Structure-based mutation analysis TSD

No citations, I don't read THAT many books :P - Have some music instead ;)

The journal of this task can be found here here.

Structure preparation

<figtable id="tbl:struct_comp">

Table 1: Comparison of experimental parameters for the two resolved structures of UniProt entry P06865. The parameters are important for choosing the structcture used for the structure based mutation analysis. Coverage is the sequence residues covered by the structure according to UniProtKB. This however is based on the SEQRES data. It should be noted that there is an unresolved region (residues 75 to 88) in both structures.

PDB-ID:Chain	Coverage	Resolution (Å)	R-value	R-free	pH
2gjx:A	23-529	2.8	0.270	0.288	5.5
2gk1:A	23-529	3.25	0.277	0.322	5.5

</figtable>

There are two structures resolved for the HEXA_HUMAN reference sequence used for the course of this practical. The struture of this Uniprot entry is found in the alpha-chains of the PDB-IDs and 2gjx and 2gk1 both form the same publication <ref name="hexa_pdb_ref">Lemieux, M., Mark, B., & Cherney, M. (2006). Crystallographic Structure of Human beta-Hexosaminidase A: Interpretation of Tay-Sachs Mutations and Loss of GM2 Ganglioside Hydrolysis. Journal of molecular biology, 359(4), 913-29. doi:10.1016/j.jmb.2006.04.004</ref>. Unfortunately a 14 residue stretch towards the N-terminus of the protein (residues 75 to 88) is unresolved in both structures. However as previously shown the alpha subunit of Hex A consists of two domains. The N-terminal domain, Glyco_hydro_20b, is not involved in catalysis. Therefore, to evade problems with the missing backbone in the course of this task, the structure was truncated to contain only the C-terminal, catalytic domain, Glyco_hydro_20. In concordance with previous tasks and based on the experimental data in for both structures, shown in <xr id="tbl:struct_comp"/>, 2gjx was chosen as the reference structure. Details on the alteration of the structure according to the measures described above can be found in the journal.

TODO mention, that this unforntunately does not include the (pseudo-)ligand and we therefore have to make assumptions on the hydrogen bonds...

Mutations

As only disease causing SNPs were assigned, the week before, some mutations had to be replaced. Additionally the reference PDB structure limited the possibilities, as only the second domain was retained for the following analysis. Thus an almost new set of SNPs was chosen: R178H, R178C, P182L, D207E, S293I, F434L, L451V, E482K, L484Q, E506D. Their position in the 3D-structure of HEXA is shown in <xr id="fig:snpsOnstr"/>.

<figure id="fig:snpsOnstr">

SNPs highlighted on the HexA subunit structure. The mutations are displayed in red and the cut out domain is displayed in grey. The active site is displayed in orange and the important residues in yellow.

</figure>

The chosen mutations are displayed in the context of the remaining domain, the active site and important residues. For a detailed description of the important residues, see introduction . There are 5 mutations occurring in loop regions and 5 in helix elements. One intersection between mutations and important residues is at position 207 where there is a mutations from D to E. At the other important residue within the mutations, namely position 178 two mutants were chosen for the SNP set. Mutations of the important residues are expected to have a severe effect on the protein function.

For the new mutation set the biochemical properties are listed in <xr id="tab:biochem"/>. They have been assembled according to the sequence based mutation analysis.

<figtable id="tab:biochem">

Table 1: Biochemical properties

Mutation	Wildtype			Mutant			Grantham score
	Hydrophobicity	Volume	Charge	Hydrophobicity	Volume	Charge
R178H	-4.5 (polar)	173.4 (bulky)	positive	-3.2 (polar)	153.2 (bulky)	neutral	29
R178C	-4.5 (polar)	173.4 (bulky)	positive	2.5 (polar)	108.5 (small)	neutral	180
P182L	-1.6 (nonpolar)	112.7 (small)	neutral	3.8 (nonpolar)	166.7 (bulky)	neutral	98
D207E	-3.5 (polar)	111.1 (small)	negative	-3.5 (polar)	138.4 (bulky)	negative	45
S2931	-0.8 (polar)	89.0 (tiny)	neutral	4.5 (nonpolar)	166.7 (bulky)	neutral	142
S434L	2.8 (nonpolar)	189.9 (bulky)	neutral	3.8 (nonpolar)	166.7 (bulky)	neutral	22
L451V	3.8 (nonpolar)	166.7 (bulky)	neutral	4.2 (nonpolar)	140.0 (small)	neutral	32
E482K	-3.5 (polar)	138.4 (bulky)	negative	-3.9 (polar)	168.6 (bulky)	positive	56
484	3.8 (nonpolar)	166.7 (bulky)	neutral	-3.5 (polar)	143.8 (bulky)	neutral	113
E506D	-3.5 (polar)	138.4 (bulky)	negative	-3.5 (polar)	111.1 (small)	negative	45

</figtable>

Map all the 10 mutations onto the crystal structure. Color the mutants differently than the rest of the protein and create a snapshot for the wiki. If applicable find out whether the mutations are close to the active site, a binding interface or other important functional sites. Visualize this and describe it properly.

Molecular Mechanics

<figtable id="tbl:pymolstruc">

Overview of all SNPs: Comparison wildtype and mutant structure. The wildtype is colored light green (or if it is a important residue yellow respectively), the mutant red and polar contacts are displayed in blue.

R178H	R178C	P182L	D207E	S293I
F434L	L451V	E482K	L484Q	E506D

</figtable> Since these are only the manually chosen side chain conformation, selected from the ones given by PyMOL, most of the analysis can be found in the sections below based on the predictions by SCWRL and FoldX. Here a few general things:

--

In the following, compare wild type (WT) and mutant structures.

Now that you should have a clear idea of the WT and mutant proteins we will try to calculate some energies. Always calculate the energy for the wild type and mutants – then substract/compare.

SCWRL and FoldX

<figtable id="tbl:pymolstruc">

Table TODO: Overview of all SNPs in the 3D-structure. Compared are the wildtype as well as the SCRWL and FoldX results on the mutation. The wildtype is colored light green (or if it is a important residue yellow respectively), the SCRWL modelling purple and the foldX modelling cyan. The polar contacts are displayed in blue.

R178H	R178C	P182L	D207E	S293I
F434L	L451V	E482K	L484Q	E506D

</figtable>

R178H/C

R178 is one of the important residues. As can be seen in the according figure in <xr id="tbl:pymolstruc"/> it could form hydrogen bonds with the neighbouring residues D207 and E462. In addition it seems reasonable that these two neighbouring residues with the negative charges somehow coordinate the positive charges placed between them. More importantly R178 has already been shown to form a hydrogen bond with the ligand.

The mutant amino acid histidine, depending on the pH value, can also carry a positive charge. The reported optimum pH value for Hex A is around 4.4, in these conditions, histidine is sure to contain a positive charge <ref name="phforhexa">Grebner,E.E. et al. (1986) Two abnormalities of hexosaminidase A in clinically normal individuals. American journal of human genetics, 38, 505-14.</ref><ref name="histcharge">when is his pos charged, alberts TODO</ref>. However the larger problem under is that under these conditions the formation of a hydrogen bond with the ligand is very unlikely. This is reinforced by the recessed positioning of the residue relative to the active site. Even for the case of SCWRL (purple), where the hydrogen is included (and the ring flipped compared to FoldX), the distance could already be too long for coordination of the ligand. There are slight clashes of both the SCWRL as well as the FoldX structures with D175 however these are very minor, fixable and clearly not the main problem here.

For the mutation to cysteine, the problem is much more pronounced. There is no doubt that formation of the hydrogen bond to the ligand is not possible anymore due to the increase in distance. Clashes do not exist, but would actually be hard to create in any case.

In conclusion the modelling of both mutation side chains is mostly as good as possible and shows only minor differences between the two methods. The orientation of the histidine ring is however more convincing in the SCWRL result.

P182L

<figure id="fig:p182surface">

Surface conditions in the reference structure near wildtype residue P182. The Hex A alpha subunit (2gjx:A) is shown in green, the beta subunit (2gjx:B) in blue. P182 is highlighted in orange. As can be seen, there is no interaction between the two subunits at the position of interest and the mutation to leucine should not have an effect dimer formation.

</figure> P182 is on the surface of the Hex A alpha subunit and proline act as a helix breaker. Breaking of the helix however is mandatory in this case, since the ensuing loop continues straight towards the active site, which is orthogonal to the direction of the alpha-helix. If a mutation of proline should lead to an elongation of the alpha-helix this would very likely have an effect on the protein's function since R178, found in the following loop, could not be placed accurately any more. However the mutation to leucine must not necessarily lead to this and as such is not necessarily disease-causing.

Another consideration should be made about the changed protein surface, which is slightly larger with leucine at position 182. This is the side of alpha subunit, which interacts with the beta-subunit of Hex A and is therefore important to consider, however, as <xr id="fig:p182surface"/> shows, there is a cavity at this position and the change to leucine should not have an effect on the interaction of the subunits.

There are differences between the sidechain placement in FoldX and SCWRL, however under the given surrounding conditions and due to the short sidechains none of the two can be considered better than the other. The only notable difference is that the small clashes seem to be better handled in the placement by SCWRL.

D207E

<figure id="fig:d207ishard">

Local packing in the reference structure near wildtype residue D207. Shown as spheres are, from left to right, H262, D027 and R178. The mutation D207E elongates the sidechain to a degree where it cannot be placed anywhere anymore without creating clashes or loosing H262 as a protein donor.

</figure> D207 is another residue considered important, since it forms a hydrogen bond with the "substitute ligand" NGT (c.f. important residues), however this is the same atom that is also reached by R178. The original publication of the crystal structure suggests, that R178 forms hydrogen bond with the ligand, while D207 interacts with the neighbouring residue H262, that acts as protein donor.

The mutant amino acid glutamate has chemical features very similar to the ones of the wildtype, which means that the interaction with histidine is in theory still possible. However the sidechain is longer which in this particular case is very problematic. The side chain placement of FoldX is basically an elongation of the wildtype. While this keeps the charged group close to the H262 there is clearly a clash with R178 that is not easily resolved. SCWRL places the side chain further away, which better evades the clashes, however to fully resolve them a further tilt of the charged group would bring into to close proximity to another neighbouring histidine. Additinally this placement is too far away from H262 to allow any interaction. As can be seen in <xr id="fig:d207ishard"/>, the space usage in the wildtype is near-perfect and there seems to be no choice for a functional placement of the mutant without introducing larger changes in the backbone that might have strong effects on the surrounding residues. Given that this is in the active site, the effects would likely be disease-causing.

E506D

Investigate the local hydrogen-bonding network using pymol[1] – also check for potential clashes (when sidechains are too close to each other). Are you introducing hydrophilics to the core or hydrophobics to the protein surface? Are there any holes introduced to the  protein due to the mutations?
Foreach of the mutations also a new structure will be created. Note down all of the energies, but also use these structures in the next steps.

Compare the scwrl and foldx structures in Pymol and superimpose them. What are the differences?

Minimise

 What happens regarding the energy? 

Gromacs

Analyze the minimization of the system with the following command: g_energy -f FILE.edr -o energy_1.xvg. Do the analysis for Bond, Angle and Potential. The xvg graphs can be viewed with xmgrace and in the print settings you can choose eps output, the print and convert to pdf.