Energy Calculations for 4-Hydroxy-3-Methylbutyl Diphosphate

Elissa Williams

Modeling of Protein-Small Molecule Complexes; The Protein Isopentenyl-Diphosphate Delta-Isomerase And the Hetero Compound 4-Hydroxy-3-MethylButyl Diphosphate

Introduction

In the bacterium Escherichia coli, the protein Isopentenyl-Diphosphate Delta-Isomerase (PDB ID Code: 1NFZ) is a catalytic enzyme which transforms the compound isopentenyl pyrophosphate (IPP) into its isomer dimethylallyl pyrophosphate (DMAPP). IPP and DMAPP are activated isoprenes, compounds which serve vital functions in bacteria. IPP and DMAPP are the precursors to over 50,000 compounds in bacteria such as signaling molecules and components of the plasma membrane. The IPP and DMAPP Isomerase protein is found in a variety of bacteria in addition to E. coli. The hetero compound, 4-Hydroxy-3-MethylButyl Diphosphate (HET Code: EIP), is an irreversible inhibitor of the protein Isopentenyl-Diphosphate Delta-Isomerase and covalently binds to the protein when it is in an epoxy form. Experiments in which EIP is bound to 1NFZ and crystallized have lead researchers to believe that the key amino acid residues involved in isomerization of IPP to DMAPP are Glu-116, Tyr-104, and Cys-67.

1 Elissa Williams

Part I: Choosing a Protein-Hetero compound Complex and Extracting the Hetero compound

In Part I of the Protein Modeling Assignment, a protein and a hetero compound were selected. The selected hetero compound and protein were complexed non-covalently to one another. The hetero compound which was chosen for the Protein Modeling Assignment was 4- Hydroxy-3-MethylButyl Diphosphate (HET Code: EIP). The protein chosen for the Protein Modeling Assignment, which complexes with the hetero compound, was Isopentenyl-Diphosphate Delta-Isomerase (PDB ID Code: 1NFZ). A PDB file of the complexed hetero compound and protein was obtained from the RCSB Protein Data Bank website and downloaded using the computer program DS Visualizer. The hetero compound was then extracted from the protein, Figure 1, in DS Visualizer.

Figure 1: The Hetero compound. The Hetero compound 4-Hydroxy-3-MethylButyl Diphosphate from Isopentenyl-Diphosphate Delta-Isomerase.

2 Elissa Williams

Part II: Displaying the Protein-Hetero compound Complex

For Part II of the Protein Modeling Assignment, the PDB file downloaded in DS Visualizer was displayed in a different format in order to obtain a more visually pleasing image of the hetero compound complexed within the protein. As seen in Figure 2, the protein is in a solid ribbon format in gray, blue, magenta, and green colors. The hetero compound is complexed in the right subunit of the protein and is in ball and stick style colored by element.

Figure 2: The Protein-Hetero compound Complex. The Hetero compound 4-Hydroxy-3- MethylButyl Diphosphate Complexed within Isopentenyl-Diphosphate Delta-Isomerase.

3 Elissa Williams

Part III: Energy Calculations for 4-Hydroxy-3-MethylButyl Diphosphate

For Part III of the Protein Modeling Assignment, the extracted hetero compound file, created in DS Visualizer, was imported into Chem3D Ultra. Chem3D Ultra was then used to compute the steric energy calculations of the extracted hetero compound. These energy calculations are titled “Single Point Energy Calculations” in Table 1. The extracted hetero compound, as seen in Chem3D Ultra, is the left hand image in Figure 3. An energy minimization, or geometry minimization, was performed on the hetero compound. An image of the resulting energy minimized hetero compound is the right hand image in Figure 3. Chem3D Ultra was used to compute the steric energy calculations of the energy minimized hetero compound. These energy calculations are titled “Minimized Energy Calculations” in Table 1. A discussion was written which compares the steric energy terms for the extracted hetero compound and the energy minimized hetero compound.

Energy Term Single Point Energy Calculations(kcal/mol) Minimized Energy Calculations(kcal/mol) Stretch 0.2573 1.0014 Bend 21.038 5.5746 Stretch-Bend -0.0283 0.4464 Torsion 2.5847 2.152 Non-1,4 VDW 6.564 -0.3777 1,4 VDW 8.7138 9.4056 Charge/Charge -94.5526 -103.3567 Charge/Dipole 12.2533 5.2264 Dipole/Dipole -9.7998 -8.185 Total -52.9698 -88.113 Table 1: Energy Calculations. Chem3D Energy Calculations for the extracted and energy minimized forms of 4-Hydroxy-3-MethylButyl Diphosphate.

4 Elissa Williams

Figure 3: The Hetero compound with and without Energy Minimization. The Structure of 4- Hydroxy-3-MethylButyl Diphosphate when it is complexed within Isopentenyl-Diphosphate Delta- Isomerase (Left Figure) and Chem3D Energy Minimized 4-Hydroxy-3-MethylButyl Diphosphate (Right Figure).

5 Elissa Williams

Part III Discussion: Comparison of Steric Energy Terms before and After Energy Minimization of the Hetero compound

All of the calculated energy terms for the hetero compound, 4-Hydroxy-3-MethylButyl Diphosphate, changed when a MM2 energy minimization was performed on the hetero compound. Not only do the values in Table 1 reflect the differences in energy of the two structures of the hetero compound but also it is evident in Figure 3 that 4-Hydroxy-3-MethylButyl Diphosphate was rotated, bent, and twisted after the energy minimization. The total energy of the hetero compound decreased ~35kcal/mol after the energy minimization.

The stretch energy term, which represents the energy associated with any bonds that are distorted from optimal length, increased ~0.7kcal/mol once the energy minimization was performed. The bend energy, the energy associated with bond angles that are deformed from their optimal values, was 21.038kcal/mol for the non-energy minimized hetero compound and 5.5746kcal/mol for the energy minimized hetero compound. The stretch-bend energy or the energy required to stretch two bonds in a bond angle when the bond angle is compressed, increased ~0.4kcal/mol for the energy minimized structure of 4-Hydroxy-3-MethylButyl Diphosphate. The torsion energy decreased ~0.4kcal/mol for the energy minimized hetero compound. Torsion energy is the energy of deformed torsional angles in a compound. The through-space interactions between atoms which are more than three bonds away from each other are referred to as non-1,4 van der Waals energy. The non-1,4 van der Waals energy for 4-Hydroxy-3-MethylButyl Diphosphate decreased ~6kcal/mol when the compound was energy minimized. The 1,4 van der Waals energy, non-bonded interactions between atoms separated by two bonds, increased slightly less than 1kcal/mol for the energy minimized hetero compound. The charge/charge energy, interactions between charged atoms, decreased ~9kcal/mol when the hetero compound was energy minimized. The charge/dipole energy, interactions between charged atoms and transiently forming dipoles, decreased from 12.2533kcal/mol for the non-energy minimized hetero compound to 5.2264kcal/mol for the energy minimized hetero compound. The dipole/dipole energy, representative of the interactions between bond dipoles, decreased ~1kcal/mol when 4-Hydroxy-3- MethylButyl Diphosphate was energy minimized.

For both the non-energy minimized form of 4-Hydroxy-3-MethylButyl Diphosphate and the energy minimized form, the charge/charge energy term contributed the most to the total steric energy of the hetero compound. The stretch-bend energy contributed least to the total steric energy for the non-energy minimized hetero compound. The non-1,4 van der Waals energy contributed least to the total steric energy of the minimized energy form of 4-Hydroxy-3-MethylButyl Diphosphate.

6 Elissa Williams

Part IV: Superimposing the Extracted and the Energy Minimized Hetero compound

For Part IV of the Protein Modeling Assignment, Chem3D Ultra performed an overlay of the extracted hetero compound and the energy minimized hetero compound. The resulting image is shown below (Figure 4). This image clearly demonstrates that the diphosphate and the hydroxyl group of the energy minimized compound have been rotated and the four carbon chain is slightly less bent in the energy minimized hetero compound.

Figure 4: Overlay of Extracted and Energy Minimized Hetero compound. A superimposed image of 4-Hydroxy-3-MethylButyl Diphosphate in the Extracted and Energy Minimized Form.

7 Elissa Williams

Part V: Protein-Ligand Interactions

In Part V of the Protein Modeling Assignment, the wiring diagram of the protein, Isopentenyl-Diphosphate Delta-Isomerase, was obtained from the PDBSum website. The wiring diagram displays all the amino acids in one subunit of the protein and indicates, with red dots, which amino acid residues interact with the hetero compound. The wiring diagram is Figure 5 displayed below. The ligplot of the hetero compound, Figure 6, was also obtained from the PDBSum website. The ligplot is a 2D visualization of the amino acid residues in the protein which interact with the hetero compound. Figure 7, displayed next to Figure 6 for easy comparison, was generated in DS Visualizer and also demonstrates the hetero compound and the interacting amino acid residues. To obtain Figure 7, the interacting amino acid residues shown in the ligplot from the PDBSum website were selected along with the hetero compound in the downloaded PDB file in DS Visualizer. Only these amino acid residues and the hetero compound were displayed in a new tab in DS Visualizer and the hydrogen bonds between the residues and the hetero compound were generated using DS Visualizer. I had much trouble aligning Figure 7 to match the ligplot, Figure 6. The hetero compound is closely aligned in both figures; however, some amino acid residues are not in the same location in Figure 6 and 7. The PDB file in DS Visualizer was stylized to demonstrate only the protein in gray solid ribbon format, Figure 8. The hetero compound was then displayed in ball and stick format within the gray solid ribbon depiction of the protein, Figure 9. The protein, hetero compound, and interacting amino acid residues, no hydrogen bonds are shown, were obtained in DS Visualizer and is Figure 10 below. Also, the protein, hetero compound, and interacting amino acid residues, including hydrogen bonds are depicted in Figure 11.

8 Elissa Williams

Figure 5: The Wiring Diagram. An image of the primary and secondary structure of Isopentenyl- Diphosphate Delta-Isomerase complexed with the hetero compound 4-Hydroxy-3-MethylButyl Diphosphate.

The above figure is the wiring diagram for the primary and secondary structure of Isopentenyl- Diphosphate Delta-Isomerase complexed with the hetero compound 4-Hydroxy-3-MethylButyl Diphosphate. The red dots demonstrated on this figure represent the amino acid residues which interact with the hetero compound. The blue dots in the figure represent the amino acid residues which interact with a metal ion. The red rectangles demonstrate amino acid residues which are in the protein’s catalytic site. This figure was obtained from the PDBSum website using the search term “1NFZ.”

9 Elissa Williams

Figure 6 (Left Figure): The Ligplot. An image of the Ligplot of the hetero compound 4-Hydroxy- 3-MethylButyl Diphosphate complexed within the protein Isopentenyl-Diphosphate Delta- Isomerase.

The above left figure is the “Ligplot” of 4-Hydroxy-3-MethylButyl Diphosphate complexed within the protein Isopentenyl-Diphosphate Delta-Isomerase. This “ligplot” demonstrates the interactions between the amino acids of the protein and the hetero compound. The following amino acids interact with the hetero compound: Cys 118, Glu 116, Tyr 104, Lys 55, Ala 34, Glu 114, Arg 51, Cys 67, Arg 83, Glu 87, Gly 68, Lys 21, and His 69. The green dashed lines in the figure represent hydrogen bonds and the amino acids depicted as eyelashes interact hydrophobically with the hetero compound. The “ligplot” was obtained from the PDBSum website.

Figure 7 (Right Figure): Interactions between the Hetero compound and the Amino Acids. An image of the hetero compound, 4-Hydroxy-3-MethylButyl Diphosphate interacting with key Amino Acid Residues.

The above right diagram demonstrates the amino acids, shown in yellow stick style, which interact with the hetero compound, shown in ball and stick style. The green dashed lines represent hydrogen bonds between the hetero compound and amino acids or between amino acids. This figure was generated using DS Visualizer.

10 Elissa Williams

Figure 8: The Protein. An image of the protein Isopentenyl-Diphosphate Delta-Isomerase.

The above picture is a solid ribbon representation of the protein Isopentenyl-Diphosphate Delta- Isomerase. This picture was generated in DS Visualizer.

11 Elissa Williams

Figure 9: The Protein and the Hetero compound. An image of the protein Isopentenyl- Diphosphate Delta-Isomerase complexed with the hetero compound 4-Hydroxy-3-Methylbutyl Diphosphate.

The above picture is a solid ribbon representation of the protein Isopentenyl-Diphosphate Delta- Isomerase complexed with the hetero compound 4-Hydroxy-3-MethylButyl Diphosphate which is shown in ball and stick format. This picture was generated using DS Visualizer.

12 Elissa Williams

Figure 10: The Protein, Hetero compound, and Interacting Amino Acids. An image of the hetero compound, 4-Hydroxy-3-Methylbutyl Diphosphate, interacting with the amino acids of the protein Isopentenyl-Diphosphate Delta-Isomerase.

13 Elissa Williams

Figure 11: The Hetero compound complexed within the Protein and Interacting with Amino Acid Residues. An image of the protein Isopentenyl-Diphosphate Delta-Isomerase complexed with the hetero compound 4-Hydroxy-3-MethylButyl Diphosphate and interacting with the amino acid residues of the protein.

The above schematic shows the protein, Isopentenyl-Diphosphate Delta-Isomerase, complexed with the hetero compound 4-Hydroxy-3-MethylButyl Diphosphate. The protein is shown in gray ribbon and the hetero compound is the gray, orange, and red ball and stick figure on the left hand side of the protein. The schematic demonstrates the key amino acids which interact with the hetero compound (shown in yellow) and the hydrogen bonds between the hetero compound and the amino acid residues are the green dashed lines. This figure was generated using DS Visualizer.

14 Elissa Williams

Part VI: Table of Hetero Compound and Amino Acid Interactions

For Part VI of the Protein Modeling Assignment, the interacting amino acid residues, which were identified in the ligplot in Part V of the assignment, were analyzed. The analysis was constructed into a table, Table 2: Hetero compound and Amino Acid Interactions. The interacting amino acids were defined along with the atoms in the hetero compound which interact with the amino acid residues. The type of interaction is also explained in Table 2 below. It is important to note that the ligplot, Figure 6 above, and the 3D ligplot generated in DS Visualizer, Figure 7, were used and often referred to in order to complete this part of the assignment.

Table 2: Hetero compound and Amino Acid Interactions. A table defining the type of interactions between the key amino acid residues of the protein and the hetero compound, 4- Hydroxy-3-MethylButyl Diphosphate. Amino Acid Residue Atoms in the Hetero Type of Interaction Compound which interact with the Residue cys 118 - OH (alcohol group) The sulfur group of the amino acid interacts with the alcohol of the hetero compound through dipolar and hydrogen bonding interactions. glu 116 -OH (alcohol group) The carboxylate group of the amino acid is able to hydrogen bond with the hetero compound’s alcohol group. tyr 104 - OH (alcohol group) The alcohol group of the amino acid is able to hydrogen bond with the hetero compound’s alcohol group. + lys 55 - Two oxygen atoms of the The charged NH3 group of the terminal phosphate group amino acid is able to form a -2 (PO4) charged or electrostatic interaction with two oxygen atoms of the terminal phosphate group of the hetero compound. These oxygen atoms have a partial negative charge. ala 34 - CH3 (methyl group) The methyl group of the amino acid interacts with the methyl group of the hetero compound through van der Waals forces. glu 114 -OH (alcohol group) The carboxylate group of the amino acid is able to hydrogen bond with the hetero compound’s alcohol group.

15 Elissa Williams

+ arg 83 - Three oxygen atoms of The NH2 and the NH2 group of the terminal phosphate the amino acid forms four -2 group (PO4) hydrogen bonds with three oxygen atoms of the terminal phosphate group in the hetero compound. glu 87 - Two oxygen atoms of the The carboxylate group of the terminal phosphate group amino acid interacts -2 (PO4) electrostatically with two oxygen atoms of the terminal phosphate group of the hetero compound. gly 68 - The internal oxygen of the Two hydrogens of the amino acid O3P - O - CH2 bond form weak, polar interactions with the internal oxygen of the hetero compound. + lys 21 - Two oxygen atoms of the The charged NH3 group of the terminal phosphate group amino acid is able to form a -2 (PO4) charged or electrostatic interaction with two oxygen atoms of the terminal phosphate group of the hetero compound. These oxygen atoms have a partial negative charge. his 69 - Two oxygen atoms of the The two NH groups in the amino terminal phosphate group acid form hydrogen bonds with -2 (PO4) the oxygen atoms of the terminal phosphate of the hetero compound. + arg 51 - Two oxygen atoms of the The NH2 and the NH2 group of internal phosphate group the amino acid forms four -2 (PO4) hydrogen bond with two oxygen atoms of the internal phosphate group in the hetero compound. cys 67 - OH (alcohol group), - The The sulfur group of the amino internal oxygen of the acid interacts with the alcohol of O3P - O - CH2 bond the hetero compound through dipolar and hydrogen bonding interactions. The hydrogens of the amino acid form weak polar interactions with the internal oxygen of the hetero compound.

16 Elissa Williams

Part VII: Bibliographic Information

In order to complete Part I-VI of the Protein Modeling Assignment, the following sources of information were used:

1. The RCSB Protein Data Bank was used to obtain the Protein-Hetero compound PDB File: H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne: The Protein Data Bank. Nucleic Acids Research, 28 pp. 235-242 (2000).

2. Information regarding the Protein-Hetero compound complex was found in the journal article which first reported the Protein-Hetero compound structure: Wouters, J., Oudjama, Y., Barkley, S.J., Tricot, C., Stalon, V., Droogmans, L., Poulter, C.D., Journal of Biological Chemistry, 2003, 278, 11903-11908. "Catalytic Mechanism of Escherichia coli Isopentenyl Diphosphate Isomerase Involves Cys-67, Glu-116, and Tyr- 104 as Suggested by Crystal Structures of Complexes with Transition State Analogues and Irreversible Inhibitors". http://www.jbc.org/cgi/reprint/278/14/11903

3. The PDBSum Website, http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/, was used to obtain the wiring diagram and Ligplot of the Protein-Hetero compound: Laskowski R A, Chistyakov V V, Thornton J M (2005). PDBsum more: new summaries and analyses of the known 3D structures of proteins and nucleic acids. Nucleic Acids Res., 33, D266-D268.