Difference between revisions of "Gaucher Disease: Task 09 - Structure-based mutation analysis"
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The following figure visualizes the five residues we are going to mutate on the reference structure, 2V3E, chain B. |
The following figure visualizes the five residues we are going to mutate on the reference structure, 2V3E, chain B. |
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+ | <figure id="pymol_mutations" > |
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− | <gallery widths=550px heights=300px caption="PyMol visualization of five residues of the structure 2V3E, chain B, we are going to mutate as described in Table 2." perrow="2"> |
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+ | <gallery widths=550px heights=300px caption="" perrow="2"> |
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File:2V3E_B_mutations_ligands_effect_colored_mesh_spheres-labeled.png|The five residues to be mutated mapped onto the structure 2V3E, chain B. Those residues are represented as green (not disease causing S77R and N141S) and red (disease causing G241E, N409S and L483P) spheres. The ligands bound to the structure at three binding sites during its resolution (BMA, FUC, NAG and NND) are represented as spheres in different shades of orange. Neither of the five residues lies in the proximity of one of the binding sites. |
File:2V3E_B_mutations_ligands_effect_colored_mesh_spheres-labeled.png|The five residues to be mutated mapped onto the structure 2V3E, chain B. Those residues are represented as green (not disease causing S77R and N141S) and red (disease causing G241E, N409S and L483P) spheres. The ligands bound to the structure at three binding sites during its resolution (BMA, FUC, NAG and NND) are represented as spheres in different shades of orange. Neither of the five residues lies in the proximity of one of the binding sites. |
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File:2V3E_B_mutations_ligands_effect_colored_mesh_spheres-other_side-labeled.png| Same as the first figure, only view from the other side of the protein. |
File:2V3E_B_mutations_ligands_effect_colored_mesh_spheres-other_side-labeled.png| Same as the first figure, only view from the other side of the protein. |
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</gallery> |
</gallery> |
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+ | <small>'''<caption>''' PyMol visualization of five residues of the structure 2V3E, chain B, we are going to mutate as described in Table 2.</caption></small> |
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+ | </figure> |
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As can be seen on the image, none of the residues to be mutated lies in the proximity of one of the three binding sites. (See also the second subfigures of the figure galleries in the next subsection for close up on the residues and their hydrogen bonds.) However, four of the residues lie within a secondary structure element (beta sheet or helix) and one - Glycine 241 - in a turn |
As can be seen on the image, none of the residues to be mutated lies in the proximity of one of the three binding sites. (See also the second subfigures of the figure galleries in the next subsection for close up on the residues and their hydrogen bonds.) However, four of the residues lie within a secondary structure element (beta sheet or helix) and one - Glycine 241 - in a turn |
Revision as of 22:41, 4 September 2013
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Contents
Preparation
1. Choice of a structure to work with
We chose the structure 2V3E, chain B, which has the following properties:
<figtable id="2V3E">
PDB-ID | Resolution (Å) | Chain | Covered residues (UniProt seq.) | Missing residues (ATOM seq.) | Covered residues (ATOM seq.) | R-Value(obs.) | R-Free | pH | Temperature (K) |
---|---|---|---|---|---|---|---|---|---|
2V3E | 2.0 | A/B | 40-536 (92.7%) | A: 31, (498-503), B: (-1), (498-503) | A: -1-30, 32-497, B: 0-497 | 0.163 | 0.220 | 7.5 | 100 |
</figtable>
For more information about other candidates and the missing residues, see the lab journal.
2. Visualization of the mutations to work with
We selected the following five mutation from the mutations selected in for this task:
<figtable id="mutations">
Reference | Codon change | Codon Number (UniProt) | Codon Number (PDB) | Amino Acid change | Polarity | Charge (pH) | Disease causing? |
---|---|---|---|---|---|---|---|
rs368786234 | AGC ⇒ AGA | 77 | 38 | Ser ⇒ Arg (S77R) | polar ⇒ polar | neutral ⇒ positive | FALSE |
rs374003673 | AAT ⇒ AGT | 141 | 102 | Asn ⇒ Ser (N141S) | polar ⇒ polar | neutral ⇒ neutral | FALSE |
CM992894 | GGA ⇒ GAA | 241 | 202 | Gly ⇒ Glu (G241E) | nonpolar ⇒ polar | neutral ⇒ negative | TRUE |
CM880036 | AAC ⇒ AGC | 409 | 370 | Asn ⇒ Ser (N409S) | polar ⇒ polar | neutral ⇒ neutral | TRUE |
CM870010 | CTG ⇒ CCG | 483 | 444 | Leu ⇒ Pro (L483P) | nonpolar ⇒ nonpolar | neutral ⇒ neutral | TRUE |
</figtable>
The following figure visualizes the five residues we are going to mutate on the reference structure, 2V3E, chain B.
<figure id="pymol_mutations" >
The five residues to be mutated mapped onto the structure 2V3E, chain B. Those residues are represented as green (not disease causing S77R and N141S) and red (disease causing G241E, N409S and L483P) spheres. The ligands bound to the structure at three binding sites during its resolution (BMA, FUC, NAG and NND) are represented as spheres in different shades of orange. Neither of the five residues lies in the proximity of one of the binding sites.
PyMol visualization of five residues of the structure 2V3E, chain B, we are going to mutate as described in Table 2. </figure>
As can be seen on the image, none of the residues to be mutated lies in the proximity of one of the three binding sites. (See also the second subfigures of the figure galleries in the next subsection for close up on the residues and their hydrogen bonds.) However, four of the residues lie within a secondary structure element (beta sheet or helix) and one - Glycine 241 - in a turn near a helix. This implies that exchange of these residues with others with different functional groups, polarity and charge could lead to destruction of some hydrogen bonds within or between the secondary structures (e.g. Asparagine 141). This might lead to structural changes and even to destruction of the secondary structures or important blocks of secondary structure elements. Moreover, an exchange with a side chain of a bigger size might lead to clashed with proximate residues (e.g. with the loop near the Serine 77).
3. Creation of mutated structures
We used SQWRL4 to create the five mutated structures. (See lab journal.) The mutated residues in comparison to the native residues, the hydrogen of the mutants and possible clashes are shown in the following figures.
S77R
Serine 77 (PDB 38) is colored magenta, it's chain is in the stick representation and all the backbone residues are in the cartoon representation (the same for the following subfigures). Serine 77 lies in the middle of a beta strand flanked by two other strands in an anti-parallel order. It forms two hydrogen bonds with a residue in a neighboring strand.
According to this images, we would also predict the mutation S77R as not disease causing.
N141S
Asparagine 141 (PDB 102)is located in a helix and forms four hydrogen contacts: two with the main-chain carboxyl- and amino-groups with two helix amino acids (one in each direction) and two with the side chain. One of the contacts of the Asparagine side chain also binds to the same residue in the helix as the Asparagine amino-group binds. The second hydrogen bond of the Asparagine side chain is formed with the side chain of a residue in the neighboring helix. Maybe this bond plays a stabilizing role between the two helices.
Hydrogen bonds formed by the mutant Serine 141. A contact that was formed between the native Asparagine side chain and a side chain pointing from a neighboring helix (showed in sticks) is missing, as well as a second contact with a residue in the same helix. Instead, the mutated Serine side chain forms a contact with another residue in the same helix.
The mutation N141S is benign, therefore the missing stabilizing contact between the two helices is probably not so important.
G241E
As no changes in the hydrogen bonds or clashes etc. can be noticed, we would predict the mutation G241E as benign. However, it is a disease causing mutation. Maybe the effect is caused by the polarity and negative charge of the mutant Glutamate, instead of the nonpolar and neutral Glycine.
N409S
Asparagine 409 (PDB 370) is located in a helix. It builds five hydrogen bonds: four with the main chain and one with the side chain. Two of the main chain contacts are formed by the carboxyl-group: one with a helix residue and one with a residue located in a proximate beta-strand beginning, the teo binding partners are separated by a short turn. Two other main chain hydrogen bonds are built by the amino-group and two carboxyl-groups of an amino acid in the helix (from the other side). The fifth bond is formed between the side chain amino-group and one of the caroboxy-groups of the last mentioned helix residue.
We would predict the mutation N409S as non-effect. Due to the fact that all hydrogen bonds remain conserved in the mutant and that no clashes etc. could be detected, we would predict the mutation G241E as benign. Moreover, the both amino acids, Asparagine and Serine, are both polar and neutral. Nevertheless, if is disease causing.
L483P
Comparison of wild type (wt) Leucine 483 (magenta) and mutant Proline (orange). The mutant Proline is a circle amino acid, which has very different properties than the hydrophobic Leucine. Is leads to a shift in the main chain conformation "backwards", so that the beginning of the neighboring strand is shifted.
Leucine 483 (PDB 444) lies at the very beginning of a beta strand. There is another beta strand in the proximity on the right, which is followed by three others, forming a beta sheet. However, the Leucine does not form any contact with the neighboring strand, as its side chain is hydrophobic and point in another direction. The Leucine forms an interesting hydrogen bond with its amino-group to the side chain of a residue located in the neighboring turn. After this loop another beta-strand follows.
We would predict the mutation L483P as having an effect - and so it is.
Energy comparisons
foldX
We applied foldX for the five mutations to predict new structures with energies.
<figtable id="scwrl_foldx">
SCWRL | FoldX | |||||||
---|---|---|---|---|---|---|---|---|
Mutant | Energy | Mutant-WT | Predicted effect | Energy Mutant | Energy WT | Mutant-WT | Predicted effect | Observed effect |
WT | 386.356 | |||||||
S77R | 386.706 | 0.350 | FALSE | 20.90 | 23.25 | -2.35 | FALSE | FALSE |
N141S | 390.966 | 4.610 | TRUE | 28.81 | 28.84 | -0.03 | FALSE | FALSE |
G241E | 394.319 | 7.963 | TRUE | 32.30 | 29.31 | 2.99 | TRUE | TRUE |
N409S | 391.73 | 5.374 | TRUE | 29.08 | 27.77 | 1.31 | TRUE | TRUE |
L483P | 424.715 | 38.359 | TRUE | 38.40 | 35.55 | 2.85 | TRUE | TRUE |
</figtable>
We decided to regard all differences between the mutant and WT energies higher than 1 as significant. SCWRL identifies the three disease causing mutations as such, however also one not disease causing mutation (N141S) is predicted falsely as effect causing, but with the smallest positive difference (higher 1) of 4.61. Interestingly the most prevalent Gaucher disease mutation, L483P, has a highest energy change of 38.359. FoldX makes a correct prediction in all cases, the two benign mutations even have a small negative energy change, however the disease causing mutations have all only small energy increase (1.31 - 2.99). The mutation L483P does not have the highest energy increase.
Next, we superimposed the SCWRL and foldX structures in Pymol and compared them in the following figures.
Mutation G241E. In foldX the conformation of the Glutamate side chain is a little bit different, but it has no meaning, as it lies on the outside of the protein. Also the binding partner side chain has a little bit different angle, so that two hydrogen bonds, instead of of only one in SCWRL, are formed.
Minimise
We applied minimise for all mutant structures produced by SCWRL and foldX and the WT structure. We used the output of one minimisation for another run as input 5 times for each structure. The <xr id="minimise"/> summarizes the resulting energies:
<figtable id="minimise">
Method | Mutation | Round 1 | Round 2 | Round 3 | Round 4 | Round 5 |
---|---|---|---|---|---|---|
- | WT | -12360.0 | -11661.9 | -12079.7 | -11636.9 | -11630.6 |
SCWRL | S77R | -3482.3 | -12111.1 | -11724.2 | -11509.3 | -11260.2 |
N141S | -3593.4 | -12199.7 | -11787.7 | -11568.1 | -11328.3 | |
G241E | -3548.4 | -12163.9 | -11792.4 | -11593.1 | -11381.2 | |
N409S | -3631.0 | -12214.2 | -11801.9 | -11594.4 | -11347.0 | |
L483P | -3469.4 | -11900.1 | -11610.9 | -11665.8 | -11330.6 | |
foldX | S77R | -12297.0 | -11892.1 | -12037.2 | -11707.4 | -11703.1 |
N141S | -12359.7 | -11961.2 | -11983.4 | -11550.9 | -11515.9 | |
G241E | -12356.6 | -11944.8 | -12103.6 | -11782.5 | -11764.0 | |
N409S | -12389.9 | -11966.0 | -12117.7 | -11845.4 | -11808.9 | |
L483P | -12335.4 | -11925.2 | -12096.9 | -11771.4 | -11771.3 |
</figtable>
For the WT, the lowest energy is reached after the first iteration of minimise. This is because the structure is already correct and therefore optimal. Interestingly, the lowest energy of the SQWRL mutants is reached after two minimise iterations, whereas for the foldX mutants the minimal energy is reached already after the first iteration. This may be explained by the fact that foldX already performs a minimisation step. Moreover, foldX minimal energies are always lower, than SCWRL minimal energies.
Another interesting observation is that after reaching the lowest energy in the second minimise iterations in the WT and the foldX mutants, the energy rises constantly in the consequent runs. This probably means that the minimal local energy is reached in the second iteration and then the computation of the gradient at the minimal point can only lead to a higher energy value, maybe to another local minimum with a higher energy. A different pattern happens during the minimisation of the SCWRL mutants: after reaching the local minimal energy already after the first iteration, in the second iteration the energy rises, but in the third iteration it falls a little again, but not into the same local minimum as in the first iteration, indicating that a second "suboptimal" local minimum is found. After the forth iteration the energy rises again.
We compared the mutant structures in Pymol again after the minimise runs reaching the best energies: after the second round for SQWRL and the first round for foldX (the following figure).
To conclude, SQWRL and foldX are both pretty good tools for calculating the energy difference and the structural change of a protein after a point mutation. There is almost no difference between the resulting structures, in particular the conformations of the mutated residues and their environment, between the two programs.