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
Reference	--	247.944	1
Mutation 2 [M35T]	35	252.324	1,017665279
Mutation 3 [S65C]	65	246.695	0,994962572
Mutation 4 [I105T]	105	250.833	1,011651825
Mutation 5 [Q127H]	127	252.368	1,017842739
Mutation 6 [A176V]	176	280.381	1,130823896
Mutation 7 [T217I]	217	260.189	1,049386152
Mutation 8a [C282Y]	282	389.539	1,571076533
Mutation 8b [C282S]	282	255.859	1,031922531

• The energy is normalized by the wild-type structure. A value larger then 1 means that the energy is increased compared to the wild-type. A value smaller 1 shows a decreased energy.

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	Energy normalized
Reference	--	-3724.153777	1
Mutation 2 [M35T]	35	-5020.465319	1,348082174
Mutation 3 [S65C]	65	-5040.815685	1,353546601
Mutation 4 [I105T]	105	-5028.869826	1,35033893
Mutation 5 [Q127H]	127	-5031.137220	1,350947765
Mutation 6 [A176V]	176	-4957.946411	1,331294761
Mutation 7 [T217I]	217	-5037.718631	1,352714988
Mutation 8a [C282Y]	282	-2596.778899	0,697280256
Mutation 8b [C282S]	282	-5017.057355	1,347167076

• The energy is normalized by the wild-type structure. A value larger then 1 means that the energy is increased compared to the wild-type. A value smaller 1 shows a decreased energy.

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: Foldx -runfile runfile.txt > output.txt

Results:

Mutation	Position	Energy	Energy normalized
Reference	--	169.51	1
Mutation 2 [M35T]	35	208.08	1,227538198
Mutation 3 [S65C]	65	206.66	1,219161111
Mutation 4 [I105T]	105	210.39	1,241165713
Mutation 5 [Q127H]	127	205.04	1,209604153
Mutation 6 [A176V]	176	214.31	1,264291192
Mutation 7 [T217I]	217	208.15	1,227951153
Mutation 8a [C282Y]	282	242.23	1,429001239
Mutation 8b [C282S]	282	215.50	1,271311427

• The energy is normalized by the wild-type structure. A value larger then 1 means that the energy is increased compared to the wild-type. A value smaller 1 shows a decreased energy.

Gromacs

• We used the -ignh mode to ignore all hydroxen atom.
• As forcefield, we chosed the AMBER03, XXX and XXX model.

Energy for the Wild-Type

Energy	Average	Err.Est.	RMSD	Tot-Drift (kJ/mol)
Bond	848.392	380	-nan	-2320.49
Angle	2707.42	22	-nan	-96.9545
Potential	-32380.6	1200	-nan	-7696.01

References

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