Structure-based mutation analysis (Phenylketonuria)

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Summary

In Task 8 the sequence of PAH was used for finding mutational effects, now the structure will be taken for these analysis. But how to find out, if a mutation changes the structure? Therefore, one calculates the energy of all atoms for the wildtype and the mutated structure and compares the results for changes. There are two different methods for this calculations given: Quantum Mechanics (QM) and Molecular Mechanics (MM). In QM the energy of all electrons in a protein is calculated. It is one of the most accurated methods, but it is very time consuming. In MM the energy of a system is calculated as a function of nuclear positions. It is very fast and easy to calculate, but it ignores electronic motions and is not as accurate as QM. Since QM is too time intensive and the results of MM are nearly as good as the ones calculated with QM, we use MM for the further analysis. Molecular Mechanics uses force fields for the energy calculation, which is defined as a sum of terms. The terms are non-bonded (electrostatic and Van-der-Waals) and bonded (Bond stretching, Angle stretching, bond rotation) interactions. For the structure based mutation analysis the SCWRL and FoldX webserver were used.

Structure selection

Lab journal

In some Tasks before, we used the protein structure of 2PAH as reference, but now we have to check some more constraints for the protein structure selection:

• Structure with the highest resolution (small Å value),
• smallest R-factor,
• highest coverage,
• pH-value ideally near physiological pH of 7.4 and
• no gaps (missing residues) included in the structure, so a consecutive numbering of residues should be given.

To check which protein structure to use for further analysis, we compared the constraint data for all sequences given in the PAH (P00439) Uniprot entry. <figtable id="pro-struc">

Protein	Method	Resolution(Å)	R-factor	pH	Gaps	Chain	Positions	Coverage %
1DMW	X-ray	2.00	0.20	6.80	-	A	118-424	67,92
1J8T	X-ray	1.70	0.20	6.80	-	A	103-427	71.90
1J8U	X-ray	1.50	0.16	6.80	-	A	103-427	71.90
1KW0	X-ray	2.50	0.22	6.80	-	A	103-427	71.90
1LRM	X-ray	2.10	0.21	6.80	-	A	103-427	71.90
1MMK	X-ray	2.00	0.20	6.80	-	A	103-427	71.90
1MMT	X-ray	2.00	0.21	6.80	-	A	103-427	71.90
1PAH	X-ray	2.00	0.18	6.80	-	A	117-424	68.14
1TDW	X-ray	2.10	0.21	6.80	-	A	117-424	68.14
1TG2	X-ray	2.20	0.21	6.80	-	A	117-424	68.14
2PAH	X-ray	3.10	0.25	7.00	136LEU-143ASP	A/B	118-452	74.12
3PAH	X-ray	2.00	0.18	6.80	-	A	117-424	68.14
1ANP	X-ray	2.11	0.20	6.80	-	A	104-427	71.68
4PAH	X-ray	2.00	0.17	6.80	-	A	117-424	68.14
5PAH	X-ray	2.10	0.16	6.80	-	A	117-424	68.14
6PAH	X-ray	2.15	0.17	6.80	-	A	117-424	68.14

...

</figtable> All proteins were found with the X-ray diffraction method. In <xr id="pro-struc"/> we can see, that the structure of 1J8U has a better resolution value as well as R-factor than the other structures. Although 2PAH has a better pH-value, a higher coverage and even two chains, however, the structure includes one gap. For this reason as well as the better R-factor and higher resolution value, we have chosen the structure of 1J8U (no gaps) for further analysis. Moreover, the structure includes the second highest coverage and also a very good pH-value.

The structure of 1J8U as well as its ligands are shown in the <xr id="1j8u"/> below. The binding sites which belong to the ligands are shown in <xr id="bindingsite"/>.

</figure> </figure>

3D structure of 1J8U (green) in cartoon style with its two ligands H4B - C₉H₁₅N₅O₃ (blue) and FE(II) (grey).

Structure of 1J8U (green) in cartoon style with zoom to the ligands H4B - C₉H₁₅N₅O₃ (blue) with corresponding binding site (red) and FE(II) (grey) with corresponding binding site (orange). The binding sites are shown in sticks and their belonging surface structure.

<figure id="1j8u">

<figure id="bindingsite">

Visualisation of used mutations

Following five mutations from the previously selected mutations in Task8 are mapped to the crystal structure:

Substitution	Prediction	Database
Gln172His	neutral	dbSNP
Ala259Val	non-neutral	HGMD
Thr266Ala	non-neutral	dbSNP
Phe392Ser	non-neutral	dbSNP
Pro416Gln	non-neutral	HGMD

Gln172His

<figure id="Q172H">

Mutation of glutamine (yellow) to histidine (purple) at position 172 of 1J8U (green).

</figure> ...

Ala259Val

<figure id="A259V">

Mutation of alanine (yellow) to valine (purple) at position 172 of 1J8U (green).

</figure> ...

Thr266Ala

<figure id="T266A">

Mutation of threonine (yellow) to alanine (purple) at position 172 of 1J8U (green).

</figure> ...

Phe392Ser

<figure id="F392S">

Mutation of phenylalanine (yellow) to serine (purple) at position 172 of 1J8U (green).

</figure> ...

Pro416Gln

<figure id="P416Q">

Mutation of proline (yellow) to glutamine (purple) at position 172 of 1J8U (green).

</figure> ...

Mutated structure creation

SCWRL4

SCWRL4 (Side-chain Conformation Prediction With Rotamer Library) predicts protein side-chain conformations. Therefore, it uses a backbone-dependent rotamer library. .. <ref name="scwrl4"> Georgii G. Krivov1, Maxim V. Shapovalov1 and Roland L. Dunbrack Jr. (2009): "Improved prediction of protein side-chain conformations with SCWRL4". Proteins Vol.77(4):778-95. doi:10.1002/prot.22488</ref> There is also an online SCWRL Server available. It is easy to use, accuracy and very fast. After generating the mutated structures with SCWRL, we compared the results to the wildtype structure in Pymol and checked if only the mutated side chain or another one has been changed. In the observation, only the mutated side chain was changed.

FoldX

...

Energy comparisons

<figtable id="scwrl">

SCWRL results
Type	Energy	Energy Mutation / Energy Wildtype	Prediction
WT	164.210	1.00	-
Q172H	169.699	1.03	x
A259V	197.235	1.20	x
T266A	167.116	1.02	x
F392S	171.409	1.04	x
P416Q	169.007	1.03	x

Comparison of the SCWRL results between the wildtype and the mutant structures. In the first column the type (mutation or wildtype) is given, then the resulting total minimal energy of the graph from the SCWRL results. In the third column this energy is divided through the wildtype resulting energy, to check the difference between this two types, and in the last column the prediction of the SCWRL results is represented. </figtable> ...

Minimise

In the table below, the energy for all five runs of the minisation are given. Since the SCWRL output could not be minimised, we only can see the difference between the wildtype (WT) and the five mutation structures constructed with foldX. <figtable id="minimise">

minimisation run
Type	1	2	3	4	5
WT	-7516.27	-7524.20	-7291.36	-7133.71	-6996.34
Q172H	-7514.27	-7504.92	-7281.60	-7131.31	-7023.56
A259V	-7469.61	-7462.48	-7221.58	-7065.94	-6951.32
T266A	-7536.77	-7523.38	-7298.14	-7165.29	-7084.60
F392S	-7511.51	-7528.61	-7290.01	-7132.75	-7010.52
P416Q	-7556.57	-7542.79	-7299.39	-7151.21	-7040.37

...

</figtable> The energies of the wildtype and the mutated structures is very similar and is per run increasing slightly. Only for the structures of the wildtype and the mutation F392S has the second run a small decreased value.

References

<references/>