Structure based mutation analysis of GBA

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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 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 8: GROMACS Energy for the AMBER03 forcefield with nsteps 10.
Figure 9: GROMACS Energy for the AMBER03 forcefield with nsteps 100.
Figure 10: GROMACS Energy for the AMBER03 forcefield with nsteps 500.
Figure 11: GROMACS Energy for the AMBER03 forcefield with nsteps 1000.
Figure 12: 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 13: GROMACS Energy for the CHARMM27 forcefield with nsteps 10.
Figure 14: GROMACS Energy for the CHARMM27 forcefield with nsteps 50.
Figure 15: GROMACS Energy for the CHARMM27 forcefield with nsteps 100.
Figure 16: GROMACS Energy for the CHARMM27 forcefield with nsteps 500.
Figure 17: GROMACS Energy for the CHARMM27 forcefield with nsteps 1000.
Figure 18: 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 19: GROMACS Energy for the OPLS/AA forcefield with nsteps 10.
Figure 20: GROMACS Energy for the OPLS/AA forcefield with nsteps 50.
Figure 21: GROMACS Energy for the OPLS/AA forcefield with nsteps 100.
Figure 22: GROMACS Energy for the OPLS/AA forcefield with nsteps 500.
Figure 23: GROMACS Energy for the OPLS/AA forcefield with nsteps 1000.
Figure 24: 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 25: 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 26: 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 27: 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 28: 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 29: 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 30: 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 31: 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 32: 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 33: 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 34: GROMACS Bond Energy for the tenth mutation with AMBER03 forcefield and nsteps 500.



Discussion

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