Task 9: Structure-based mutation analysis

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Lab_Journal_Hemochromatosis_Task9

Structure Selection

PDB ID	Res [A]	R-value (obs)	pH	missing residues	coverage
1A6Z	2.60	2.33	6.5	1-3	83.4%
1DE4	2.80	2.31	8.0	1-3	83.4%

From the two available structures, we chose 1A6Z, because it has a slightly higher resolution and a nearly identical resolution compared to 1DE4. On the downside, 1A6Z was resolved at a pH value of 6.5, which is more distant to the physiological pH than the resolution pH of 1DE4. 1A6Z was chosen nevertheless, because it was used in all previous tasks, in order to keep consistency.

Mutations

Mutation	Disease causing ?
Val53Met	Yes
His63Asp	Yes
Met97Ile	No
Thr217Ile	No
Cys282Tyr	Yes

<figtable id="mut_overview" >

Figure 1: The PDB structure 1A6Z of HFE_HUMAN with the MHC I domain colord in limegreen and the Ig C1-set domain colored in blue. The disease causing mutations are shown in red and the non-disease causing in orange. The cysteine, that is the disulfide bridge binding partner of C282, is colored in white.

</figtable>

Structure Mutation using SCWRL

Val53Met

<figtable id="53_mut">

Figure 2: Wild type(grey) and mutant(red) residues for position 53.

</figtable> Figure 2 shows that the mutation to Methionin does not change the polar contacts of the residue. But Methionin extends further into the binding pocket than Valin, which might disturb the structure of the binding pocket and inhibit the binding process.

His63Asp

<figtable id="63_mut">

Figure 3: Wild type (grey) and mutant (red) residues for position 63.

Regarding the polar contacts of residue 63, there is no change, because both variants do not exhibit polar interactions (Figure 3). Also, the mutation lies in a loop region and does not disturb an ordered secondary structure. But nevertheless, the mutation is disease causing, which might be due to the fact that the loop where it is located is still part of the binding interface to ferritin and that the change from an aromatic, mainly uncharged residue to a negatively charged residue disturbs this interface.

Met97Ile

<figtable id="97_mut">

Figure 4: Wild type(grey) and mutant(red) residues for position 97.

Both variants show the same polar contacts, which are only the intra-backbone hydrogen bonds that stabilize the alpha helix (Figure 4). The isoleucin is slightly smaller than methione, but the residue stays uncharged and nonpolar and thus, although it is located directly at the binding interface to ferritin, the mutatiion is neutral.

T217I

<figtable id="217_mut">

Figure 5: Wild type(grey) and mutant(red) residues for position 217.

While the change from a polar to a non-polar side chain reduces the number of hydrogen bonds at this loop and thus the stability of the loop (Figure 5), it is not enough to affect the function of the protein, because the loop is not near a binding interface or an essential structural part.

C282Y

<figtable id="282_mut">

Figure 6: Wild type(grey) and mutant(red) residues for position 282. The disulfide bridge binding partner of C282 is shown in white.

Although for this mutation, the polar contacts to the neighbouring beta sheet remain unchanged (Figure 6), it is evident why this mutation is the major cause for hemochromatosis. The replacement of cysteine with tyrosine causes a break of the disulfide bond that connects the two beta sheets of the Ig c1-set domain. Consequently, the structure of this domain is destabilized, which probably inhibits the formation of the HFE,ferritin,beta-micorglobulin complex. It is also noteworthy, that scwrl does not change the conformation of the cysteine binding partner, resulting in a clash of tyrosine and cysteine.

Comparison of SCWRL and FoldX

<figtable id="fx_scwrl" style="border-width: 0px">

Figure 7: Comparison between mutations performed by SCWRL(green) and foldx(cyan). For the C282Y mutation, the disulfide bridge binding partner is shown in white.

V53M: Practically, there is no difference between the rotamers generated by the two programs.	H63D: In this case, there is also nearly no difference between the placements of the programs.	M97I: As in the two cases before, the results do not differ significantly.
T217I SCWRL: For this mutation, the rotamer of the mutant differs, but this has nearly no effect, since the side chain sticks out of the protein and has no interaction partner inside of the protein. The major difference betwen the programs in this case is, that FoldX minimises the protein after a suitable rotamer has been found and thus changes the conformation of the surrounding residues. In this particular case, the minimisation results in a weakening or loss of the polar interactions with neighbouring residues.	T217I FoldX: See neighbouring image description.
C282Y SCWRL: In this case, the two rotamers do again not differ significantly, but the minimisation step from FoldX resolves the clash between the partner cysteine and the tyrosine.

Minimisation

Table 3: The energies after each minimisation iteration are shown for all mutations and the wildtype. For each minimisation series, the step that yields the lowest energy is marked in green.

Method	Mutation	Iter. 1	Iter. 2	Iter. 3	Iter. 4	Iter. 5
-	WT	-3724.2	-5003.5	-5118.4	-5198.3	-5301.4
scwrl	V53M	-5022.7	-5295.7	-5154.7	-5272.5	-5260.4
	H63D	-4940.5	-5212.8	-5084.4	-5190.7	-5189.6
	M97I	-5025.3	-5291.5	-5146.5	-5247.7	-5246.2
	T217I	-5037.7	-5307.9	-5171.54	-5277.0	-5269.3
	C282Y	-2596.7	-5107.7	-5037.12	-5159.1	-5191.7
foldx	V53M	-5323.9	-5544.4	-5450.0	-5377.1	-5436.9
	H63D	-5284.4	-5493.8	-5437.6	-5364.6	-5454.7
	M97I	-5264.6	-5482.1	-5405.7	-5343.1	-5255.8
	T217I	-5275.8	-5492.3	-5416.5	-5343.9	-5431.5
	C282Y	-3376.9	-5217.0	-5194.0	-5231.1	-5290.4

The results of the minimisation runs (Table 3), nearly all have their lowest energy after iteration 2. The only exception to this are the C282Y mutant structures and the wildtype structure. The reason for this probably is, that the local minimum is already reached after iteration 2 and the computation of the gradient at a local minimum points to a higher energy state. The method "overshoots" its target. In the case of two consecutive energy increases, the following reasoning might apply: The minimisation first finds one local minimum that it then leaves to reach a second local minimum. But from the second local minimum, it does not find its way back to the first one, but finds a third one with even higher energy instead. The wildtype and the C282Y mutant structures probably reach a local minimum only after the 5th iteration.

When comparing the energies of the mutant structures between the two programs after the first iteration, it stands out, that the energies of the FoldX mutants are always significantly lower than those of the SCWRL mutants. This is due to the additional minimisation step carried out by FoldX. Interestingly, the overall best energy of every FoldX mutant structure is lower than that of the corresponding SCWRL structure.

<figure id="282_min2">

Figure 8: The conformation of the C282Y mutants is shown after 2 iterations of minimisation. The SCWRL mutant strucure is shown in green and the FoldX structure in cyan.

</figure>

Figure 8 shows that the rotamers for the tyrosine and cysteine residues generated by FoldX and SCWRL are nearly identical after minimisation.

In conclusion, it can be said that FoldX produces better initial results than SCWRL, but after minimisation, there is nearly no difference between the two methods.