Structure-based mutation analysis (PKU)

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Revision as of 14:01, 23 June 2012 by Hollizeck (talk | contribs) (results)

Short task description

This week, we will introduce mutations in the known tertiary structure of our protein and calculate and compare the potential energy of mutant and wildtype protein with different methods. See the complete task description for details. Our journal might be found here.

Finding the right structure

As proposed we searched the UNIProt entry for our protein and then selected the entry with the highest resolution and the lowest r-Value. In our case this is 1J8U which is the protein in a complex with its cosubstrate BH4. IN the following we will only use this structure, but we also list the results we found. <figtable id="tab:uniprotresult">

Table with results from UNIProt with r-Value inserted
Entry Method Resolution (Å) r-Value Chain Positions PDBsum
1DMW X-ray 2.00 0.200 A 118-424 [»]
1J8T X-ray 1.70 0.197 A 103-427 [»]
1J8U X-ray 1.50 0.157 A 103-427 [»]
1KW0 X-ray 2.50 0.220 A 103-427 [»]
1LRM X-ray 2.10 0.211 A 103-427 [»]
1MMK X-ray 2.00 0.199 A 103-427 [»]
1MMT X-ray 2.00 0.213 A 103-427 [»]
1PAH X-ray 2.00 0.176 A 117-424 [»]
1TDW X-ray 2.10 0.206 A 117-424 [»]
1TG2 X-ray 2.20 0.213 A 117-424 [»]
2PAH X-ray 3.10 0.251 A/B 118-452 [»]
3PAH X-ray 2.00 0.175 A 117-424 [»]
4ANP X-ray 2.11 0.204 A 104-427 [»]
4PAH X-ray 2.00 0.169 A 117-424 [»]
5PAH X-ray 2.10 0.163 A 117-424 [»]
6PAH X-ray 2.15 0.171 A 117-424 [»]

</figtable> In <xr id="tab:uniprotresult"/> there are all results according to which we selected 1J8U to be our reference for this weeks task. The corresponding line is marked in yellow. Unfortunately, as we discovered in the preparation of SCWRL, the coverage in not from 103 to 427 for 18JU but only from 118 to 427.

1J8U

In order to know the structure of the protein and its important residues, we have a look at its structure with PyMol and visualize the BH4 and the Fe-ion with the most important residues.

<figure id="fig:1J8Uwhole">
Rendering of the overall structures of 1J8U using PyMol. The protein is colored cyan overall, whereas the Fe-atom is colored red and the important residues are shown as sticks and colored in the element-based fashion. Binding is shown with yellow strokes, if the distance is bigger than 1.5 Å
</figure>
<figure id="fig:1J8Uclose">
Rendering of a close-up of the structures of 1J8U using PyMol. The protein is colored cyan overall, whereas the Fe-atom is colored red and the important residues are shown as sticks and colored in the element-based fashion. Binding is shown with yellow strokes, if the distance is bigger than 1.5 Å
</figure>

<figure id="fig:structuremoving">

Structure of 1J8U with both ligands Iron (red) and BH4 (element coded color) with their binding sites orange and yellow respectively. BH4-binding sites are van-der-Waal's based and hydrogen bond based, whereas iron is covalently bound (orange)

</figure>

Mutations

As you probably know from last weeks dataset the first two mutations are located before residue 103 and therefore not contained in the structure. We changed them to mutations, which we think are interesting from a structural view. We propose the following dataset, chosen mostly from well known SNPs from OMIM. They include mutations causing no reported effect, the mild related hyperphenylalaninemia (reduced activity, but functional enzyme) and phenylketonuria.

SNP effect prediction validation
ARG158GLN disease causing
Unknown.jpeg
GLN172HIS non-disease
Unknown.jpeg
ARG243GLN disease causing
Unknown.jpeg
LEU255SER disease causing
Unknown.jpeg
MET276VAL non-disease
Unknown.jpeg
ARG297CYS disease causing
Unknown.jpeg
New.jpeg
ALA322GLY hyperphenylalaninemia
Unknown.jpeg
GLU330ASP disease causing
Unknown.jpeg
New.jpeg
GLY337VAL disease causing
Unknown.jpeg
ARG408TRP disease causing
Unknown.jpeg

ARG158GLN

<figure id="fig:ARG158GLNmutation">

The bindingsites are colored according to the overview picture above the mutation (ARG158GLN)is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>This mutation, which is diseasecausing is in a moderate distance to the important binding sites (<xr id="fig:ARG158GLNmutation" />). A clear explanation, why this has an effect on the protein can not be found from the structure alone. But even with the results from last weeks Task, we just had to rely on the clashes which occurred when we estimated the structure.

GLN172HIS

<figure id="fig:ARG158GLNmutation">

The bindingsites are colored according to the overview picture above the mutation (GLN172HIS) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>In <xr id="fig:ARG158GLNmutation" /> one can see the big distance between the binding sites and the mutation location. Since we know that this is harmless, one would tend to say, that its clear, due to the distance, but as we know, that we also have mutations in a big distance which cause the disease, we are bound to say we do not know the mechanism.

ARG243GLN

<figure id="fig:ARG243GLNmutation">

The binding sites are colored according to the overview picture above the mutation (ARG243GLN) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>This is the second mutation of this kind, which means a change from arginine to glycine. As the other mutation, this one is disease causing. But in difference to the other case, this one is rather close to the binding areas (see <xr id="fig:ARG243GLNmutation" /> )

LEU255SER

<figure id="fig:LEU255SERmutation">

The binding sites are colored according to the overview picture above the mutation (LEU255SER)is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>When one compares this mutation ( in <xr id="fig:LEU255SERmutation" />) to the reference structure in <xr id="fig:structuremoving" /> one can see, that one of the yellow residues (BH4 binding site) is mutated. And as one would expect this mutation is harmful.

MET276VAL

<figure id="fig:MET276VALmutation">

The binding sites are colored according to the overview picture above the mutation (MET276VAL) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>As the mutation shown in <xr id="fig:MET276VALmutation" /> is rather far from any important binding site, one would expect it to be of little effect for the protein, which is the case for this mutation.

ARG297CYS

<figure id="fig:ARG297CYSmutation">

The binding sites are colored according to the overview picture above the mutation (ARG297CYS) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>As the last mutation, this one is located quite far from any annotated sites ( <xr id="fig:ARG297CYSmutation" />), however it is located in the kink of two helices, and therefore the expectation which this mutation might have on the protein tends towards disease causing, which is actually the right choice.

ALA322GLY

<figure id="fig:ALA322GLYmutation">

The binding sites are colored according to the overview picture above the mutation (ALA322GLY) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure> The mutation, which affects one of the amino acids responsible for the BH4-binding, is shown in <xr id="fig:ALA322GLYmutation" /> and as one would guess is disease causing.

GLU330ASP

<figure id="fig:GLU330ASPmutation">

The binding sites are colored according to the overview picture above the mutation (GLU330ASP) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>As the last mutation in <xr id="fig:ALA322GLYmutation" /> this mutation affects a binding site (this time iron) and is disease causing.

ARG408TRP

<figure id="fig:ARG408TRPmutation">

The binding sites are colored according to the overview picture above the mutation (ARG408TRP) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>In <xr id="fig:ARG408TRPmutation" /> one can again see a mutation which is pretty far from the annotated sites, but still is disease causing.


SCWRL

preparations

We use the repairPDB script to extract the sequence from our model file. Afterwards we do some bashing to get the sequence in small letters but for the mutation, which is in capital. <source lang="bash"> $ repairPDB 1J8U.pdb -seq > 1J8U.seq $ tr '[:upper:]' '[:lower:]' < 1J8U.seq > lower18JU.seq $ sed 's/^\(.\{<pos-118>\}\)<original in small letter>\(.*\)/\1<target in capital letter>\2/' lower18JU.seq > lower18JUmut<pos>.seq </source>

usage

Afterwards, we used a little bashscript which just applies all SCWRL-calls after another. In the following you see one example: <source lang="bash"> $ /opt/SS12-Practical/scwrl4/Scwrl4 -i ../1J8U.pdb -s lower18JUmut158.seq -o mut158.pdb > mut158.log </source>

results

The following pictures show the structure optimized by SCWRL. The mutant is colored in red for this residue as the wildtype is colored in green, as the structure apart from this residue is unchanged, the color is chosen automatically by PyMol. Polar bonds are colored respectively to mutant and wildtype, but note, that there might be overlaps, where only one color is shown.

foldX

THe other approach we used is foldX

preparation

We use the run.txt from the example at the test and adapt the list and individual list as its shown in the [[ |journal]]

usage

<source lang="bash">

scwrl -runfile run.txt

</source>

results

As we already have results from the SCWRL run, we just compare those with the new results from foldX. The comparison with the sourounding structures is left for the reader as an exercise. The residue placed by SCWRL is colored in blue whereas the foldX residue is orange. THe surrounding structure is colored randomly by PyMol.

minimise

preparation

To use minimise we have to clear the structure of hydrogen atoms and solvent which we again used the repairPDB script <source lang="bash"> $ repairPDB 1J8U.pdb -noh -nosol > 1J8U_pure.pdb </source>

usage

Afterwards a little script is used to apply the minimization five times and each time using the output as input.

  • usage:

<source lang="bash"> $ runAllMinimiser.sh <filestem> </source>

results

Just to see, the differences between the results of one run and several using the output again as input, we produce this little animated .gif, which is basicly all the results from the 18JU.pdb on till the fifth minimise result. <figure id="fig:helixminimise">

Superimposition of all structures derived from minimise for 1J8U which means the wildtype. For a better comparison first the wildtype is shown in blue. Then it will get transparent and the first result of minimize is shown in teal. Then the transparent wildtype disappears and the first result will get transparent and the second result will show. This continues until the final result which is red. Then the animation will start again.

</figure> In <xr id="fig:helixminimise" /> one can see that minimise shifts the start of the helix further down in the picture, and once it reaches a certain point, the start of the helix is converted to a coiled region instead. A similar effect can be found if one compares sheets in the output of the different minimise runs. Here a twisting of the sheets takes place, which can bee seen in <xr id="fig:sheetminimise" /> <figure id="fig:sheetminimise">

Structure of 1J8U before (cyan) and after (red) minimise. The red structure is the result of five consecutive runs of minmise, and shows a twist in the orientation of the sheet.

</figure>

Gromacs

Choose scwrl structures for now, they are better in general

fetchPDB: just wget for gzipped PDBfile

repairPDB -jprot would do the trick.

choose Amber03 (1), CHARMM27 (8) and GROMOS96 53a6 (13) force fields

script to execute pdb2gmx

all GROMOS ff's constantly throws errors (and do not work with tip3p) choose opslaa instead

TODO: time runs > 16000 steps