Normal Mode Analysis (PKU)

Short Task Description

This week, we will perform a normal mode analysis of our wildtype protein. We will calculate visualize large coordinate movements to identify e.g. domains and try to watch ligand interaction. For this, we will use the webnm@ and elNemo webservers. See the task description for details, a journal of commands and scripts, if necessary, can be found here.

Normal Mode Analysis

In normal mode analysis (NMA) the protein is modeled as harmonically oscillating system to e.g. describe conformational changes. Normal modes are much faster to calculate than a molecular dynamics simulation, especially as usually very few low-frequency modes suffice to describe a proteins motions.<ref name=ElNemo>Karsten Suhre and Yves-Henri Sanejouand; ElNémo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Research Volume 32, Issue suppl 2</ref> Often, models do not include the sidechains of a protein so the effect of mutations can not be detected. Also, non-harmonic motions are not observable which limits the insights gained from NMA in comparison to the much more detailed molecular dynamics simulation.

There are 14 unmutated structures of human phenylalanine hydroxylase, some of them with different ligands e.g. the co-factor tetrahydrobiopterin, the co-factor and thienylalanine (thienylalanine is a receptor antagonist to the substrate phenylalanine) or the co-factor and norleucine. As can be seen from <xr id="tab:rmsd"/> of RMSD values below, unfortunately they are all in the same conformation. There are also phosphorylated and unphosphorylated structures of PheOH of rattus norvegicus (1PHZ and 2PHM in <xr id="tab:rmsd"/>) but they also are virtually identical with a RMSD of 0.330. When we submit 1J8U and e.g. 1MMT, the structures with the highest RMSD, we get an overlap of the high frequence modes and the 'conformational change' of over 0.82, which is neither surprising nor of much value for the analysis as the structural changes are very small and limited to some displaced loops. Therefore we cannot compare different conformations, but use the 1J8U structure with and without ligand in the analysis.

<figtable id="tab:rmsd"> RMSD of human phenylalanine hydroxylase structures in PDB to the 1J8U reference structure

PDB ID	RMSD to 1J8U
1J8T	0.111
1DMW	0.150
1KWO	1.583
1LRM	0.167
1MMK	1.586
1MMT	1.640
1PAH	0.278
2PAH	0.590
3PAH	0.288
4PAH	0.283
5PAH	0.286
6PAH	0.280
4ANP	0.220
1PHZ (rattus n.)	0.380
2PHM (rattus n.)	0.409

</figtable>

WEBnm@

<figure id="fig:mode7">

Animation of mode 7 from WEBnm@. Original pdb-structure is colored green. BH4 is colored yellow and the Fe2+ ion is colored in red. The mesh represents the model gained from WEBnm@.

</figure> <figure id="fig:mode8">

Animation of mode 8 from WEBnm@. Original pdb-structure is colored green. BH4 is colored yellow and the Fe2+ ion is colored in red. The mesh represents the model gained from WEBnm@.

</figure> <figure id="fig:mode9">

Animation of mode 9 from WEBnm@. Original pdb-structure is colored green. BH4 is colored yellow and the Fe2+ ion is colored in red. The mesh represents the model gained from WEBnm@.

</figure>

elNemo

ElNemo computes ten perturbed models for the first five non-trivial normal modes of the input protein as a standard and perturbations for the lowest 25 modes on request in a separate queue. There is no fixed limit to the size or number of modes but too long running jobs will eventually be killed. The calculation uses a cutoff value to determine which atom-atom interactions are kept in the elastic network model and another parameter to determine how many residues are treated as rigid body to speed up calculation appropriately.

1J8U (without ligand)

Modes 7, 9 and 10 show rather random motions of the protein that are hard to evaluate. Mode 11 and especially mode 8 could show an opening of the catalytic subunit to allow access to the active site. Mode 8 is also the only one, where the termini of the sequence do not move. As the crystal structure is truncated on both ends, the strong movement other modes show there is probably arbitrary .

Mode 7

<figure id="fig:1J8U_nm7">

The crystal structure of 1J8U in transparent green and the normal mode 7 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U_nm7_fluc">

The relative fluctuations of the C alpha atoms of 1J8U computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 7.

</figure>

Mode 8

<figure id="fig:1J8U_nm8">

The crystal structure of 1J8U in transparent green and the normal mode 8 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U_nm8_fluc">

The relative fluctuations of the C alpha atoms of 1J8U computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 8.

</figure>

Mode 9

<figure id="fig:1J8U_nm9">

The crystal structure of 1J8U in transparent green and the normal mode 9 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U_nm9_fluc">

The relative fluctuations of the C alpha atoms of 1J8U computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 9.

</figure>

Mode 10

<figure id="fig:1J8U_nm10">

The crystal structure of 1J8U in transparent green and the normal mode 10 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U_nm10_fluc">

The relative fluctuations of the C alpha atoms of 1J8U computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 10.

</figure>

Mode 11

<figure id="fig:1J8U_nm11">

The crystal structure of 1J8U in transparent green and the normal mode 11 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U_nm11_fluc">

The relative fluctuations of the C alpha atoms of 1J8U computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 11.

</figure>

1J8U and BH4 ligand

We included the ligand tetrahydrobiopterin and the FE(II) molecule in the normal mode analysis. Interestingly, except mode 9 the modes including the ligand appear to be inverted to the ones without ligand. The fluctuation matrices are almost identical but inversely colored, that means, 'opening' motions are now closing motions and vice versa. Modes 7 and 8 are additionally switched. As several crystal structures with different ligands are available (cf. our introduction), we know that different ligands influence the conformation only little. This applies for the wildtype protein at least, as there are disease causing mutations that respond to large doses of tetrahydrobiopterin with restored activity.

Mode 7

<figure id="fig:1J8U+BH4_nm7">

The crystal structure of 1J8U with the BH4 ligand in transparent green and the normal mode 7 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U+BH4_nm7_fluc">

The relative fluctuations of the C alpha atoms of 1J8U with the BH4 ligand computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 7.

</figure>

Mode 8

<figure id="fig:1J8U+BH4_nm8">

The crystal structure of 1J8U with the BH4 ligand in transparent green and the normal mode 8 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U+BH4_nm8_fluc">

The relative fluctuations of the C alpha atoms of 1J8U with the BH4 ligand computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 8.

</figure>

Mode 9

<figure id="fig:1J8U+BH4_nm9">

The crystal structure of 1J8U with the BH4 ligand in transparent green and the normal mode 9 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U+BH4_nm9_fluc">

The relative fluctuations of the C alpha atoms of 1J8U with the BH4 ligand computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 9.

</figure>

Mode 10

<figure id="fig:1J8U+BH4_nm10">

The crystal structure of 1J8U with the BH4 ligand in transparent green and the normal mode 10 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U+BH4_nm10_fluc">

The relative fluctuations of the C alpha atoms of 1J8U with the BH4 ligand computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 10.

</figure>

Mode 11

<figure id="fig:1J8U+BH4_nm11">

The crystal structure of 1J8U with the BH4 ligand in transparent green and the normal mode 11 movement computed by ElNemo in blue.

</figure> <figure id="fig:1J8U+BH4_nm11_fluc">

The relative fluctuations of the C alpha atoms of 1J8U with the BH4 ligand computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 11.

</figure>

1PHZ

<figure id="fig:1PHZ_nm7_fluc">

The relative fluctuations of the C alpha atoms of 1PHZ computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 7. The smaller C-terminal binding domain is ound in the upper left quadrant, the larger catalytic domain in the lower right quadrant

</figure>

<figure id="fig:1PHZ_nm7">

The relative fluctuations of the C alpha atoms of 1PHZ computed by ElNemo. Red or green dots indicate those residues for which the distance significantly increases or decreases respectively in movement related to mode 7. The smaller C-terminal binding domain is ound in the upper left quadrant, the larger catalytic domain in the lower right quadrant

</figure> To see if independent movement of the domains is observable, we submitted another structure that contains the catalytic domain and the binding domain of phenylalanine hydroxylase, 1PHZ. Indeed show the matrices of fluctuation of the C alpha backbone of the low frequency modes almost exclusively the movement of the domains as unit and little intra-domain movement. In <xr id="fig:1PHZ_nm7_fluc"/> the large uncolored area in the nine lower right squares indicates how the catalytic domain moves collectively as rigid unit, the smaller mostly uncolored area in the upper left square shows the less rigid but still coordinated binding domain. See the animation in <xr id="fig:1PHZ_nm7"/> for a nice visualization.

References

<references/>