Structure-based mutation analysis Gaucher Disease

Cystral structure

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

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>

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 residues which are chosen from a rotamer library. <xr id="fig:scwrl"/> denotes the results.

1: H60R
2: V172I
3: E111K
4: L197P
5: W209R
6: L470P
7: W312C
8: A384D
9: D443N
10: R44S

Rotamers of SNPs from <xr id="tab:mutations"/>. Blue: wildtype; Red: rotamer SCWRL. </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.

Nr	Mutation	Wildtype		Mutatant		Clashes	Structural change
Nr	Mutation	H-bonds	Hydrophobicity	H-bonds	Hydrophobicity	Clashes	Structural change

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>

We further noticed that SCRWL changed the backbone at some positions which led to different secondary structure assignments (<xr id="fig:scwrl_ss"/>). The positions at which the deviations could be observed were independent from the mutated sites.

Seconary structure elements of 2nt0_A (grey) compared to secondary structure elements of models built by SCRWL.

</figure>

FoldX

The superposition of the rotamer configurations predicted by FoldX and SCWRL are shown in <xr id="fig:foldx"/>. The predictions of both tools differed in case of four mutations. In case of H60R, the side-chain orientation of arginine predicted by FoldX forms two instead one hydrogen bonds to T741 and might therefore impact the protein structure more the the orientation of SCRWL. In case of A384D, the romater of FoldX might be more stable than the one of SCWRL since it has a higher distance to the surrounding residues. In case of D443N we prefer the prediction of SCWRL which is closer to the wildtype configuration. For the same reason we prefer the prediction of FoldX in case of R442. For the subsequent GROMACS analysis, we hence chose the FoldX model in case of mutation number 8 and 10 and the SCWRL models for all all other mutations.

1: H60R
2: V172I
3: E111K
4: L197P
5: W209R
6: L470P
7: W312C
8: A384D
9: D443N
10: R44S