Difference between revisions of "Structure-based mutation analysis"
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A damaging mutation within a turn. Here a polar amino acid is exchanged into an aromatic one. Gromacs is the only tool which shows a decreasing energy. The other tools show an increasing energy. As this mutation is not within a structural element, and the mutation is a damaging one, the reason for the change in function is most likely either a functional residue or a change in the flexibility. As we have no information about functional residues, we assume a change in flexibility is the reason for the damage. |
A damaging mutation within a turn. Here a polar amino acid is exchanged into an aromatic one. Gromacs is the only tool which shows a decreasing energy. The other tools show an increasing energy. As this mutation is not within a structural element, and the mutation is a damaging one, the reason for the change in function is most likely either a functional residue or a change in the flexibility. As we have no information about functional residues, we assume a change in flexibility is the reason for the damage. |
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− | '''Mutation 6''' |
+ | '''Mutation 6 [A176V]''' |
'''Mutation 7''' |
'''Mutation 7''' |
Revision as of 19:48, 28 August 2011
Contents
TODO
add all references/quotes
DISCUSSION!
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.
All stereochemistrical properties of the structure are shown in Figure 1.<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 in Figure 2. 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 (Figure 2). The mutations are shown in detail in the table below.
The structure shown in green is the reference structure, the white one is the mutation and the yellow the overlap between the structure of the mutation and reference (please have first a glance at the PyMOL pictures to see where the mutation acutally is because all SCWRL mutation structures are shown completly in white). It is clearly visible that the sidechains/rotamers used by SCWRL are often very different to these introduced by PyMOL. PyMOL does only that the sidechains of the mutated amino acid but SCWRL even recalculates sidechains of non mutated amino acids because of non allowed clashes between them. SCWRL introduces mostly small directional changes to prohibit these clashes. We believe the rotamers used by SCRWL are the more correct ones, because of the fact that SCRWL does also check for possible clashes and that PyMOL is more a visulization tool.
Energy comparison
SCWRL
SCWRL predicts protein side-chain confirmations given a fixed backbone. We are using SCWRL4<ref>Georgii G. Krivov, Maxim V. Shapovalov, and Roland L. Dunbrack, Jr.: Improved prediction of protein side-chain conformations with SCWRL4</ref> 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
- cmd:
Results:
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 than 1 means that the energy is increased compared to the wild-type. A value smaller 1 shows a decreased energy.
Only mutation 3 shows an decreased energy level which means that this mutation is able to occur more often because it is favoured. All mutations expect 8a are placed around an energy level of 1 what means that mutations should occur at the same amout in population as the reference. Therefore it is very astounding that mutation 8a has the highest increased energy level altough it is the mutation which causes most of all hemochromatosis cases.
Minimise
Minimise is able to minimise the energy of a model.
Usage:
- remove all hydrogen and water atoms from the pdb files with repairPDB
- cmd:
repairPDB 1A6ZA_mutant_x.pdb -nosol > ./repair_pdb/1A6ZA_mutant_x_clean.pdb
- cmd:
- 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
- cmd:
Results:
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 than 1 means that the energy is increased compared to the wild-type. A value smaller 1 shows a decreased energy.
The result seems to almost a correct one. According to the energy levels all mutations except 8a occur less than the reference structure because they have an significant increased energy level. But it is not very clear, why mutation 8a shows a radical decreased energy level, which implies that this mutation will occur much more often in the population as the reference structure. This can not be correct and thus should be counted as an error by minimise.
FoldX
FoldX<ref>Joost Schymkowitz, Jesper Borg, Francois Stricher, Robby Nys, Frederic Rousseau, and Luis Serrano: The FoldX web server: an online force field</ref> 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
- cmd:
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.
The result seems to be a mixture of the result of SCWRL and Minimise. All mutations have an increased energy level, which means they do not occur as often as the reference. But again mutation 8a has the highest energy level, which seems to some strange behaviour associated with this mutation.
Gromacs
Gromacs<ref>David Van Der Spoel, Erik Lindahl, Berk Hess, Gerrit Groenhof, Alan E. Mark, Herman J. C. Berendsen: GROMACS: Fast, flexible, and free</ref> is a software suite for chemical simulations, developed at the University of Groningen in the early 1990s.
- We used the
-ignh
mode to ignore all hydroxen atom. - As forcefield, we chosed the AMBER03, CHARMM27 and AMBERGS model, the correspinding energie curves are shown below in Figure 3 to Figure 6.
Energy table for the AMBER03 forcefield.
Mutation | Total Energy Bond | Difference Bond | Total Energy Angle | Difference Angle | Total Energy Potential | Difference Potential |
---|---|---|---|---|---|---|
Wild-Type | 848,392 | 1 | 2707,42 | 1 | -32380,6 | 1 |
[M35T] | 774,332 | 0,912705447 | 2707,35 | 0,999974145 | -32738 | 1,011037473 |
[S65C] | 695,044 | 0,819248649 | 2699,65 | 0,997130109 | -32872,2 | 1,01518193 |
[I105T] | 0 | 0 | 0 | 0 | 0 | 0 |
[Q127H] | 835,05 | 0,984273779 | 2782,21 | 1,027624085 | -32213,2 | 0,994830238 |
[A176V] | 760,573 | 0,896487709 | 2734,66 | 1,010061239 | -33030,1 | 1,020058307 |
[T217I] | 727,061 | 0,8569871 | 2712,54 | 1,001891099 | -32609,5 | 1,007069048 |
[C282Y] | 851,692 | 1,003889711 | 2754,62 | 1,017433571 | -31431,1 | 0,970676887 |
[C282S] | 852,244 | 1,004540354 | 2706,07 | 0,99950137 | -32036,4 | 0,989370178 |
Wild-Type force field comparisson
Forcefield | Bond | Angle | Potetial |
---|---|---|---|
AMBER03 | 848,392 | 2707,42 | -32380,6 |
CHARMM27 | 1064,95 | --- | -37356,1 |
AMBERGS | 724.545 | 2785.47 | -40390.8 |
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 |
Energy for the Mutation [M35T]
Energy | Average | Err.Est. | RMSD | Tot-Drift (kJ/mol) |
---|---|---|---|---|
Bond | 774.332 | 300 | 2156.92 | -1864.67 |
Angle | 2707.35 | 16 | 130.232 | -51.7455 |
Potential | -32738 | 1100 | 3905.23 | -7119.34 |
Energy for the Mutation [S65C]
Energy | Average | Err.Est. | RMSD | Tot-Drift (kJ/mol) |
---|---|---|---|---|
Bond | 695.044 | 230 | 1873.95 | -1383.66 |
Angle | 2699.65 | 9.8 | 113.651 | -42.4234 |
Potential | -32872.2 | 890 | 3424.15 | -5775.47 |
Energy for the Mutation [I105T]
For this mutation, gromacs faild to calculate energies.
Energy for the Mutation [Q127H]
Energy | Average | Err.Est. | RMSD | Tot-Drift (kJ/mol) |
---|---|---|---|---|
Bond | 835.05 | 370 | -nan | -2189.82 |
Angle | 2782.21 | 20 | -nan | -94.6097 |
Potential | -32213.2 | 1100 | -nan | -7254.92 |
Energy for the Mutation [A176V]
Energy | Average | Err.Est. | RMSD | Tot-Drift (kJ/mol) |
---|---|---|---|---|
Bond | 760.573 | 290 | -nan | -1676.2 |
Angle | 2734.66 | 20 | -nan | -125.203 |
Potential | -33030.1 | 1000 | -nan | -6485.15 |
Energy for the Mutation [T217I]
Energy | Average | Err.Est. | RMSD | Tot-Drift (kJ/mol) |
---|---|---|---|---|
Bond | 727.061 | 260 | 1980.17 | -1567.84 |
Angle | 2712.54 | 12 | 119.206 | -50.0057 |
Potential | -32609.5 | 980 | 3633.19 | -6435.18 |
Energy for the Mutation [C282Y]
Energy | Average | Err.Est. | RMSD | Tot-Drift (kJ/mol) |
---|---|---|---|---|
Bond | 851.692 | 370 | 2500.48 | -2249.54 |
Angle | 2754.62 | 25 | 158.776 | -152.741 |
Potential | -31431.1 | 2100 | 16296.8 | -13588.8 |
Energy for the Mutation [C282S]
Energy | Average | Err.Est. | RMSD | Tot-Drift (kJ/mol) |
---|---|---|---|---|
Bond | 852.244 | 380 | 2424.32 | -2374.34 |
Angle | 2706.07 | 24 | 145.766 | -110.811 |
Potential | -32036.4 | 1200 | 4277.91 | -7896.74 |
Discussion
TODO: still missing..
Mutation 2 [M35T]
This mutation is a non damaging mutation. The energy level in all cases differ just a bit, but in all cases, we can see an increase of the energy level. While Methionine can form a sulfid bond, Threonine is able to form a hydrogen bond. Thus, the mutated amino acid can easly stabilized by the surrounding amino acids. And also, as this mutation is within a beta sheet a hydrogen bond can stabilize the sheet additionally. And as these mutation is a non damaging one, Mehtionin at position 35 is also not a functional residue.
Mutation 3 [S65C]
In this case, serin is changed to cystein. Compared to Mutation one, here an amino acid which can form a hydrogen bond is changed into one which is able to form a sulfur bond. As the mutation is also part of a beta sheet, a missing hydrogen bond destabilizes the structure. SCWRL evaluated this with a slightly decreas and the other tools with an increase. As the mutation is at the end of a beta sheet, it is possible, that the length of the sheet is just decreased, and the corresponding turn is increased in length.
Mutation 4 [I105T]
This mutation is part of a helix and while this mutation is first of all a change in size, the structure of the helix could be damaged by the mutation. The helix is bended and so the parallel helices are no longer parallel. And as we assmue this is a functional component like a binding site, the function of the protein is disturbed. This may also be the reason why Gromacs was not able to calculate the energy of this model.
Mutation 5 [Q127H]
A damaging mutation within a turn. Here a polar amino acid is exchanged into an aromatic one. Gromacs is the only tool which shows a decreasing energy. The other tools show an increasing energy. As this mutation is not within a structural element, and the mutation is a damaging one, the reason for the change in function is most likely either a functional residue or a change in the flexibility. As we have no information about functional residues, we assume a change in flexibility is the reason for the damage.
Mutation 6 [A176V]
Mutation 7
Mutation 8a
Mutation 8b
References
<references />