Fabry:Homology based structure predictions

Fabry Disease » Homology based structure predictions

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The following analyses were performed on the basis of the α-Galactosidase A sequence. Please consult the journal for the commands used to generate the results.

Dataset preparation

<figtable id="tab:datasetHHpred"> Dataset HHpred

pdb ID	E-value	Identity in %
> 80% sequence identity
3hg3	8.6e-90	100
40% - 80% sequence identity
1ktb	4.2e-85	53
< 30% sequence identity
3cc1	5.5e-74	25
1zy9	3.1e-48	13
3a24	7.8e-40	17
2xn2	5.3e-37	15
2d73	5.7e-36	14
3mi6	1.4e-31	15
2yfo	9.1e-30	13
2f2h	2.7e-20	17
2g3m	2.2e-20	16
3nsx	6e-20	13
3lpp	2.2e-18	15
3l4y	1.9e-18	15
3top	3.6e-18	12
2xvl	3.2e-18	16
2x2h	4.9e-16	13

</figtable>

<figtable id="tab:datasetCOMA"> Dataset COMA

pdb ID	E-value	Identity in %
> 80% sequence identity
-	-	-
40% - 80% sequence identity
1ktb	1.7e-61	52
< 30% sequence identity
3lrk	1.2e-66	23
3a21	2.7e-65	26
1szn	3.7e-59	22
3cc1	5.2e-58	19
1zy9	1.7e-39	9
3mi6	4.3e-38	11
2yfn	4.4e-35	10
2d73	1.9e-32	9
3a24	5.6e-30	10
1xsi	1.9e-12	10
2g3m	2.4e-11	10
3pha	2.9e-10	6
3lpo	4.7e-09	8
2x2h	8.2e-09	8
3mo4	1.2e-08	7
2xvg	2.4e-08	8
3ton	4.3e-08	8
2xib	1e-07	7
3eyp	1.6e-06	8
3k1d	3.5e-06	9
2zwy	8.8e-06	9
3gza	1.8e-05	8
3m07	2.3e-05	7
1eh9	0.00013	6
1gvi	0.00035	8
1aqh	0.00039	5
1mwo	0.00058	7
3vmn	0.0018	9
1bf2	0.0019	6
3aml	0.0019	8

</figtable>

We performed a HHpred as well as a COMA search, to generate three distinct datasets. Since COMA did not find any homologue structures with a similarity above 41% (see <xr id="tab:datasetCOMA"/>), we used the dataset created with the HHpred search and the script described in the journal. Hereby we found one structure with a similarity above 80%, one with a similarity between 40 and 80% and 15 with sequence similarity below 30%, of which 14 had a similarity of under 20% (see <xr id="tab:datasetHHpred" />). All HHpred matches had an E-value below 1e-15, for the COMA homologues we tried a less strict threshold of 0.002.
In most of the cases we used the structures 3hg3, 1ktb and 3cc1 for modelling, because either they are the only representatives in their class, or in the case od 3cc1, the sequence identity did not seem too low.
In this case it is important to mention, that although the identity of 3hg3 is 100%, it is not the pdb structure annotated for the AGAL protein, but the structure of the substrate bound catalytic mechanism, hence the high similarity.
1ktb is the X-ray structure for the already mentioned α-N-acetylgalactosiminidase in chicken, which in future might be used for enzyme replacement therapy in the treatment of Fabry Disease.
The last one of the frequently used structures, 3cc1, is the x-ray structure of a putative α-N-acetylgalactosiminidase in in Bacillus Halodurans.

Calculation of models

Modeller

Default settings

<figtable id="tab:pics_1R46_3HG3"> ...

Model 1 (red), created with Modeller with the template 3HG3, superimposed on the x-ray structure of α-Galactosidase A (green)

Model 1 (red) superimposed on the x-ray structure of α-Galactosidase A (green) and the structure of 3HG3 (yellow)

</figtable>

<figtable id="tab:pics_1R46_1KTB"> ...

Model 2 (red), created with Modeller with the template 1ktb, superimposed on the x-ray structure of α-Galactosidase A (green)

Model 2 (red) superimposed on the x-ray structure of α-Galactosidase A (green) and the structure of 1ktb (yellow)

</figtable>

<figtable id="tab:pics_1R46_3CC1"> ...

Model 3 (red), created with Modeller with the template 3CC1, superimposed on the x-ray structure of α-Galactosidase A (green)

Model 3 (red) superimposed on the x-ray structure of α-Galactosidase A (green) and the structure of 3CC1 (yellow)

</figtable>

Multiple templates

<figtable id="tab:pics_1R46_multi1"> ...

Model Multi1 (red) (templates 3HG3 and 1KTB), superimposed on the x-ray structure of α-Galactosidase A (green)

Model Multi1 (red) superimposed on the x-ray structure of α-Galactosidase A (green) and the structure of 3HG3 (yellow) and 1KTB (orange)

</figtable>

<figtable id="tab:pics_1R46_multi2"> ...

Model Multi 2 (red), created with Modeller on basis of the templates 3HG3, 1KTB and 3CC1, superimposed on the x-ray structure of α-Galactosidase A (green)

Model Multi 2 (red) superimposed on the x-ray structure of α-Galactosidase A (green) and the structure of 3HG3 (yellow), 1KTB (orange) and 3CC1 (lightorange)

</figtable>

<figtable id="tab:pics_1R46_multi3"> ...

Model Multi 3 (red), created with Modeller on basis of the templates 3CC1, 3ZSS and 3A24, superimposed on the x-ray structure of α-Galactosidase A (green)

Model Multi 3 (red) superimposed on the x-ray structure of α-Galactosidase A (green) and the structure of 3CC1 (yellow), 3ZSS (orange) and 3A24 (lightorange)

</figtable>

<figtable id="tab:pics_1R46_multi4"> ...

Model Multi 4 (red), created with Modeller on basis of the templates 3CC1 and 3HG3, superimposed on the x-ray structure of α-Galactosidase A (green)

Model Multi 4 (red) superimposed on the x-ray structure of α-Galactosidase A (green) and the structure of 3CC1 (yellow) and 3HG3 (orange)

</figtable>

Edited Alignment input

<figtable id="tab:pics_1R46_3HG3_CHAS"> ...

Model CHAS (red), with active site shifted right to next D (7 and 1 positions) in 2d alignment file, superimposed on the x-ray structure of α-Galactosidase A (green)

For comparison with Model CHAS and CHAS 2, Model 1 (orange) which was basis for the edited alignments, superimposed on α-Galactosidase A (green)

Model CHAS 2 (red), with active site shifted right to next D (7 and 1 positions) in both alignment files, superimposed on the x-ray structure of α-Galactosidase A (green)

</figtable>

<figtable id="tab:pics_1R46_3HG3_CHAS3"> ...

Model CHAS 3 (red), with active site shifted right to next D (7 and 1 positions) in both alignment files and the substrate binding region (position 203-207) forced to be consecutive, superimposed on of α-Galactosidase A (green)

For comparison with Model CHAS 3, Model 1 (orange) which was basis for the edited alignments, created with Modeller on basis of the templates 3HG3, superimposed on the x-ray structure of α-Galactosidase A (green)

</figtable>

Evaluation

<figtable id="tab:compare"> Comparison of apo and complex structure

Superimposed structures of 1R46 (blue) and 1R47 (green) in cartoon representation. Obviously, the structures do not differ much.

Comparison of the residues invoked in the binding of α-galactose in the apo structure (blue) and the complex structure (green)

</figtable>

<figure id="fig:GAL:1R47">

Residues involved in the binding of α-galactose in 1R47 source

</figure>

As an initial step of the evaluation, we compared the apo structure 1R46 and the complex structure (with bound α-galactose) 1R47. Since the alignment of both the chains A of 1R46 and 1R47 in Pymol (see <xr id="tab:compare"/>) revealed a RMSD value of 0.248 and the comparison of the position and direction of the residues involved in the binding of the sugar (see <xr id="fig:GAL:1R47"/>) do not differ significantly, we used only the 1R46 structure for vizualisation, but computed all values and statistics for both structures.
In the right figure in <xr id="tab:compare"/>, the residues Asp92A, Asp93A, LYS168A, ARG227A and ASP231A are depicted in sticks representation (thicker); they are responsible for the binding of the sugar in the complex structures, which is shown in magenta. Clearly, one can see not much difference in this region between 1R46 and 1R47.

TM-score

<figtable id="tab:TMscore_1R46"> TM-score

Model	Number of residues in common	RMSD of the common residues	TM-score	GDT-TS-score	GDT-HA-score
output_TMscore/out/1R46_3HG3::1R46.B99990001.out	390	1.115	0.9841	0.9667	0.8558
output_TMscore/out/1R46_1KTB::1R46.B99990001.out	390	2.098	0.9596	0.9071	0.7635
output_TMscore/out/1R46_3CC1::1R46.B99990001.out	390	22.707	0.4087	0.2699	0.1814
output_TMscore/out/1R46_CHAS::1R46.B99990001.out	390	1.115	0.9841	0.9667	0.8558
output_TMscore/out/1R46_CHAS2::1R46.B99990001.out	390	15.292	0.4651	0.3622	0.3038
output_TMscore/out/1R46_CHAS3::1R46.B99990001.out	390	15.292	0.4651	0.3622	0.3038
output_TMscore/out/1R46_MULTI1::1R46.B99990001.out	390	0.575	0.9938	0.9910	0.9128
output_TMscore/out/1R46_MULTI2::1R46.B99990001.out	390	12.625	0.7364	0.6949	0.6404
output_TMscore/out/1R46_MULTI3::1R46.B99990001.out	390	21.196	0.2048	0.0673	0.0314
output_TMscore/out/1R46_MULTI4::1R46.B99990001.out	390	10.798	0.7405	0.6737	0.5833
output_TMscore/out/1R46_Model_2.out	390	0.512	0.9950	0.9917	0.9218
output_TMscore/out/1R46_Model_3.out	390	1.551	0.9660	0.9032	0.7538

</figtable>

<figtable id="tab:TMscore_1R47"> TM-score

Model	Number of residues in common	RMSD of the common residues	TM-score	GDT-TS-score	GDT-HA-score
output_TMscore/out/1R47_3HG3::1R46.B99990001.out	390	1.119	0.9840	0.9654	0.8519
output_TMscore/out/1R47_1KTB::1R46.B99990001.out	390	2.093	0.9600	0.9083	0.7647
output_TMscore/out/1R47_3CC1::1R46.B99990001.out	390	22.713	0.4092	0.2731	0.1821
output_TMscore/out/1R47_CHAS::1R46.B99990001.out	390	1.119	0.9840	0.9654	0.8519
output_TMscore/out/1R47_CHAS2::1R46.B99990001.out	390	15.290	0.4652	0.3635	0.3019
output_TMscore/out/1R47_CHAS3::1R46.B99990001.out	390	15.290	0.4652	0.3635	0.3019
output_TMscore/out/1R47_MULTI1::1R46.B99990001.out	390	0.575	0.9938	0.9897	0.9115
output_TMscore/out/1R47_MULTI2::1R46.B99990001.out	390	12.609	0.7363	0.6942	0.6378
output_TMscore/out/1R47_MULTI3::1R46.B99990001.out	390	21.191	0.2058	0.0679	0.0314
output_TMscore/out/1R47_MULTI4::1R46.B99990001.out	390	10.793	0.7405	0.6744	0.5846
output_TMscore/out/1R47_Model_2.out	390	0.515	0.9950	0.9923	0.9231
output_TMscore/out/1R47_Model_3.out	390	1.532	0.9667	0.9058	0.7545

</figtable>

RMSD with SAP

<figure id="fig:RMSD_1ktb">

Depiction of the RMSD (green) of Model 2 (magenta) from Modeller and 1R46 (cyan)

</figure>