Difference between revisions of "Canavan Task 7 - Structure-based mutation analysis"

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==Playing with Gromacs!==
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Revision as of 10:42, 24 June 2012

A mermaid found a swimming lad,
Picked him for her own,
Pressed her body to his body,
Laughed; and plunging down
Forgot in cruel happiness
That even lovers drown.

William Butler Yeats

Protocol

Further information can be found in the protocol.

Mapping of mutations

<figure id="aspa_mut_overview">

<xr nolink id="aspa_mut_overview"/>
ChainA of the aspa structure is shown in cartoon representation. Chain B is shown in surface representation to emphasize the dimer interface. The intermediate substrate analog is shown in blue. Disease causing mutations are shown in red and non-disease causing SNPs are colored in orange.

</figure>

We use the same mutations for this analysis as for the sequence based mutation analysis.

As a struture we used 2O4H which was crystallized as a dimer (see protocol for further info). For our analysis we will only consider chainA.

In <xr id="aspa_mut_overview"/> you can see an overview of the Aspa structure with the mutations mapped onto the structure.



<figtable id="mutation_vis_table">

<xr nolink id="mutation_vis_table"/> Description of the structural environment of each mutation and visualization of the pymol mutation and the scwrl mutation output.
Mutation Comment Pymol Scwrl
E285A E285 is located in the binding pocket with a distance of 3.6 A to the substrate analog of 2O4H. It does not interact with the substrate, but has hydrogen bonds interactions to T118 and the backbone of Y288. These hydrogen bonds can not be established by the mutant residue alanine.
There are no differences between the Pymol mutation and the scwrl output.
<figure id="e285a_hb" >
<xr nolink id="e285a_hb"/>
</figure>
<figure id="e285a_scwrl" >
<xr nolink id="e285a_scwrl"/>
</figure>
A305E A305 is located at the end of the 13th beta sheet at the C-terminus of the protein. In figure <xr nolink id="a305e_crowded"/> the mutated residue glutamic acid is shown in red. The space at this position is rather crowded, so that alanine as a small residue fits very well in this position. Glutamic acid instead, hardly finds space and overlaps with neighboring residues.
There is a slight difference in orientation for the carboxyl group between the pymol and scwrl output. Although this group is rotated in the scwrl output, there will still be steric clashes with neighboring residues.
<figure id="a305e_crowded">
<xr nolink id="a305e_crowded"/>
</figure>
<figure id="a305e_scwrl">
<xr nolink id="a305e_scwrl"/>
</figure>
G123E G123 is located at the beginning of the fourth beta strand in Aspartoacylase. This strand is not buried but solvent accessible. In <xr nolink id="g123e_space"/>, the mutated residue E123 is presented in red. Eventhough glutamic acid is much larger than glycin, there is enough space and no clashes occur. Furthermore, Glycin is not involved in any interactions.
There is only a slight difference in orientation for glutamic acid. But this change in orientation should not have a large influence, since there is enough space to neighboring residues and no interactions with them are possible.
<figure id="g123e_space">
<xr nolink id="g123e_space"/>
</figure>
<figure id="g123e_scwrl">
<xr nolink id="g123e_scwrl"/>
</figure>
R71H R71 is located at the end of the fourth helix in the active site of the enzyme. It is involved in substrate binding via one Hbond. It also forms other Hbonds with an active water molecule and D68. In <xr nolink id="r71h_hbonds"/> the mutated residue H71 is presented in red and the intermediate substrate analog in blue. H71 is not able to build the Hbonds with the substrate or neighboring residues as does R71.
Pymol and scwrl output the same conformation for histidine. In both cases the mutated residue is not able to form those Hbonds that arginine could form.
<figure id="r71h_hbonds">
<xr nolink id="r71h_hbonds"/>
</figure>
<figure id="r71h_scwrl">
<xr nolink id="r71h_scwrl"/>
</figure>
R71K R71 is located at the end of the fourth helix in the active site of the enzyme. It is involved in substrate binding via one Hbond. It also forms other Hbonds with an active water molecule and D68. In <xr nolink id="r71k_hbonds"/> the mutated residue K71 is presented in red and the intermediate substrate analog in blue. K71 almost has the same shape as R71 and might also be able to form an Hbond to the substrate or D68.
There is no difference between the pymol and scwrl mutation result. Lysine is not able to form Hbonds in this specific orientation, but still it might be possible.
<figure id="r71k_hbonds">
<xr nolink id="r71k_hbonds"/>
</figure>
<figure id="r71k_scwrl">
<xr nolink id="r71k_scwrl"/>
</figure>
K213E K213 is located on the loop connecting the N-, and C-terminal of the enzyme. It is on the surface of the protein, far away from the binding site or the dimer interaction site. Change of colour (sorry, schlechte Absprache im Team ;-)): In <xr nolink id="k213e_pymol"/>, the mutated residue K213E as computed by PyMol is presented in blue and the reference structure and residue in green. The mutated residue Glutamic Acid is able to form an HBond with the neighbouring helix. <xr nolink id="k213e_scwrl"/> shows the mutated residue in as computed by scwrl. <figure id="k213e_pymol">
<xr nolink id="k213e_pymol"/>
</figure>
<figure id="k213e_scwrl">
<xr nolink id="k213e_scwrl"/>
</figure>
V278M V278M is located on a loop in the C-terminal of the enzyme. It is also on the surface of the protein, far away from the binding site or the dimer interaction site. In <xr nolink id="v278m_pymol"/>, the mutated residue V278M as computed by PyMol is presented in red and the reference structure and residue in green. Both the mutated and the original residue are part of a beta-sheet and form two HBonds each. <xr nolink id="v278m_scwrl"/> shows the mutated residue as computed by scwrl. <figure id="v278m_pymol">
<xr nolink id="v278m_pymol"/>
</figure>
<figure id="v278m_scwrl">
<xr nolink id="v278m_scwrl"/>
</figure>
M82T M82T is located on a loop in the N-terminal of the enzyme. It is on the surface of the protein, not close to the interaction site, but in the neighbourhood of the dimer interaction site. In <xr nolink id="m82t_pymol"/>, the mutated residue M82T as computed by PyMol is presented (colours as before). This residue does not form any HBonds. <xr nolink id="m82t_scwrl"/> shows the mutated residue as computed by scwrl. <figure id="m82t_pymol">
<xr nolink id="m82t_pymol"/>
</figure>
<figure id="m82t_scwrl">
<xr nolink id="m82t_scwrl"/>
</figure>
E235K This residue is again located on a loop in the C-terminal region of the enzyme. It also lies on the surface of the protein, in close neighbourhood to the dimer interaction site. In <xr nolink id="e235k_pymol"/>, the mutated residue E235K as computed by PyMol is presented (colours as before). This residue points out into the solvent and does not form any intra-molecular HBonds. <xr nolink id="e235k_scwrl"/> shows the mutated residue as computed by scwrl. <figure id="e235k_pymol">
<xr nolink id="e235k_pymol"/>
</figure>
<figure id="e235k_scwrl">
<xr nolink id="e235k_scwrl"/>
</figure>
I270T This residue is located on a beta-strand in the C-Terminal region of the protein where it forms HBonds to a neighbouring beta-strand. It also lies on the surface of the protein, and also in close neighbourhood to the dimer interaction site. In <xr nolink id="i270t_pymol"/>, the mutated residue I270T as computed by PyMol is presented (colours as before). <xr nolink id="i270t_scwrl"/> shows the mutated residue as computed by scwrl. <figure id="i270t_pymol">
<xr nolink id="i270t_pymol"/>
</figure>
<figure id="i270t_scwrl">
<xr nolink id="i270t_scwrl"/>
</figure>

</figtable>



Comparing SCWRL4 and FoldX mutants

<figtable id="mutation_vis_table">











<xr nolink id="mutation_vis_table"/> Description of the structural environment of each mutation and visualization of the pymol mutation and the scwrl mutation output.
Mutation Comment Superposition
E305A There is only a slight difference in the orientation of the carboxyl group of the mutated residue E305. In both cases E305 is able to form a hydrogen bond to Gln138. A305 was not able to build this interaction. Furthermore, E305 clashes in both cases with C218 and I137. A305 did not clash.
A305e compare scwrl fold.png
E235K Foldx and Scwrl chose different rotamers for K235. This position is solvent exposed on the surface and no interaction partners are in reach. Lysine is positively charged and therefore suitable for a mutation on the surface of a protein, where it can interact with water molecules.
E235k compare scwrl fold.png
E285A Alanine has no rotamers and therefore there is no difference for the foldx and scwrl output. Alanine is not able to form hbonds with T118 as E285 does.
E285a compare scwrl fold.png
G123E G123 had no interactions with neighboring residues or with the solvent. E123 has the same rotamer in the foldx and scwrl model. In the case of foldx, the residue Y153 is moved slightly, so that E123 can form an Hbond with its hydroxyl group. The substitution of E for G is furthermore suitable for the solvent exposed position for interaction with water molecules.
G123e compare scwrl fold.png
I270T Both methods chose the same rotamer for isoleucine which also identically matches the position of the native isoleucine residue. Neither isoleucine nor threonine can perform interactions with neighboring residues. The nearest residue being P232 at a distance of 3.9 A.
I270t compare scwrl fold.png
K213E Foldx and scwrl chose different rotamers for this solvent exposed residue. No neighboring residues are in close proximity to perform interactions. The mutant residue glutamic acid is also able to form Hbonds with water molecules of the solvent though.
K213e compare scwrl fold.png
M82T Both methods found the same rotamer that has no interactions to other residues. M82 also has no interactions to neighboring residues. This position is solvent exposed. Yet, M82 as well as the mutant residue threonine are hydrophobic and do not interact with solvent.
M82t compare scwrl fold.png
R71H The histidine rotamer that scwrl chose is able to form an hbond with asp68, whereas the slightly different orientated foldx rotamer is not. Yet the native residue R71 can form three interactions: with asp68, the substrate and one coordinated water molecule of the binding site. and foldx chose different rotamers that are both do not form any Hbonds. This is a difference to the scwrl result for the R71H mutant that could at least form one hbond. This result is surprising, since lysine is far more similar to arginine than histidine and we expected similar possible interactions for arginine and lysine.
R71h compare scwrl fold.png
R71k Scwrl and foldx chose different rotamers that both do not form any Hbonds. This is a difference to the scwrl result for the R71H mutant that could at least form one hbond. This result is surprising, since lysine is far more similar to arginine than histidine and we expected similar possible interactions for arginine and lysine.
R71k compare scwrl fold.png
V278M Scwrl and foldx chose methionine rotamers that are orientated into opposing directions. foldx chose the rotamer that is oriented towards the inner of the protein and scwrl chose the rotamer that is oriented towards solvent. In this case, one can consider the foldx rotamer more reasonable since methionine is a fairly hydrophobic residue.
V278m compare scwrl fold.png

</figtable>


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Given a 4-letter pdb ID, it downloads it using wget.