53.1. Detecting Competition between a Ligand and Water

The observed X-ray crystallographic structures of enzymes are often used in order to design novel inhibitors. However at a later stage of the research, when the X-ray structure of the enzyme+inhibitor complex has been determined, it sometimes turns out that the ligand does not bind as predicted but has found an alternative binding mode. This can happen because there are positions, which may be called Disfavoured Sites in some receptor clefts from which a specific chemical group seems to be excluded because water would interact better.

In the current release of GRID there is a directive which switches the Programme into Reverse Mode for the detection of such disfavoured places. When GRID is used in this Reverse Mode it computes a balance sheet for the Probe interacting with bulk water on the one hand, and the Probe interacting with its receptor cleft on the other.

In this Tutorial, the active site of the enzyme dihydrofolate reductase (DHFR Brookhaven file 1RG7) will be investigated. The DHFR pdb structure and the inhibitor methotrexate (MTX) are included in the Tutorial directory. Move to the tutorial directory typing:

cd greater06

and start Greater:

Greater

now add the target molecule selecting Targets->Add single target from the menu or pressing the Add single button, insert 1RG7.pdb in the input box and select "Automatic" as filtering level.

Some "warnings" are normally produced when importing this structure. However the filtering method is able to cope with that and the status menu will turn to "ready".

Now prepare the GRID run first choosing the probes for the computation; we will use probes N2, O:: and DRY.

Next define a box for the computation; select Method->Define box size from the menu, uncheck the "automatic" checkbox and put the following values in the input boxes:

then set the following GRID directives:

in the "box" section put the value "2" for NPLA,

in the "advanced" section put the value "1" for ALMD.

Start the GRID run in the usual way.

Once the computation has finished, try to visualise the GRID map using the View button or the Targets->View fields menu command. As usual more information can be found in the User manual page for Gview.

Gview automatically shows the protein structure and the GRID contours for the first DRY probe. Change to the N2 Molecular Interaction Field (N2 is a flat nitrogen with two hydrogens used to mimic the amino groups of the methotrexate).

In order to do this, click on Edit->Select then click on Field and select the N2 field (the second one).

Try to reproduce the content of the Figures reported below.

These Figures show the observed structure of the binding cleft occupied by the inhibitor methotrexate (not used in the calculation).

To change the energy levels, select from the Edit menu of programme Gview:

Edit->Field style

Move the values up to -9.0 Kcal and then press Exit twice.

The attractive blue regions predict the positions where a -NH2 group will bind the protein residues at -9.0 kcal/mol.

To import the MTX inhibitor, click in the Gview graphic windows in:

File->Open and then select the mtx.pdb from the dialogue).

In order to change the stile of the MTX representation click on:

Edit->Selection select mtx.pdb file in the box, press edit style and change the rendering style to sticks and the color to atom type. Then press Exit and again Exit from Selection window.

To display the atom name press ESC in the Gview graphic window (the actual cursor will change to "arrow" style), then click on methotrexate atoms.

View->Toggle mode->put the arrow in the selected atom and click Press ESC button to exit.

As you see the predicted regions correspond to the experimental one of the MTX.

To display the contribution to the Molecular Interaction Field produced by individual protein atoms, select the tab "Field" and click "atom contribution" (atom type color).

The blue regions of NH2-protein attractive interaction will be now mainly coloured in red. This is due to the oxygens of the active site interacting with the NH2 probe. Select the random color by clicking on "atom contribution" (contrast color).

The regions of NH2-protein attractive interaction will be coloured in different colours now, showing that different residues are giving a cooperative interaction with the NH2 groups of methotrexate. For example, the N3 atom of the MTX inhibitor makes strong hydrogen bonds to three atoms of two residues, such as 856 OG1 oxygen of Thr113 and 178 and 179 OD1 OD2 oxygens of Asp27.

The amino substituent N6 of MTX donates a couple of short strong hydrogen bonds to a pair of backbone carbonyl oxygen atoms (34 O of Ile5 and 700 O of Ile94).

Try to visualise the other Molecular Interaction Fields such as those produced by carboxyl probe and the hydrophobic field. You should be able to reproduce these figures below.

Carboxy (O::) probe	Hydrophobic (DRY) probe