Structure based mutation analysis of GBA

Introduction

A detailed list of the ten mutations analyzed in this section, can be found in Task 6.

The different tools and the corresponding steps, applied in this analysis are described in this workflow for reasons of clarity.

Structure Selection

To carry out a structure-based analysis of the mutations chosen in Task 7 a crystal structure had to be chosen. According to Uniprot 19 different crystal structures of glucocerebrosidase exist. The table below shows the six different structures with a resolution of or better than 2 Angstrom. 2NT0 is chosen as template for the analysis carried out in this section, as no residues are missing, the R-value is quite low, and it has the best resolution among the structures without missing residues. Only incomplete structures have been resolved near the physiological pH (7.4), therefore a structure resolved at a more acid pH had to be chosen. The structure can either be downloaded from the PDB website or by using the script fetchpdb, which validates the ID and downloads the corresponding structure.

PDB ID	Resolution [Å]	R-factor	Coverage	pH	# Missing Residues (A/B)
1OGS	2.00	0.195		4.6	0
2NT0	1.79	0.181	.	4.5	0
2V3D	1.96	0.157		6.5	9/8
2V3E	2.00	0.163		7.5	7/7
2V3F	1.95	0.154		6.5	8/14
3GXI	1.84	0.193		NULL	0

Mutation Mapping

The ten positions at which the mutations analyzed in this task take place, are hilighted in the structure of 2NT0 shown in Figure 1. As already mentioned in Task 5 and 6, one can clearly see that two mutations are next to the active site residues Glu235 and Glu340, namley the positions 120 and 311. The wildtype residues at these positions (Arg120 and His311) are known to form hydrogen bonds with the active sites and should therefore be quite important for function and structure. <ref>Kim et al., Crystal Structure of the Salmonella enterica Serovar Typhimurium Virulence Factor SrfJ, a Glycoside Hydrolase Family Enzyme. Journal of Bacteriology, 2009, p. 6550-6554, Vol. 191, No. 21 </ref> The other eight mutation positions are located all over the protein.

Figure 1: 2NT0 with hilighted mutation positions (red) and active site residues (blue).

Figure 2: Close-up of active site of 2NT0 with hilighted mutation positions (red) and active site residues (blue).

SCWRL

SCRWL4 was applied ten times, once for each mutation. The resulting conformations of the mutants are visualized in Figure 3. For this representation the hydrogens of the SCWRL mutant structures have been removed to simplify the comparison with the other two structures. Figure 3 additionally shows the wildtype amino acids and the mutants created with the mutagenesis method of pymol. The conformations, created with SCWRL4 and pymol vary greatly. Only in mutation 9 they seem to be quite similar. Figure 4 shows a superposition of the wild type protein and the mutated proteins in cartoon representation. This shows that SCWRL did not only change the mutant residues, but also changed some beta sheets at the bottom of the structure (shown in green). This fact may be due to the different polar interactions of the mutants.

Figure 3: Wildtype amino acids (red) and mutations created with SCWRL (green) and pymol mutagenesis (orange) hilighted on the structure of 2NT0.

Figure 4: Cartoon representation of 2NT0, chain A (gray) superimposed with the resulting structures of SCWRL (green).

The table below shows the residues forming polar interactions with the mutant/wild type amino acid. The polar interactions can be seen in Figure 3 as well, but one may not clearly distinguish, whether an interaction is only formed by e.g. the wild type or by wild type and mutant. To determine the interaction partners, the tool Pymol was used [actions -> find -> polar contacts -> to other atoms in object].

Mutation	Wild Type	Pymol	SCWRL
1	S8	S8 F9	S8
2	K413 Y418	Y22	K413 Y418
3	T471		G62
4	G83 M85 S177 D282 N234 E340	G83 M85	G83 M85 S177 S339
5	S181 L185	S181 L185	S181 L185
6	L268 H273 H274	L268 H273 H274	L268 H274
7	S366 Y373 V375	S366 Y373 V375	S366 Y373 V375
8	D282 D283 R285 S339	D283 R285	D282 R285 Y313 E340
9	L420	L420	L420
10	T482	T482 V468	T482 Minimise Gromacs Energy Comparison SCWRL Mutation 1 2 3 4 5 6 7 8 9 10 Minimal Energy 351.329 348.659 350.017 355.416 473.454 364.148 352.615 362.604 354.98 375.976 FoldX The total energies calculated with FoldX are shown in the table below. The differences between the wild type and the different mutant structures have been calculated and are listed in the table as well. Mutation Total Energy Difference WT -372.60 0 1 -225.82 -146.78 2 -228.18 -144.42 3 -226.97 -145.63 4 -226.38 -146.22 5 -196.84 -175.76 6 -224.01 -148.59 7 -228.48 -144.12 8 -217.29 -155.31 9 -221.71 -150.89 10 -218.65 -153.95 Minimise Gromacs Discussion