Project 03
Assignment A

Dihydrofolate reductase (DHFR) is a key enzyme in synthesizing tetrahydrofolate, an essential cofactor required for synthesizing purines, thymidylate, and certain amino acids. By catalyzing the reduction of dihydrofolate to tetrahydrofolate, DHFR facilitates critical reactions necessary for cell growth and proliferation.

Consequently, DHFR is a pivotal target for antibiotics, particularly against bacterial pathogens, and chemotherapeutic agents aimed at rapidly dividing cancer cells. DHFR inhibitors typically function through competitive inhibition, binding to the enzyme's active site and preventing substrate access. These inhibitors often structurally resemble the natural substrate, dihydrofolate, allowing effective competitive binding. Notable DHFR-targeting drugs include methotrexate, used in chemotherapy; trimethoprim, widely used as an antibiotic; and pyrimethamine, an antiprotozoal medication.

Although the enzyme's structure is conserved across species, variations exist that determine its specificity, affinity, and susceptibility to inhibitors. Understanding these structural differences helps researchers design targeted therapies against pathogenic organisms while minimizing effects on human enzymes. In this assignment, you will use PyMOL to retrieve, visualize, and analyze DHFR structures from three sources: humans ( Homo sapiens ), bacteria ( Staphylococcus aureus ), and protozoa ( Toxoplasma gondii ).

Q01 ¶

Points: 20

Retrieve the DHFR structures for Homo sapiens, Staphylococcus aureus, and Toxoplasma gondii using their respective PDB IDs. Download the structures in .pdb format from the Protein Data Bank and load them into PyMOL. You can also use the fetch command with the PDB IDs.

• Homo sapiens (UniProt ID: P00374 , PDB ID: 4M6K )
• Staphylococcus aureus (UniProt ID: P0A017 , PDB ID: 3FRD )
• Toxoplasma gondii (UniProt ID: Q07422 , PDB ID: 4EIL )

Once loaded, remove all water molecules to clean up the structures.

Examine the Full Validation Report for each structure on the PDB website. Assess the structural quality of different chains within each PDB file and determine which chain is the highest quality. This evaluation should be based on geometric validation statistics, particularly the "Quality of Chain" table found in the "Overall quality at a glance" section. Select the chain that has the fewest geometric outliers and the highest percentage of quality markers.

Once you have determined the best chain for each structure, record your selections for later analysis. Your should describe the criteria you used to evaluate chain quality and justify your selection for each species.

PUT YOUR EXPLANATIONS HERE

In [ ]:

HS_CHAIN = ""
SA_CHAIN = ""
TG_CHAIN = ""

HS_CHAIN = "" SA_CHAIN = "" TG_CHAIN = ""

Q02 ¶

Points: 20

Using PyMOL, select all DHFR-relevant ligands in each structure and expand the selection by 8 Angstroms to capture the surrounding residues. Ensure your selection is properly constrained to a single model, as multiple structures might be included by default. If necessary, refine the selection by excluding other unintentionally included objects.

After defining your selections, rename them appropriately for clarity (e.g., 4M6K-8ang ).

After making these selections, you should align your 3FRD and 4EIL selections to your 4M6K selections. For example, if your selections were 4M6K-8ang and 4EIL-8ang , you would run the following command at the bottom of PyMOL>

align 4EIL-8ang, 4M6K-8ang

Your PyMOL session should look like this after aligning both.

Note that your colors may vary based on the order you loaded your structures in.

Upon execution, the alignment output will include the RMSD values, which quantify the structural deviation between the aligned molecules.

Match: read scoring matrix.
Match: assigning 187 x 171 pairwise scores.
MatchAlign: aligning residues (187 vs 171)...
MatchAlign: score 128.000
ExecutiveAlign: 85 atoms aligned.
Executive: RMSD =    0.918 (62 to 62 atoms)
Executive: object "aln_4M6K_to_Ligand-8-ang" created.

The Executive: RMSD = 0.918 (62 to 62 atoms) line will contain the RMSD for for that object. Put these values as the variables below where SA_RMSD is the 3FRD RMSD with respect to 4M6K .

In [ ]:

SA_RMSD = 0.0
TG_RMSD = 0.0

SA_RMSD = 0.0 TG_RMSD = 0.0

Q03 ¶

Points: 20

Switch the visualization mode to a licorice or stick representation for the ligands and active site residues in PyMOL. We will carefully observe the interactions between these residues and the ligands.

You can use PyMOL commands to easily select residues. For example,

select model 4M6K and resi 30

would select Glu30 on the human DHFR. Then you can run to center PyMOL on the selection.

center sele

Please fill out the following dictionary where the keys are 4M6K residues and the values will be used to store the amino acid three-letter abbreviation and index. We filled out the first one for Glu30 to show you an example.

In [ ]:

residues_aligned = {
    "Glu30": {"3FRD": "Asp27", "4EIL": "Asp31"},
    "Phe31": {"3FRD": "", "4EIL": ""},
    "Arg32": {"3FRD": "", "4EIL": ""},
    "Tyr33": {"3FRD": "", "4EIL": ""},
    "Lys68": {"3FRD": "", "4EIL": ""},
    "Thr136": {"3FRD": "", "4EIL": ""},
}

residues_aligned = { "Glu30": {"3FRD": "Asp27", "4EIL": "Asp31"}, "Phe31": {"3FRD": "", "4EIL": ""}, "Arg32": {"3FRD": "", "4EIL": ""}, "Tyr33": {"3FRD": "", "4EIL": ""}, "Lys68": {"3FRD": "", "4EIL": ""}, "Thr136": {"3FRD": "", "4EIL": ""}, }

Q04 ¶

Points: 20

Using your structural analysis, examine the following amino acid residues and answer the corresponding questions regarding their conservation, structural importance, and potential effects of mutations. Explain based on their biophysical properties and functional roles in the enzyme. Note that these residues labels are with respect to 4M6K .

Part A ¶

Consider Glu30 and Thr136 . Why might these residues be conserved across species, while Tyr33 is not? Explain based on their biophysical properties and functional roles in the enzyme.

PUT YOUR ANSWER HERE

Part B ¶

Is Phe31 conserved in all three DHFR structures? What biophysical properties are shared between these structures? Discuss its role in structural stability and/or ligand interactions.

PUT YOUR ANSWER HERE

Part C ¶

Arg32 is mutated into a different amino acid in one or more species. Evaluate whether this mutation significantly impacts enzyme function, considering the role of arginine and its possible substitutions.

PUT YOUR ANSWER HERE

Part D ¶

Examine Lys68 and discuss whether mutations at this position would be significant. Compare this to the Arg32 mutations and determine whether one mutation has a greater potential impact than the other.

PUT YOUR ANSWER HERE

Q05 ¶

Points: 20

Using PyMOL, select the FOL or DHF ligands in each of the DHFR protein structures and expand the selection to include all residues within 4 Angstroms. Ensure that only the specific object is included in your selection, avoiding extraneous structures or molecules.

For each selection, identify hydrogen bonds and electrostatic interactions by following these steps:

1. Click on Find → Polar Contacts → To Other Atoms in Object.
2. Observe the dashed yellow lines representing polar contacts.
3. Count the total number of polar contacts and document the values for each structure in the dictionary below.

The ligand residue names and indices for each structure are as follows:

• 4M6K : FOL201
• 3FRD : DHF300
• 4EIL : FOL703

In [ ]:

n_polar_contacts = {
    "4M6K": 0,
    "3FRD": 0,
    "4EIL": 0,
}

n_polar_contacts = { "4M6K": 0, "3FRD": 0, "4EIL": 0, }

Next, analyze pi-pi interactions:

1. Click on Find → Pi Interactions → All.
2. Identify any pi-pi stacking interactions between the ligand and aromatic protein residues.
3. Record a list of protein residues (in the same format as residues_aligned ) involved in pi-pi interactions for each structure. If none are present, document an empty list.

In [ ]:

pi_pi_interactions = {
    "4M6K": [],
    "3FRD": [],
    "4EIL": [],
}

pi_pi_interactions = { "4M6K": [], "3FRD": [], "4EIL": [], }