Task 7: Structure-based mutation analysis

1 Task description
2 Selection of protein structure
3 Mapping mutations to the structure
- 3.1 I65T
- 3.2 R71H
- 3.3 R158Q
- 3.4 R261Q
- 3.5 T266A
- 3.6 P275S
- 3.7 T278N
- 3.8 P281L
- 3.9 G312D
- 3.10 R408W
4 Binding Site
5 Introducing mutations to 1J8U
- 5.1 Introducing mutations to 1J8U with SCWRL
- 5.2 SCWRL versus PyMol: Comparison of the rotation of the side chains
6 Hydrogen bond network of the WT and mutants
- 6.1 I65T
- 6.2 R71H
- 6.3 R158Q
- 6.4 R261Q
- 6.5 T266A
- 6.6 P275S
- 6.7 T278N
- 6.8 P281L
- 6.9 G312D
- 6.10 R408W
7 FoldX
- 7.1 Execution
- 7.2 Results
8 Minimise
9 Gromacs
- 9.1 Mutations
- 9.2 Timerun
10 Final results and discussion

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):

Figure 1: Summary of experimental details for structure 1J8U

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:

Figure 2: visualization of catalytic site residues and position of selected mutations

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.

Binding Site

Figure 3: Binding site of the compounds visualized in orange

To get the binding sites of the two compounds (red) we selected the residues (orange) within 6 Angstrom range of the compounds.

Binding residues:

245

247-251

254-255

263-266

281

285-286

290

307

321-322

325-326

330

345-346

Figure 4: Binding site of the compounds visualized in orange, mutated residues in range of the binding site are in white and residues out of range of the binding site are colored in blue

In this case the residues in range of the compounds are orange, the mutated residues out of range are blue and the mutated residues in range are white. The residues 266 and 281 are affected by our mutations and in range of the compounds.

Introducing mutations to 1J8U

Introducing mutations to 1J8U with SCWRL

We had to employ several steps to introduce our mutated residues to our structure with SCWRL:

1. extract amino acid sequence from PDB file

/apps/scripts/repairPDB 1J8U.pdb -seq > 1J8U_seq.txt

2. convert all upper case residues to lower case

vim 1J8U_seq.txt

:%s/.*/\L&/g

3. create one sequence file for each mutation and put the residue to mutate as an uppercase letter

4. execute SCWRL for each mutation

/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R158Q.txt -o 1J8U_R158Q.pdb | tee scwrl_r158q.out

/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R261Q.txt -o 1J8U_R261Q.pdb | tee scwrl_r261q.out

/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_T266A.txt -o 1J8U_T266A.pdb | tee scwrl_t266a.out

/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_P275S.txt -o 1J8U_P275S.pdb | tee scwrl_p275s.out

/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_T278N.txt -o 1J8U_T278N.pdb | tee scwrl_t278n.out

/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_P281L.txt -o 1J8U_P281L.pdb | tee scwrl_p281l.out

/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_G312D.txt -o 1J8U_G312D.pdb | tee scwrl_g312d.out

/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R408W.txt -o 1J8U_R408W.pdb | tee scwrl_r408w.out

SCWRL versus PyMol: Comparison of the rotation of the side chains

I65T

Could not compare the side chain of this mutation since this position is not included in 1J8U.