Difference between revisions of "Structure based mutation analysis of GBA"

Revision as of 14:32, 14 August 2011

Introduction

After the sequence based mutation analysis, as described in Task 6, a structure based mutation analysis of ten mutations (listed in the table below) was carried out. For the analysis, several different tools have been used. TThe different tools and the corresponding steps, applied in this analysis are described in more detail in this workflow for reasons of clarity.

Nr.	SNP ID/Accession Number	Database	Position including SP	Position without SP	Amino Acid Change	Codon Change
1	CM081634	HGMD	49	10	Gly - Ser	cGGC-AGC
2	rs74953658, CM050263	dbSNP, HGMD	63	24	Asp - Asn	tGAC-AAC
3	rs1141820	dbSNP	99	60	His - Arg	CAC - CGC
4	CM880035	HGMD	159	120	Arg - Gln	CGG-CAG
5	rs80205046, CM041347	dbSNP, HGMD	221	182	Pro - Leu	CCC - CTC
6	rs74731340, CM970620	dbSNP, HGMD	310	271	Ser - Asn	AGT - AAT
7	CM880036	HGMD	409	370	Asn - Ser	AAC-AGC
8	CM993703	HGMD	350	311	His - Arg	CAT-CGT
9	rs80020805, CM052245	dbSNP, HGMD	455	416	Met - Val	cATG-GTG
10	rs113825752	dbSNP	509	470	Leu - Pro	CTT - CCT

Structure Selection

To carry out a structure-based analysis of the mutations chosen in Task 6, a crystal structure had to be chosen. According to Uniprot there are 19 different crystal structures of glucocerebrosidase available. 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

Figure 1 shows the positions of the analyzed mutations in the original structure of 2NT0. 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 mutations at 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. The amino acid properties of the different mutant and wildtype structures have already been analysed in Task 6.

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).

Minimise

Figure 5 shows the interesting positions with hilighted mutants and wild type residues of the pdb files obtained with Minimise after having applied the steps as indicated in the workflow. The hydrogens of the structures have been removed as well.

Figure 5: Wildtype amino acids (red) and mutations (green) created with Minimise. Polar interactions are shown with dotted lines.

Gromacs

Figure 6 shows the interesting positions with hilighted mutants and wild type residues of the pdb files obtained with Gromacs after having applied the steps indicated in the workflow. The hydrogens of the structures have been removed as well.

Figure 6: Wildtype amino acids (red) and mutations (cyan) created with Gromacs. Polar interactions are shown with dotted lines.

Polar Interactions

Polar interactions are crucial for the structue, and therefore function of a protein. A mutation of a single amino acid could therefore alter the features and appearance of a protein. Analyzing polar interactions may therefore help to determine whether a mutation is damaging or neutral. If the polar interactions, which are present in the wild type structure, are still formed after the mutation, the mutation may be tolerated, otherwise it should be damaging.

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, Figure 5 and Figure 6 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	PDB Wild type	Pymol Mutagenesis	SCWRL	Minimise		Gromacs
Mutation	PDB Wild type	Pymol Mutagenesis	SCWRL	Wild Type	Mutation	Wild Type	Mutation
1	S8	S8 F9	S8	S8	S8	S8	S8
2	K413 Y418	Y22	K413 Y418	K413 Y418	K413 Y418	K413 Y418	K413 Y418
3	T471		G62	T471	G62	T417
4	G83 M85 S177 D282 N234 E340	G83 M85	G83 M85 S177 S339	G83 M85 S177 D282 N234 E340	G83 M85 S177 S339	G83 M85 S177 D282 N234 E340	G83 M85 S177
5	S181 L185	S181 L185	S181 L185	S181 L185	S181 L185	S181 L185	S181 L185
6	L268 H273 H274	L268 H273 H274	L268 H274	L268 H273 H274	L268 H274	L268 H273 H274	L268 H274 T267
7	S366 Y373 V375	S366 Y373 V375	S366 Y373 V375	S366 Y373 V375	S366 Y373 V375	S366 Y373 V375	S366 Y373 V375
8	D282 D283 R285 S339	D283 R285	D282 R285 Y313 E340	D282 D283 R285	D283 R285 Y313 E340 N234 E235	D282 D283 R285 S339	D283 R285 Y313 E340
9	L420	L420	L420	L420	L420	L420	L420
10	T482	T482 V468	T482	T482	T482	T482	T482

Mutations 1, 2, 5, 7, 9 and 10 show in most cases (SCWRL, Minimise, Gromacs) the same polar interactions as the wild type and should therefore not have a big influence. The other mutations (3,4,6 and 8) form however additional bonds or miss some interactions which are present in the wild type. Interestingly, the most common mutation found in Gaucher Disease forms the same polar interactions as the wildtype.

Clashes or Holes

Furthermore it was analyzed whether the mutations cause clashes or holes in the protein structure. The following table shows whether the mutations created/minimized with Pymol, SCWRL, Minimise and Gromacs lead to structural inconsistency compared to the wildtype glucocerebrosidase (2NT0).

Mutation	Pymol Mutagenesis	SCWRL	Minimise	Gromacs
1	no but different surface	no	no	no but different surface
2	no	no	no	no
3	no	no but different surface	no but different surface	no
4	no	no	no	no but different surface
5	no	no	no	no
6	no but different surface	no but different surface	no but different surface	no but different surface
7	no	no	no	no
8	no	no but different surface	no	no
9	no	no but different surface	no	no but different surface
10	no	no but different surface	no but different surface	no

None of the mutations lead to clashes or holes in the protein structure, but some of the mutations cause a different surface. Mutation 6 leads to a different surface, no matter whith which tool they were created or minimized. This is quite interesting as Serine and Asparagine are structurally and chemically very similar. Regarding the polar interactions, they are not identical.

Overall one would assume that the surface od the resulting protein is changed if a mutation leads to different polar interactions and if the mutations takes place inside a structural element. Mutation 1 leads to a different surface when build with Pymol Mutagenesis and Gromacs. The mutated residue is part of a secondary structural element, the mutated amino acid has a different polarity than the wildtype and the Pymol Mutagenesis build forms an additional hydrogen bond. These facts could explain the different surface. Mutation 2 does not lead to a different surface. This is quite interesting regarding the Pymol Mutagenesis Build, as this one forms completely different hydrogen bonds than the wildtype. The fact that the mutated residue is not part of a secondary structural element and that Asparagine and Aspartic Acid have a similar molar mass could be an explanation for the not altered surface.