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

From Bioinformatikpedia
(G241E)
(2. Visualization of the mutations to work with)
 
(33 intermediate revisions by the same user not shown)
Line 42: Line 42:
 
|[http://www.pdb.org/pdb/files/2V3E.pdb 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
 
|[http://www.pdb.org/pdb/files/2V3E.pdb 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
 
|}
 
|}
<center><small>'''<caption>''' 2V3E, chain B, the chosen reference structure of GBA sequence P04062.</caption></small></center>
+
<center><small>'''<caption>''' Properties of 2V3E, chain B, the chosen reference structure of GBA sequence P04062.</caption></small></center>
 
</figtable>
 
</figtable>
   
Line 53: Line 53:
 
<figtable id="mutations">
 
<figtable id="mutations">
 
{|class="colBasic2"
 
{|class="colBasic2"
! Reference || Codon change || Codon Number (UniProt) || Codon Number (PDB) || Amino Acid change || Polarity || Charge (pH) || Disease causing?
+
! Reference || Codon number (UniProt) || Codon number (PDB) || Codon change || Amino acid change || Polarity || Charge (pH) || Disease causing?
 
|-
 
|-
| [http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=368786234 rs368786234]|| AG<span style="color:#FF0040">'''C'''</span> ⇒ AG<span style="color:#FF0040">'''A'''</span> || 77 || 38 || Ser ⇒ Arg (S77R) ||polar ⇒ polar || neutral ⇒ positive || FALSE
+
| [http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=368786234 rs368786234]|| 77 || 38 || AG<span style="color:#FF0040">'''C'''</span> ⇒ AG<span style="color:#FF0040">'''A'''</span> || Ser ⇒ Arg (S77R) ||polar ⇒ polar || neutral ⇒ positive || FALSE
 
|-
 
|-
| [http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=374003673 rs374003673]|| A<span style="color:#FF0040">'''A'''</span>T ⇒ A<span style="color:#FF0040">'''G'''</span>T || 141 || 102 || Asn ⇒ Ser (N141S) || polar ⇒ polar || neutral ⇒ neutral || FALSE
+
| [http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=374003673 rs374003673]|| 141 || 102 || A<span style="color:#FF0040">'''A'''</span>T ⇒ A<span style="color:#FF0040">'''G'''</span>T || Asn ⇒ Ser (N141S) || polar ⇒ polar || neutral ⇒ neutral || FALSE
 
|-
 
|-
| CM992894|| G<span style="color:#FF0040">'''G'''</span>A ⇒ G<span style="color:#FF0040">'''A'''</span>A|| 241 || 202 || Gly ⇒ Glu (G241E) || nonpolar ⇒ polar || neutral ⇒ negative || TRUE
+
| CM992894|| 241 || 202 || G<span style="color:#FF0040">'''G'''</span>A ⇒ G<span style="color:#FF0040">'''A'''</span>A|| Gly ⇒ Glu (G241E) || nonpolar ⇒ polar || neutral ⇒ negative || TRUE
 
|-
 
|-
| CM880036|| A<span style="color:#FF0040">'''A'''</span>C ⇒ A<span style="color:#FF0040">'''G'''</span>C || 409 || 370 || Asn ⇒ Ser (N409S) || polar ⇒ polar || neutral ⇒ neutral || TRUE
+
| CM880036|| 409 || 370 || A<span style="color:#FF0040">'''A'''</span>C ⇒ A<span style="color:#FF0040">'''G'''</span>C || Asn ⇒ Ser (N409S) || polar ⇒ polar || neutral ⇒ neutral || TRUE
 
|-
 
|-
| CM870010|| C<span style="color:#FF0040">'''T'''</span>G ⇒ C<span style="color:#FF0040">'''C'''</span>G || 483 || 444 || Leu ⇒ Pro (L483P) || nonpolar ⇒ nonpolar || neutral ⇒ neutral || TRUE
+
| CM870010|| 483 || 444 || C<span style="color:#FF0040">'''T'''</span>G ⇒ C<span style="color:#FF0040">'''C'''</span>G || Leu ⇒ Pro (L483P) || nonpolar ⇒ nonpolar || neutral ⇒ neutral || TRUE
 
|}
 
|}
 
<center><small>'''<caption>''' Selected mutations of GBA sequence P04062. Mapping of the UniProt positions onto the PDB ATOM sequence is given. </caption></small></center>
 
<center><small>'''<caption>''' Selected mutations of GBA sequence P04062. Mapping of the UniProt positions onto the PDB ATOM sequence is given. </caption></small></center>
Line 89: Line 89:
 
<gallery widths=300px heights=200px 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, its 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_mutant_38R_hb.png| Hydrogen bonds formed by the mutant Argenine 77.
+
File:2V3E_B_mutant_38R_hb.png| Hydrogen bonds formed by the mutant Argenine 77 remain conserved, as they are formed by the main chain.
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 its side chain 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>
 
<small>'''<caption>''' PyMol visualization of the mutation '''S77R''' (PDB 38) of the structure 2V3E, chain B, with SCWRL.</caption></small>
Line 102: Line 102:
 
<gallery widths=300px heights=200px perrow="3">
 
<gallery widths=300px heights=200px perrow="3">
 
File:2V3E_B_mutation_N102S_sticks.png| Comparison of wild type (wt) Asparagine 141 (magenta) and mutant Serine (orange). The mutated side chain is a little shorter and points towards another direction.
 
File:2V3E_B_mutation_N102S_sticks.png| Comparison of wild type (wt) Asparagine 141 (magenta) and mutant Serine (orange). The mutated side chain is a little shorter and points towards another direction.
File:2V3E_B_N102_hb.png| 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.
+
File:2V3E_B_N102_hb.png| 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.
 
File:2V3E_B_mutant_102S_hb_2diff.png| 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.
 
File:2V3E_B_mutant_102S_hb_2diff.png| 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.
 
File:2V3E_B_mutant_102S_hb_spheres_missing_contact.png| Spheres representation of the mutant Serine and the missing contact with the neighboring helix.
 
File:2V3E_B_mutant_102S_hb_spheres_missing_contact.png| Spheres representation of the mutant Serine and the missing contact with the neighboring helix.
Line 114: Line 114:
 
====G241E====
 
====G241E====
 
<figure id="G241E">
 
<figure id="G241E">
<gallery widths=300px heights=200px perrow="3" align="center">
+
<gallery widths=300px heights=200px perrow="3">
 
File:2V3E_B_mutation_G202E_sticks.png| Comparison of wild type (wt) Glycine 241 (magenta) and mutant Glutamate (orange). The mutated residue has a long side chain.
 
File:2V3E_B_mutation_G202E_sticks.png| Comparison of wild type (wt) Glycine 241 (magenta) and mutant Glutamate (orange). The mutated residue has a long side chain.
 
File:2V3E_B_G202_hb.png| Glycine 241 (PDB 202) lies in a turn in the proximity of a helix end and in front of an another helix. Its amino-group forms a hydrogen bond to a residue of the latter helix.
 
File:2V3E_B_G202_hb.png| Glycine 241 (PDB 202) lies in a turn in the proximity of a helix end and in front of an another helix. Its amino-group forms a hydrogen bond to a residue of the latter helix.
File:2V3E_B_mutant_202E_hb.png| The mutant Glutamate 241 maintains the same hydrogen bond (formed by the main chain) with a residue in a neighboring helix.
+
File:2V3E_B_mutant_202E_hb.png| The mutant Glutamate 241 maintains the same hydrogen bond (formed by the main chain) with a residue in a neighboring helix. The residue lies on the surface and the bigger side chain points to the outside of the protein.
 
</gallery>
 
</gallery>
<center><small>'''<caption>''' PyMol visualization of the mutation '''G241E''' (PDB 202) of the structure 2V3E, chain B, created with SCWRL.</caption></small></center>
+
<small>'''<caption>''' PyMol visualization of the mutation '''G241E''' (PDB 202) of the structure 2V3E, chain B, created with SCWRL.</caption></small>
 
</figure>
 
</figure>
   
Line 125: Line 125:
   
 
====N409S====
 
====N409S====
  +
<figure id="N409S" >
<gallery widths=300px heights=200px caption="PyMol visualization of the mutation '''N409S''' (PDB 370) of the structure 2V3E, chain B, created with SCWRL." perrow="3">
 
  +
<gallery widths=300px heights=200px perrow="3">
 
File:2V3E_B_mutation_N370S_sticks.png| Comparison of wild type (wt) Asparagine 409 (magenta) and mutant Serine (orange). The mutated side chain is a little bit smaller, but points towards the same direction.
 
File:2V3E_B_mutation_N370S_sticks.png| Comparison of wild type (wt) Asparagine 409 (magenta) and mutant Serine (orange). The mutated side chain is a little bit smaller, but points towards the same direction.
File:2V3E_B_N370_hb.png| 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.
+
File:2V3E_B_N370_hb.png| 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 two 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.
File:2V3E_B_mutant_370S_hb.png| Hydrogen bonds formed by the mutant Serine 409. The five hydrogen bonds described in the figure with the Asparagine 409 contacts are conserved.
+
File:2V3E_B_mutant_370S_hb.png| Hydrogen bonds formed by the mutant Serine 409. The five hydrogen bonds described in the figure on the left with the Asparagine 409 contacts are conserved.
 
</gallery>
 
</gallery>
  +
<small>'''<caption>''' PyMol visualization of the mutation '''N409S''' (PDB 370) of the structure 2V3E, chain B, created with SCWRL.</caption></small>
We would predict the mutation N409S as non-effect.
 
  +
</figure>
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. <!-- Do I oversee something?-->
 
  +
  +
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 N409S as non-effect. Moreover, the both amino acids, Asparagine and Serine, are both polar and neutral. Nevertheless, this mutation is annotated as disease causing. <!-- Do I oversee something?-->
   
 
====L483P====
 
====L483P====
  +
<figure id="L483P" >
<gallery widths=300px heights=200px caption="PyMol visualization of the mutation '''L483P''' (PDB 444) of the structure 2V3E, chain B, created with SCWRL." perrow="3">
 
  +
<gallery widths=300px heights=200px perrow="3">
File:2V3E_B_mutation_L444P_sticks.png| 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.
 
  +
File:2V3E_B_mutation_L444P_sticks.png| Comparison of wild type (wt) Leucine 483 (magenta) and mutant Proline (orange). The mutant Proline is a cycle 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.
File:2V3E_B_L444_hb.png| 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.
 
  +
File:2V3E_B_L444_hb.png| 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 points 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.
File:2V3E_B_mutant_444P_hb_sticks_missing_contact.png| The mutant Proline 483 does not form a hydrogen bond with the residue in the loop, like the Leucine main chain amino-group does (see the corresponding figure), because Proline lacks a free amino-group because of the loop formation.
 
  +
File:2V3E_B_mutant_444P_hb_sticks_missing_contact.png| The mutant Proline 483 does not form a hydrogen bond with the residue in the loop, like the Leucine main chain amino-group does (see the corresponding figure). The reason for this is that Proline lacks a free amino-group due to the cycle formation.
 
</gallery>
 
</gallery>
We would predict the mutation L483P as having an effect - and so it is.
+
<small>'''<caption>''' PyMol visualization of the mutation '''L483P''' (PDB 444) of the structure 2V3E, chain B, created with SCWRL.</caption></small>
  +
</figure>
  +
  +
We would predict the mutation L483P as having an effect - and so it is according to annotations.
   
 
==Energy comparisons==
 
==Energy comparisons==
Line 169: Line 176:
 
</figtable>
 
</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.
+
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, it also predicts one not disease causing mutation (N141S) as effect causing, thought with the smallest positive difference (higher 1) of 4.61. Interestingly, the most prevalent Gaucher disease mutation according to OMIM, L483P (L444P in OMIM), 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 a small energy increase (1.31 - 2.99). In foldX the mutation L483P does not have the highest energy increase, in the contrary to SCWRL.
   
 
Next, we superimposed the SCWRL and foldX structures in Pymol and compared them in the following figures.
 
Next, we superimposed the SCWRL and foldX structures in Pymol and compared them in the following figures.
   
  +
<figure id="scwrl_vs_foldx" >
<gallery widths=300px heights=200px caption="Comparison between the mutants created with SCWRL (gray protein, orange mutated residue, yellow polar contacts) and foldX (lime protein, cyan mutated residue, dark cyan polar contacts)." perrow="3">
 
  +
<gallery widths=300px heights=200px perrow="3">
 
File:2V3E_B_mutant_38R.png| Mutation S77R. There is almost no difference in the conformation. However, foldX predicts two additional hydrogen bonds between the Argenine side chain: one to a residues in a proximate loop and the second is a big Tyrosine side chain from the same beta strand.
 
File:2V3E_B_mutant_38R.png| Mutation S77R. There is almost no difference in the conformation. However, foldX predicts two additional hydrogen bonds between the Argenine side chain: one to a residues in a proximate loop and the second is a big Tyrosine side chain from the same beta strand.
File:2V3E_B_mutant_102S.png| Mutation N141S. There is almost no difference in the conformation and the same hydrogen bond are predicted.
+
File:2V3E_B_mutant_102S.png| Mutation N141S. There is almost no difference in the conformation and the same hydrogen bonds are predicted.
File:2V3E_B_mutant_202E.png| 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.
+
File:2V3E_B_mutant_202E.png| 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 only one in SCWRL, are formed.
File:2V3E_B_mutant_370S.png| Mutation N409S. SCWRL and foldX agree in the conformation of the mutated and the binding residues and the hydrogen bond, apart from tiny shifts.
+
File:2V3E_B_mutant_370S.png| Mutation N409S. SCWRL and foldX agree in the conformation of the mutated and the binding residues, including the hydrogen bonds formed between them, apart from tiny shifts.
File:2V3E_B_mutant_444P.png| Mutation L483P. Also here SCWRL and foldX predict nearly the same conformation of the Proline (and the residue which forms a hydrogen bond in WT with the Leucine).
+
File:2V3E_B_mutant_444P.png| Mutation L483P. Also here SCWRL and foldX predict nearly the same conformation of the Proline (and the residue which forms a hydrogen bond in WT with the Leucine).
 
</gallery>
 
</gallery>
  +
<small>'''<caption>''' Comparison between the mutants created with SCWRL (gray protein, orange mutated residue, yellow polar contacts) and foldX (lime protein, cyan mutated residue, dark cyan polar contacts).</caption></small>
  +
</figure>
   
 
===Minimise===
 
===Minimise===
Line 216: Line 226:
 
|-
 
|-
 
|}
 
|}
<center><small>'''<caption>''' 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.</caption></small></center>
+
<center><small>'''<caption>''' Energies produced by each of five consecutive runs of the program '''minimise''', applied on the WT structure and the five mutant structures calculated by SQWRL and foldX. The lowest energies for each structure are marked gold.</caption></small></center>
 
</figtable>
 
</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.
+
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 those calculated by SCWRL.
   
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.
+
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, however it falls a little again in the third iteration, thought not into the same local minimum as in the first iteration. This indicates 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).
+
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 (<xr id="minimise"/>).
   
  +
<figure id="minimise" >
<gallery widths=300px heights=200px caption="Comparison between the mutants created with SCWRL after two minimise iterations (gray protein, orange mutated residue, yellow polar contacts) and foldX after one minimise iteration (lime protein, cyan mutated residue, dark cyan polar contacts)." perrow="3">
 
  +
<gallery widths=300px heights=200px perrow="3">
File:2V3E_B_S38R_minimised-scwrl_vs_foldx.png| Mutation S77R. There is almost no difference in the conformation for this and also the following mutations.
 
  +
File:2V3E_B_S38R_minimised-scwrl_vs_foldx.png| Mutation S77R.
 
File:2V3E_B_N102S_minimised-scwrl_vs_foldx.png| Mutation N141S.
 
File:2V3E_B_N102S_minimised-scwrl_vs_foldx.png| Mutation N141S.
 
File:2V3E_B_G202E_minimised-scwrl_vs_foldx.png| Mutation G241E.
 
File:2V3E_B_G202E_minimised-scwrl_vs_foldx.png| Mutation G241E.
Line 232: Line 243:
 
File:2V3E_B_L444P_minimised-scwrl_vs_foldx.png| Mutation L483P.
 
File:2V3E_B_L444P_minimised-scwrl_vs_foldx.png| Mutation L483P.
 
</gallery>
 
</gallery>
  +
<small>'''<caption>''' Comparison between the mutants created with SCWRL after two minimise iterations (gray protein, orange mutated residue) and foldX after one minimise iteration (lime protein, cyan mutated residue). There is almost no difference in the conformations of all the mutations. </caption></small>
  +
</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.
+
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 in the resulting structures between the two programs, in particular the predicted conformations of the mutated residues and their environments are very similar.

Latest revision as of 00:52, 30 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
Properties of 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 number (UniProt) Codon number (PDB) Codon change Amino acid change Polarity Charge (pH) Disease causing?
rs368786234 77 38 AGC ⇒ AGA Ser ⇒ Arg (S77R) polar ⇒ polar neutral ⇒ positive FALSE
rs374003673 141 102 AAT ⇒ AGT Asn ⇒ Ser (N141S) polar ⇒ polar neutral ⇒ neutral FALSE
CM992894 241 202 GGA ⇒ GAA Gly ⇒ Glu (G241E) nonpolar ⇒ polar neutral ⇒ negative TRUE
CM880036 409 370 AAC ⇒ AGC Asn ⇒ Ser (N409S) polar ⇒ polar neutral ⇒ neutral TRUE
CM870010 483 444 CTG ⇒ CCG 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="S77R" >

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

<figure id="N141S" >

PyMol visualization of the mutation N141S (PDB 102) of the structure 2V3E, chain B, created with SCWRL. </figure>

The mutation N141S is benign, therefore the missing stabilizing contact between the two helices is probably not so important.

G241E

<figure id="G241E">

PyMol visualization of the mutation G241E (PDB 202) of the structure 2V3E, chain B, created with SCWRL. </figure>

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

<figure id="N409S" >

PyMol visualization of the mutation N409S (PDB 370) of the structure 2V3E, chain B, created with SCWRL. </figure>

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 N409S as non-effect. Moreover, the both amino acids, Asparagine and Serine, are both polar and neutral. Nevertheless, this mutation is annotated as disease causing.

L483P

<figure id="L483P" >

PyMol visualization of the mutation L483P (PDB 444) of the structure 2V3E, chain B, created with SCWRL. </figure>

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

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, it also predicts one not disease causing mutation (N141S) as effect causing, thought with the smallest positive difference (higher 1) of 4.61. Interestingly, the most prevalent Gaucher disease mutation according to OMIM, L483P (L444P in OMIM), 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 a small energy increase (1.31 - 2.99). In foldX the mutation L483P does not have the highest energy increase, in the contrary to SCWRL.

Next, we superimposed the SCWRL and foldX structures in Pymol and compared them in the following figures.

<figure id="scwrl_vs_foldx" >

Comparison between the mutants created with SCWRL (gray protein, orange mutated residue, yellow polar contacts) and foldX (lime protein, cyan mutated residue, dark cyan polar contacts). </figure>

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 minimise, 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 those calculated by SCWRL.

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, however it falls a little again in the third iteration, thought not into the same local minimum as in the first iteration. This indicates 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 (<xr id="minimise"/>).

<figure id="minimise" >

Comparison between the mutants created with SCWRL after two minimise iterations (gray protein, orange mutated residue) and foldX after one minimise iteration (lime protein, cyan mutated residue). There is almost no difference in the conformations of all the mutations. </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 in the resulting structures between the two programs, in particular the predicted conformations of the mutated residues and their environments are very similar.