Structure-Based Mutation Analysis Hemochromatosis
Riddle of the task
It took you over an hour to figure out the right combination, but the door is finally open. The sight is unbelievable. Inside the nex room lies a treasure beyond imagination: heaps of gems, gold, and jewelry. Exotic furs, marvelous paintings, and many more. You step inside to collect what should now be yours...
The moment you reach out for the first piece of treasure it vanishes into thin air. ALL of it. The treasure was just an illusion... You look around and see another entrance into the room. A collapsed one. Across the room is a person, kneeling before another door. You shout... No answer. He didn't even move. As you get closer to him you see that, whoever it was, is dead. His skin mummified due to the dry air. Next to him an old leathery backpack. You reach out to take it as you notice small fragments on the floor. They look like tiny bits of red glass. Now that you're in front of him you also see many of these splinters burried inside the person's flesh. Within the backpack you find several glass orbs: a blue one, a yellow one, a green one, an orange one, a cyan one, and a violet one. Infront of the dead man, at the bottom of the door, you notice three slots. Each of them about the size of the orbs. One of them is red, the second one orange, and the third one yellow...
Short task description
Detailed description: Structure-based mutation analysis
A protocol with a description of the data acquisition and other scripts used for this task is available here.
Structure selection and mapping of the mutations
There are only two structures available for HFE at PDB: 1a6z and 1de4. We chose 1a6z for this task as it has the better resolution (2.6 Å instead of 2.8 Å) and has only a beta-2-microglobulin in addition to HFE. In 1de4 HFE would be complexed with transferrin receptor (TFR). All of the mutations from the previous task (M35T, V53M, G93R, Q127H, A162S, L183P, T217I, R224W, E277K, and C282S) are included in the PDB structure (residues 26-297).
<xr id="mut_map"/> shows a three dimensional mapping of the mutations (red) onto 1a6zC. Glycosylation sites (cyan) and disulfide bonds (orange) are also indicated. The only such residue that is directly affected by a mutation is the disulfide bond spanned by C225 and C282 where C282 is mutated into Serine. Though Q127H, L183P, and R224W are quite close to the glycosylation site at 130 and the two disulfide bonds (C124-C187, C224-C282) and therefore might affect them indirectly.
SCWRL and FoldX
In order to analyze the effects of the mutations we have created several models with SCWRL and FoldX. These models were then superimposed onto the reference structure (1a6zC). Our analysis included changes in the hydrogen bonds, rotamer rearrangements, and surface changes (unless burried within the protein). The color codes in the following section are:
- green: reference (1a6zC)
- cyan: SCWRL wildtype
- magenta: SCWRL mutant
- orange: FoldX wildtype
- red: FoldX mutant
An overview table containing all energy values for the models and their wildtypes can be found here.
- SCWRL energy (norm.): 17.720
- FoldX energy (norm.): 7.744
The wildtype of M35T is part of a beta sheet complex in the MHC I domain and spans two hydrogen bonds to a neighboring beta sheet (cf. <xr id="M35T_pymol"/>). Both of these hydrogen bonds are preserved in the mutant model (SCWRL and FoldX). The FoldX model uses a slighty different rotamer, though, which enables it to form an additional hydrogen bond to the previous residue. This might cause an increased stability over the wildtype. The changes to the surface due to the mutation are only minor and should not cause any problems. The surface model also shows that FoldX uses a slighty different rotamer for the wildtype model. Even the energy values indicate only minor changes in the whole model. Therefore this mutation should be considered non disease causing.
- SCWRL energy (norm.): 70.349
- FoldX energy (norm.): 3.705
V53M marks the transition of a beta sheet into a turn within the MHC I domain and forms two hydrogen bonds to the beginning of the next beta sheet (cf. <xr id="V53M_pymol"/>). The mutant models both retain these bonds and do not form additional ones either. As this residue is burried within the protein, there are no changes to the surface, but the residue loses its strong hydrophobic character which would force it into the protein. Even though the rotamers used by SCWRL and FoldX differ only slightly the difference in the energy model is quite huge. While FoldX would not indicate that V53M is disease causing, SCWRL's energy change does so.
- SCWRL energy (norm.): 16.141
- FoldX energy (norm.): -3.772
G93R lies within a big alpha helix in the MHC I domain (cf. <xr id="G93R_pymol"/>). The important three hydrogen bonds for the helix stabilization are conserved in both mutant models. In the FoldX model an additional hydrogen bond within the helix structure is formed. While these changes seem harmless at first, it should also be noted that this region is supposed to be the interface for the TFR-HFE complex. This makes the changes to the surface even more severe than they would seem on their own. The much bigger arginine causes a massiv bulk on the surface which is very likely to interfere with the complex formation. Therefore this mutation should be considered disease causing, even if the energy models do not suggest this.
- SCWRL energy (norm.): 17.911
- FoldX energy (norm.): -3.676
Q127H is at the start of a coil/turn between two beta sheets in the MHC I domain (cf. <xr id="Q127H_pymol"/>). While both mutant models retain the two hydrogen bonds that stabilize this coil/turn, the FoldX model forms even an additional one, they both lose a hydrogen bond which connects Q127 and E125. Thus the indirect anchor to the previous beta sheet is lost. This might not be that severe, but one of the connected amino acids marks the glycosylation site N130 (connected by the lower hydrogen bond in the figures). With this in mind this mutation should be considered disease causing. Like in the previous mutation this is contrary to the energy models.
- SCWRL energy (norm.): 38.099
- FoldX energy (norm.): 6.643
A162S is part of a helix in the MHC I domain (cf. <xr id="A162S_pymol"/>). All wildtype hydrogen bonds are preserved in the mutant models and several new ones are formed (3 in SCWRL and 4 in FoldX). This should further stabilize the structure. Additionally the residue is buried within the protein and thus causes no changes on the surface. Even the size of the wildtype and mutant amino acids does not differ much. The only indicator for a malign mutation would be the energy change in the SCWRL model, but this has proven to be quite unreliabe in the previous mutations. Therefore this mutation should be considered non disease causing.
- SCWRL energy (norm.): 0.284
- FoldX energy (norm.): 33.996
L183P is, again, located in one of the MHC I domain's helices. Proline's effect as a helix breaker is demonstrated in <xr id="L183P_pymol"/>. Both stabilizing hydrogen bonds are lost and no new ones are formed. As mentioned before this region is interface for the TFR-HFE complex and therefore a break in one of the three big helices should be considered to be disease causing, even though this particular residue is not on the surface or the protein. This is also the first FoldX model to show a big energy change. Maybe FoldX's energy model is a better indicator than SCWRL's.
- SCWRL energy (norm.): 48.103
- FoldX energy (norm.): 9.312
- Warning: Highly hydrophobic amino acid on the surface!
- SCWRL energy (norm.): 53.506
- FoldX energy (norm.): 2.104
- SCWRL energy (norm.): 122.406
- FoldX energy (norm.): 25.854
- SCWRL energy (norm.): 34.746
- FoldX energy (norm.): 46.690
Next we used Minimise to minimize (lame pun...) the energy for each of the 31 models created with SCWRL (10 mutations + WT) and FoldX (10 mutations and wildtypes). Each model was consecutively minimized five times (i.e. the output from the previous iteration was used as input for the next one). A table with the absolute energy values can be found here.
The median energy change per iteration in relation to the first iteration is shown in <xr id="energy_gain"/>. It clearly demonstrates that too many iterations not only fail to improve the model, but make it even worse. For the FoldX models only the second iteration makes the models better, every iteration thereafter makes the models worse than they were after the first one. The SCWRL models stop to improve after the third iteration. After the fifth iteration they are about as good as after the first iteration.
title = PBSA minimization in vacuum cpp = /usr/bin/cpp define = -DFLEXIBLE -DPOSRES implicit_solvent = GBSA integrator = steep emtol = 1.0 nsteps = 500 nstenergy = 1 energygrps = System ns_type = grid coulombtype = cut-off rcoulomb = 1.0 rvdw = 1.0 constraints = none pbc = no
We used this .mdp file for evaluating all energies. For more information regarding the arguments read this.
The output of g_energy can be seen in the following pictures. These are only for chosen models. The total amount of pictures can be found here. The final energies can also be found on that page.