Structure-based mutation analysis Gaucher Disease

Cystral structure

<figtable id="tab:mutations">

PDB	Res [Å]	R value	Coverage	pH
2nt0	1.80	0.18	96% (40-536)	4.5
3gxi	1.84	0.19	96% (40-536)	5.5
2v3f	1.95	0.15	96% (40-536)	6.5
2v3d	1.96	0.16	96% (40-536)	6.5
1ogs	2.00	0.18	96% (40-536)	4.6

The 5 crystral structures of glycosylceramidase with the highest resolution. The physiological lysosomal pH value is 4.5. 2nt0 was selected for the analysis. </figtable>

Mutations

<figtable id="tab:mutations">

Nr	Pos P04062	Pos 2nt0_A	From	To	Disease causing
1	99	60	H	R	No
2	211	172	V	I	No
3	150	111	E	K	Yes
4	236	197	L	P	Yes
5	248	209	W	R	Yes
6	509	470	L	P	No
7	351	312	W	C	Yes
8	423	384	A	D	Yes
9	482	443	D	N	No
10	83	44	R	S	No

Mutations used for the structure-based mutation analysis. </figtable>

<figure id="fig:mutations">

2nt0_A with the selected mutations of <xr id="tab:mutations"/>. Blue: wildtype residues; Red: mutant residues; Orange: active site residues E235 and E340.

</figure>

SCWRL

We employed SCWRL <ref name="scwrl">Qiang Wang, Adrian A. Canutescu, and Roland L. Dunbrack, Jr.(2008). SCWRL and MolIDE: Computer programs for side-chain conformation prediction and homology modeling. Nat Protoc.</ref> for substituting the wildtype residues listed in <xr id="tab:mutations"/> by the corresponding mutatant residue which is chosen from a rotamer library. <xr id="fig:scwrl"/> denotes the results.

<figure id="fig:scwrl">

• 

  1: H60R

• 

  2: V172I

• 

  3: E111K

• 

  4: L197P

• 

  5: W209R

• 

  6: L470P

• 

  7: W312C

• 

  8: A384D

• 

  9: D443N

• 

  10: R44S

SNPs of <xr id="tab:mutations"/> introduced by SCWRL. Blue: wildtype residue; Red: mutant residue. </figure>

None of rotamers chosen by SCWRL clashed with another side-chain or the backbone. We further could not observer changes in the secondary structure or the formation cavities. The hydrogen bonding network changed in case of mutation number 1, 5, 7, and 8 (cf. <xr id="tab:scwrl"/>). W209R introduces a hydrophilic arginine which forms a hydrogen bond to T180. Although not predicted by SCWRL, the arginine might impact the protein structure. W312C is located next to the active site (cf. <xr id="fig:mutations"/>) and there exists a hydrogen bond to E340. Substitution the hydrophobic tryptohphane by a hydrophlic cysteine in the vicinity of the active site might account for the disease-causing effect of this mutation.

<figtable id="tab:scwrl">

Nr	Mutation	Wildtype		Mutatant		Clashes	Structural change
		H-bonds	Hydrophobicity	H-bonds	Hydrophobicity
1	H60R	T471	Hydrophilic	G62	Hydrophilic	No	No
2	V172I		Hydrophobic		Hydrophobic	No	No
3	E111K		Hydrophilic		Hydrophilic	No	No
4	L197P		Hydrophobic		Hydrophobic	No	No
5	W209R		Hydrophobic	T180	Hydrophilic	No	No
6	L470P	T482	Hydrophobic	T482	Hydrophobic	No	No
7	W312C	E340, C342, P316	Hydrophobic	E340, C342	Hydrophilic	No	No
8	A384D		Hydrophobic	V404	Hydrophilic	No	No
9	D443N		Hydrophilic		Hydrophilic	No	No
10	R44S	S13, Y487	Hydrophilic	S13, Y487	Hydrophilic	No	No

Structure-based analysis of SNPs from <xr id="tab:mutations"/>. H-bonds: residues involved in forming hydrogen bonds (cut-off: 3.2 Å). </figtable>

References

<references/>