Molecular Dynamics Simulations TSD

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The journal for this task can be found here.

Mutations

Based on previous analyses the following two mutations were chosen for the MD analysis.
R178H because it is a disease causing mutation with a strong effect on the active site. R178C was not chosen, since there seems to be no possibility to make it work at all, while for the mutation to histidine it should be possible ot fit the residue in the binding pocket. This will still not allow coordination of the ligand, so the mutation is disease-causing for sure, but it will still be interesting to see where the sidechain is placed and what alternative interactions might arise.
The second mutation is P182L. The residue is found at the protein surface, near the binding site to the beta-subunit. However it does not seem to play a role in the interaction. It will be interesting to see how this changes due to MD simulation. In addition, it should be oberserved how the helix changes, now that the helix breaker proline is not present anymore.

Simulations

Intermediate Structures

In the AGroS pipeline several structures are created in intermediate steps. <xr id="tbl:intermed"/> lists all of these and gives an explanation of their content. Three scripts are repeatedly used:

• findBreaks.pl: Checks for breaks in the structure. Sometimes parts are unresolved e.g. because they would hinder crystallization.
• clshwtr.pl: Removes water molecules that have a Euclidian distance to the protein below a user defined cutoff.
• repairPDB.pl: PDB files tend to be highly heterogenous. This script addresses several of the common issues and for example allows to remove water atoms, hydrogens, extract only the protein part or renumber residues.

<figtable id="tbl:intermed">

Table 1:List of intermediate structures created during an AGroS run. Structures are sorted by time of creation in the pipeline beginning with the oldest ones. After the input file, only the suffixes added to the filename are denoted. The extension for all files is .pdb unless denoted otherwise. Scripts denotes the scripts involved in creation of the file and the important parameters given.

File	Description	Scripts
2gjx178H	Input structure, with waters removed	repairdPDB.sh -nohoh, clshwtr.pl with cutoff 2.6
_br	Protein part only	repairdPDB.sh -jprot
_br_0	Structure until the first break. Since there are none, it is the full structure again	findBreaks.pl
.pdb2	2gjx178H.pdb with hydrogens removed	repairPDB -noh
_repair	Indices changed to begin with 0 and DNA removed	repairPDB -nodna -offset
_repair_0	Recalculated indices, if several chains present in input	sepPDB, repairPDB -rres
_water	Waters with B-value below 15 Å extracted from the structure	repairPDB -ssw 15
_dna	DNA extracted from the structure	repairPDB -dna
_sc	Side chain placements by SCWRLv4	scwrl
_nh	Protein part only from _sc. In addition structural water and DNA (previously extracted _water and _dna files)	repairPDB -jprot
_solv_tmp	Addition of ions (Na+ and Cl-)	genion -pname NA+ -nname CL-
_solv	Remove overlapping solvents from _solv_tmp	repairPDB -cleansol
_solv.pdb2	DNA and protein part only from _solv_tmp	repairPDB -dna, repairPDB -jprot
solv_0	Input for creation of custom position restraints for several chains, if present	?
_solv_min	Minimization run with only solvent flexible. Protein backbone and sidechains are fixed	grompp, mdrun
_solv_min.pdb2	Protein only extracted from _solv_min.pdb	repairPDB -jprot
_solv_min_0	Input for creation of custom position restraints for several chains, if present	?
_solv_min2	Minimization with only backbone fixed	grompp, mdrun
_solv_min3	Another minimization with fixed backbone, this time using conjugate gradient as integrator	grompp, mdrun
step *?

</figtable>

In the following there were 5 intermediate preprocessing structures chosen and displayed for the 3 simulations, namely the structure in its native form x_br_0.pdb, the x_solv.pdb, the x_solv_min.pdb, the x_solv_min2.pdb and the final minimization structure x_solv_min3.pdb.

Wildtype

<figure id="fig:anWT">

Figure 1: Wildtype intermediate states. Displayed is the WT_br_0.pdb (red), WT_solv.pdb (purple), WT_solv_min.pdb (blue), WT_solv_min2.pdb (cyan) and WT_solv_min3.pdb (green).

</figure>

<xr id="fig:anWT"/> shows an animation of the 5 intermediates of the wildtype structure preprocessing. As expected there is almost no visible change within the different steps. Only some loops on the surface or protein ends experience tiny shifts.

R178H

<figure id="fig:anR178H">

Figure 2: R178H intermediate states. Shown is the R178H_br_0.pdb (red), R178H_solv.pdb (purple), R178H_solv_min.pdb (blue), R178H_solv_min2.pdb (cyan) and R178H_solv_min3.pdb (green). The R178H mutation is additionally colored yellow.

</figure>

The according 5 processing steps from the R178H mutation are displayed in <xr id="fig:anR178H"/>. Although the sequence is mutated the backbone stays fixed and therefore no great changes within the structures are anticipated. Some of the outer loops move slightly but otherwise the structure stays rigid.

<figure id="fig:ancloseWT">

Figure 3: Enlargement of the R178H mutation.

</figure>

<xr id="fig:ancloseWT"/> shows a close-up of the mutation during the different steps. The br_0 structure stands out insofar as it is deprived of H atoms. Within the first minimization step the side chain is slighty twisted so it fits better into its surrounding. By minimization step 3 the ring has reached an optimal position within the pocket. There are no clashes and from these illustrations alone the mutation would possibly not be considered deleterious.

P182L

<figure id="fig:anP182L">

Figure 4: P182L intermediate states. Depicted is the P182L_br_0.pdb (red), P182L_solv.pdb (purple), P182L_solv_min.pdb (blue), P182L_solv_min2.pdb (cyan) and P182L_solv_min3.pdb (green). The P182L mutation is additionally colored yellow.

</figure>

<xr id="fig:anP182L"/> shows the animation of the intermediate structures for the P182L mutation. The protein again keeps its conformation and expresses only slight side chain movements just like the illustrations above.

<figure id="fig:ancloseP182L">

Figure 5: Close-up of the side chain changes of the P182L mutation.

</figure>

The enlargement of the changes around position 182, see <xr id="fig:ancloseP182L"/>, shows that this mutation does not lead to any clashes which is supposedly why the conformation of the side chain is almost not altered by the minimization. Within the preprocessing steps this mutation retains its status as presumably harmless.