Structure-based mutation analysis (PKU)

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 at some time.

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
GLN172HIS	non-disease
ARG243GLN	disease causing
LEU255SER	disease causing
MET276VAL	non-disease
ARG297CYS	disease causing
ALA322GLY	hyperphenylalaninemia
GLU330ASP	disease causing
GLY337VAL	disease causing
ARG408TRP	disease causing

ARG158GLN

<figure id="fig:ARG158GLNmutation">

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

</figure>

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> 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>