Experiment: PyMol Molecular Visualization

Experiment: PyMol Molecular Visualization Lysozyme and Its Inhibitors In this exercise, you will use PyMol to explore the three-dimensional structures of lysozyme and its inhibitors. The lysozyme enzyme is a small, stable protein that is found in a variety of species. Animals, insects, and plants use lysozyme as a primary agent to kill invading gram-positive bacteria by attacking the peptidoglycan components of the bacterial cell walls. Specifically, lysozyme possesses neuraminidase activity which cleaves glycosidic bonds between two carbohydrate moieties, such as N-acetylglucosamine and Nacetylmuramic acids. This action results in the degradation of the bacterial cell walls. Lysozyme is thus viewed as a natural antimicrobial agent abundant in the extra-cellular fluid and chicken eggs, cow milk, human saliva, tears, blood, and body fluids. Lysozyme binds to and hydrolyzes N-acetylglucosamine (NAG) oligosaccharides. For example, it converts NAG hexamer (NAG6) to NAG tetramer (NAG4) and NAG dimer (NAG2) as the major products, and to two NAG trimers (NAG3) as the minor product. The binding subsites in lysozyme for individual monosaccharides are defined as A, B, C, D, E, and F. NAG3 binds strongly to lysozyme, and thus becomes an inhibitor to this enzyme. Additionally, lysozyme also binds NAG2 and NAG, albeit much weaker. Lysozyme’s substrate binding site, a cleft formed by several amino acid residues, is somewhat complementary to NAG3 in terms of size and shape. The specificity of this interaction is attributed to the extensive hydrogen bond formation between lysozyme residues and the NAG3 molecule as well as aromatic stacking. Such hydrogen bond formation is believed to contribute to the large, negative H value obtained by calorimetry. In this exercise, you will use PyMol to visualize the binding site of lysozyme and its inhibitors. You will need to perform modeling in order to answer the provided questions.

HAZARDS There are no particular hazards associated with this computer lab.

Procedure The educational version of PyMol can be requested at http://pymol.org/educational/. A useful introduction for beginners can be found in the PyMol Wiki web site at http://www.pymolwiki.org/index.php/ Practical_Pymol_for_Beginners. The Protein Data Bank (PDB) IDs for lysozyme/NAG3 and lysozyme/NAG complexes are 1HEW and 1JA7, respectively, and can be accessed at http://www.rcsb.org/pdb/. The PyMol program consists of two separate windows: a Menu window, containing the menu toolbar and a command line, and a Viewer window. The Viewer window also consists of two parts: “structure display” on the left and “command buttons” on the right. A screenshot for the launched program is shown in Figure 1S.

Menu

Command Lines

Command buttons

Figure 1S. The PyMol program. The individual windows in Figure 1S are zoomed in for Figures 2S and 3S. To open the 1HEW file, select File from Menu followed by Open (i.e. File  Open*). To display the protein sequence, do Display  Sequence.

Figure 2S. The Menu Window and its Submenu.

Figure 3S. The Viewer Window. After opening the 1HEW file and displaying its sequence, the display is defaulted to show all atoms including all other small molecules (ex. water).

Figure 3S shows HEW lysozyme in the Viewer. Use the left button on the mouse to rotate the structure and the right button to zoom the structure in or out. The water molecules can be hidden from display by clicking on H (Hide in Command Buttons)  Water.** To group the polypeptide only into a selection, select the whole peptide sequence excluding NAG3 and rename the selection to, for example, lysozyme*** (shown as (lysozyme) in Command Buttons) by selecting A (Action)  Rename Selection (Figure 4S).

Note: Save your session by clicking File  Save Session so you won’t lose any operations you just performed.

Figure 4S. Grouping Lysozyme into a Selection.

To display lysozyme in Ribbon presentation (Cartoon in the program), first choose H  Everything. This action will hide the peptide but not NAG3. Then choose S (Show)  Cartoon. One also can change color by selecting C (Color)  By SS  Helix Sheet Loop. An example is shown in Figure 5S.

Figure 5S. Coloring Lysozyme by its Secondary Structure. The selections can be extended to NAG3, disulfide bridges, key catalytic residues, etc. as shown in Figure 6S. For example, Figure 7S was generated by selecting S  Surface in lysozyme Selection.

Figure 6S. Selections are created with the Command Buttons. The background was changed to white by selecting Display  Background  White.

Figure 7S. Surface Presentation of Lysozyme depicting the NAG3 Binding Cleft.

Once selections are created, one could display the essential information by selecting or deselecting selection(s), changing models, coloring and labeling. For example, Figure 8S specifically shows the hydrogen bond network among moieties of NAG3 and its surrounding residues. To identify those amino acid residues, first make sure that the (1HEW) selection is always selected (it is gray when selected and black when deselected) or nothing will show. Those residues can be found by selecting A  Find  Polar Contact  To Other Excluding Solvent from the (NAG3) selection. Select by clicking on those residues forming hydrogen bond(s) (yellow dashed lines) and rename it to (residueHB) selection. Next, do S  Sticks, C  By Elements, and L (Label)  Residues. Perform the same procedure for selection (NAG3). Figure 8S shows an example after such operation. Note the color of the dashed lines for hydrogen bonds can be changed in (NAG3_polar_conts) as previously described. The properties of dashed lines can be changed in order to make hydrogen bonds more clear. This can be done by choosing Setting  Edit All; use keyword “dash” to filter out the desired setting. Place the desired numbers for the gap, length, and width.

Figure 8S. The Hydrogen Bond Network among NAG3 and Lysozyme Amino Acid Residues.

PyMol contains similar features with that of RasMol, which allows one to measure the distance between two atoms. To use this, choose Wizard  Measurement and pick the first and second atoms according to the instructions on the screen. The distance in Å will be displayed on the screen (make sure that the colors of the background and text exhibit good contrast). To compare the structure of lysozyme/NAG3 and lysozyme/NAG complexes, both structures can be loaded to the program using Open in Menu and aligned using command “align 1JA7, 1HEW” in the command line to minimize the Root Mean Square Deviation or Distance (RMSD). RMSD is very useful for evaluation of homologous structures (i.e. identical protein sequence in 1HEW and 1JA7). In general, suitable corresponding atoms have been chosen and the molecules have been translated and rotated as region body to obtain the best match.1 Mathematically, RMSD measures the average distance between atoms of two superimposed proteins and can be shown as RMSD = SQRT[{SUM(dii)2}/N]

eq. 1

where dii is the distance between the ith atom of structure 1 and the ith atom of structure 2, and N is the number of atoms matched in each structure. To align and compute a RMSD for the polypeptide backbone only, use the command “align 1HEW and name n+ca+c+o, 1JA7 and name n+ca+c+o”. The RMSD value is 0 Å for identical structures and it increases as the two structures become different. A RMSD less than 3 Å is generally small and is considered to exhibit no significant conformational change as a whole. In this practice, you will see a variety of information in the command line section following command execution such as (i)

Number of alignment atoms and RMS value before refinement.

(ii)

Number of refinement cycles and their RMS values, including the number of atoms rejected.

(iii)

Number of alignment atoms and RMS value after refinement.

*

Notation for operations in Menu (Bold font) Notation for operations in Command Buttons (Underlined font) *** Notation for Selection (Italicized font) **

Thermodynamics and Structural Change It is clear that the specificity of lysozyme to NAG3 or NAG is governed by the formation of a hydrogen bond network between the ligand and receptor. However, it is a daunting task to derive thermodynamic parameters from macromolecular structural changes. In general, one could calculate the standard Gibbs free energy change or standard free energy of the reaction (Go) for binding based on structures of ligand-unbound receptor, ligand, and ligand-bound receptor. Go is actually a balance between the changes in bond strength (ex. Ho) and the entropy of the system (∆So) including water molecules. The derivations of thermodynamic and kinetic parameters from structures encounter major problems given that structures are rarely determined in identical experimental conditions. Furthermore, proteins have to be viewed as dynamic structures, rather than

static, as implied by crystal structure techniques. Despite the observed differences, the structures obtained from X-ray crystallography and solution-based nuclear magnetic resonance (NMR) generally agree each other, while the binding mechanisms are not necessarily always consistent. So is a value that can only be derived from the known Go and Ho values. On the other hand, Ho can be either directly measured by calorimetry or calculated through the van’t Hoff equation. ITC and differential scanning calorimetry (DSC) are only two examples of techniques that directly measure H. The calculation for thermodynamic parameters based on structure is beyond that of the current theoretical and experimental approaches. Although the lysozyme system has been well studied in the past years, similar problems are applied. Recall that the heat evolved (Q), detected by ITC allows for the determination of H, which is the internal energy change (E) measured at constant P, provided that the volume change (V) in aqueous solution is negligible. The equation is shown as: H = E + (PV) = E + PV ≈ E = H

eq. 2

If we assume that hydrogen bond formation in the complex is the only single contribution to the heat enthalpy change in our case, then HNAG3 (Ho for lysozyme/NAG3 formation) is the change between formed and broken hydrogen bonds in the ligand-bound and unbound states of lysozyme. Without ligand binding, water molecules are likely to form hydrogen bonds with both the protein and ligand components. Thus, a hydrogen bond inventory2 can be applied to calculate the predicted H (HNAG3(Pred)) by the following: HNAG3(Pred) = H(breaking H-bond(s) to water) – H(forming H-bonds in complex) – H(forming water to water H-bond(s)).

eq. 3

Note that the first term in the equation includes all hydrogen bonds forming from water molecules to lysozyme and NAG3. The second term includes all hydrogen bond formation, including those not necessarily located in the binding interface. If one assumes that the strength of hydrogen bonds is similar in water-water and also in receptor/ligand-water, the above equation can further be broken down. HNAG3(Pred) = n1 H(breaking one H-bond to water) – n2 H(forming one H-bond in complex) – n3 H(forming one H-bond between water molecules)

eq. 4

where n1, n2, and n3 are the number of hydrogen bonds involved in each term in eq. 3. It is clear that even with this assumption it becomes difficult to determine the exact numbers (i.e. n1, n2, and n3) of hydrogen bonds for each term in eq. 4. Furthermore, the energies required to form hydrogen bonds are still unclear in solution. There are studies showing that water to water hydrogen bond formation enthalpies range from 2-10 kcal/mol (or 8-40 kJ/mol). However, hydrogen bonds are not created equally because the strength is influenced by several factors including electrostatic attraction, polarization, covalency (directional formation),

dispersive attraction, and repulsion. Without detailed information of the hydrogen bond network including that with water molecules, the predicted value of H from eq. 4 can only be viewed qualitatively rather than quantitatively. In this exercise, we assume that the enthalpy of the reaction determined by ITC is primarily attributed from the six or three hydrogen bonds formed in the binding interface of lysozyme and NAG3 or NAG, respectively, as suggested by literature. Note that one of the hydrogen bonds seen in lysozyme/NAG has to be excluded because its formation is unlikely. It is clear that such an assumption is naive, thus the exercise should be viewed as an education tool rather than for purposes of a research approach.

Now create the custom views to work through the following exercises. You will need to upload the Session files to Digital DropBox in Blackboard and include screenshots in your reports.

For Exercises 1-9 and 7-8,10 use the structures of lysozyme/NAG3 (1HEW) and lysozyme/NAG (1JA7), respectively to answer the following questions. (1) Show the three dimensional structure of lysozyme/NAG3 and highlight the  helix and  structures using different colors. To easily see the structure, the side chains should be omitted for clear presentation. (2) Rotate your structure and determine what category lysozyme belongs to based on the Structural Classification of Proteins (SCOP) definition. (3) Highlight (with a different color or presentation model) the cysteine and cystine residues in the structure. How many of them exist? Rationalize their existence to lysozyme’s high thermal stability (the melting temperature (Tm) = ~ 80oC). (4) Present the lysozyme structure as a surface model with the NAG3 molecule as a stick model (or space model) so you can see how the inhibitor fits into the protein binding pocket. Roughly determine which subsites of lysozyme it occupies. When you assign subsites, start from the direction that the inhibitor occupies. (5) Indicate the orientation of the NAG3 molecule by specifying its reducing and non-reducing ends. (6) With a literature search and your biochemistry textbook, indicate the catalytic amino acid residues in your model. What is the distance between Glu35 (and Asp53) to the closed anomeric carbon? Do those residues have contact with the inhibitors? Use the distance criteria of 3.4 - 4.2 Å for nonbonded atoms. Highlight those residues. (7) Determine the amino acid residues that form hydrogen bonds with NAG3 and NAG. Highlight those residues and hydrogen bonds between the enzyme and substrate. For better presentation, omit the side chains except for the residues forming hydrogen bonds and show the protein as a cartoon model. Indicate which residue(s) use their peptide backbones for binding. (8) Is there any significant structural change observed between lysozyme/NAG3 and lysozyme/NAG? What is the RMSD value for the alignment and how about the alignment for the protein backbone (shown in Menu after performing alignment)? Indicate which region(s) have major changes by visually inspecting the structures.

(9) Search the PDB database for HEW lysozyme without a bound inhibitor. Compare it with lysozyme/NAG3. Perform a RMSD calculation. Is this a lock-and-key or induce-fit model for protein recognition? Justify your answer. (10) Based on the structure of lysozyme/NAG, explain why lysozyme does not bind glucose. Assume that it binds on the same site as NAG, but the different functional groups it carries might impair hydrogen bond formation.

References (1) Coutsias, E. A.; Seok, C.; Dill, K. A. Using quaternions to calculate RMSD. J. Comput. Chem. 2004, 25, 1849-1857. (2) Baldwin, R. In search of the energetic role of peptide hydrogen bonds. J. Biol. Chem. 2003, 278, 1758117588.