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).

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 5 shows the interesting positions with hilighted mutants and wild type residues of the pdb files obtained with Gromacs after having applied the steps as 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

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	PDB Wild type	Pymol Mutagenesis	SCWRL	Minimise		Gromacs
				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

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

The total energies calculated with minimise 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	-12263.635255	0
1	-10405.258800	1858.37645
2	-10354.081889	1909.55337
3	-10313.900667	1949.73459
4	-10415.502839	1848.13242
5	-8558.228610	3705.40664
6	-10397.436817	1866.19844
7	-10427.599844	1836.03541
8	-3611.581531	8652.053719
9	-10030.430566	2233.20469
10	-10300.108046	1963.52721

Gromacs

Wildtype

Correlation between nsteps and runtime of mdrun

The following table shows the measured time for mdrun for different nsteps and force fields. It also shows the steps it really took to calculate the lowest energy. With AMBER03 and CHARMM27 it took less than 500 steps, so the values for 500, 1000 and 5000 nsteps should be similar. The same you can see in figure 5.

nsteps	AMBER03	CHARMM27	OPLS/AA
10	real: 0m4.615s user: 0m2.910s sys: 0m0.230s Steps: 10	real: 0m10.760s user: 0m2.760s sys: 0m0.150s Steps: 10	real: 0m3.801s user: 0m2.280s sys: 0m0.320s Steps: 10
50	error occured	real: 0m34.086s user: 0m10.830s sys: 0m0.260s Steps: 50	real: 0m13.042s user: 0m9.200s sys: 0m0.620s Steps: 50
100	real: 0m29.400s user: 0m22.060s sys: 0m0.640s Steps: 100	real: 0m23.616s user: 0m19.410s sys: 0m0.590s Steps: 100	real: 0m25.184s user: 0m18.330s sys: 0m0.740s Steps: 100
500	real: 1m9.519s user: 0m53.700s sys: 0m1.010s Steps: 242	real: 3m15.607s user: 1m1.790s sys: 0m1.070s Steps: 307	real: 3m51.264s user: 1m7.870s sys: 0m1.590s Steps: 500
1000	real: 2m9.160s user: 0m52.990s sys: 0m1.190s Steps: 242	real: 3m22.812s user: 1m2.120s sys: 0m1.170s Steps: 307	real: 5m9.851s user: 1m38.390s sys: 0m1.860s Steps: 732
5000	real: 2m13.786s user: 0m53.020s sys: 0m1.030s Steps: 242	real: 3m15.591s user: 1m2.730s sys: 0m1.560s Steps: 307	real: 5m5.631s user: 1m37.240s sys: 0m1.830s Steps: 732

The following plot shows the correlation between nsteps and the runtime of mdrun. We therefore used the real time.

Figure 7: Correlation between nsteps and the runtime of mdrun for different force fields

Force Fields: AMBER03, CHARMM27 and OPLS/AA

AMBER03

Bond

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	6809.8	5000	-nan	-25601.3 (kJ/mol)	read 9 time 10.000
100	1664.38	790	-nan	-5142.49 (kJ/mol)	read 78 time 100.000
500	1037.65	300	2925.39	-1749.57 (kJ/mol)	read 181 time 234.000
1000	1037.65	300	2925.39	-1749.57 (kJ/mol)	read 181 time 234.000
5000	1037.65	300	2925.39	-1749.57 (kJ/mol)	read 181 time 234.000

Figure 6: GROMACS Energy for the AMBER03 forcefield with nsteps 10.

Figure 7: GROMACS Energy for the AMBER03 forcefield with nsteps 100.

Figure 8: GROMACS Energy for the AMBER03 forcefield with nsteps 500.

Figure 9: GROMACS Energy for the AMBER03 forcefield with nsteps 1000.

Figure 10: GROMACS Energy for the AMBER03 forcefield with nsteps 5000.

Angle

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	5270.93	620	-nan	-2356.46 (kJ/mol)	read 9 time 10.000
100	4402.05	140	-nan	-783.551 (kJ/mol)	read 78 time 100.000
500	4355.86	41	324.958	-111.52 (kJ/mol)	read 181 time 234.000
1000	4355.86	41	324.958	-111.52 (kJ/mol)	read 181 time 234.000
5000	4355.86	41	324.958	-111.52 (kJ/mol)	read 181 time 234.000

Potential

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	-37523.9	5600	-nan	-30866.3 (kJ/mol)	read 9 time 10.000
100	-46525.6	1600	-nan	-10705.8 (kJ/mol)	read 78 time 100.000
500	-48425.9	950	3935.69	-6022.96 (kJ/mol)	read 181 time 234.000
1000	4355.86	41	324.958	-111.52 (kJ/mol)	read 181 time 234.000
5000	4355.86	41	324.958	-111.52 (kJ/mol)	read 181 time 234.000

CHARMM27

Bond

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	9134.93	5400	14834.3	-27981.2 (kJ/mol)	read 7 time 10.000
50	3624.43	2500	-nan	-12676.4 (kJ/mol)	read 38 time 49.000
100	2537.64	1200	-nan	-6312.93 (kJ/mol)	read 78 time 100.000
500	1805.15	230	3077.4	-1388.65 (kJ/mol)	read 232 time 305.000
1000	1805.15	230	3077.4	-1388.65 (kJ/mol)	read 232 time 305.000
5000	1805.15	230	3077.4	-1388.65 (kJ/mol)	read 232 time 305.000

Figure 11: GROMACS Energy for the CHARMM27 forcefield with nsteps 10.

Figure 12: GROMACS Energy for the CHARMM27 forcefield with nsteps 50.

Figure 13: GROMACS Energy for the CHARMM27 forcefield with nsteps 100.

Figure 14: GROMACS Energy for the CHARMM27 forcefield with nsteps 500.

Figure 15: GROMACS Energy for the CHARMM27 forcefield with nsteps 1000.

Figure 16: GROMACS Energy for the CHARMM27 forcefield with nsteps 5000.

Angle

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	6896.32	910	1922.48	-4990.23 (kJ/mol)	read 7 time 10.000
50	5450.29	580	-nan	-2853.11 (kJ/mol)	read 38 time 49.000
100	5247.34	270	-nan	-1273.53 (kJ/mol)	read 78 time 100.000
500	5207.89	36	491.191	-2.16444 (kJ/mol)	read 232 time 305.000
1000	5207.89	36	491.191	-2.16444 (kJ/mol)	read 232 time 305.000
5000	5207.89	36	491.191	-2.16444 (kJ/mol)	read 232 time 305.000

Potential

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	60.8518	14	30.4721	70.4929 (kJ/mol)	read 7 time 10.000
50	109.833	16	-nan	105.768 (kJ/mol)	read 38 time 49.000
100	146.801	22	-nan	148.136 (kJ/mol)	read 78 time 100.000
500	244.525	41	83.6506	293.282 (kJ/mol)	read 232 time 305.000
1000	244.525	41	83.6506	293.282 (kJ/mol)	read 232 time 305.000
5000	244.525	41	83.6506	293.282 (kJ/mol)	read 232 time 305.000

OPLS/AA

Bond

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	6116	4300	10637.4	-21181.9 (kJ/mol)	read 9 time 9.000
50	2501.41	1100	-nan	-7804.83 (kJ/mol)	read 39 time 49.000
100	1629.77	770	-nan	-4729.34 (kJ/mol)	read 78 time 99.000
500	913.547	110	1889.7	-645.765 (kJ/mol)	read 394 time 500.000
1000	890.429	71	1573.48	-408.773 (kJ/mol)	read 569 time 727.000
5000	890.429	71	1573.48	-408.773 (kJ/mol)	read 569 time 727.000

Figure 17: GROMACS Energy for the OPLS/AA forcefield with nsteps 10.

Figure 18: GROMACS Energy for the OPLS/AA forcefield with nsteps 50.

Figure 19: GROMACS Energy for the OPLS/AA forcefield with nsteps 100.

Figure 20: GROMACS Energy for the OPLS/AA forcefield with nsteps 500.

Figure 21: GROMACS Energy for the OPLS/AA forcefield with nsteps 1000.

Figure 22: GROMACS Energy for the OPLS/AA forcefield with nsteps 5000.

Angle

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	4183.1	280	629.187	-1564.39 (kJ/mol)	read 9 time 9.000
50	3703.5	150	-nan	-897.133 (kJ/mol)	read 39 time 49.000
100	3659.93	87	-nan	-356.71 (kJ/mol)	read 78 time 99.000
500	3723.98	27	152.63	156.454 (kJ/mol)	read 394 time 500.000
1000	3737.09	24	128.576	130.478 (kJ/mol)	read 569 time 727.000
5000	3737.09	24	128.576	130.478 (kJ/mol)	read 569 time 727.000

Potential

nsteps	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
10	-82897	5100	11916.1	-26801.7 (kJ/mol)	read 9 time 9.000
50	-89732.6	2100	-nan	-15085.3 (kJ/mol)	read 39 time 49.000
100	-92256.7	1800	-nan	-11818.5 (kJ/mol)	read 78 time 99.000
500	-95730.2	780	3011.34	-4980.18 (kJ/mol)	read 394 time 500.000
1000	-96174.9	660	2594.71	-4302.38 (kJ/mol)	read 569 time 727.000
5000	-96174.9	660	2594.71	-4302.38 (kJ/mol)	read 569 time 727.000

Mutations

Mutation	Total Energy Bond	Difference Bond	Total Energy Angle	Difference Angle	Total Energy Potential	Difference Potential
WT	1037.65	0	4355.86	0	-48425.9	0
1	1274.92	237.27	4255.25	-100.61	-47765.7	660.2
2	1456.65	419	4236.44	-119.42	-47077.9	1348
3	1231.56	193.91	4183.17	-172.69	-48600.7	-174.8
4	1338.99	301.34	4240.62	-115.24	-47096.1	1329.8
5	1478.43	440.78	4311.75	-44.11	77967	126392.9
6	1308.78	271.13	4244.41	-111.45	-47639.3	786.6
7	1201.46	163.81	4258.22	-97.64	-47883.1	542.8
8	1628.70	591.05	4182.29	-173.57	-40120	8305.9
9	1421.28	383.63	4230.01	-125.85	-47052.5	1373.4
10	1514.09	476.44	4283.65	-72.21	-46575.7	1850.2

Mutation 1: Gly - Ser (Pos. 10)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1274.92	500	3967.13	-3038.46 (kJ/mol)	read 306 time 398.000
Angle	4255.25	28	265.491	71.602 (kJ/mol)	read 306 time 398.000
Potential	-47765.7	1500	6978.27	-9464.84 (kJ/mol)	read 306 time 398.000

Figure 23: GROMACS Bond Energy for the first mutation with AMBER03 forcefield and nsteps 500.

Mutation 2: Asp - Asn (Pos. 24)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1456.65	680	4628.95	-4212.43 (kJ/mol)	read 223 time 292.000
Angle	4236.44	39	306.459	-37.8896 (kJ/mol)	read 223 time 292.000
Potential	-47077.9	1800	8037.84	-11650.4 (kJ/mol)	read 223 time 292.000

Figure 24: GROMACS Bond Energy for the second mutation with AMBER03 forcefield and nsteps 500.

Mutation 3: His - Arg (Pos. 60)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1231.56	460	3764.8	-2712.35 (kJ/mol)	read 340 time 440.000
Angle	4183.17	25	249.669	93.2215 (kJ/mol)	read 340 time 440.000
Potential	-48600.7	1400	6639.68	-8746.39 (kJ/mol)	read 340 time 440.000

Figure 25: GROMACS Bond Energy for the third mutation with AMBER03 forcefield and nsteps 500.

Mutation 4: Arg - Gln (Pos. 120)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1338.99	580	4237.53	-3506.39 (kJ/mol)	read 267 time 350.000
Angle	4240.62	32	282.449	33.6883 (kJ/mol)	read 267 time 350.000
Potential	-47096.1	1600	7399.52	-10330.7 (kJ/mol)	read 267 time 350.000

Figure 26: GROMACS Bond Energy for the fourth mutation with AMBER03 forcefield and nsteps 500.

Mutation 5: Pro - Leu (Pos. 182)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1478.43	700	4792.41	-4089.05 (kJ/mol)	read 351 time 449.000
Angle	4311.75	35	287.097	-52.7644 (kJ/mol)	read 351 time 449.000
Potential	77967	130000	2.2001e+06	-760308 (kJ/mol)	read 351 time 449.000

Figure 27: GROMACS Bond Energy for the fifth mutation with AMBER03 forcefield and nsteps 500.

Mutation 6: Ser - Asn (Pos. 271)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1308.78	550	4136.28	-3263.06 (kJ/mol)	read 281 time 364.000
Angle	4244.41	32	277.965	43.6715 (kJ/mol)	read 281 time 364.000
Potential	-47639.3	1600	7314.5	-10123.2 (kJ/mol)	read 281 time 364.000

Figure 28: GROMACS Bond Energy for the sixth mutation with AMBER03 forcefield and nsteps 500.

Mutation 7: Asn - Ser (Pos. 370)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1201.46	430	3661.19	-2529.9 (kJ/mol)	read 360 time 461.000
Angle	4258.22	26	246.136	110.294 (kJ/mol)	read 360 time 461.000
Potential	-47883.1	1300	6455.75	-8370.77 (kJ/mol)	read 360 time 461.000

Figure 29: GROMACS Bond Energy for the seventh mutation with AMBER03 forcefield and nsteps 500.

Mutation 8: His - Arg (Pos. 311)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1628.7	850	5363.95	-5160.93 (kJ/mol)	read 269 time 351.000
Angle	4182.29	42	309.833	-56.0265 (kJ/mol)	read 269 time 351.000
Potential	-40120	9100	96292.1	-57418.1 (kJ/mol)	read 269 time 351.000

Figure 30: GROMACS Bond Energy for the eighth mutation with AMBER03 forcefield and nsteps 500.

Mutation 9: Met - Val (Pos. 416)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1421.28	650	4522.24	-4029.74 (kJ/mol)	read 234 time 307.000
Angle	4230.01	38	300.113	-29.8853 (kJ/mol)	read 234 time 307.000
Potential	-47052.5	1700	7880.56	-11371.6 (kJ/mol)	read 234 time 307.000

Figure 31: GROMACS Bond Energy for the ninth mutation with AMBER03 forcefield and nsteps 500.

Mutation 10: Leu - Pro (Pos. 470)

Energy	Average	Err. Est.	RMSD	Tot-Drift (kJ/mol)	Last energy frame
Bond	1514.09	740	4792.64	-4608.11 (kJ/mol)	read 208 time 275.000
Angle	4283.65	49	321.438	-149.588 (kJ/mol)	read 208 time 275.000
Potential	-46575.7	1900	8664.13	-12790.7 (kJ/mol)	read 208 time 275.000

Figure 32: GROMACS Bond Energy for the tenth mutation with AMBER03 forcefield and nsteps 500.