Difference between revisions of "Task 9 (MSUD)"

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[[Task 9 Lab Journal (MSUD)|Lab journal]]
 
[[Task 9 Lab Journal (MSUD)|Lab journal]]
  +
   
 
For this task we have chosen 5 mutations from HGMD and dbSNP. 2 of them are neutral mutations which do not have functional changes over the protein structure. Following are the 5 mutations:
 
For this task we have chosen 5 mutations from HGMD and dbSNP. 2 of them are neutral mutations which do not have functional changes over the protein structure. Following are the 5 mutations:
  +
   
 
<table border=1>
 
<table border=1>
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| 2BEW || X-ray || 1.79 || A || 46-445 || 0.147 || 5.5 || 301ARG-307ASN
 
| 2BEW || X-ray || 1.79 || A || 46-445 || 0.147 || 5.5 || 301ARG-307ASN
 
|-
 
|-
| 1V1R || X-ray || 1.8 || A || 46-445 || 0.158 || 5.5 || 222ASN-231SER,286TYR-314HIS
+
| 1V1R || X-ray || 1.80 || A || 46-445 || 0.158 || 5.5 || 222ASN-231SER,286TYR-314HIS
 
|-
 
|-
| 2BEV || X-ray || 1.8 || A || 46-445 || 0.139 || 5.5 || 301ARG-307ASN
+
| 2BEV || X-ray || 1.80 || A || 46-445 || 0.139 || 5.5 || 301ARG-307ASN
 
|-
 
|-
 
| 1U5B || X-ray || 1.83 || A || 46-445 || 0.156 || 5.8 || 301ARG-306VAL
 
| 1U5B || X-ray || 1.83 || A || 46-445 || 0.156 || 5.8 || 301ARG-306VAL
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| 2BEU || X-ray || 1.89 || A || 46-445 || 0.171 || 5.5 || 301ARG-307ASN
 
| 2BEU || X-ray || 1.89 || A || 46-445 || 0.171 || 5.5 || 301ARG-307ASN
 
|-
 
|-
| 1OLU || X-ray || 1.9 || A || 46-445 || 0.161 || 5.5 || 26ASN-30GLY,287ARG-313ASP
+
| 1OLU || X-ray || 1.90 || A || 46-445 || 0.161 || 5.5 || 26ASN-30GLY,287ARG-313ASP
 
|-
 
|-
| 1V16 || X-ray || 1.9 || A || 46-445 || 0.132 || 5.5 || 288ILE-313ASP
+
| 1V16 || X-ray || 1.90 || A || 46-445 || 0.132 || 5.5 || 288ILE-313ASP
 
|-
 
|-
 
| 1V11 || X-ray || 1.95 || A || 46-445 || 0.139 || 5.5 || 288ILE-313ASP
 
| 1V11 || X-ray || 1.95 || A || 46-445 || 0.139 || 5.5 || 288ILE-313ASP
 
|-
 
|-
| 1V1M || X-ray || 2 || A || 46-445 || 0.13 || 5.5 || 289GLY-313ASP
+
| 1V1M || X-ray || 2.00 || A || 46-445 || 0.13 || 5.5 || 289GLY-313ASP
 
|-
 
|-
| 1X80 || X-ray || 2 || A || 46-445 || 0.161 || 5.8 || 287ARG-313ASP
+
| 1X80 || X-ray || 2.00 || A || 46-445 || 0.161 || 5.8 || 287ARG-313ASP
 
|-
 
|-
| 1X7X || X-ray || 2.1 || A || 46-445 || 0.149 || 5.8 || 287ARG-313ASP
+
| 1X7X || X-ray || 2.10 || A || 46-445 || 0.149 || 5.8 || 287ARG-313ASP
 
|-
 
|-
 
| 1OLX || X-ray || 2.25 || A || 46-445 || 0.161 || 5.5 || 301ARG-307ASN
 
| 1OLX || X-ray || 2.25 || A || 46-445 || 0.161 || 5.5 || 301ARG-307ASN
 
|-
 
|-
| 1DTW || X-ray || 2.7 || A || 46-445 || 0.224 || 7.5 || 301ARG-314HIS
+
| 1DTW || X-ray || 2.70 || A || 46-445 || 0.224 || 7.5 || 301ARG-314HIS
 
|}
 
|}
   
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File:2bff_m82l.png|Local environment of residue 82. Original methionine is replaced by leucine.
 
File:2bff_m82l.png|Local environment of residue 82. Original methionine is replaced by leucine.
 
File:2bff_a222t.png|Local environment of residue 222. Original alanine is replaced by threonine.
 
File:2bff_a222t.png|Local environment of residue 222. Original alanine is replaced by threonine.
File:2bff_c264w.png|Local environment of residue 264. Original cysteine is replaced by tryptophan. Clearly, the mutant residue tryptophan clashes with the binding site (<span style="color:green;">Green</span>) for ion binding.
+
File:2bff_c264w.png|Local environment of residue 264. Original cysteine is replaced by tryptophan. Clearly, the mutant residue tryptophan clashes with residues near the binding site (<span style="color:blue;">Blue</span>). The smallest atomic distance we have found is 1.2 &Aring;.
 
File:2bff_r346h.png|Local environment of residue 346. Original arginine is replaced by histidine.
 
File:2bff_r346h.png|Local environment of residue 346. Original arginine is replaced by histidine.
 
File:2bff_i361v.png|Local environment of residue 361. Original isoleucine is replaced by valine.
 
File:2bff_i361v.png|Local environment of residue 361. Original isoleucine is replaced by valine.
 
</gallery>
 
</gallery>
   
  +
<!--
 
===Mutant structures (SCWRL)===
 
===Mutant structures (SCWRL)===
  +
-->
   
 
===Energy comparisons===
 
===Energy comparisons===
   
 
====FoldX====
 
====FoldX====
  +
<table border=1>
  +
<tr style="font-weight: bold;"><td>Mutation</td><td>PDB</td><td>SD</td><td>total</td><td>energy</td><td>Backbone</td><td>Hbond</td><td>Sidechain</td><td>Hbond</td><td>Van der Waals</td></tr>
  +
<tr><td>M82L</td><td>pdb2bff_1</td><td>0</td><td>0.08</td><td>0.01</td><td>-0.05</td><td>0.46</td><td>-0.1</td><td>0</td><td>0.44</td></tr>
  +
<tr><td>A222T</td><td>pdb2bff_2</td><td>0</td><td>1.22</td><td>0.17</td><td>-1.17</td><td>-0.7</td><td>0.02</td><td>1.48</td><td>-0.51</td></tr>
  +
<tr><td>C264W</td><td>pdb2bff_3</td><td>0</td><td>34.77</td><td>-0.16</td><td>-0.15</td><td>-1.99</td><td>-0.09</td><td>2.88</td><td>-2.95</td></tr>
  +
<tr><td>R346H</td><td>pdb2bff_4</td><td>0</td><td>1.55</td><td>1.28</td><td>1.9</td><td>1.36</td><td>-0.57</td><td>-3.03</td><td>0.73</td></tr>
  +
<tr><td>I361V</td><td>pdb2bff_5</td><td>0</td><td>0.78</td><td>-0.02</td><td>0.01</td><td>0.54</td><td>-0.02</td><td>-0.29</td><td>1.06</td></tr>
  +
</table>
  +
  +
Resulted structural models were compared to the structures calculated by SCWRL. By using alignment tool of PyMOL, we did not find any global deviation between the structures. Sequentially, structures produced by SCWRL have some residues missing. Following table shows the sequential difference.
  +
  +
<table border=1>
  +
<tr style="font-weight: bold;"><td>Mutation</td><td>RMSD(&Aring;)</td><td>Missing residues in SCWRL</td></tr>
  +
<tr><td>M82L</td><td>0.0</td><td>SER76,HIS96,MET146,LEU325,SER326</td></tr>
  +
<tr><td>A222T</td><td>0.0</td><td>SER76,HIS96,MET146,LEU325,SER326</td></tr>
  +
<tr><td>C264W</td><td>0.0</td><td>SER76,HIS96,MET146,LEU325,SER326</td></tr>
  +
<tr><td>R346H</td><td>0.0</td><td>SER76,HIS96,MET146,LEU325,SER326</td></tr>
  +
<tr><td>I361V</td><td>0.0</td><td>SER76,HIS96,MET146,LEU325,SER326</td></tr>
  +
</table>
  +
  +
In 3 mutant structures the mutation residues have different side-chain conformation between results of FoldX and SCWRL. The other 2 mutant structures do not show such difference.
  +
  +
<gallery perrow=3 widths="330" heights="200" caption="Mutation residues with different side-chain conformations (FoldX in red, SCWRL in blue)">
  +
File:M82L-foldx-scwrl.png|Slightly difference between side-chain conformations of LEU82 in mutant '''M82L'''(FoldX in <span style="color:red">red</span>, SCWRL in <span style="color:blue">blue</span>)
  +
File:C264W-foldx-scwrl.png|Difference between side-chain conformations of TRP264 in mutant '''C264W'''(FoldX in <span style="color:red">red</span>, SCWRL in <span style="color:blue">blue</span>). They show completely different orientation of the heterocyclic ring in tryptophane.
  +
File:R346H-foldx-scwrl.png|Slightly difference between side-chain conformations of HIS346 in mutant '''R346H'''(FoldX in <span style="color:red">red</span>, SCWRL in <span style="color:blue">blue</span>)
  +
</gallery>
   
 
====Minimise====
 
====Minimise====
   
  +
The enegies of each Minimise run for the wild type and mutant structures are given in the following table. For the mutant structures there was only an output for structures calculated with FoldX. For input structures from SCWRL, Minimise gave no output (probably there was a problem with gaps).
  +
  +
  +
{| class='wikitable' border='1' style='width:400px'
  +
! run !! 1 !! 2 !! 3 !! 4 !! 5
  +
|-
  +
| WT || -17591 || -17424 || -16955 || -16792 || -16578
  +
|-
  +
| M82L FoldX || -17981 || -17722 || -17298 || -17001 || -16809
  +
|-
  +
| A222T FoldX || -17940 || -17708 || -17297 || -17001 || -16800
  +
|-
  +
| C264W FoldX || -15606 || -17724 || -17319 || -17164 || -16897
  +
|-
  +
| R346H FoldX || -18014 || -17769 || -17339 || -17049 || -16853
  +
|-
  +
| I361V FoldX || -18010 || -17684 || -17313 || -17012 || -16799
  +
|}
  +
  +
  +
A reduction in the energy can only be observed for C264W, and only for the second recursive run. All other energies increase slightly with every run, but are overall similar to each other.
  +
  +
<!--
 
====Gromacs====
 
====Gromacs====
  +
-->
  +
  +
== Discussion ==
  +
  +
* For the disease causing mutations (C264W and R346H), FoldX and SCWRL come to different results regarding the side chain conformation. This gives an indication, that the substituted amino acids do not fit into the local environment compared to the wild type structure. So they change the structure of the protein and hence can alter its function.
  +
* The tryptophane that replaces a cysteine in C264W reaches into an ion binding site and so might interfere with binding of that ion, resulting in inhibition of enzymatic activity.
  +
* We did not observe any minimization when running Minimise on the WT and mutant structures (except for C264W), the energy rather stagnated around a value of about -17000. Probably the structures are already in an energy minimum and can not be minimized any more, which does not mean that the mutant structures keep the functionality of the wild type.

Latest revision as of 19:21, 9 August 2013

Results

Lab journal


For this task we have chosen 5 mutations from HGMD and dbSNP. 2 of them are neutral mutations which do not have functional changes over the protein structure. Following are the 5 mutations:


DatabaseAccession_codeMutationPathogenic
dbSNPrs11549938M82LFalse
dbSNPrs141086188A222TFalse
HGMDCM045934C264WTrue
HGMDCM062448R346HTrue
dbSNPrs61736656I361V False


Selection of structure model

The following table shows an overview of all structures that are available for our protein:


Entry Method Resolution [Å] Chain Positions R-value pH gaps
2BFD X-ray 1.39 A 46-445 0.15 5.5 289GLY-292SER,293THR-313ASP
2BFF X-ray 1.46 A 46-445 0.15 5.5 301ARG-305GLU
1X7Y X-ray 1.57 A 46-445 0.15 5.8 287ARG-313ASP
2BFC X-ray 1.64 A 46-445 0.144 5.5 288ILE-313ASP
2BFE X-ray 1.69 A 46-445 0.15 5.5 289GLY-291HIS,293THR-313ASP
1X7Z X-ray 1.72 A 46-445 0.154 5.8 301ARG-306VAL
1X7W X-ray 1.73 A 46-445 0.148 5.8 287ARG-313ASP
2BFB X-ray 1.77 A 46-445 0.145 5.5 287ARG-313ASP
2BEW X-ray 1.79 A 46-445 0.147 5.5 301ARG-307ASN
1V1R X-ray 1.80 A 46-445 0.158 5.5 222ASN-231SER,286TYR-314HIS
2BEV X-ray 1.80 A 46-445 0.139 5.5 301ARG-307ASN
1U5B X-ray 1.83 A 46-445 0.156 5.8 301ARG-306VAL
1WCI X-ray 1.84 A 46-445 0.149 5.5 301ARG-309TRP
1OLS X-ray 1.85 A 46-445 0.172 5.5 292SER-295ASP,298SER-300TYR,301ARG-308TYR
2J9F X-ray 1.88 A/C 46-445 0.171 5.5 300TYR-313ASP
2BEU X-ray 1.89 A 46-445 0.171 5.5 301ARG-307ASN
1OLU X-ray 1.90 A 46-445 0.161 5.5 26ASN-30GLY,287ARG-313ASP
1V16 X-ray 1.90 A 46-445 0.132 5.5 288ILE-313ASP
1V11 X-ray 1.95 A 46-445 0.139 5.5 288ILE-313ASP
1V1M X-ray 2.00 A 46-445 0.13 5.5 289GLY-313ASP
1X80 X-ray 2.00 A 46-445 0.161 5.8 287ARG-313ASP
1X7X X-ray 2.10 A 46-445 0.149 5.8 287ARG-313ASP
1OLX X-ray 2.25 A 46-445 0.161 5.5 301ARG-307ASN
1DTW X-ray 2.70 A 46-445 0.224 7.5 301ARG-314HIS


Unfortunately all structures contain gaps that span positions 302-304 (corresponding to 347-349 in the reference sequence), so we cannot create a composite structure, that does not contain this gap. We chose 2BFF because it has the smallest gap, a good resolution and a low R-value. It is not resolved at physiological pH, but the only structure with pH 7.5 (1DTW) has a bad resolution. The RMSD between 2BFF and 1DTW is about 0.3, so the different pH does not lead to a different structure and therefore the low pH at that 2BFF was resolved should not be a problem.


Visualization of mutant structures

The mutagen tool of PyMOL was used to introduce mutations to the protein structure. Mutations and their neighboring residues are visualized and shown in following figures.


Energy comparisons

FoldX

MutationPDBSDtotalenergyBackboneHbondSidechainHbondVan der Waals
M82Lpdb2bff_100.080.01-0.050.46-0.100.44
A222Tpdb2bff_201.220.17-1.17-0.70.021.48-0.51
C264Wpdb2bff_3034.77-0.16-0.15-1.99-0.092.88-2.95
R346Hpdb2bff_401.551.281.91.36-0.57-3.030.73
I361Vpdb2bff_500.78-0.020.010.54-0.02-0.291.06

Resulted structural models were compared to the structures calculated by SCWRL. By using alignment tool of PyMOL, we did not find any global deviation between the structures. Sequentially, structures produced by SCWRL have some residues missing. Following table shows the sequential difference.

MutationRMSD(Å)Missing residues in SCWRL
M82L0.0SER76,HIS96,MET146,LEU325,SER326
A222T0.0SER76,HIS96,MET146,LEU325,SER326
C264W0.0SER76,HIS96,MET146,LEU325,SER326
R346H0.0SER76,HIS96,MET146,LEU325,SER326
I361V0.0SER76,HIS96,MET146,LEU325,SER326

In 3 mutant structures the mutation residues have different side-chain conformation between results of FoldX and SCWRL. The other 2 mutant structures do not show such difference.

Minimise

The enegies of each Minimise run for the wild type and mutant structures are given in the following table. For the mutant structures there was only an output for structures calculated with FoldX. For input structures from SCWRL, Minimise gave no output (probably there was a problem with gaps).


run 1 2 3 4 5
WT -17591 -17424 -16955 -16792 -16578
M82L FoldX -17981 -17722 -17298 -17001 -16809
A222T FoldX -17940 -17708 -17297 -17001 -16800
C264W FoldX -15606 -17724 -17319 -17164 -16897
R346H FoldX -18014 -17769 -17339 -17049 -16853
I361V FoldX -18010 -17684 -17313 -17012 -16799


A reduction in the energy can only be observed for C264W, and only for the second recursive run. All other energies increase slightly with every run, but are overall similar to each other.


Discussion

  • For the disease causing mutations (C264W and R346H), FoldX and SCWRL come to different results regarding the side chain conformation. This gives an indication, that the substituted amino acids do not fit into the local environment compared to the wild type structure. So they change the structure of the protein and hence can alter its function.
  • The tryptophane that replaces a cysteine in C264W reaches into an ion binding site and so might interfere with binding of that ion, resulting in inhibition of enzymatic activity.
  • We did not observe any minimization when running Minimise on the WT and mutant structures (except for C264W), the energy rather stagnated around a value of about -17000. Probably the structures are already in an energy minimum and can not be minimized any more, which does not mean that the mutant structures keep the functionality of the wild type.