Rs1800430
Contents
General Information
| SNP-id | rs1800430 |
| Codon | 399 |
| Mutation Codon | Asn -> Asp |
| Mutation Triplet | AAC -> GAC |
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Sequence-based Mutation Analysis
Pysicochemical Properities
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.
| Asn | Asp | consequences |
| polar, small, hydrophilic, negatively charged | polar, small, hydrophilic, negatively charged | Both amino acids have the same properities and therefore we suggest that an exchange of these two amino acids do not destroy the protein structure and function |
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Visualisation of the Mutation
In the next step, we created the visualization of the muation with PyMol. Therefore we created a picture for the original amino acid, for the new mutated amino acid and finally for both together in one picture whereas the mutation is white colored. The following pictures display that the mutated amino acid Aspartic acid looks very different to Asparagine. They both agree in the first part of the rest but then go complety in opposite directions. Otherwise both rests fork at the end. All in all, the different directions prevail and will therefore probably cause big structural changes which will cause effects on the protein function.
| picture original aa | picture mutated aa | combined picture |
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Subsitution 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 accoding 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 Asparagine to Aspartic acid has very high values that agree with the most frequent value for all three substitution matrices. Therefore, according to all matrices a mutation at this position will probably not cause structural changes which can affect functional changes.
| PAM 1 | Pam 250 | BLOSOUM 62 | ||||||
| value aa | most frequent substitution | rarest substitution | value aa | most frequent substitution | rarest substitution | value aa | most frequent substitution | rarest substitution |
| 36 | 36 (Asp) | 0 (Cys, Met) | 7 | 7 (Asp) | 2 (Cys, Leu, Phe, Trp) | 1 | 1 (Asp, His, Ser) | -4 (Trp) |
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PSSM Analysis
Besides, we looked additional at the position specific scoring matrix (PSSM) for ouer 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 Asparagine to Aspartic acid at this position is in the middle between the value for the rarest substitution and the value for the most frequent substitution. Therefore, the value from PSSM is not realy significant and we are not able to determine effects on the protein.
| PSSM | ||
| value aa | most frequent substitution | rarest substitution |
| 0 | 2 | -2 |
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. Here we can see, that almost all other mammalians have another amino acid or a gap on this position. Therefore, the mutation on this position is not highly conserved and a mutation there will cause probably no huge structural and functional changes for the protein.
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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 drastical the mutation can be. In this case the tools disagree. JPred predict at the position of the mutation a coil and PsiPred predicted there a alpha-helix. For JPred a 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 drastical change of the protein structure and its function is unlikly for the JPred prediction. For PsiPred a mutation at the beginning of the alpha helix could inhibit the formation if this alpha helix. Therefore, we think that a this mutation would probably cause a extreme change in the protein structure and function.
JPred: ...CCCCCCCEEEEEECCCCCCCCHHHHHHHHHCCCCEEECCCCCCCCCCCCCCCCCC... PsiPred: ...CCCCCCCCEEEEECCCCCCCHHHHHHHHHHCCCEEEECCCCCCCCCCCCCCCCCC...
Comparison with the real Structure:
Afterwards we also visualize the position of the muation (red) in the real 3D-structure of PDB and compare it with the predicted secondary structure. The visualisation 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 mutationposition disagree with the position of the predicted secondary structure from JPred, but agrees with the position from PsiPred. The visualization displays the mutation at the beginning of an alpha-helices. This means a mutation will probably cause structural changes combined with functional changes of the protein, because it can prevent the formation of this alpha-helix.
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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 Aspartic acid which is the real mutation in this case. SNAP has a result that the exhange from Asparagine to Aspartic acid at this position is neutral with an average accuracy of 60%. This means that this certain mutation on this position cause likely no structural and functional changes of the protein.
| Substitution | Prediction | Reliability Index | Expected Accuracy |
| D | Neutral | 1 | 60% |
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 mutationposition 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, there are five substitutions that are not tolerated: Tryptophan, Tyrosine, Phenylalanine, Cysteine and Methionine. The substitution to Aspartic acid is tolerated at this position. This means that this mutation at this position is probably neutral and will not cause any 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.
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SIFT Table
Threshold for intolerance is 0.05.
Amino acid color code: nonpolar, uncharged polar, basic, acidic.
Capital letters indicate amino acids appearing in the alignment, lower case letters result from prediction.
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PolyPhen2 Prediction
Finally, we also regarded the PolyPhen2 prediction for this muation. This prediction visualizes have strongly demaging the mutation probably will be. Therefore it gives the result for two possible cases: HumDiv and HumVar. HumDiv is a prefered model for evaluation rare allels, dense mapping of regions identified by genome-wide assiociation studies and analysis of neutral selection. In contrast, HumVar is a prefered model for diagnostic of Mendelian diseases which require distinguishing mutations with drastic effects from all remaining human variations including abundant mildly deleterious allels. 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 benign. This means that the mutation is neutral and will probably not damage the structure and the function of the protein.
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Structure-based Mutation Analysis
Mapping onto Crystal Structure
Color declaration:
- red: position of mutation
- green: position of active side
- yellow: position of glycolysation
- cyan: position of Cystein
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 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 aa | picture mutated aa | combined picture |
The pictures show, that the structure of the different amino acids is very different. Although these two amino acids have very similar pysicochemical properties, they differ in their structure. The orientation of the residues is different and therefore it is possible, that these changes lead to missing or additional H-Bonds.
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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 | -155.11 | - |
The energy value of the mutated structure is lower than the energy value of the orignal structure. Therefore this means, that the structure is more rigid than the original structure. 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 | 100.72 | -0.72 |
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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 | -9505.864181 |
In this case the engery value of the mutated structure is a little bit higher than the engery value of the original structure. This means that the mutated structure is not that rigid as the original one. 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 | 98.91 | 1.09 |
With the ratio the difference between the energy of the original and mutated structure is about 1%.
Comparing Structure:
This tool also gives as output a pdb file with the position of the original and the mutated amino acid.
| picture original aa | picture mutated aa | combined picture |
On the pictures we can see, that the orientation of the residues is different. The result is similar to them of SCWRL.
Visualization of H-bonds and Clashs:
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 structure | H-bonds near the mutation | Clashes of the mutation |
In both cases there are no H-bonds with other residues of the protein or with the backbone. Therefore, if this mutation lead to a loss of function of the protein it can not explained by missing or additional H-bonds.
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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 | 938.527 | 210 | 2287.3 | -1208.35 |
| Angle | 3201.09 | 59 | 240.737 | 389.204 |
| Potential | -48693.9 | 870 | 3377.76 | -5762.65 |
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 | 79.43 | 20.57 |
The result of Gromacs is consistent with the result of minimise. In this case, the original structure has a lower energy value than the mutated structure, which means that the mutated structure is more stable than the mutated structure.
Comparing Structure:
Gromacs also offers pictures of the mutated amino acids which can be seen in the following section.
| picture original aa | picture mutated aa | combined picture |
The two pictures are similar to the result of SCWRL and minimise.
Visualization of H-bonds and Clashs:
| H-bonds of the original structure | H-bonds near the mutation | Clashes of the mutation |
Neither the original nor the mutated structure have any H-bonds with the rest of the protein. The mutation also do not cause any clashes with the rest of the protein.
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