Structure & Function of Macromolecules 2009
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Ranking of HIV-protease inhibitors
using AutoDock
1. Task
Calculate possible binding modes and estimate the binding free energies for 1–3
inhibitors of HIV-protease.
You will learn:
• Some of the theory behind automated docking.
• To use a docking program that is used by scientists all over the world.
• To analyze interactions between a ligand and a receptor.
2. A (very) short introduction to AutoDock
AutoDock is a program that is designed to dock small molecules to a receptor of known
structure. With this program, you can find possible binding sites for ligands and estimate
how well they bind to the receptor.
In the AutoDock calculations, the receptor is held rigid while the ligand is free to rotate,
translate, and change conformation. A modified genetic algorithm (Lamarckian genetic
algorithm) is used to identify possible binding modes for the ligand and, during docking,
a force field energy function evaluates how well it fits in the receptor. After docking, a
semi-empirical energy function is used to estimate the binding free energies for the
ligand.
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3. The receptor: HIV-protease
Open a terminal window. (right click on desktop and choose ‘open terminal’)
Copy files to your home directory using
cp –r
/ibg/courses/1MB280/ProtEng/
.
Open a terminal window and go into to the copied directory (cd ProtEng). Start
AutoDock tools by writing adt. Load the receptor in ADT: File  Read Molecule.
Choose HIVprotease.pdb. Take a close look at the protein using the mouse (See
below). Also color the protein using: Color  by Atom Type. Try to identify possible
binding site!
Try to locate a possible binding site for the ligand!
Mouse:
Right button:
Middle button:
Middle button+shift:
Right button+shift:
translation in x,y-direction
rotate
translation in z-direction
changes slab
Use Edit  Delete  Delete Molecule to remove the protein from the screen.
Now close ADT.
Figure 1. HIV-protease.
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4. The inhibitors: 1hef, 1heg or 1hvj
The pdb files for the ligands are located in the directories: 1hef, 1heg and 1hvj. Move to
one of these directories (cd 1xxx)and start adt. Load the ligand using: Ligand 
Input Molecule  Read Molecule. (change file type to ‘*.pdb’)
Note that 1hef_lig and 1heg_lig should have an N3+ atom in one end: Edit  Atoms
 Edit Type, click the nitrogen and then choose N3+. Add hydrogens to the chosen
ligand by using Edit  Hydrogens  Add. Save the ligand in its directory as a pdb-file:
File  Save  Write PDB. Name the ligand file:ligand.pdb
Use Edit  Delete  Delete Molecule to remove the molecule from the screen. Make
sure that all files have been saved in the correct directory, i.e. the directory of the chosen
ligand.
Figur 2. 1heg.
Make sure that the ADT window is empty.
Ligand
The module Ligand assigns partial charges, rotatable bonds, aromatic carbons, and
identifies polar hydrogens.
•
Reload ligand.pdb using: Ligand  Input Molecule  Read Molecule
- Partial charges are calculated.
- Polar hydrogens are identified.
- Aromatic carbons are identified.
•
Ligand  Define Rigid Root  Automatically.
•
Ligand  Rotatable Bonds  Define Rotatable Bonds
Let amide bonds be rigid. All other bonds should be flexible. Look at the screen
to identify the bond which will be kept rigid. Make sure that all (and only) amide
bonds are ‘Non-rotatable’ (magenta bonds).
•
Ligand  Write PDBQ…
The above information is written to a file. Name it: ligand.out.pdbq
Now remove the ligand from the window! (Edit  Delete  Delete Molecule)
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Grid
AutoDock uses a grid to calculate the energy of a ligand in the receptor. The grid is
calculated before the docking calculation is initialized, which saves computation time.
•
Load HIV-protease using: Grid  Macromolecule  Read Macromolecule
Choose HIVprotease.pdb (The file is located one directory up)
-
•
Kollman charges are calculated.
Solvation parameters are assigned.
The molecule is saved as a .pdbqs file. Name it: HIVprotease.pdbqs
Choose HIV-protease using: Select  Direct Select  Molecule list
Use File  Save  Write PDBQS to save the file in the directory of the
chosen ligand with the name: HIVprotease.pdbqs.
Now press the button: clear selection
•
Identify the atom types of the ligand using Grid  Set Map Types  By
Reading Formatted File. Choose ligand.out.pdbq
These atoms will be used as probes in the calculation of AutoDock’s grid maps.
The window AutoDpf Ligand Parameters shows the identified atom types.
Press Accept.
•
Now you will define the volume where the AutoDock will try to dock the ligand.
The total number of grid points should be approximately 300 000.
Press Grid  Set Grid and use the scroll bars to define a suitable position and
size of the box. (Do not change the Spacing!) When you are satisfied with the
choice of your box, close the window using: File  Close saving current.
•
Grid  Set Other Options
Don’t change anything here.
•
Grid  Write Gpf
Save this file in the directory of the chosen ligand with the name:
HIVprotease.gpf
Run
•
Run  Start AutoGrid
With this command you start the program autogrid3, which creates the atom
maps that are used in the docking of the inhibitor.
Write /opt/bin/autogrid3 in the window Program Pathname and then
press enter. Use Launch to start the calculation. Calculating the grid maps takes
approximately 60 s and a log file with the name HIVprotease.glg is
written.
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Docking
Now we will start the actual docking calculation.
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Docking  Set Macromolecule  Choose Macromolecule
Choose HIV protease.
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Docking  Set Ligand Parameters  Choose Ligand
Choose your ligand.
The window AutoDpf Ligand Parameters shows your chosen parameters.
Click Accept.
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Docking  Set Search Parameters  Genetic Algorithm Parameters
Everything should be default here except:
- Number of GA Runs: 100
- Maximum Number of energy evaluations: 700 000
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Docking  Set Search Parameters  Local Search Parameters:
Click Accept.
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Docking  Set Docking Run Parameters:
Everything should be set to default except:
- RMS Cluster Tolerance (Angstrom): 2.0
Click Accept.
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Docking  Write DPF  GALS.dpf
Save this file with the name: HIVprotease.dpf
Run
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Run  Start AutoDock
With this command, the program AutoDock, which performs the actual docking
calculation, is started. Write /opt/bin/autodock3 in the window Program
Pathname and then press enter . Use Launch to start the calculation.
This calculation takes a few hours to complete. You can look in the
HIVprotease.dlg file (use tail –100 HIVprotease.dlg) to make sure
that the calculation has started.
While the calculation is running in the background, set up a new docking for another
ligand (close ADT and start from the beginning). The results will be analyzed during your
next practical. When you leave, do not log out! Instead, lock your computer by clicking
‘actions’ and then ‘lock computer’ on your desktop.
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5. Analyzing the results
Go to the directory containing the ligand. Start ADT by typing adt.
Analyze
•
Analyze  Docking Logs  Read Docking Log
Reads the docking results.
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Analyze  Molecules  Show Macromolecule
Shows the receptor (HIV-protease).
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Analyze  Results
In Get Output and Show Histogram the results from the docking are
displayed.
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Analyze  Conformations  Show Conformations
Shows all docked conformations.
In Analyze there are also many other ways to analyze the docking results. Explore!
For example, the hbondCommands module can be used to show hydrogen bonds.
Load it by choosing hbondCommands in File  Load Module and press Load
Module. To show these hydrogen bonds Build must be used.
6. Some interesting questions to discuss
•
Why and how do these ligands bind to HIV-protease?
•
Which ligand would you suggest as a possible inhibitor? How would you
improve it?
•
How does the Lamarckian genetic algorithm work? What assumptions
have you made in your calculations? How would you further improve the
accuracy of your results?
7. References
Morris, G. M., Goodsell, D. S., Halliday, R.S., Huey, R., Hart, W. E., Belew, R. K. and Olson, A. J., ”Automated Docking Using a
Lamarckian Genetic Algorithm and and Empirical Binding Free Energy Function”, J. Comp. Chem., 19: 1639-1662, (1998).
Murthy, K. H., Winborne, E. L., Minnich, M. D., Culp, J. S., Debouck, C., ”The crystal structures at 2.2-A resolution of
hydroxyethylene-based inhibitors bound to human immunodeficiency virus type 1 protease show that the inhibitors are
present in two distinct orientations”, J. Biol. Chem., 267, 22770-22778, (1992).
Hosur, M. V., Bhat, T. N., Kempf, D. J., Baldwin, E. T., Liu, B. S., Gulnik, S., Wideburg, N. E., Norbeck, D. W., Appelt, K.,
Erickson, J. W., ”Influence Of Stereochemistry On Activity and Binding Modes For C(2) Symmetry-Based Diol Inhibitors
Of HIV-1 Protease”, J. Am. Chem. Soc., 116 847 (1994).
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