HisH System Setup

Exercise 1: Using VMD to investigate the hisH active site

To get an introduction to the system, load in the structure HisHmoe_start.pdb into VMD. Select the ligand glutamine and the catalytic triad (Cys84, His178 and Glu180).

Although the glutamine can be attached in VMD, we will use MOE to make the covalent bond and determine what force field parameters are missing.

Exercise 2: System setup in MOE

For the following three exercises, we will use the molecular modelling program MOE (Molecular Operating Environment). Although the exercise makes exclusive use of the widely-used CHARMM22 empirical force field, any of the calculations may be repeated and applied to other available parameter sets (e.g AMBER, OPLS-AA, etc.).

Exercise 2, step 1: Starting MOE

To run moe, type moe at the UNIX command prompt.

Exercise 2, step 2: Loading the protein

Load the starting PDB structure HisHmoe_start.pdb into Moe by clicking on:
File: Open and click OK
Check at the load options: Center molecule and click OK
In MOE we will work with the Main window and the Sequence Editor. The Main window contains pull-down menus and buttons for the major function groupings. Often there are multiple ways a particular feature of the program can be accessed. For example, you can open the Sequence Editor with the buttons on the right or by selecting it under the Window pulldown.

Figure 2: This is what your hisH system should look like in MOE.

You should see something similar to the screen image in figure 2. The Sequence Editor is a very powerful tool to select residues, hide them or render them in different representations.

Exercise 2, step 3: Changing the representation of an object

In the Sequence Editor, click with the left mouse button on the ligand GLN to select it. After it is selected, right mouse click on it. A popup menu will appear. To make the selection:
Atoms: Select (the atoms will turn pink)
Render : Ball and Stick
Now glutamine should be rendered differently than the rest of the residues. Click in the Main window in the open space and the pink arrowheads will disappear (indicating the ligand is deselected).

Exercise 2, step 4: Highlighting CYS84, HIS178 and the ligand

Let's focus in on the catalytic triad to make our work easier. First, we will hide the protein:
Click on Chain 1 to select the entire protein chain
Right click: Atoms: Select
Right click: Atoms: Hide Now only the ligand is shown.
With VMD, you have already determined the residues of the catalytic triad as well as the residues that comprise the docking site for the glutamine ligand. You can access the residue CYS84 rapidly in the Sequence Editor by clicking on them with the left mouse button. The residue will highlight in the Sequence Editor and clicking with the right mouse button shows you the various options you may apply to the selection. Right click: Atoms: Show Now you should see just CYS84 and the ligand.
Repeat this to select HIS178
For selecting atoms by element, hybridization or other chemical properties the Selection pull-down menu is more suitable and you will see examples for using it. With the Render pull-down you can do the same as you have experienced with the pop menus. Play around with different selections and rendering modes to get a feel for the program.

Exercise 2, step 5: Adding missing hydrogens

To add the hydrogens, click on:
In the MAIN window, click on Edit: Add hydrogen
It is important to remember that MOE adds hydrogens by guessing and does not intrinsically know anything about protonation states of the various amino acids. For example, histidine residues can be in a charged or neutral state depending on the local environment within the protein. This property is very often exploited by enzymes in order to carry out biochemical reactions (e.g. as in our example of the catalytic triad of hisH). Since it is important from a functional perspective, when we are building our model we must ensure the model takes the relevant biochemical information into account.

When performing a full system preparation, it is important to check the protonation state of every residue in the protein. However for this particular exercise we will restrict ourselves only to the docking site in hisH.

Figure 3: Making a bond in MOE: note the atoms to be bonded are shown in pink (indicating they are selected).

Exercise 3: Connecting the ligand to the protein: MOE

Now we want to make the covalent bond between glutamine and the CYS84 residue of hisH:
In MAIN window, click on Builder
In MAIN window, click on Label: Name
Now we will build the bond between the sulfur and carbon:
In MAIN window, click on the atom labelled SG
to select the second atom, press SHIFT and click on CDG
click on Bond in the Builder window.
We want to model the system after step 2 of the mechanism (see Figure 1). To do this, we need to delete some atoms. We need to delete the atom NH2 and its hydrogens (doing this will "hydrolize" the ammonia away!). To delete: In MAIN window, click on atom NH2
Now in Builder, click Delete. The attached hydrogens will also be deleted.
Finally select all hydrogens and delete them (we will add the hydrogens back later with another program).
In MAIN window, click Selection : Elements : Hydrogen
In MAIN window, click Edit: Delete
Save your structure:
File: Save as:
Give your file a name, and select Format: PDB.
Now we need to close the session:
File: Close
In case you have problems making this file, we have provided you with the structure HisHmoe_cyg.pdb.