Structure-based mutation analysis BCKDHA

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Revision as of 16:21, 10 August 2011 by Demel (talk | contribs) (Structure selection)

Structure selection

In order to perform a structure based mutation analysis an appropriate protein structure is needed. Therefore one has to check whether the resolution of the structure is high enough, that means the Å-value should be small. The R-factor (reliability factor or residual factor) is another measure to access the quality of a structure. The R-factor rates how well the crystallographic model predicts the observed data from experimental X-ray diffraction. The smaller the R-factor the better. The coverage


The following table presents the PDB structures for BCKDHA to date. For each structure the resolution in [Å], the R-factor, coverage and ph-value are listed as well.

PDB id resolution [Å] R-factor coverage ph-value
1DTW 2.70 0.224 7.5*
1OLS 1.85 0.172 5.5
1OLU 1.90 0.161 5.5
1OLX 2.25 0.161 5.5
1U5B 1.83 0.156 5.8
1V11 1.95 0.139* 5.5
1V16 1.90 0.132* 5.5
1V1M 2.00 0.130* 5.5
1V1R 1.80 0.158 5.5
1WCI 1.84 0.149 5.5
1X7W 1.73 0.148 5.8
1X7X 2.10 0.149 5.8
1X7Y 1.57 0.150 5.8
1X7Z 1.72 0.154 5.8
1X80 2.00 0.161 5.8
2BEU 1.89 0.171 5.5
2BEV 1.80 0.139 5.5
2BEW 1.79 0.147 5.5
2BFB 1.77 0.145 5.5
2BFC 1.64 0.144 5.5
2BFD 1.39* 0.150 5.5
2BFE 1.69 0.150 5.5
2BFF 1.46 0.150 5.5
2J9F 1.88 0.171 5.5

The asteriks-marked values indicate that these structures were resolved with the asked experimental quality. As one can see, none of the structures fulfills all conditions.

Furthermode, we could not use any of the PDB structures for BCKDHA because all of them had gaps in the secondary structure which means that some residues were missing. So we took the structure which has the less gaps: 1U5B

  • resultion: 1.83
  • R-factor: 0.156
  • ph-value: 5.8

This structure has to be modified with some programms to close the gaps. Additionally the first residues which are in BCKDHA misses in 1U5B thats why the start position corresponds to position 6 of the BCKDHA -PDB sequence.

As we can see none of the values corresponds to the demands because it was asked for a structure which has a very small R-factor, a pH of 7.4 and a high resolution.

Mapping of the mutations on the crystal structure

Structure of BCKDHA. Violet: mutations, orange: thiamine pyrophosphate binding sites, yellow: metal binding sites.


Hydrogen bonds are interactions between an hydrogen atom and an electronegative atom. Electronegative atoms which often take part in hydrogen bonds are oxygen, nitrogen and fluorine (not present in amino acid side chains). They serve as a hydrogen bond acceptor, whereas a hydrogen bond donor is a electronegative atom bonded to a hydrogen atom. Hydrogen bonds are essential for the three-dimensional structures of proteins. They play a important role in the formation of helices and beta-sheets and cause proteins to fold into a specific structure.

Showing hydrogen bonds with Pymol: A -> find -> polar contacts -> within selection The respective amino acids were colored by element, s.t. oxygen is red, nitrogen is blue, hydrogen is white and sulfur is yellow.

M82L
Hydrogen Bonds for methionine on pos 82 in the wild type structure
Hydrogen Bonds for leucine on pos 82 in the mutated type structure

Comparing the two figures for the wildtype and the mutated amino acid on position 82, no change in the hydrogen bonding network can be observed. This is due to the similar physiochemical properties of these two amino acids. No atom which could serve as additional hydrogen-bond donor or acceptor was introduced or removed.

Q125E
Hydrogen Bonds for glutamine on pos 125 in the wild type structure
Hydrogen Bonds for glutamic acid on pos 125 in the mutated type structure

The substitution from glutamine to glutamic acid changes the side chain properties completely. A NH2 group is substituted by a negatively charged oxygen. The NH2 which served in the wildtype structure as a hydrogen bond acceptor is not present any more, so the hydrogen bonding network changed for this substitution.

Y166N
Hydrogen Bonds for tyrosine on pos 166 in the wild type structure
Hydrogen Bonds for asparagine on pos 166 in the mutated type structure

Although tyrosine and asparagine both could play a role in the hydrogen bonding network, no hydrogen bond is formed for position 166. Therefore this substitution has no influence on the hydrogen bonding network of the protein.

G249S
Hydrogen Bonds for glycine 249 in the wild type structure
Hydrogen Bonds for serine on pos 249 in the mutated type structure

Introducing a serine on position 249 leads to the formation of several additional hydrogen bonds. Two of the newly established bonds are due to the new hydroxy group which is very likely to participate in hydrogen bonds. Another additional hydrogen bond is formed using the nitrogen atom as a hydrogen bond acceptor.

C264W
Hydrogen Bonds for cysteine on pos 264 in the wild type structure
Hydrogen Bonds for tryptophan on pos 264 in the mutated type structure

Although the amino acids cysteine and tryptophan have very different structures and chemical properties, no change in the hydrogen bonding network occurs.

R265W
Hydrogen Bonds for arginine on pos 265 in the wild type structure
Hydrogen Bonds for tryptophan on pos 265 in the mutated type structure

The mutation from arginine to tryptophan leads to a drastic change in the hydrogen bonding network. Arginine, which contains three nitrogen atoms in its side chain is removed and therefore three hydrogen bond acceptors are missing in the mutated protein.

I326T
Hydrogen Bonds for isoleucine on pos 326 in the wild type structure
Hydrogen Bonds for threonine on pos 326 in the mutated type structure

The mutation from isoleucine to threonine doesn't have an influence on the hydrogen bonding network, although the oxygen atom of threonine could serve as an additional hydrogen bond donor.

F409C
Hydrogen Bonds for phenylalanine on pos 409 in the wild type structure
Hydrogen Bonds for cysteine on pos 409 in the mutated type structure

The phenylalanine side chain in the wildtype protein does not participate in any hydrogen bonds. The mutation to serine doesn't introduce new hydrogen bonding donors or acceptors, therefore the mutation has no effect on the hydrogen bonding network.

Y438N
Hydrogen Bonds for tyrosine on pos 438 in the wild type structure
Hydrogen Bonds for asparagine on pos 438 in the mutated type structure

The hydrogen bond donor property of the amino acid on position 438 is maintained but the bond seems to be between different sidechains now. This substitution also disturbs the hydrogen bonding network of our protein.

Comparison energies

SCWRL

Before we could use SCWRL we first had to get the sequence of our model: repairPDB bckdha.pdb -seq >> bckdha.seq

When we have the sequence we have to make one file for each mutation. In these files we copied the bckdha.seq and changed the sequence to lower case letters. Then we add the mutation in an upper case letter.

To run SCWRL we used the command: scwrl -i bckdha.pdb -s mutation1.seq -o mutation1Model.pdb


Total minimal energy of the graph

Position Energy
M82L 642.213
Q125E 616.85
Y166N 616.293
G249S 633.378
C264W 805.257
R265W 710.647
I326T 619.424
F409C 617.305
Y438N 615.951

foldX

To use foldX we first build a runscript. It is important to change values of <Temperature> and <pH> to the values of the used protein. These values can be found on the pdb side . Additionally we had to create one file with all PDB Ids each in a new line (list.txt). We used the command Foldx -runfile run.txt > Stout.txt to run the programm.


total energy difference
wildtype 401.00 0
M82L 437.88 -36.88
Q125E 431.77 -30.77
Y166N 432.24 -31.24
G249S 432.22 -31.22
C264W 488.43 -87.43
R265W 460.43 -59.43
I326T 432.94 -31.94
F409C 433.33 -32.33
Y438N 431.56 -30.56

After using foldx we have the total energy for the wiltype protein and for each mutation. The value of the wildtype protein is 401.00 which is already a high value. This means that the protein is quite instabile. To find out which mutation has a high influence on the protein we look at the energies and especially on the difference between the energy of the mutated protein and the wildtype protein. All of the mutated proteins have a much higher energy than the unmutated protein which means that these proteins are less stable. We can see in the table that the proteins can be divided into two groups. The first group has an energy difference of about 31 and the other group has a much higher difference.

Minimise

It is important to remove the hydrogens and water before using the programm. For this we used the new version of repairPDB of the virtualbox. The programm can be started with the command: repairPDB bckdha.pdb -nosol out.pdb > Stout.txt
It is also possible to use the old version but then the command is: repairPDB bckdha.pdb -nosol -noh out.pdb > Stout.txt
It is useful to save the output in a file because it includes the energy.


total energy difference
wildtype -2485.452755 0
M82L -4253.174790 1767.722015
Q125E -4080.989512 1595.536757
Y166N -4354.495238 1869.042483
G249S -4280.043000 1794.590245
C264W -3745.313620 1259.860865
R265W -3989.790625 1504.33787
I326T -4317.105618 1831.652863
F409C -4358.528143 1873.075388
Y438N -4339.778964 1854.326209


Minimise calculates the energy for a mutation by building a new model for each mutation. And then it calculates the energy for the whole mutated model. To find out if there is a difference between the wildtype and the model that is calculated by Minimise. The aim by comparing the mutated models with the wildtype is to find out if there is a structural change caused by a mutation. We superposed each mutated protein with the wildtype and focused on the mutated position. In the pictures there are always the superposed structures. In the wildtype pictures the structure of the unmutated residue is bold and in the mutated pictures the structure of the mutated residue is bold. So we can compare the two pictures to see if there is a change in the structure caused by the mutation on this residue.


mutation wildtype structure mutated structure
M82L
wildtype
mutation M82L
Q125E
wildtype
mutation Q125E
Y166N
wildtype
mutation Y166N
G249S
wildtype
mutation G249S
C264W
wildtype
mutation C264W
R265W
wildtype
mutation R265W
I326T
wildtype
mutation I326T
F409C
wildtype
mutation F409C
Y438N
wildtype
mutation Y438N

Gromacs

The first part describes general background information for gromacs as well as how to run those programs. The second part contains the result description and analysis.

General

1. fetchpdb

The fetch-pdb script first checks, if it was called with an valid PDB-id. If the entered PDB code has 4letters, the script tries to download the pdb-file from the server. The successfully downloaded folder gets unzipped and everything except the actual pdb file is removed.

2. repairPDB

For repairPDB the following options are available:

-offset value offset the residue numbering
-chain char change Chain ID
-ratom renumber Atoms
-rres renumber Residues
-noh remove hydrogens
-het no change of HETATM to ATOM for AA
-seq returns protein sequence from AA in pdb file
-seqrs protein sequence from SEQRES entries
-nosol just Protein, no solvent OR
-ssw cutoff print only waters with B-value below cutoff OR
-cleansol remove overlapping solvent for GROMACS


We run repairPDB using the following command:

repairPDB bckdha_mod.pdb -noh -nosol > bckdha_clean.pdb

Using this command we removed hydrogens and solvent from our pdb to get just the protein.

3. SCWRL

SCWRL was executed using the following command:

scwrl -i bckdha_mod.pdb -s extractedPDB.seq -o bckdha_scwrl.pdb

SCWRL returned a pdb including HETATOMS. These solvent atoms needed to be removed before continuing.

4.pdb2gmx

use clean pdb without HEATOMS

pdb2gmx -f bckdha_clean.pdb -o bckdha.gro -p bckdha.top -water tip3p -ff amber03

5. MDP

title = PBSA minimization in vacuum
cpp = /usr/bin/cpp
define = -DFLEXIBLE -DPOSRES
implicit_solvent = GBSA
integrator = steep
emtol = 1.0
nsteps = 500
nstenergy = 1
energygrps = System
ns_type = grid
coulombtype = cut-off
rcoulomb = 1.0
rvdw	 = 1.0
constraints = none
pbc = no

adjust nsteps for the time vs steps analysis

integrator a steepest descent algorithm for energy minimization.
emtol tolerance for steep integrator:the minimization is converged when the maximum force is smaller than this value
nsteps maximum number of steps to integrate or minimize, -1 is no maximum
nstenergy frequency to write energies to energy file (last energies are always written)
energygrps groups to write to energy files
ns_type Which atoms to check when construction a new neighbor list
coulombtype which coulomb-type to use, e.g. Cut-off, PME, Ewald
rcoulomb distance for the Coulomb cut-off
rvdw Van-der-Waals cut-of (distance for the LJ or Buckingham cut-off)
constraints constraints for bonds, e.g bonds are presented by a potention, all bonds are converted to constraints,...
pbc Remove the periodicity (make molecule whole again)

6. grompp

grompp -v -f bckdha.mdp -c bckdha.gro -p bckdha.top -o bckdha.tpr

7. System Minimization

mdrun -v -deffnm bckdha 2> mdrun_out.txt

8. Analyzation

g_energy -f bckdha.edr -o energy_1.xvg

Analysis

Wildtype analysis: nsteps vs time

The table below shoes the running time for mdrun depending on different values for nsteps. It also lists the real number of steps carried out to calculate the energy.

steps time (real) [s] time (user) [s] time (sys) [s] performed steps
50 5.453 4.730 0.120 50
100 10.393 9.210 0.240 100
500 36.419 30.660 0.780 338
1000 5.261 4.390 0.130 47
2000 10.564 8.500 0.290 93
3000 10.661 8.840 0.230 96
4000 2.620 2.010 0.140 21
5000 3.693 3.300 0.100 35

The following plot shows the correlation between nsteps and the running time for mdrun

nsteps vs running time for mdrun

Interestingly, the running time is not dependent on the number of nsteps, but just on the number of really performed steps. There is a linear dependency between the calculation time and the number of performed steps. The number of performed steps however is not correlating with the value for nsteps. It is not obvious why the number of performed steps varies so extremely given a certain value for nsteps.

Wildtype analysis: force fields

The different force fields chosen for this task were:

  • AMBER03
GROMACS Energy for the AMBER03 forcefield using the wildtype bckdha structure.
  • CHARMM27
GROMACS Energy for the CHARMM27 forcefield using the wildtype bckdha structure.
  • OPLS-AA
GROMACS Energy for the OPLS-AA forcefield using the wildtype bckdha structure.

Bond Analysis

Force Field Average Err. Est. RMSD Tot-Drift (kJ/mol)
AMBER03 3072.83 2200 -nan -13100.2
CHARMM25 3180.46 1700 7382.72 -9958.05
OPLS 2780.55 2100 -nan -11542.6

Angle Analysis

Force Field Average Err. Est. RMSD Tot-Drift (kJ/mol)
AMBER03 3616.97 230 -nan -1295.57
CHARMM25 5018.38 490 1646.81 -2783.35
OPLS 3271.23 340 -nan -1889.98

Potential Analysis

Force Field Average Err. Est. RMSD Tot-Drift (kJ/mol)
AMBER03 2.67001e+07 2.6e+07 -nan -1.60382e+08
CHARMM25 487.479 97199.742 673.043
OPLS 2.38353e+07 2.4e+07 -nan -1.39932e+08

Mutation analysis

M82L

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2518.71 1700 6337.97 -10023.3
Angle 3642.41 270 638.624 -1479.34
Potential 5.16e+06 5.1e+06 7.47e+07 -3.13e+07

Q125E

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2519.85 1700 6351.32 -10027.5
Angle 3626.21 260 618.433 -1418.24
Potential 5.23e+06 5.2e+06 7.5e+07 -3.17e+07

Y166N

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 3029.19 2200 -nan -12529.5
Angle 3654.58 280 -nan -1486.71
Potential 7.95e+06 7.8e+06 -nan -4.67e+07

G249S

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2775.97 2000 6761.45 -11375.2
Angle 3682.24 300 670.885 -1625.24
Potential 5.96e+06 5.0e+06 8.02e+07 -3.61e+07

C264W

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 3186.75 2300 -nan -13603.2
Angle 3831.06 370 -nan -2070.89
Potential 3.41e+07 3.3e+07 -nan -2.03e+08

R265W

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2473.43 1700 6385.14 -9741.04
Angle 3726.4 330 827.187 1803.54
Potential 5.36e+06 5.3e+06 7.68e+07 -3.26e+07

I326T

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 3214.03 2300 7364.47 -13490.1
Angle 3738.44 310 698.943 -1792.01
Potential 7.29e+06 6.9e+06 8.86e+07 -4.38e+07

F409C

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2341.69 1600 6048.14 -9087.07
Angle 3597.89 240 594.267 -1309.54
Potential 4.68e+06 4.7e+06 7.12e+07 -2.85e+07

Y438N

Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 3141.2 2300 -nan -13216.1
Angle 3672.66 290 -nan -1550.04
Potential 8.33e+06 8.1e+06 -nan -4.94e+07

Links

go back to Maple syrup urine disease main page

go back to Task 6 Sequence based mutation analysis

go to Task 8 Molecular Dynamics Simulations

go to Reference Sequence BCKDHA