2021 DOCK tutorial 1 with PDBID 1HW9

Introduction

DOCK

DOCK 6 is one of the many tools available to computational biologists that predicts ligand binding geometries and interactions. The functions of DOCK 6 are diverse and have several general applications. A primary use of the program involves a virtual screening of thousands of molecules for an intended purpose. These purposes can include database screenings for molecules that inhibit enzyme activity, bind a particular protein, or even bind to larger complexes. As more versions of the program are released, new features are added such as the inclusion of solvation and receptor flexibility considerations in its calculations.

IHW9

IHW9 is the PDB code for the catalytic complex between human HMG-CoA reductase (HMGR) and Simvastatin. HMGR is considered a rate-controlling enzyme in the metabolic pathway responsible for the biosynthesis of cholesterol. Inhibitors of HMGR, known as statins, are often prescribed as treatment therapies for high cholesterol patients. While statins inhibit the catalytic effect of HMGR, they also provide other positive biochemical effects such as the stimulation of bone growth and anti-inflammatory responses. Studying statin binding using this complex can potentially aid in the discovery of drugs capable of producing these off-target effects.

Directory Preparation

Before even beginning the DOCK prep, it's much easier to create the directories we'll be using throughout the calculation now rather than later. To begin, make a directory using the PDB code provided on the website. This makes organizing the files cleaner and easier to follow.

  mkdir 1HW9

Once the directory is made, enter it as we will be creating several more directories within this directory.

  cd 1HW9

Inside the 1HW9 directory, you should make several additional directories with the following titles. You do not have to follow the same nomenclature provided here as these are simply examples. However, the names you ultimately choose should be indicative of the information you will be keeping in them. This makes finding files much easier later on in the DOCK process.

  001_structure  002_surface_spheres  003_gridbox  004_dock  005_virtual_screen  006_virtual_screen_mpi  007_cartesianmin  008_rescore

You can confirm the presence of these directories within 1HW9 by using the "ls" command.

Receptor Preparation

Downloading and Opening 1HW9

The first file we need to get started is the 1HW9 PDB file from the website. If you head over to the Protein Data Base website, you'll notice a search bar located at the top of the page. Simply type in "1HW9" into that search bar and it will take you to the corresponding page. You can also search using the name of the complex, but since we already have the corresponding code, this makes things much easier.

Once on the webpage, you'll notice a breadth of information regarding the protein complex. Take note of the information you see and where you can access this information such as relevant literature links and images. At the right of your screen, you'll see a "Download Files" tab which you should click on.

  Download Files -> PDB format -> (Your_Specified_Folder)

Since you are now the proud owner of the 1HW9 PDB file, you can begin opening it up in Chimera. Once Chimera is open, go to the top left and click:

  File -> Open -> (Path_to_Specified Folder) -> 1HW9.pdb

Once open, the program should show you something that looks like the above image. This is known as a ribbon diagram and visualizes several properties of the HMGR-Sim complex. Using the ribbon diagram and Chimera, we can ascertain several defining characteristics such as specific residues, rotatable bonds, and protein chains. The image at the moment looks like a lot, but you would never run a calculation with all this information at once. We need to isolate several components first to make the image easier to follow and calculations easier to perform.

DOCK Prep of the Protein Receptor

While having the entire complex at our disposal is nice, it doesn't do much good to have all chains present during the calculations. This spends unnecessary computational resources and time. Instead, we can isolate one of the chains that incorporates the interaction between HMGR and Simvastatin. Our focus will be on Chain A of the complex. In order to visualize the chain:

  Select -> Chain -> A

Notice, however, that this does not remove Chains B, C, and D from our view. We need to perform this action manually.

  Select -> Invert (Selected Molecules) -> Actions -> Atoms/Bonds -> Delete

After deletion, the only thing remaining should be Chain A of the original file. It is within your best interest to save this session as you'll need to come back to this file when preparing the ligand. Doing so will prevent you from having to do the above deletion step again.

  File -> Save Session As ->

Now that the session has been saved, we can go ahead and isolate the receptor. While we've isolated Chain A, you'll notice that Simvastatin is still located on the molecule. In order to remove everything besides HMGR, we need to do the following:

  Select -> Residue -> ADP -> Actions -> Atoms/Bonds -> Delete
  Select -> Residue -> HOH -> Actions -> Atoms/Bonds -> Delete
  Select -> Residue -> SIM -> Actions -> Atoms/Bonds -> Delete

Notice that not only did we remove Simvastatin, but we also removed any ADP and water molecules. These are not vital to the calculations and can be removed as such. The only item remaining should be the isolated receptor.

The isolated receptor can now be saved as a mol2 file which we will again modify shortly after saving.

  File -> Save Mol2 -> 1HW9_rec_noH.mol2

We now need to actually prepare the molecule for future DOCK calculations. This can be done using Chimera to add both charge and hydrogens to the receptor. Fortunately, there is a very useful command in Chimera that can do both in one step.

  Tools -> Structure Editing -> DOCK prep

Follow the onscreen prompts and command windows to fully prep the receptor. This would be a great time to review the literature present on the PDB website. Consulting the literature, you'll notice the neutral charge on the receptor molecule. It's important to note that this charge needs to be the same charge that you assign the receptor molecule in Chimera during the dock prep. After you're finished, the receptor should look like this.

  Atoms/Bonds -> Show

Now that the receptor is prepped, we can save it and move on to isolating and prepping the ligand. This file should be saved as a separate mol2 file.

  File -> Save Mol2 -> (Destination) -> 1HW9_rec_wH.mol2

Ligand Preparation

Isolating the Ligand

Open the session in Chimera we previously saved that consisted of Chain A with both the receptor and Simvastatin ligand. This time around, we'll be using the session file to isolate the ligand instead of the receptor.

  Select -> Residue -> SIM -> Select -> Invert (Selected Molecules) -> Actions -> Atoms/Bonds -> Delete

Similar to the receptor, we'll need to save this ligand as its own mol2 file.

  File -> Save Mol2 -> (Destination) -> 1HW9_lig_noH.mol2

DOCK Prep of the Ligand

We now need to prepare the ligand file for DOCK as well. This will be done in the exact same manner as the DOCK prep for the receptor.

  Tools -> Structure Editing -> DOCK Prep

Again, follow the onscreen prompts to complete this step. However, you'll notice that Simvastatin ligand will have a net charge of -1. This needs to be confirmed using the literature on the PDB website. If we look at the residues at the binding pocket, we'll see the presence of arginine and lysine residues. This would indicate that that ligand here is most likely in a deprotonated state.

Save this file under a different name using the previous nomenclature.

  File -> Save Mol2 -> (Destination) -> 1HW9_lig_wH.mol2

At this point in time, make sure that all previously generated files have been moved to the 001_structure directory we created at the beginning of this tutorial.

Surface Generation and Selection of Spheres

Visualizing the Surface

Chimera is great because it can create an even better visualization of the surface of the receptor protein. The program covers the expected surface of the protein with a series of spheres. In doing so, we create an image that easily visualizes the binding pocket of the protein. In order to create this surface:

  File -> Open -> 1HW9_rec_wH.mol2 -> Actions -> Surface -> Show

The blue surface in the image represents the ligand. It's great to visualize both as you can see where these two molecules are interacting.

Of course, we need to save this surface without the ligand for future calculations. This surface will aid in finding exactly where the ligand-receptor interaction is occuring.

  Tools -> Structure Editing -> Write DMS -> 1HW9_rec_surface.dms

Once saved, please move the .dms file to the 002_surface_spheres directory created earlier.

Sphere Selection

We now need to visualize the empty space that exists on the surface of the protein. These empty spaces represent possible locations for binding to occur. Several properties can be modified when generating the surface spheres such as the minimum difference between the spheres and surface, and maximum/minimum sphere radii. Ideally, we want to avoid steric clashing so we'll set this property to 0. Additionally, we don't want the generated spheres to be abnormally large or abnormally small. As such, we'll set the boundaries for these properties to 4 (max) and 1.4 (min).

Before running the command to generate the spheres, we need to create an input file using the properties previously described. For programming purposes, please name the input file and output file as you see it here.

  vi INSPH

Let's set up the file with the desired properties. Do not copy the comments into your file

  1HW9_rec_surface.dms      -> your input file we made a step before
  R                         -> R flag specifies external sphere generation
  X                         -> Specifies all surface points
  0.0                       -> Steric clashing
  4.0                       -> Max sphere radii
  1.4                       -> Min sphere radii
  1HW9_receptor_noH.sph     -> Output file

We can now use our input file to run the Linux sphere generation command "sphgen"

  sphgen -i INSPH -o OUTSPH

This will generate the .sph file we specified in the INSPH input file. If you followed exactly, 1HW9_receptor_noH.sph should appear in your directory. Let's open it in Chimera to visually confirm the success.

The above image represents the generated spheres on the surface of our protein receptor. If you only open the sphere file, you'll only see the colored spheres in Chimera. Opening up the mol2 file of the receptor will show you where these spheres are in reference to the surface of the receptor.

Notice, however, that some of these surface spheres are relatively useless for the docking calculations. We only want to focus on the spheres that are present within the binding pocket of the receptor. Luckily, there's a command we can use to narrow down the spheres that are within 10 angstroms of Simvastatin.

  sphere_selector 1HW9_receptor_noH.sph 1HW9_lig_wH.mol2 10.0

Box and Grid Generation

DOCK calculations can be computationally expensive, so it's important to perform as many actions as possible to reduce the amount of computation power required. One such step is the generation of an energy grid. DOCK uses the grid to calculate an energy score between one ligand pose and the receptor. Any poses or interactions that occur outside this grid are simply ignored.