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.

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"/>.

<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

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

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?

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.

FoldX

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.