Task 7: Structure-based mutation analysis
Contents
Task description
A detailed task description can be found here.
Selection of protein structure
We had the following choice of reference structures for PAH:
| Entry | Method | Resolution (A) | Chain | Positions |
|---|---|---|---|---|
| 1DMW | X-Ray | 2.00 | A | 118-424 |
| 1J8T | X-Ray | 1.70 | A | 103-427 |
| 1J8U | X-Ray | 1.50 | A | 103-427 |
| 1KW0 | X-Ray | 2.50 | A | 103-427 |
| 1LRM | X-Ray | 2.10 | A | 103-427 |
| 1MMK | X-Ray | 2.00 | A | 103-427 |
| 1MMT | X-Ray | 2.00 | A | 103-427 |
| 1PAH | X-Ray | 2.00 | A | 117-424 |
| 1TDW | X-Ray | 2.10 | A | 117-424 |
| 1TG2 | X-Ray | 2.20 | A | 117-424 |
| 2PAH | X-Ray | 3.10 | A/B | 118-452 |
| 3PAH | X-Ray | 2.00 | A | 117-424 |
| 4PAH | X-Ray | 2.00 | A | 117-424 |
| 5PAH | X-Ray | 2.10 | A | 117-424 |
| 6PAH | X-Ray | 2.15 | A | 117-424 |
All these structures have in common that they did not solve the structure of the whole PAH protein. They only solve the catalytic domain of PAH, the missing parts are the tetramerisation domain and the regulatory domain which are located at the N- and C- terminal ends. In addition, there is no complete true apo structure available either. All structures have at least a Fe2+ atom bound. Because of this we thought it might be better if we select a structure which has all reaction components or at least most of them bound in the catalytic site in order to get a good picture of the binding site configuration. Though, only 1KW0 and 1MMK fulfilled the constrains that all reaction components are bound.
In the end we did not select 1KW0 or 1MMK, we decided us for the structure 1J8U which is complexed with Fe2+ and BH4 (5,6,7,8-TETRAHYDROBIOPTERIN). Only. This has simple reasons. First of all it has the lowest resolution (1.5 Angstrom) and secondly we already used this structure in previous task as our reference structure for PAH. So we think to keep our experiments more consistent we should stay with this structure. Furthermore, we identified this structure to have no gaps and it solves the complete catalytic domain (as all available structures). Also, the R-Value looked good to us which is 0.157.
To sum it up our selected structure 1J8U has the following experimental metrics (taken from PDBe):
Mapping mutations to the structure
We identifier the following functional residues and catalytic sites with the help of UniProt entry P00439 and Catalytic Site Atlas. We looked for catalytic sites in the structure of 1J8U.
We identified the following functional residues and catalytic sites:
- HIS 285, functional part: side chain (from CSA)
- HIS 290 (from UniProt)
- GLU 330 (from UniProt)
- SER 349, functional part: side chain (from CSA)
In the following picture we can see the Fe+2 atom as a brown sphere, BH4 as a cloud of green blue and red spheres, the location of the mutated residues in orange (mutation I65T and R71H are not included) and the four identified catalytic sites in yellow:
I65T
This mutation is not part of our structure but we would say probably no effect on catalytic site because it is too far away.
R71H
This mutation is not part of our structure but we would say probably no effect on catalytic site because it is too far away.
R158Q
Probably no effect on catalytic site because it is too far away.
R261Q
Probably no effect on catalytic site because it is too far away.
T266A
No direct influence on catalytic site residue. However, this residue is located what we would define as the catalytic center.
P275S
Probably no effect on catalytic site because it is too far away.
T278N
No direct influence on catalytic site residue. However, this residue is located what we would define as the catalytic center. ´
P281L
Probably direct influence on catalytic site residue HIS 285. In addition, this residue is located what we would define as the catalytic center.
G312D
Probably no effect on catalytic site because it is too far away.
R408W
Probably no effect on catalytic site because it is too far away.
