HINT 2.30 Manual: Chapter 6S

LESSON 8: Optimizing the Position and Orientation of a Small Ligand

This lesson shows how you may be able to use HINT to improve molecular models that include small ligands such as water. In this case we will analyze a single water molecule that was located crystallographically at the active site of the HIV-1 protease/A74704 complex. This water has already been protonated and optimized via molecular mechanics methods, so we will look at the optimization two ways, first by optimizing the water as it is, and second, by removing the hydrogens, adding them back (randomly) and optimizing the resulting water molecule. It would be best for this lesson if there were no molecules or backgrounds from previous lessons currently active in SYBYL. If you are entering the HINT Tutorial at this point, follow the instructions in Step 1 of Lesson 1.

Prepare the HIV-1 Protease and Ligand

Read in the HIV-1 Protease molecule (hiv1.mol2) and ligand (a74704.mol2) into M1 and M2, respectively. Partition each as before (Lesson 3, step 3 and Lesson 5, step 3.)

Identifying and Characterizing Crystallographic Water(s)

Many crystal structures are reported with the positions of a number of water molecules. Some of these water molecules can be clearly identified as significant contributors to the overall structure of the biomolecule as they, for example, bridge between two subunits, mediate interactions between an enzyme and its inhibitor, or are present in a functionally important location in the biomolecule. Other water molecules are less important, as they are just benignly interacting at the surface of a protein, or interacting only with other waters.

No universal rule has been developed to quickly ascertain the function of a water molecule, other than visual inspection. However, that is a difficult task before the hydrogens have been added and optimized for interaction. However, simple distance relationships such as water molecules within 4 Angstroms of both subunits are potentially bridging, etc. can be proposed. We are putting forward this new function of HINT to facilitate in understanding water.

Optimize a Single Water Molecule
If you examine the structure for the HIV-1 Protease/A74704 complex you will note a single water molecule (opposite of the functional -OH of the inhibitor) that has been read in as part of the inhibitor. To optimize this water, do the following: From the eslc, Hint, HintTable menu, pulldown the Optimize Ligand... command to activate the Small Molecule Ligand Optimization dialog. The first step is to select the ligand molecule. Do that in this case by pressing the Select Ligand (atom) button and then picking the Oxygen [atom M2(107)] of the water. Next, review the Partition Options for Ligand:; the defaults of Partition Method: Calculate, Hydrogen Treatment: Essential, and Polar Proximity: Via Bond should be fine. The default Distance Function, as described previously, for interaction calculations should be used.
Next the Optimization Control Parameters should be set. First, the Cut Off Radius is a much more important choice here than previously as it determines which "site" atoms are to be used for the calculations. This will significantly impact the speed of the optimization calculations. Generally use around 6 for Cut Off Radius. Use Van der Waals Limit of 1.0 as usual. The Translation Limit is the distance we are allowing the ligand to move in its search for an optimum location. As this water is pretty well locked in, enter 0.2 as the Translation Limit. The Convergence is the how tightly we wish to determine the water's location. Use 0.02 as the Convergence. Finally, Speed is a loosely defined parameter related to how fast the optimization engine finds a solution. Low speeds in the HINT 2.30 version of this function are very slow, so choose 4 for Speed.
The Site Selection portion of the setup is to identify what molecules (which have already been Partitioned, see step 1 above) are to be included in the "site" definition. In this case both M1 and M2 are part of the site. Choose these by selecting the check boxes for (Partitioned) Site Molecule 1 and (Partitioned) Site Molecule 2 and choosing the appropriate Sybyl Molecular Areas under the two active push buttons.
Now Press OK. The calculation may take as long as 6-7 minutes and the following will appear in the Sybyl Text window:

Initial Ligand Score: -93.368896 Iteration 1, TestScore = 19.692627 Iteration 2, TestScore = 19.692627 Iteration 3, TestScore = 22.194580 Iteration 4, TestScore = 22.194580 Iteration 5, TestScore = 22.194580 Iteration 6, TestScore = 22.194580 Final Search of Ligand Rotation... Final Ligand Score: 26.199219

Then, the new coordinates for the water will be transferred to Sybyl and its position will be modified on the screen.
Optimize a Poorly Oriented Water Molecule
Read the original A74704 molecule into M3. Now, from the Sybyl Build/Edit pulldown select Delete, Atom.... Pick the two hydrogens on the water [M3(108) and M3(109)]. and press OK. Add these hydrogens back with the Build/Edit, Add, Hydrogens command to Molecular Area M2. Now, re-partition the A74704 molecule in M3. Re-run the Small Ligand Optimization command as before. Most of the parameters should be as you left them. You will need to: 1) pick the water Oxygen as the center of the small ligand, and 2) select M1 and M3 as the site molecules.
Now Press OK.
```
Initial Ligand Score: -12753.454102

Iteration 1, TestScore = -191.313721
Iteration 2, TestScore = -104.167725
Iteration 3, TestScore = -104.167725
Iteration 4, TestScore = -104.167725
Iteration 5, TestScore = -104.167725

Final Search of Ligand Rotation...

Final Ligand Score: -74.254639
```
This result is not identical to the one obtained by starting with the previously optimized water molecule, but visual inspection indicates they are very similar. If you allow a second cycle, you can increase the score to 20.5, with even better overlap with the M2 water. NOTE: You must re-partition M3 before the second (or any suceeding) cycles. The algorithm used to determine the members of the "site" will not function properly otherwise. Also note that one of the reasons that M2 and M3 do not match more exactly is that M3 has a generic H-O-H angle, about 7^o different from that of M2.