MODELING PROTEINLIGAND INTERACTION:

A GATEWAY TO DISEASE TREATMENT

Kristen Procko and Jennifer Truong
In this exercise, you will use the molecular modeling program PyMOL to facilitate
your understanding of biochemical literature. Specifically, you will read a journal
article in which the potential binding interactions are discussed for a proteinligand
complex. You will then model the interactions conferred in the article to delineate how
binding occurs on a molecular level and learn the techniques necessary to model
proteins using PyMOL.

INTRODUCTION:
An understanding of proteinligand interaction is critical to an appreciation of
biochemical processes, drug interactions, and disease states. Proteins play vital roles in
cellular functions, including transport, metabolism, and control of cell growth.
Misfolding of proteins is associated with diseases including cystic fibrosis,
phenylketonuria, Alzheimers disease, and Parkinsons disease.1 Proteins can also be
targets for disease treatment. Medicinal chemists and biologists use structure-based drug
design to formulate novel drugs based on the knowledge of how ligands bind to proteins.
Advances in protein crystallography have paved the way for structure-based drug
discovery. One example, in the development of protease inhibitors for the treatment of
HIV, X-ray crystallography played a critical role.2 Retroviral protease (HIV PR) is an
enzyme encoded by the HIV-I genome that was identified as a drug target in early studies
toward the treatment of HIV infection. Based on the genomic sequence of the virus, it
was hypothesized that HIV PR was similar to a eukaryotic family of aspartic proteases
related to pepsin. Early crystal structures confirmed that the active site of the protein is
indeed very similar to those of pepsin-like proteases. Once synthetic inhibitors of HIV PR
were established, cocrystals of the protease and these compounds revealed that the
enzyme undergoes a substantial conformational change upon binding, and that binding to
each inhibitor was similar; the functional groups of the bound inhibitors aligned well with
respect to each other. A water molecule in the active site was identified that was
coordinated to both the ligand and protein, which prompted the development of novel
urea-based inhibitors that could capitalize on this interaction.2
Protein crystallography is essential to the development of drugs through rational
design. Because this technique will most likely be used to guide the design of a drug that
you prescribe or administer to a patient, we will examine binding on a molecular level
using the modeling program PyMOL.

ABOUT PYMOL:
PyMOL is a molecular modeling program that is particularly effective for the
construction and 3D visualization of macromolecules, including proteins and protein
ligand complexes. This program is available to students free of charge at
http://www.pymol.org/ under download.

PyMOL can be used to visualize .pdb files, which contain a refinement of a

crystal structure. A refinement is a 3D structure of a protein that is fit based on electron
density maps obtained directly via X-ray crystallography. The .pdb files are available
from the protein data bank, http://www.pdb.org: a resource for biological macromolecule
structure. Journal articles that publish protein crystal structures provide .pdb files
through this database.
When a crystal structure is solved and published, the authors may comment on
structural elements of the protein, including sequence information, elements of the
secondary and tertiary structure, the residues of the protein that potentially interact with
the ligand, and the presence of water molecules in the active site. In this exercise, you
will learn to use PyMOL in conjunction with a journal article to gain a better
understanding of the relationship between protein structure and binding, and to help you
better comprehend the biochemical literature.
In this exercise, we will study ligand binding to the mouse major urinary protein
(MUP-I). MUP-I is a transporter protein that carries lipophilic pheromones through the
aqueous environment of the mouses body.3 To begin, read the following paper, which
discusses the nuances of ligand binding to MUP-I. Download the PyMOL application and
the .pdb files of MUP-I bound to 2-sec-butyl-4,5-dihydrothiazole and MUP-I bound to 6hydroxy-6-methyl-3-heptanone.
Timm, D. E., Baker, L. J., Mueller, H., Zidek, L., Novotny, M. V. 2001. Structural basis
of pheromone binding to the mouse major urinary protein (MUP-I). Protein Sci., 10:
9971004.

I. THE BASIC CONTROLS

A. Opening a file:
1. Navigate to the protein data bank and find the desired file. The database allows
one to search by author, protein keyword, PDB ID, or EC number.
2. Find the protein of interest and click on the link. Note: When searching by author,
the title that comes up may not be the title of the article; in this case, look for the
name of the protein or proteinligand complex of interest.
3. On the left hand side of the screen, click Download Files and download PDB
text
4. Click to open the file in PyMOL directly. Alternatively, the file may be saved to
your computer, and then opened.
Note: If the files are saved, you can open multiple files within the same PyMOL window
to create a session with multiple complexes.
B. Moving and manipulating the whole image:

Rotate the image Click left mouse button and drag

Zoom Click right mouse button and drag

Clipping/Cross-sectional view scroll with mouse wheel. This control basically

shaves away layers of the protein so that you can better visualize structural
features that may be blocked by other parts of the protein.
Moving the entire complex click with mouse wheel and drag complex to
desired position. Note: Not every mouse is compatible with this function.

C. The control buttons:

Use these buttons to manipulate the way you view the structure:

[A]ctions Use to rename or delete selections, or to center the complex for ease
of viewing
[S]how Use to display the image in different ways (i.e. as cartoon, sticks, or
mesh surface). Try each of these viewing options. Note: The structural features
will show up on top of each other. In order to show them one at a time, use this in
conjunction with the hide command below.
[H]ide Use to hide elements available under the show command; also may be
used to hide entire selections.
[L]abel Use to show residue names or differentiate chains
[C]olor Use to change the color of a selected object as a whole or by element

D. Entering Command Prompts:

Certain tasks in PyMOL require or can be simplified by a typed command. In this
exercise, text commands will be provided as they are required. When you see PyMOL>
this means to enter the command that is written after the > symbol. Additional
commands are provided in various PyMOL tutorials, which are available online.4
Commands are entered in the following window, which will henceforth be called the
terminal:

E. Rendering and Saving Images:

Whenever you see the symbol , you must produce a screenshot of the image
you create and provide this file to your instructor. In order to do this, you must render the
image to provide for a well-defined picture prior to saving. The commands are as
follows:
3

1. PyMOL>ray 600,400
This will create a rectangular image that is 600 x 400 pixels. (lxh)
2. Click File>Save image as png in the terminal
At some point, you may need to prepare a PyMOL file at high resolution for a
publication. You may wish to save the image at a certain dpi. If you need to do so, use the
following command:
PyMOL>png<space>\users\<username on computer>\<folder you want image in>\<file
name><space>dpi=300
Ex: PyMOL>png \users\Jenny T\desktop\protein1 dpi=300

II. ANALYSIS OF MUP-ILIGAND INTERACTION

2-SEC-BUTYL-4,5-DIHYDROTHIAZOLE
When reading Structural basis of pheromone binding to the mouse major urinary
protein (MUP-I) you should mark the sections in which the binding of ligand and protein
are discussed. We will use excerpts from their discussion to better understand how to
model protein structure and binding. For example, on p. 998, the authors discuss the
secondary structure of the protein:
Excerpt 1:
MUP-I contains a nine-stranded antiparallel -sheet (Fig. 2), which forms an eightstranded -barrel structure Continuous hydrogen bonding between strands in the barrel is maintained by the two portions of the first -strand Two 310 helices occur in
the N-terminal third of the molecule, and a single -helix and a short 310 helix occur near
the C-terminal.
A. Show the protein as a cartoon and change it to a color of your choice. You will
need to hide everything and then show the cartoon. Commands are as follows (note that
you may also accomplish this using the control buttons:
1. PyMOL>hide everything
2. PyMOL>show cartoon
Identify the -helices, the -strands, and the -strands that contribute to the -barrel.
Excerpt 2:
Both pheromones are bound within the hydrophobic environment at one end of the barrel, formed by the side chains of Phe56, Leu58, Leu60, Ile63, Leu72, Phe74, Met87,
Val100, Tyr102, Phe108, Ala121, Leu123, Leu134, and Tyr138.

B. Show the side chain residues within the active site of the protein.
One way to do this is using the sequence. Note that the sequence corresponds to
the primary structure of the protein:
1. Under Display select Sequence [on]
2. Select each residue individually by clicking on it.
Selecting multiple amino acids at once will group them all together as one
selection. Manipulating the selection through the control buttons will not affect
the remainder of the protein structure, only the selection itself.
3. Right-click a residue within the sequence display to open up a menu in order to
choose how to display it (show as sticks, change the color)
You can also use the following commands to show the side chain. This may be simpler as
the residue numbers are listed in the paper.
1.
2.
3.
4.

PyMOL> select active, (resi 56,58,60,63,72,74,87,)

PyMOL> show stick, active
PyMOL> zoom active
Color the residues by element so that the carbons are a contrasting color with
respect to the protein to indicate that these comprise the hydrophobic pocket.

If you do not wish to further operate on the selection, you can delete the selection through
the actions button. This will not affect the changes youve already made to the
selection, it will only prevent you from further operating on it as a group.
Excerpt 3:
The outermost side chains of the ligand binding site (Met87, Phe56, and Leu58) are in
van der Waals contact with the Tyr102 side chain, which caps the entrance to the interior
of the -barrel.
C. Use the measurement wizard to show the van der Waals interactions. Show that
the distanced measured is significant to these interactions.
1. Find the residues by selecting them using the sequence, and then orient the
molecule in a way that shows them both clearly.
2. Deselect the residues.
3. In the terminal, click Wizard Measurement
Distance is selected by default. Clicking on this box will allow you to change the
type of measurement.
4. Click on two atoms. The measured value (in ) between the two atoms will be
displayed. Each measurement will become a separate object selection.
(Accepted bond lengths for van der Waals interactions are between 3.64.5 . If
the values that you obtain are not within the accepted value, delete that
measurement and try again.)
5. When finished, click Done
5

6. After finalizing all your measurements, click on Create New Object and change
to Merge with Previous. This will add all measurements into one object group.
7. Redo all measurements and delete the individual measurements from step 4.
Note: Although this method may seem roundabout, we group the measurements
this way because it is easier to trace over a group accurately. In PyMOL, if youre
inaccurate with one measurement when in merge with previous mode, it is
difficult to undoyou essentially have to delete the whole selection and try again.
8. To hide measured values and only display the dotted line:
PyMOL> hide labels
Excerpt 4:
Two water molecules are also present in the ligand binding site, interacting with the
Tyr138 hydroxyl group and the carbonyl oxygen atoms of Phe56 and Leu58.
D. Display the active site water molecules. Use the following commands:
1. PyMOL>select active_water, ((resi 56,58,138)around 3.5) and(resn HOH)
This command will find water molecules with hydrogen bonding to the
designated residues within 3.5.
2. PyMOL>show spheres, active_water
3. PyMOL>alter active_water, vdw=0.5
4. PyMOL>rebuild
5. PyMOL>color blue, active_water
These commands will display the water as a sphere. Command 3 reduces the radius of the
sphere that has been created. Command 5 colors the sphere blue.
E. Use the instructions for measuring distances (Part C) to show the hydrogen
bonds between the residues listed and the active waters.
Accepted bond lengths for hydrogen bonds are between 2.83.0 , with a minimum bond
length of 2.6 and a maximum bond length of 3.6 .
Excerpt 5:
SBT binds in a specific orientation with the five-membered thiazoline ring proximal to
the entrance to the active site and the branched sec-butyl group oriented toward the
bottom of the -barrel
F. Display the ligand and proteinligand interactions.
With the sequence open, the ligand will be given a three-letter designation, which
in this case is TZL. Right-click on this to open a menu to select how the ligand is
displayed. Alternatively, you can use the following commands to set the ligand as a
separate object, which can be useful for manipulation.
1. PyMOL> select ligand, resn TZL
2. PyMOL> show sticks, ligand

Excerpt 6:
The branched electron density indicates that the SBT ethyl group is located on the side
of the ligand-binding pocket formed by Leu60, Leu72, and Phe74. Whereas the methyl
carbon atom makes potential van der Waals interactions with side-chain carbon atoms of
Ala121, Leu123, and Leu134, the ethyl group is separated by at least 4.6 from the
nearest carbon atom. The unoccupied space around the ethyl group could account for the
reported binding of SBT analogs (i.e., iso-butyl-thiazole) to MUP-I
3. Using the measurement wizard, show van der Waals interactions between the
ligand and Ala 121, Leu 123, and Leu 134.
Excerpt 7:
The thiazole S1 atom is located in potential van der Waals contact with the Leu72,
Phe108, and Val100 side chains
4. Using the measurement wizard, show van der Waals interactions between the
ligand and the residues listed.
Excerpt 8:
The N3 atom is located 2.9 from the water molecule bound to the Phe56 carbonyl
oxygen atom. Thus, binding may involve a water-mediated hydrogen bond to the SBT N3
atom as part of a solvent-inaccessible hydrogen-bond network.
5. Using the measurement wizard, show the hydrogen bonding interactions between
the ligand, water molecules, and appropriate residues.

6-HYDROXY-6-METHYL-3-HEPTANONE
Excerpt 9:
The closest contacts between nonhydrogen atoms in HMH and MUP-I again involve the
side chains of Phe56, Leu58, Leu60, Phe74, Met87, Val100, Tyr102, Phe108, Ala121,
Leu123, and Tyr138 at distances of 2.9-4.1
1. Display the protein as a cartoon.
2. Display the residues within the active site as sticks. Color the residues by atom.
3. Display the ligand as sticks. Color the ligand so that it contrasts well with the
protein.
Excerpt 10:
HMH binding also involves the hydrogen bond network described above, with the
ketone oxygen serving as a hydrogen bond acceptor in the place of the thiazole N3 atom.
The hydroxyl oxygen atom serves as a potential hydrogen bond donor to the Tyr138
hydroxyl oxygen.
4. Use the measurement wizard to show the hydrogen bonds between the residues
and the ligand.

G. Align the two protein-ligand complexes (SBT and HMH)

You can name each protein/protein-ligand complex at this point. Say that we
have protein A bound to ligand A, which we name PEP1 and protein A bound to ligand
B, which we call PEP2. As long as these proteins are part of the same session, we can
align them.When we align, the first file name we type moves, and the second file stays
stationary. In this example, we will pretend that the corresponding residues do not have
the same number. Say that PEP1 begins at residue 18, while PEP2, although the same
protein, begins at residue 1. (A reason that this may occur is due to different tags on the
ends of the proteins. These tags facilitate purification.) Do not start the alignment at the
first residue.
1. PyMOL> align PEP1 & i. 20-171 & n. n+c+o+ca,PEP2 & i. 3-153 & n. n+c+ca+o
Note:
i. = residue
n. = atom type (n = nitrogen, c = carbon, o = oxygen, ca = backbone)
The order of the atom type does not matter when using this command.
2. Select the aligned proteins by clicking on both of their names.
Bonus: Locate and show measurements for all residues that may be involved in van der
Waals interactions with HMH.
1

Dobson, C. M. The structural basis of protein folding and its links with disease. Phil.
Trans. R. Soc. Lond. B 2001, 356, 133145.
2
Wlodawer, A. and Vondrasek, J. Inhibitors of HIV-I protease: A major success of
structure-based drug design. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 24984.
3
Novotny, M. Pheromones, binding proteins and receptor responses in rodents. 2003,
31, 117122.
4
The following URLs link to a some of the many PyMOL tutorials available online:
http://137.189.50.96/kbwong/teaching/pymol/pymol_tutorial.html
http://www.ebi.ac.uk/~gareth/pymol/
http://www.rubor.de/anlagen/PyMOL_Tutorial.pdf
http://www.carlyhuitema.com/pymol_tutorial.html