mutation analysis]

---

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	-151.61	-

In this case, the energy of the mutated structure has a higher energy, but the difference is not that much. Therefore the protein is a little bit more unstable, than the original protein, but it could be possible, that the protein works also with this mutated amino acid.

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	98.44	1.56

Back to [Structure-based mutation analysis]

---

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	-9480.968602

As before, in this case the mutated structure has a higher energy than the original structure. To have the possibility to compare the energy values of the different tools, we calculate again the ratio between these two values.

Ratio Original	Ratio mutated protein	Differences
100	98.65	1.35

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 Histidine	Figure 16: Amino acid Aspartate	Figure 17: Picture which visualize the mutation

If we compare these pictures (Figure 15, Figure 16, Figure 17) with the pictures created by SCWRL (Figure 13, Figure 14, Figure 15), it is possible to see a little difference in the position of the Asparagine (Figure 13 and Figure 16). But the difference is not very strong.

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 Asparagine residue.

H-bonds of the original amino acid	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 of the mutated amino acid

The mutated structure (here colored in red in Figure 19) has no H-Bonds with any other residues. This is the same behavior as we can see for the original amino acid on Figure 18, which also have no H-bonds with any other amino acids in the protein. Furthermore, we can see that the mutated amino acid has no clashes with other amino acids, which is visualized in Figure 20. Therefore, the protein has not to fold in another way, because of clashes.

Back to [Structure-based mutation analysis]

---

Gromacs Energy Comparison

The last tool we used for the energy analysis was Gromacs.

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	1126.89	410	-nan	-2542.54
Angle	3090.93	48	-nan	248.857
Potential	-48160.5	1200	-nan	-8000.95

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	Ratio mutated amino acid	difference
100	78.56	21.44

Comparing Structure:

Gromacs also offers the user some pictures with the mutated amino acid.

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

As we have already seen, the mutated amino acids is much more smaller than the original amino acid (Figure 21, Figure 22 and Figure 23). Therefore there is no problem that the mutated amino acid needs more space and therefore, the protein has to change in another way.

Visualization of H-bonds and Clashes:

To check if there are missing H-Bonds because of the smaller amino acid, we visualized the H-Bonds in both structures.

H-bonds of the original structure	H-bonds of the mutated structure	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

As we can see in Figure 24 above, there is no H-bond between the original amino acid and the rest of the protein. Furthermore, there is also no H-Bond between the mutated amino acid and the rest of the structure, which can be seen in Figure 25. Moreover, there are no clashes between the mutated amino acid residue and the rest of the protein (Figure 26).

Back to [Structure-based mutation analysis]

---