Tutorial

Exploring Protein-ligand Binding

March 31, 2016

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Exploring Protein-ligand Binding

This tutorial takes you through how protein-ligand binding can be explored using CLC Drug
Discovery Workbench. The development of inhibitors of cyclin-dependent kinase 2 (CDK2) is used
as an example.
Typically, a drug target is a specific key protein involved in a particular metabolic or signaling
pathway specific to a disease condition. For a drug (ligand) to hit the target with high affinity and
specificity, it should be complementary in shape and electrostatics to the active region of the
target protein. 3D modeling of the protein-ligand interaction obtained via docking simulations is
therefore an invaluable tool in the process of drug design. It helps you to

Understand the mechanism of ligand binding to the target.

Understand why one compound binds more strongly than another.

Spawn design ideas for improved binders.

Test design ideas before proceeding to synthesis.

Example data relating to this tutorial can be imported from the Help menu (Help | Import Example
Data). The files for this tutorial are found in CLC_Data/Example Data/Protein-ligand docking.

You can work quickly through the tutorial, just doing the actions listed with blue background.
The header titles through the tutorial match the titles of the blue boxes in figure 1.
Notice, that in the end of the tutorial you can find "Good to know..." overviews ... about
molecule data in the workbench, ... about modeling protein-ligand interactions in the
workbench I and ... about modeling protein-ligand interactions in the workbench II.

Import Protein Structure

Molecular docking is performed against a target protein structure. The Protein Data Bank holds
more than 90 thousand protein structures. To find the most relevant structure to use, you can
go to www.pdb.org and search for it, or use the built in Search for PDB Structures at NCBI option
from the Download menu.
For this tutorial you should download the PDB file with ID 1KE5 (see figure 2).

Go to the Download menu and select Search for PDB Structures at NCBI...
Write the PDB ID 1KE5 in the search field and press Start search.
Double click on the row that appears.

The molecule structures listed in the PDB file will then open in a Molecule Project, showing the
content in a 3D view (see figure 3).

Save the Molecule Project, by clicking the Save button in the Toolbar
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Figure 1: Schematic of the tutorial workflow. The Molecule Project is central in the work. It contains
molecule structures that can be visualized in 3D. Molecule Tables and Docking Results Tables
contain rows of molecules with 2D depictions. Example file names are shown in italic.

or select Save in the File menu. A Molecule Project file named 1KE5 appears in the Navigation
Area.
The protein target and other molecules imported with the protein can be visualized using the
Project Tree and visualization options available in the Side Panel. Sequence alignment and
analysis tools can also be used to find out more about the protein. This is described in the
Explore your Protein tutorial.

Find Binding Pockets

A molecular docking is aimed at a specific region of the target, expected to be the binding site.
You may know the location of this site based on the position of ligands or cofactors co-crystallized
with the protein structure, or positions of amino acids known to participate in the binding. If you
do not know the binding site, you can use the Find Binding Pockets tool found in the Toolbox.
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Figure 2: The built-in Search for PDB Structures at NCBI option.

Figure 3: The content of the PDB file 1KE5 imported to a Molecule Project (this visualization is
saved as view 'Just downloaded' on the example file 1KE5).

Invoke the Find Binding Pockets tool found in the Drug Design folder in the Toolbox.
Step 1: Select the Molecule Project 1KE5 as input.
Step 2: Leave the parameters at their defaults setting.
Step 3: Choose to Open the results and click Finish.
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Info box: Druggable binding pockets

Analysis of 5600 protein-ligand structures from the PDB has revealed that 95 % of binding
sites are within one of the three largest solvent-accessible pockets found on the protein [Li
et al., 2008]. In the Find Binding Pockets tool, the lower limit for pocket volumes can
be specified. Pockets with good drug-binding properties are typically, but not always,
compact [Cheng et al., 2007]. The tool will also look for more exposed pockets, if 'Include
more exposed pockets' is checked (see figure 4).

Figure 4: Parameter settings for the Find Binding Pockets tool.

Now some binding pockets will appear in the Project Tree.

Use the check boxes in the Project Tree to display the found binding pockets one by one.

In this case we already knew where to locate the binding site, as the protein structure came with
a co-crystallized inhibitor. As expected, we see that the largest pocket returned from the Find
Binding Pocket tool overlaps with the inhibitor (LS1) position as seen in figure 5.

Figure 5: Binding pocket indicated with green spheres. The co-crystallized ligand seen in ball-and-
sticks representation (this visualization is saved as view 'Largest pocket' on the example file '1KE5
pockets found').

Setup Binding Site

Click the Setup Binding Site button found below the Project Tree in the Side Panel.
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This will raise the Setup Binding Site dialog box as seen in figure 6.

Figure 6: The Setup Binding Site dialog box with the binding site volume indicated as a transparent
green sphere.

The binding site volume to target with the dockings should be specified first. The center of the
site can be based on co-crystallized ligands, binding pockets found using the Find Binding Pockets
tool, or on a set of atoms selected in the 3D view. In this example, we have a co-crystallized
ligand, LS1, which we can use as center. It is important to make sure that the binding site
sphere/volume is set large enough, so that all ligands you plan to dock against the protein can
fit inside it. For this example, the default radius of 13 is fine.
An automatic binding site setup is performed inside the binding site volume. The setup can be
inspected and manually altered via the dialog box. Be aware that all molecules included in the
binding site setup, i.e. protein chains, cofactors, nucleic acids, and water, are treated as rigid
entities during the docking, and the ligands docked to the binding site will not be allowed to
overlap with these molecules. You can go through the categories in the dialog box, and inspect
the settings and e.g. try changing the protonation of one of the amino acids.
Clicking the Help button ( ( )) in the bottom of the dialog box will take you to a detailed
description of the Binding Site Setup dialog box in the manual.

Info box: Binding Pockets

The same algorithm is used to find binding pockets in the Find Binding Pockets tool and
in the Setup Binding Site dialog box. In the case of the Find Binding Pockets tool, pockets
are found on the union of all protein chains in the Molecule Project. In the case of the
Setup Binding Site dialog box, pockets are found on the union of all molecules included
in the binding site setup.

For this example, the automatic binding site setup should be fine, so no changes to the setup
are needed.

Press the OK button.

Your Molecule Project now has a Binding Site Setup included, which appears in the Project Tree,
and your protein target is now ready for docking. You can always inspect and change the Binding
Site Setup of the Molecule Project by re-invoking the Setup Binding Site dialog box.
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Validate Setup
Inspect Co-crystallized Ligand
When a protein structure is downloaded from the Protein Data Bank, it will often come with a co-
crystallized ligand. This ligand appears in the Ligands category in the Project Tree after download.
It is always a good idea to begin your docking studies with a docking of the co-crystallized ligand,
to check if the docking simulation can recreate the correct binding mode for this known binder.
To model protein-ligand binding, it is important that the representation of the molecules is
physically and chemically sound. Preparing a molecule for docking thus means taking care
that it is represented with the proper connections between atoms, bond orders, hydrogen atom
positions, and atom hybridizations. As much of this information as possible is taken from the
input (in this case the PDB file). The rest is automatically assigned by the workbench based on
the given input. Using the ball'n'sticks visualization ( ) for the ligand molecule, the assignment
of bond connections, bond orders, and hydrogen atom positions are clearly visualized in the 3D
view (see figure 7). The assigned atom hybridizations can be inspected from the Property viewer
in the Side Panel, when holding the mouse over an atom.

Figure 7: The co-crystallized ligand showing the representation automatically assigned based on
the PDB input.

In the Issues list ( ), potential issues with the representation of the molecules in the Molecule
Project are listed (figure 8).

Right-click in an empty space in the 3D view and select Show | Issues.

Sort the issues after molecule by clicking the header of the Molecule column.

Selecting issues in the list will zoom to and highlight the involved atoms in the 3D view. No
issues are found related to the ligand on import, but in the next section, corrections will be made
to the ligand representation, that will make issues appear in the list.

Correct the Ligand Representation

If there is something you wish to change about the representation of a molecule, you can
manually adjust the atom and bond properties via the context menu on the atom in focus in the
3D view. In this case, the chemical group -N=CH2 does not represent the molecule added to the
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Figure 8: The Issues list of the Molecule Project seen in split-view with the co-crystallized inhibitor.

protein. In the paper describing the protein structure [Bramson et al., 2001], the chemical group
of the inhibitor is described as -NH-CH3 . The reason why the workbench has assigned a double
bond in this location is the S-N-C angle, which is very different from what would be expected for
a group with single bonds. You can measure this angle by clicking the three atoms S-N-C while
holding down Ctrl (Cmd on Mac), then the angle will be shown in the Property viewer found in the
Side Panel (129 degrees).
You should now change the representation of the chemical group. When changing bonds for an
atom, the neighboring atoms are listed with their atom names, such as N33 for a nitrogen. The
atom names can be displayed by selecting the ligand in the Project Tree and clicking the Label
button below the Project Tree.

Move the mouse pointer to the terminal carbon atom and right-click.
Set hybridization | SP3
Set hydrogen count | 3
Set bond order for | Bond to N33 | Single
Then move the mouse pointer to the nitrogen and right-click.
Set hybridization | SP3
Set hydrogen count | 1

While making the changes to the representation, you should see issues appear and disappear
in the Issues list - as seen in figure 9. In the end you should have no issues left related to the
ligand molecule.
Even when there are no issues left on the molecule, you should make sure the representation
adheres to your knowledge about the molecule.

Save the Molecule Project.

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Figure 9: Chemistry issue appearing in the Issue list while changing molecule representation.

Now the ligand is ready to be docked to the binding site.

Fast Track Docking

The molecules found in the Ligands category in the Project Tree can be docked to the Binding
Site Setup of the Molecule Project, if such is present.

Select the co-crystallized ligand by clicking on it in the Project Tree.

Click the 'Dock Ligand' button found below the Project Tree (see figure 10).

The docking simulation will now start in the background. When the docking is done, the best
scoring binding mode, found by the docking simulation, will appear in the Docking results category
in the Project Tree.
The docking simulation is stochastic, and you will not get exactly the same binding mode returned
for each time it is run, even with the same input.

Use the checkboxes in the Project Tree to display the Docking result LS1 together with the
co-crystallized ligand LS1, to compare their binding modes as exemplified in figure 11.
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Figure 10: Select one or more ligands in the Project Tree and press Dock Ligand.

Figure 11: Comparing the binding mode of the co-crystallized ligand with the docking result (this
visualization is saved as view 'Binding modes compared' on the example file '1KE5 setup validated').
You can right-click quick-style buttons (or click-hold) to get menus with available color schemes.

Figure 11 shows the co-crystallized ligand in ball-and-sticks representation and the binding mode
returned from the docking of the ligand in sticks representation with brown carbon atoms. The
core of the ligand, where the important protein interactions are found, are accurately established
in the docking result.
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Info box: Failure to re-establish known binding mode

If a co-crystallized ligand does not establish its known binding mode in the docking, the
setup of the binding site and the preparation of the ligand should be re-evaluated. Also,
the co-crystallized ligand can be extracted from the Molecule Project using the Extract
Ligands tool in the Toolbox. This will put the ligand in a Molecule Table that can be used
as input for the Dock Ligands tool found in the Toolbox and described in the following.
That will give access to tune parameters related to the sampling of binding modes.

Inspect Docking Result

To study how the protein interacts with the ligand, select either the ligand LS1 or the docking
result LS1 from the Project Tree, and invoke the right-click context menu. Pick Binding Site
Interactions | Show Hydrogen Bonds to display protein residues forming hydrogen bonds to the
ligand, with the hydrogen bonds shown as blue dashed lines. Use Binding Site Interactions | Hide
Hydrogen Bonds to hide them again.
Pick Binding Site Interactions | Create Interacting Atoms Group to generate an atom group
consisting of protein residues, and molecules included in the binding site setup, which have at
least one heavy atom within 5 of a ligand heavy atom. The atom group appears in the Atom
groups category in the Project Tree. It can be un-displayed and the visualization changed using
the quick-style buttons found in the bottom of the Project Tree, just like for molecule entries.
Individual residues of particular interest can also be selected from the atom group or from
the hydrogen bond visualization. Double-click on an atom to select all atoms in that residue.
Invoke the context-menu on the Current selection in the Project Tree, and pick Create group from
selection. Then a new atom group will appear, only containing the selected residue, and the atom
group showing all residues around the ligand can be hidden.

Import Small Molecules

We can now go on to import other molecules that we wish to see binding to the target protein.
Molecules you wish to see binding to the binding site can be pasted directly into the Molecule
Project as SMILES strings, which is handy if you have sketched the molecule in a 2D sketching
program (see the Dock Ligands from a 2D Molecule Sketch tutorial). You can also import molecules
with 3D coordinates to the workbench as a Molecule Table or add them to the Molecule Project
holding the binding site setup. In both cases, the file formats SDF, Mol2 and PDB are supported.
If the molecule is imported using the Add Molecules to Molecule Project... importer, it will appear
in the Ligands category in the Project Tree, and can be docked the same way as the co-crystallized
ligand. Alternatively, the molecule can be imported to a Molecule Table, and docked to the
Binding Site Setup in the Molecule Project using the Dock Ligands tool found in the Toolbox.
This is what we will do now.
Using the Standard Import or Import molecules with 3D coordinates... options, molecule structures
saved in Mol2 or SDF format can be imported to a Molecule Table from your computer file system.
For this tutorial, an SDF file with three ligands has been imported (example file: 3compounds).
The compounds are taken from the same inhibitor study as the protein crystal structure [Bramson
et al., 2001]. The co-crystallized inhibitor is compound 98 in that study, and the three compounds
in the example file are compound 32, 72, and 90.
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Inspect Molecules
Double-click the file 3compounds in the Example Data in the Navigation Area to open it. Then
right-click on a row and select Show | Issues, to check that there are no issues associated with
the molecules. In this case, no issues are found, and the Issues list can be closed again.
The molecules are visible as 2D depictions in the table view, and each entry holds information
about atom coordinates in 3D space. To see the molecules in 3D, click the Select View action
in the table Side Panel. Then choose to view the molecules in 'New molecule project'. You can
look at the molecules in 3D, by selecting the rows in the table. Close the 3D view, showing the
molecules from the table.

Dock Ligands

Invoke the Dock Ligands tool wizard from the Toolbox.

In Step 1, select the Molecule Table 3compounds, holding the ligands to dock, as input.
In Step 2, select the Molecule Project with the Binding Site Setup as 'Binding site', and
leave the other parameters at their default values (figure 12). Note, the wizard will always
remember the parameters you used last. To return to the default settings, click the
arrow-button in the lower left corner.
In Step 3, choose to Open the results, and click Finish.

Figure 12: The default parameters for molecular docking.

The docking will then start, and the process can be followed from the Processes tab in the
Toolbox panel. The docking simulation will output the results in a Docking Results Table that will
open when the docking is done.
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Info box: The Dock Ligands tool versus the Dock Ligand button.
Invoking the Dock Ligands tool or clicking the Dock Ligand button result in the same
operations, the only difference is how the input is selected, the output presented, and if
parameters can be customized.

Dock Ligands tool in Toolbox: A number of parameters can be customized, e.g. the
level of sampling. Ligands should be in Molecule Tables, and all molecules in the
tables selected as input are docked. Docking results are presented in a Docking
Results Table.

Dock Ligand button in Side Panel: No parameters can be customized, the default
values are used. Only ligands in the same Molecule Project as the Binding Site
Setup can be docked. Only selected ligands are docked. Docking results are directly
added to the Molecule Project.

Inspect Docking Results

The docking simulation outputs a 3D model of how each ligand might bind to the protein (the
binding mode), as well as a scoring of this binding (see the Info box: Scoring a Binding Mode).
No matter if the docking has been carried out from inside a Molecule Project, or via the Dock
Ligands tool like in this case, both the binding mode and the score are easily accessible.
The Dock Ligands tool outputs a Docking Results Table where 2D sketches of the docked ligands
can be seen, together with information on the scores of the ligand binding modes.

Make sure the Molecule Project holding the Binding Site Setup is open in the View Area.
From the Side Panel of the docking results table, use the Select View action to connect
with the Molecule Project containing the Binding Site Setup.
Make sure the protein is visible in the Molecule Project.
Browse through the table, to see the generated protein-ligand complexes in the 3D view.
Use the options in the table Side Panel to show or hide hydrogen bonds or nearby protein
atoms for the docked ligands.
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Info box: Scoring a Binding Mode

A particular binding mode of a ligand in the protein binding pocket is connected with a
score. The molecular docking simulation searches through numerous potential binding
modes of the ligand in the pocket, and the one resulting in the best score is returned from
the docking. The score mimics the potential energy change, when the protein and ligand
come together. This means that a very negative score corresponds to a strong binding
and a less negative or even positive score corresponds to a weak or non-existing binding.

Score = Stargetligand + Sligand

The Starget-ligand term is a sum over contributions from all heavy atom contacts between the
ligand and the molecules included in the binding site setup. It scores the complementarity
between the binding site and ligand by rewarding and punishing different types of heavy
atom contacts (inter atom distance below ~5.5 ). The target-ligand score is split in
three types of contributions in the Docking Results Table; Hydrogen bond score, Metal
interaction score, and Steric interaction score.
Rewarded contacts Punished contacts
Hydrogen bond interactions Too close contacts (clashes)
Lone-pair - metal ion interactions Hydrogen bond donor-donor contacts
Non-polar interactions Hydrogen bond donor-metal contacts
Hydrogen bond acceptor-acceptor contacts
Contacts between non-polar atoms and hydrogen
bond donors and acceptors

The Sligand term punishes internal heavy atom clashes in the ligand and strain resulting
from unfavorable bond rotations. This contribution to the score is listed in the Docking
Results Table as Ligand conformation penalty. As the score is a sum over contributions,
a large ligand can get a better score than a small one, simply due to its size. When
comparing scores for different molecules, this effect has to be considered and kept in
mind.

As the search for best binding mode is stochastic, the final binding mode, and thus the score,
will not be identical between docking simulations. The score mimics the potential energy change
on ligand binding, but cannot be directly compared to any experimentally measured value. The
main purpose of the binding mode score is to return the most likely binding mode of a particular
ligand, the second purpose is to compare binding strength between ligands, which is a much
harder task. In the table below is shown the measured inhibitory effect (Kinase IC50 ) of the four
compounds included in this tutorial [Bramson et al., 2001]. Compound 98 is the co-crystallized
ligand.
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Compound Kinase IC50 (nM) Score

90 4.3 -66.95
32 13 -71.07
98 560 -57.82
72 >10,000 -52.92
The docked ligands appear as guests in the Project Tree of the Molecule Project. If you prefer to
keep one or more of the bound ligand conformations in the Molecule Project, for future reference
and inspiration, molecules selected in the table can simply be copied to the Molecule Project
using the Copy Selected to Project action in the table Side Panel. The selected table entries will
then appear in the Docking results category in the Project Tree, and will be saved together with
the Molecule Project. The score of the result will be shown in the Property Viewer palette in the
Side Panel, when the docking result is selected in the Project Tree.

Continue the Exploration

Inspired by the structural model of the ligand binding, you can sketch up new ideas for binders,
import them into the workbench and dock these new molecules to the same Binding Site Setup.
You can also use the Ligand Optimizer, found below the Project Tree in the Molecule Project, to
play around with ligand modifications in the binding site.
Maybe, it is also worth playing around with the Binding Site Setup, maybe include some water
molecules in the binding site, or try to use another protein structure as target, if available. There is
a section in the Drug Design chapter of the manual on Improving docking and screening accuracy,
which describes aspects to consider regarding Binding Site Setup, ligand representation, and the
docking simulation itself.
You can also make the Molecule Project, including the Binding Site Setup and a selection of
interesting docking results, available for other researchers on the project, so that they can inspect
and potentially continue the protein-ligand docking studies on their own. If you wish to continue
the modeling exploration of the protein-ligand complexes using other software, Molecule Tables
and Molecule Projects can be exported in Mol2 format.
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References
[Bramson et al., 2001] Bramson, H. N., Corona, J., Davis, S. T., Dickerson, S. H., Edelstein, M.,
Frye, S. V., Gampe, R. T., Harris, P. A., Hassell, A., Holmes, W. D., Hunter, R. N., Lackey,
K. E., Lovejoy, B., Luzzio, M. J., Montana, V., Rocque, W. J., Rusnak, D., Shewchuk, L., Veal,
J. M., Walker, D. H., and Kuyper, L. F. (2001). Oxindole-based inhibitors of cyclin-dependent
kinase 2 (cdk2): Design, synthesis, enzymatic activities, and x-ray crystallographic analysis.
Journal of Medicinal Chemistry, 44(25):4339--4358. PMID: 11728181.

[Cheng et al., 2007] Cheng, A. C., Coleman, R. G., Smyth, K. T., Cao, Q., Soulard, P., Caffrey,
D. R., Salzberg, A. C., and Huang, E. S. (2007). Structure-based maximal affinity model
predicts small-molecule druggability. Nat Biotechnol, 25(1):71--75.

[Li et al., 2008] Li, B., Turuvekere, S., Agrawal, M., La, D., Ramani, K., and Kihara, D. (2008).
Characterization of local geometry of protein surfaces with the visibility criterion. Proteins,
71(2):670--683.
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Good to know...

... about molecule data in the workbench

Molecule data can be stored in either Molecule Projects ( ) or Molecule Tables ( ).
Molecule Project (3D view)

Content: Molecule 3D structures listed as Project Tree entries, molecule data, Binding Site
Setup, binding pockets, custom atom groups, and connection to protein sequences.
Molecule Table

Content: Molecule 3D structures listed as table entries and molecule data.

Connect to a Molecule Project to see molecules in 3D

Molecules selected in table show up as 'guests' in the connected Molecule Project (indicated by
red square above).
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Good to know...

... about modeling protein-ligand interactions in the workbench I

Dock and Screen Ligands
Molecule interactions are modeled and analyzed between ligands and a Binding Site Setup.
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Good to know...

... about modeling protein-ligand interactions in the workbench II

One-click Docking
Molecule interactions are modeled and analyzed between ligands and a Binding Site Setup.