Structure based mutation analysis of GBA

Introduction

A detailed list of the ten mutations analyzed in this section, can be found in Task 6.

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

SCWRL is a tool, specifically designed for side-chain conformation predictions, given a fixed backbone from an experimental determined structure. <ref>G. G. Krivov, M. V. Shapovalov, and R. L. Dunbrack, Jr. Improved prediction of protein side-chain conformations with SCWRL4. Proteins (2009). </ref>

Usage

• Create pdb-File only containing chain A of the protein (e.g with pymol): 2NT0_A.pdb
• Extract sequence (e.g with repairPDB) and change all letters of protein sequence to lower case and introduce mutation in capital letters: mutantSequence.seq
  • Note, that only one mutation should be inserted per sequence -> results in 10 mutant sequence files.
• Commandline: scwrl4 -i 2NT0_A.pdb -s mutantSequence.seq -o 2NT0_mutant.pdb > scwrl.log

Results

SCRWL4 was applied ten times, once for each mutation. The resulting conformations of the mutants are visualized in Figure 3. 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). These changes are consistent in all ten mutant structures.

TODO: redo images with unprotonated strucutres ...

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).

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

Energy Calculation

FoldX

FoldX is an algorithm which provides an estimation of the importance of the interactions contributing to the stability of proteins. It was developped by the FoldX consortium now centered at the Centre de Regulació Genòmica in Barcelona. <ref>http://foldx.crg.es/about.jsp</ref>

Usage

• Create a runfile according to the example "Energy of the molecule" at the FoldX website: run.txt
  • Do not forget to adjust known information like pH and temperature.
• Create a list file containing all pdb files you want the energy to be calculated of: list.txt
• Commandline: sudo ./foldx -runfile run.txt > foldx.output

Results

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

Usage

• Remove hydrogens and waters from the pdb files with repairPDB: repairPDB mutant.pdb -nosol > mutant_nw.pdb
• Apply Minimise: minimise mutant_nw.pdb mutant_nw_min.pdb > mutant_nw_min.out

Results

Gromacs

Usage

• Step 1 - pdb2gmx
  • pdb2gmx adds hydrogens to the molecules and generates a coordinate (.gro) and a topology(.top) file.
  • Commandline: pdb2gmx -f mutant_nw.pdb -o mutant_nw.gro -p mutant_nw.top -water tip3p -ff amber03
• Step 2 - MDP file
  • Create a MDP file with the following content:

title = PBSA minimization in vacuum
cpp = /usr/bin/cpp
# Preprocessing
# -DFLEXIBLE: include flexible water in stead of rigid water into topology
# -DPOSRES: includes posre.itp into topology, used for position restraints.
define = -DFLEXIBLE -DPOSRES
# Implicit solvent - simulation with implicit solvent using the Generalized Born formalism
implicit_solvent = GBSA
# Run control - steepest descent algorithm for energy minimization
integrator = steep
# Run control - tolerance
emtol = 1.0
# Run control - maximum number of steps to integrate or minimize
nsteps = 500
# Output - frequency to write energies to energy file
nstenergy = 1
# Tables - group(s) to write to energy file 
energygrps = System
# Neighbour searching - makes grid in the box and only check atoms in neighboring grid cells when constructing a new neighbor list every nstlist steps
ns_type = grid
# Electrostatics - twin range cut-off's with neighborlist cut-off rlist and Coulomb cut-off rcoulomb
coulombtype = cut-off
rcoulomb = 1.0
# VDW - distance for the LJ or Buckingham cut-off
rvdw	 = 1.0
# Bonds - no constraints except for those defined explicitly in the topology
constraints = none
# Neighbour searching - Use no periodic boundary conditions, ignore the box.
pbc = no

• Step 3 - grompp
  • grompp, the GROMACS preprocessor, prepares the system for gromacs. Therefore it reads a molecular topology file, checks the validity of the file and expands the topology from a molecular description to an atomic description.
  • Commandline: grompp -v -f mutant_nw.mdp -c mutant_nw.gro -p mutant_nw.top -o mutant_nw.tpr
• Step 4 - mdrun
  • The mdrun program is the main computational chemistry engine within GROMACS and can perform molecular dynamics simulations, Brownian Dynamics, Langevin Dynamics, Conjugate Gradient, L-BFGS, or Steepest Descents energy minimization.
  • Commandline:
• Step 5 - g_energy
  • g_energy extracts energy components or distance restraint data from an energy file and calculates Average, RMSD and drift from the simulation.
  • Commandline: g_energy -f FILE.edr -o energy_1.xvg

Results