Difference between revisions of "Gaucher Disease: Task 09 - Structure-based mutation analysis"

From Bioinformatikpedia
(2. Visualization of the mutations to work with)
(S77R)
Line 86: Line 86:
   
 
====S77R====
 
====S77R====
  +
<figure id="graph_ras" >
<gallery widths=300px heights=200px caption="PyMol visualization of the mutation '''S77R''' (PDB 38) of the structure 2V3E, chain B, with SCWRL." perrow="3">
 
  +
<gallery widths=300px heights=200px perrow="3">
 
File:2V3E_B_mutation_S38R_sticks.png| Comparison of wild type (wt) Serine 77 (magenta) and mutant Argenine (orange). The mutated residue has a longer side chain.
 
File:2V3E_B_mutation_S38R_sticks.png| Comparison of wild type (wt) Serine 77 (magenta) and mutant Argenine (orange). The mutated residue has a longer side chain.
 
File:2V3E_B_S38_hb.png| 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.
 
File:2V3E_B_S38_hb.png| 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.
Line 92: Line 93:
 
File:2V3E_B_mutant_38R_hb_spheres_no_loop_clash.png| Spheres representation of the mutant Argenine 77 (orange) shows, that it does not clash with the neighboring loop (gray spheres).
 
File:2V3E_B_mutant_38R_hb_spheres_no_loop_clash.png| Spheres representation of the mutant Argenine 77 (orange) shows, that it does not clash with the neighboring loop (gray spheres).
 
</gallery>
 
</gallery>
  +
<small>'''<caption>''' PyMol visualization of the mutation '''S77R''' (PDB 38) of the structure 2V3E, chain B, with SCWRL.</caption></small>
  +
</figure>
  +
 
According to this images, we would also predict the mutation S77R as not disease causing.
 
According to this images, we would also predict the mutation S77R as not disease causing.
   

Revision as of 22:45, 4 September 2013

<css>

table.colBasic2 { margin-left: auto; margin-right: auto; border: 1px solid black; border-collapse:collapse; }

.colBasic2 th,td { padding: 3px; border: 1px solid black; }

.colBasic2 td { text-align:left; }

/* for orange try #ff7f00 and #ffaa56 for blue try #005fbf and #aad4ff

maria's style blue: #adceff grey: #efefef

  • /

.colBasic2 tr th { background-color:#efefef; color: black;} .colBasic2 tr:first-child th { background-color:#adceff; color:black;}

</css>

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
2V3E, chain B, the chosen reference structure of GBA sequence P04062.

</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
Selected mutations of GBA sequence P04062. Mapping of the UniProt positions onto the PDB ATOM sequence is given.

</figtable>

The following figure visualizes the five residues we are going to mutate on the reference structure, 2V3E, chain B.

<figure id="pymol_mutations" >

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

<figure id="graph_ras" >

PyMol visualization of the mutation S77R (PDB 38) of the structure 2V3E, chain B, with SCWRL. </figure>

According to this images, we would also predict the mutation S77R as not disease causing.

N141S

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

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

We would predict the mutation L483P as having an effect - and so it is.

Energy comparisons

Lab journal

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
Energies of the WT structure and the five mutant structures calculated by SQWRL and foldX. FoldX minimizes the energy of the WT structure for each mutation. The energy difference between each mutant and the respective WT structure was calculated. If it is (significantly) positive, the mutation is regarded as effect causing. The observed effect of the mutation is given for comparison.

</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.

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
Energies produced by each of five consecutive runs of the program minimised, applied on the WT structure and the five mutant structures calculated by SQWRL and foldX. The lowest energies for each structure are marked gold.

</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.