Canavan Disease: Task 09 - Structure-based Mutation Analysis
Structure-based mutation analysis looks at the effects of mutations on the protein structure. This is done via the calculation of energetic changes.
Contents
Preparation
Several PDB-structures are available for aspartoacylase. To work on structure based mutation analysis, the one to choose should have some constraints: The structure should have a high resolution with a small R-factor. The pH value should be near the physiological pH and there should be no missing gaps.
There was no structure without any missing gaps, but the missing gaps were only at the beginning and end of the structure. Only one PDB-structure contained further information such as pH value. Therefore 2O4H was chosen:
It is the human brain aspartoacylase complex with intermediate analog (N- 2 phosphonomethyl-L-aspartate), with a resolution range of 2.7 Angstroms. It is one single crystal analyzed with X-ray diffraction in October 2006 using a temperature of 100 Kelvin and a pH of 6.0. Its free R value is 0.271. The missing residues range from positions 1-9 in the beginning and 311-313 in the end of the protein.
Visualization
Some mutations from the previous Task were chosen to work with. Those are displayed in <xr id="visu"></xr>:
<figtable id="visu">
Visualization of Mutations of Interest | ||||||
---|---|---|---|---|---|---|
Mutation | Description | Visualization | Sec. Struc. | Information | ||
Asn121Asp | From the previous Task it is known that Asn->Ile is disease causing. Here a detailed look on that specific mutation is of interest. | LOOP | possibly disease causing | |||
His21Pro | This mutation is definitively disease causing, since it is important for binding the zinc atom. The position is part of the active center and therefore of special interest. | LOOP | metal binding, disease causing | |||
Pro149Ala | Since Proline is known to be a HELIX breaker and this mutation is very near to a HELIX the Proline might be necessary for the structure. | LOOP near HELIX |
- | |||
Pro257Arg | According to Task 8 the mutation is expected to have no effect on the protein. | LOOP | - | |||
Thr166Ile | This position is known to be part of the binding region. It is listed to be disease causing and is exactly the changing point between LOOP and HELIX. | LOOP near HELIX |
in binding region, disease causing |
Information about functional residues were taken from Uniprot.
</figtable>
Comparisons
Comparing wild type to mutant structures no potential clashes were found in neither of the mutations. No holes were introduced to the structure. The mutations should not have a high effect on changing hydrophilicity in the core, nor hydrophobicity on the surface. Only Thr->Ile, which is part of the core region changes extremely from a more hydrophilic to a more hydrophobic amino acid. A visual comparison can be found in <xr id="engy"></xr>.
<figtable id="engy">
Comparisons on a Visual Basis | |||
---|---|---|---|
Mutation | H-bonds | SCWRL and foldX | |
Asn121Asp | |||
His21Pro | |||
Pro149Ala | |||
Pro257Arg | |||
Thr166Ile |
Comparing SCWRL (red) and foldX (cyan) is another approach
</figtable>
As it can be seen in <xr id="engy"></xr>, mutating the sidechains leads in some approaches (mutate via Pymol (blue) and create a new structure with SCWRL (red) or foldX (cyan)) to a loss of one potential hydrogen bond. However, as the residue is not located within a secondary structure element that relies on stabilization via H-bonds, the impact is not expected to be extreme. Unfortunately the approaches do not consider whether the site is a functional residue or not.
The observable differences of the sidechain conformation between Pymol, SCWRL and foldX is redundant as only one state of the sidechain dynamics is displayed at a time. The natural movement of the sites is not taken into account (compare Asn->Asp).
Energy Comparison
<figtable id="energy_comp">
Energy Comparison | ||||||
---|---|---|---|---|---|---|
Mutation | SCWRL | foldX | ||||
MT | Corr. | MT | Corr. | |||
Wild type | 400.00 | 144.46 | ||||
Asn121Asp | 403.94 | 1.01 | 145.46 | 1.01 | ||
His21Pro | 395.75 | 0.99 | 145.85 | 1.01 | ||
Pro129Ala | 401.93 | 1.00 | 148.04 | 1.02 | ||
Pro257Arg | 414.53 | 1.04 | 149.63 | 1.04 | ||
Thr166Ile | 142.25 | 1.08 | 147.35 | 1.02 |
Therefore any wider derivation from 1.0 is predicted to have an effect on the protein. Thus, only Thr->Ile would represent an effect in the SCWRL approach.
</figtable>
<xr id="energy_comp"></xr> shows that only the mutation from Threonine to Isoleucine at position 166 seems to have an effect due to the higher value presented in SCWRL. foldX would not predict any effects. The effect on the protein function that is introduced in ASPA by changing Histindine to Proline at position 21 can not be captured by the energy calculations as it does not take into account that with the missing Histidine the metal binding site is corrupt. This holds true for Threonine to Isoleucine, which is part of a substrate binding region, what is maybe the reason why it is not detected by foldX.
As it is the only mutation for which it is uncertain if it is disease causing or not, the change from Asparagine to Aspartic acid at position 121 could not be predicted as having an effect using the structure based mutation analysis as well.
The complete result list from foldX can be found in the Supplement at the end of this Task.
Minimization
To minimize the energy of the models, minimise was used. Unfortunately SCWRL was not able to produce any output. Therefore <xr id="minim"></xr> shows the different energies of foldX in five iterations whereas each iteration uses the output of the previous minimization as input. The detailed values can be found in the Supplement at the end of this Task.
<figure id="minim">
</figure>
As it can be seen in <xr id="minim"></xr> the second iteration shows the optimal energy result for the structure, with further iterations raising the level. Additionally when comparing all mutated structures, the known disease causing mutations (His21Pro and Thr166Ile) show the highest energy levels. Asn121Asp referring to the energy comparisons are predicted to have no effect. Looking at the minimization, the mutation is on the same level as His21Pro. Therefore it might be possibly disease causing. This is the same result as the assumption. A precise prediction if it is disease causing or not remains unclear.
Supplement
Tasks
- Link to Task 01: Canavan Disease
- Link to Task 02: Alignments
- Link to Task 03: Sequence-based Predictions
- Link to Task 04: Structural Alignments
- Link to Task 05: Homology Modelling
- Link to Task 06: Protein Structure Prediction from Evolutionary Sequence Variation
- Link to Task 07: Researching SNPs
- Link to Task 08: Sequence-based Mutation Analysis
- Link to Task 09: Structure-based Mutation Analysis
- Link to Task 10: Normal Mode Analysis