Structure-based mutation analysis

General

According to the UniProt entry about HFE_HUMAN are three 3D-structures of the HFE_HUMAN available, which are listed below. We have chosen the '1A6Z' because it has the best resolution, a very good R-Value (it measures the quality of the model obtained from the crystallographic data), a pH near the physiological optimum and is as good as complete. '1DE4' has a slightly better R-Value and pH, but this PDB also includes the transferrin receptor, which we do not need and do not want in our structure. Also the missing residues of chain A are the same as in the structure '1A6Z' which are only the first three positions. '1C42' is only a hypothetical model, so we exclude it from further research.

stereochemistrical properties of 1a6z

All stereochemistrical properties of the structure are shown in the figure to the right<ref>Lebrón JA, Bennett MJ, Vaughn DE, Chirino AJ, Snow PM, Mintier GA, Feder JN, Bjorkman PJ.: Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor.</ref>.

PDB	Method	Resolution (Å)	Chain	R-Value	R-Free	pH	Temperature	Completeness	Missing residues (Chain:pos)
1A6Z	X-ray	2.60	A/C	0.233	0.277	6.5	-150.15°C (123 k)	98.0	A:1-3	C:1-3	-	-	-	-
1C42	model	-	A	-	-	-	-	-	-	-	-	-	-	-
1DE4	X-ray	2.80	A/D/G	0.231	0.265	8.0	-173.15°C (100 k)	94.3	A:1-3	C:121,757-760	D:1-3	F:121,757-760	G:1-3	I:121,757-760

We used the following 7 mutations, because we were not able to map 3 of them to the 1A6ZA chain mostly because of position errors. We are using the same names as given in Task 6. Mutation 8 had to be split into two parts because of possible mutation into to different amino acids.

Mutation
Mutation 2 [M35T]
Mutation 3 [S65C]
Mutation 4 [I105T]
Mutation 5 [Q127H]
Mutation 6 [A176V]
Mutation 7 [T217I]
Mutation 8a [C282Y]
Mutation 8b [C282S]

Mapping

Because we have no annotation about the active site so we just visualized the mutations at the '1A6Z' structure. Secondly we are using the same mutations as in Task 6 and have therefore the same problemes with their visualization (only 7 of 10 visualizeable).

All mutations are scattered accross the protein with no affinity to some special region or secondary structure element. Mutations shown in red are near glycosylation positions and mutations in yellow are near disulfidbonds.

1A6ZA with 7 of 10 visualized mutations taken from task 6

Energy comparison

SCWRL

SCWRL predicts protein side-chain confirmations given a fixed backbone. We are using SCWRL version 4 released in 2009.

Usage:

• use only chain A of backbone pdb: 1A6ZA.pdb
• extract amino acid sequence and change it to lowercase: aa.txt
• introduce each mutation into on seperated aa_x.txt file as capital
  • cmd: scwrl -i 1A6ZA.pdb -s aa_x.txt -o ./mutant_pdbs/1A6ZA_mutant_x.pdb > 1A6ZA_mutant_x.txt

Results:

Robert: currently working.

Mutation	Position	Energy	Energy normalized by maximum (1 = worst, 0 = best)
Mutation 2 [M35T]	35	252.324	0.6477502894447026
Mutation 3 [S65C]	65	246.695	0.6332998749804256
Mutation 4 [I105T]	105	250.833	0.6439226881005496
Mutation 5 [Q127H]	127	252.368	0.6478632434749794
Mutation 6 [A176V]	176	280.381	0.7197764537055339
Mutation 7 [T217I]	217	260.189	0.6679408223566833
Mutation 8a [C282Y]	282	389.539	1.0
Mutation 8b [C282S]	282	255.859	0.656825118922624

Minimise

Minimise is able to minimise the energy of an model.

Usage:

• remove all hydrogen and water atoms from the pdb files with repairPDB: 1A6ZA_mutant_x_clean.pdb
  • cmd: repairPDB 1A6ZA_mutant_x.pdb -nosol > ./repair_pdb/1A6ZA_mutant_x_clean.pdb
• minimise the energy of the models:
  • cmd: minimise 1A6ZA_mutant_x_clean.pdb ./minimised_pdb/1A6ZA_mutant_x_clean_minimised.pdb > 1A6ZA_mutant_x_clean_minimised.txt

Results:

Robert: currently working.

Mutation	Position	Energy
Mutation 2 [M35T]	35	-5020.465319
Mutation 3 [S65C]	65	-5040.815685
Mutation 4 [I105T]	105	-5028.869826
Mutation 5 [Q127H]	127	-5031.137220
Mutation 6 [A176V]	176	-4957.946411
Mutation 7 [T217I]	217	-5037.718631
Mutation 8a [C282Y]	282	-2596.778899
Mutation 8b [C282S]	282	-5017.057355

FoldX

FoldX scores the importance of amino acid interactions according to the overall stability of the protein and calculates the energy.

Usage:

• create a runfile tutorial and adjust all default parameters to known (if possible): runfile.txt
• create a listfile of all pdb files that should be included in energy calculation: listfile.txt
• run foldx with runlist
  • cmd: sudo ./foldx -runfile runfile.txt > output.txt

Results:

Robert: currently working.

Gromacs

Has to be done!

References

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