Tutorial about NR functioning by Simon Folkertsma, June 2003.
Figures used in this tutorial are from Bourget et. al TiPS-October 2000 (Vol. 21)

Nuclear Receptor Functioning

Figure 1. Schematic representation of a nuclear receptor.

1) The DNA binding domain (DBD), which targets the NR to the DNA where it binds the hormone response element (HRE).
2) The ligand binding domain (LBD), which is mainly involved in dimerization and ligand binding.

The LBD shows a common helical fold of twelve helices and a sheet that consists in most cases of two short strands (Figure 2).

Figure 2. An NR LBD. Helices are numbered H1-H12, S is sheet, act = coactivator.

The transcriptional function of NRs is regulated by (lipophilic) ligands (For some NRs there is no ligand identified yet, these are the so-called orphans) and coregulators (coactivators and corepressors) that bind to the LBD. Coactivators (such as SRC-1, SRC-2, SRC-3) are proteins that normally activate transription of target genes, whereas corepressors (e.g. N-CoR and SMRT) repress the transcription. An activating compound induces a cognate surface for coactivator binding. Repositioning of the flexible helix 12 (which is the C-terminal alpha helix) is crucial in the forming of this binding site (Figure 3).

Figure 3. a) Nuclear receptor Apo-LBD (ligand binding domain without a ligand). b) Holo-LBD (ligand binding domain with an agonist bound).

You can see why this move of helix 12 is so important in more detail in Figure 4. The very conserved glutamate (E542) in this helix is positioned correctly to form a charge clamp with the conserved lysine (K362, sometimes an arginine is observed in other NRs) in the C-terminal part of helix 3. This charge clamp is very well conserved throughout the whole family. The distance between these two opposite charges is important for proper coactivator binding.

Figure 4. The charge clamp is crucial in forming the right coactivator interaction surface. Helix 12 is indicated in red, the coactivator peptide in green.

Now, let's see this in 3D by ourselves.

Nowadays there is a lot known about NRs. All these data is automatically collected, linked and updated in the NucleaRDB, which is available at www.receptors.org/NR.

Table 1: Some of the estrogen nuclear receptor crystal structures
PDB code	Name of Ligand	Type of Ligand
1A52	Estradiol	Agonist
3ERD	Diethylstilbestrol	Agonist
3ERT	4-hydroxytamoxifen	Antagonist
1ERR	Raloxifene	Antagonist

As you can see in Table one, there are more structures available for the estrogen alpha receptor (most of them have different ligands in it, or different coactivators bound). If you are interested in other NRs or in other receptors, you can download the 3D structures at the Brookhaven Protein Data Bank (www.rcsb.org). There is also a tutorial how to download these strucures. If you have time left at the end of this part, read (or listen to the narrated) tutorial here.

For now, we focus on the estrogen alpha receptor. Download 3erd.pdb and 3ert.pdb through the link in Table one. We use 3ERD (we removed one monomer, the original PDB file is a dimer), an agonist structure, to see the mechanism of agonism in more detail.

Start Sybyl from your course account directory.

>> Get 3ERD on the screen. Choose the menu File, Read..., pick your file, click OK, and Yes center the molecule.

Have a look, right mouse button to rotate, middle mouse button and the right one to zoom (If you run the application under Windows). First we have to locate the coactivator.

>> Choose the menu View, Biopolymer Display, Ribbon/Tube...

The Sequence Expression Window appears:



Two chains are present in this PDB file, use the horizontal scroll bar in this window to identify the shortest chain of the two, this is the coacivator peptide (in the PDB file only the interacting part of the coactivator is present).

>> Select the residues of the shortest chain in the Sequence Expression window.

>> Click OK, choose a color and click OK again.

The coactivator is situated at the surface of the protein. To get a clearer view on the coactivator and the interaction residues of the NR LBD, we want to delete the 'non interesting' part of the receptor.

>> Click the menu Build/Edit, Delete, Substructure...

The Substructure Expression window appears.

>> Select the residues of the coactivator chain. Click Sets... Check Sphere, and type 5 as radius, click OK, and finally click Invert (you don't want to delete your coactivator) and OK

Now we want to color the residues forming the charge clamp.

>> Click the menu View, Color, Atoms.

The Atom Expression window appears.

>> Click Substructures... Select the lysine of the charge clamp (use Figure 4...), click OK, click OK, color the residue cyan.

>> Color the glutamate of the clamp red.

Now it can be useful to use your middle mouse button, by doing this you can translate the structure on your screen.

It is clear now, that on both ends of the coactivator helix there is interaction with the two residues of the charge clamp.

Question:

What is the distance between the sidechains of these two residues? (Use Analyze, Measure, Distance... pick two atoms and the distance appears on the command line (in the terminal in which you opened Sybyl), click End in the Measure Distance window to go back to Sybyl.

The residues are important because they form hydrogen bonds with helix 12:

>> Click menu Build/Edit, Add, Hydrogens.

To view the bonds:

>> Click menu View, Display H-Bonds, Static...Click All, OK, choose WHITE, click OK, click OK (Click Yes)

Both coactivators and corepressors use a short helical motif to interact with the NR LBD. Coactivators can bind through the LXXLL sequence motif, repressors use the LXXXIXXXL/I motif. In this case the receptor interacts with a coactivator.

Question:

What is the sequence of the coactivator helix which interacts with this receptor? Use Label in View menu and select all.

You can close Sybyl by:

>> Click menu File, Exit SYBYL, click OK.


As an alternative to using Sybyl, you can have a look at the following chime page (Windows only).

Many biological questions involving nuclear receptor transactivation, ligand binding and pathological states can directly or indirectly be answered using mutant data.
At the CMBI we have created a mutation database for NRs, the Nuclear Receptor Mutation Database (NRMD), which is part of the NucleaRDB. This database contains 1095 mutations in 41 receptors, is easy to use and gives the user the opportunity to search the data in different ways.

>> Click here to go to the NRMD (alternatively you can go to the NucleaRDB. Scroll to Mutation data and click the NRMD).

Next we wanted to know what the effect is of mutations at the lysine of the charge clamp.

Question:

Search for mutation effects with the information you already have about this lysine (you know in which receptor, in which region, and you know the residue that has to be mutated). Don't search with the SWISS-PROT numbering, this can be different between different species. How is coregulator binding affected when this residue is mutated to A, D, L and R, and can you explain this with your knowledge of the amino acid properties (more about amino acids is available here)?

Now we have seen the mechanism of NR activation. The mechanism is shown in 2D in Figure 5. Now we want to know how an antagonist works.

Figure 5. 2D representation of how an agonist works.

Start Sybyl again from your course account directory.

>> Load the pdb file 3ERT.

Question:

In what position is helix 12 (which is crucial in coactivator recruitment), compare it with the previous PDB file. In other words, make a 2D representation of an antagonist structure as in Figure 5.

Look carefully at both the agonist (Diethylstilbestrol) and antagonist (4-hydroxytamoxifen) compound that we have seen in the two protein structures.

Since Sybyl cannot show the ligand properly (no bonds) you can also search the Internet to find the structures.

Question:

Looking at the antagonist compound, can you explain why helix 12 is in another position than when an agonist is bound?