LESSON 5: InterMolecular Table and Map Calculations

1. Read in the inhibitor

   Read in the inhibitor A74704. Go to the Molecule pulldown and select the Get command. Choose PDB as the Get File Type and turn Heteroatoms on. Enter a74704.pdb as the File Name; turn Reference Object on and select HIV1 as the Object Name; press Execute.

2. Prepare the inhibitor for HINT calculations

   Because this molecule was read into Hint as a PDB file, you will have to "clean it up" by adding appropriate bonds and CVFF potential types before Hint can Partition or otherwise perform calculations on it. To give us working space, first "blank" HIV1 by choosing the Blank Object command and turning Blank Operation to on for Object Name HIV1. Now use Color Molecule (Color Method: By_Atom, Molecule_Spec: A74704) and Bond_Order Molecule (Bond_Order: on, Molecule_Name: A74704) to make it easier for us to clean up A74704.

   Choose Builder from the Module Pulldown. What you need to do is ensure that the correct bond order is set for the atoms in the A74704 inhibitor. You probably note immediately that the four phenyl rings do not have the expected partial double bonds. Less evident but equally problematic is that the four carbonyls are missing the expected double bonds. From the Modify pulldown select Bond. In this command verify that Bond Operation is set to Modify_Order. With Order set to Partial Double trace around all four of the phenyl rings by selecting each bond by specifiying the end-point atoms. As you do this you should see the displayed order of the bond change. With Order set to Double change the four CO bonds to C=O. Next, select the Potentials command in the Forcefield pulldown. Choose the Potential Action of Fix, verify that the Assmbly/Mol Name is A74704 and press Execute. This operation should assign the proper potential types to the atoms of A74704.

   Corrected A74704 model

   Return the HIV1 molecule display by using the Blank Object command (Blank Operation: off, Object Name: HIV1).

3. Partition the A74704 inhibitor

   Pulldown Hint from the Module menu. Select Molecule from the Partition pulldown. The Assembly/Molecule should be A74704 and the Partition Method should be Calculate. Use Hydrogen_Treatment of Polar_Only and Via_Bonds Polar_Proximity. Press Execute. Hint calculates the LogP for A74704 to be 6.477.

4. The InterMolecular HintTable Calculation for A74704 and HIV1

   From the Setup menu pulldown the HintTable command. In this block set the parameters as follows (Interaction Select: All, Intramonomer: off, Cutoff Radius: 6.00, Van der Waals Limit: 0.90, Output Radius: 6.00, Output Value: 10.00, Output Resolution: Low, Proton Suppress: on, H-Bnd Distance X-Y 3.65). These last four parameters setup the calculation to create an interaction list that contains all interactions within 6.00 Angstrom, having an intensity (MicroInteraction Constant) of |10| or greater, and defining a hydrogen bond as an interaction between H-bond donor and H-bond acceptor atoms of 3.65 Angstrom or less. Press Execute.

   The Hint_DistFunct should be set as follows: Hydropathic Term: exp(-nr), Steric term: on, Steric/Hydro Scaler: 50, and Direction Vectors: None (Spherical). Press Execute.

   Now, choose the InterMolecular command from the HintTable pulldown. Set Molecule 1 to HIV1 and Molecule 2 to A74704. The Table Output File should be hiv1_a747_in.tbl. Choose Execute Mode to be Interactive as this calculation takes less than 30 seconds. Press Execute to begin the HintTable calculation. The resulting Total Interaction Constant (861) represents a prediction from Hint of the binding efficiency of this inhibitor in HIV1. In the absence of other information, this single number is more or less meaningless. However, correlation of Hint Interaction Constant predictions with experimental binding data for several compounds may lead to a model that can be used with new, untested molecular inhibitors. This InterMolecular HintTable calculation also creates the ASCII text file hiv1_a747_in.tbl in the current directory. You can examine this file at any time by opening a new unix window and listing or editing the file directly under unix. It may also be a good idea to print this file. In a later step of this lesson we will examine the HintTable results concurrent with results of a HintGrid calculation.

   HINT Interaction Table for HIV-1 and A74704

5. The InterMolecular HintMap Calculation for A74704 and HIV1

   In this step you will construct 3-D grid maps that enable visualization of the specific binding interactions between the A74704 inhibitor and HIV1. First, describe the Grid region for the calculation using the Grid Setup command (Grid Center: Molecule_Region, Molecule Region: A74704, Border Space: 5.00, Grid Resolution: 1.25) You will perform the calculation twice so that hydrophobic binding interactions can be distinguished from polar binding interactions. First, for the hydrophobic interactions, select the Grid_Interaction Setup command (Interaction Select: Hydrophobic, Intramonomer: off, Cutoff Radius: 6.00, Van der Waals Limit: 0.90). Then, from the HintGrid pulldown select InterMolecular. Set Molecule 1 to HIV1 and Molecule 2 to A74704. This calculation will take 20-30 minutes, so it is probably prudent to run it as a Background job (Execute Mode); set Auto_Get_Grid to on so that the results will automatically be imported into Insight when the background calculation is completed. Set Job Name as hiv1_a747_in_h and Grid File Name as hiv1_a747_in_h.grd. If you wish, enter a Job Comment before you press Execute.

   Next, for the polar interactions, select the Grid_Interaction Setup command (Interaction Select: Polar, Intramonomer: off, Cutoff Radius: 6.00, Van der Waals Limit: 0.90). Again, from the HintGrid pulldown select InterMolecular. Set Molecule 1 to HIV1 and Molecule 2 to A74704. Choose Execute Mode as Background and Auto_Get_Grid to on. Set Job Name as hiv1_a747_in_p and Grid File Name as hiv1_a747_in_p.grd. If you wish, enter a Job Comment before you press Execute.

   After both calculations have completed, the resulting HintGrids can be contoured and displayed (Contour Grid). Contour the hydrophobic interaction grid (HIV1_A747_IN_H) with +100 (green). Contour the polar interaction grid (HIV1_A747_IN_P) with -200 (red) and +200 (blue). These contours represent the significant interactions between the A74704 inhibitor and HIV1. The green regions represent hydrophobic/hydrophobic interactions that promote the binding; the blue regions represent polar/polar interactions such as acid/base, hydrogen bonding, Coulombic, etc. that promote the binding; and the red regions represent polar/polar interactions that appear to discourage the inhibitor binding. Numerical output from HintTable calculations aid in the interpretation of the Interaction HintGrids. There is also significant information in the negative portion of the hydrophobic interaction grid (contour HIV1_A747_IN_H at -200, color purple) -- these data represent hydrophobic/ polar interactions that appear to discourage binding. We advise you treat these data with care, the presence of these negative interactions is an inescapable consequence of biological interactions, and it may be tempting to extract too much significance from them.

   HINT Interaction map for HIV-1 and inhibitor A74704

6. Modify Protein Model to Improve Binding

   One interesting observation in the HIV1/A74704 system is the crucial interaction between the core hydroxyl of the substrate and the carboxylates of ASP 25 and ASP 125. Close examination of this region suggests that a better protein model may be obtained by protonating one of the two ASP residues to add a second hydrogen bond between the substrate and HIV1 in this region. Add a proton to the OD2 atom of ASP 25 (Module: Biopolymer, Residue: Replace, Residue to Replace: HIV1:25, Residue: ASP). Modify the torsion around the new O-H bond (Transform: Torsion, Measure: Distance) until the minimum distance between the new hydrogen and the substrate hydroxy oxygen (303:OX) (ca. 2.31 �) is reached. Repeat the HintTable calculation from the beginning, remembering to Partition HIV1 with the Inferred Solvent Condition so that the modified ASP 25 is properly registered. The Hint interaction constant should now be about 1424 representing an improvement of about 65 % over the original model.