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Biostatistics and Bioinformatics
Enzyme Active Site Analysis
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Biostatistics and Bioinformatics Enzyme Active Site Analysis
Description of Module Subject Name Biochemistry
Paper Name 13 Biostatistics and Bioinformatics Module Name/Title 13 Enzyme Active Site Analysis
Dr. Vijaya Khader Dr. MC Varadaraj
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1. Objectives: The learning objectives in this modules are to 1. undertake protein structure analysis using SwissPDBViewer
2. Displaying structure in stereo vision to appreciate the depth of the molecule
3. use various commands of SwissPDBViewer to detect the active site residues bound to the substrate 4. use literature information of the active site catalytic residues to complete the picture of active site
having a bound substrate
5. enhance the graphics with colours and cartoon display for appreciating the complete picture of an active site on the enzyme
2. Concept Map
3. Description
Brief Description
Download SwissPDBViewer for Structure analysis
Display Molecular Structure in Stereo Vision
Detect Active Site Residues of an Enzyme bound to Substrate
Enhance Graphics to understand role of active site residues
Summary
Use Literature Information to display Active Site residues
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The Structural analysis of an enzyme active site begins with opening a PDB file using structure analysis software. Molecular structures stored in PDB data files are visualized (i.e. viewed) using molecular visualization software, which may be an online applet in a web browser, without installing on your local computer, as we have seen in the previous module or PDB data files are visualized using a standalone software i.e. downloaded software program installed on your computer. PDB file contains the coordinates of each atom in the protein molecule, along with the atoms of ligands, if any. Upon opening a PDB file, the 3-D structure appears in the workspace of the Structure analysis software. We will use SwissPDBViewer in the present module. The basic analysis for enzyme active site includes display of molecule in stereovision, i.e. to see the molecule in real three dimensions for width, height and depth of the molecule. The molecule can be visualized in various types of molecular graphics. The most common form of graphics, where alpha helices and beta strands connected through loops are displayed in the ribbon form. The active site residues can be then displayed in solid stick graphics using information from the literature. In case the PDB structure of enzyme with bound substrate is available then, the user can detect the binding pocket residues using SwissPDBViewer Commands. In this way all the binding and catalytic residues can be displayed on the alpha helices, beta strands connected through loops. The graphics can then be enhanced to understand the mechanistic role of different residues in the active site. In this module, students will be analyzing the active site of an enzyme having bound substrate. In the module on Molecular Structure Databases, we have already seen web applets JSmol and PV at PDB site. "See in 3D" at NCBI's Entrez Structure database is available both as web browser helper application and stand alone package. The standalone programs installed in the computer have more capabilities than web browser applet or web helper applications. We will take the example of o-succinylbenzoate synthase (abbreviated as OSBS) of E.
coli for visualizing its binding pocket to ligands, o-succinylbenzoate i.e. OSB and Mg2+ ion using SwissPdbViewer software.
Therefore, the learning objectives in this module are to undertake protein structure analysis using SwissPDBViewer and to appreciate the depth of the molecule, we need to Display structure in stereo vision. Then the objective is to detect the binding pocket residues using various commands of SwissPDBViewer and also use literature information of the active site catalytic residues to complete the picture of active site having a bound substrate. Finally, we will learn to enhance the graphics display for appreciating the complete picture of an active site on the enzyme.
Concept Map
3.1. Download PDB Structure and SwissPDBViewer:
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In the last module we took the example of enzyme OSBS, i.e. o-succinylbenzoate synthase, to search PDB database and found enzyme structure 1FHV bound to its product OSB. It catalyses the dehydration of substrate to form product OSB. OSBS belongs to the enolase superfamily of enzymes, characterized by the presence of an enolate intermediate which is generated by abstraction of the alpha-proton of the carboxylate substrate. It catalyses the dehydration reaction of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1- carboxylate to produce o-succinylbenzoate. We will carry forward the same example to analyse its active site.
Visit www.rcsb.org/pdb web interface to download OSBS structure file “1FHV”. Search for “1FHV”. Open
“Download Files” dropdown list and choose “PDB Format” command.
This will open a dialog box.
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Simply check the radioButton Save File and Click OK button. This will save PDB file with 1FHV as file name in downloads folder on your local computer. This structure will be used to compute active site residues of OSBS enzyme.
Now visit, http://spdbv.vital-it.ch/ to download SwissPDBViewer. SwissPDBViewer is also known as DeepView, which is available through EXPASY server of Swiss Institute of Bioinformatics.
Download, SwissPdbViewer available at http://spdbv.vital-it.ch/disclaim.html and unZip in a separate folder, “Computer/SPDBV_4.10_PC, in the present example. The extracted archive is shown. It has executable file spdbv.exe which can be run by double click.
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Concept Map
3.2. Display Molecular Structure in Stereo Vision:
Open the folder where SwissPdbViewer files are extracted, i.e. SPDBV_4.10_PC folder on your computer. To run SwissPdbViewer, double click spdbv executable file. This will start SwissPdbViewer application and present a welcome splash screen.
Read in the information and close the welcome splash screen. Open ‘Prefs’ menu and select “General...”
command. Any command or choice in a menu, if ends with three dots, then it means that this command will open a dialog box, for conversation and selecting options.
Selecting “General...” command will open the dialog box for setting General Preferences.
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In the “general preferences” dialog box, check the choices you are interested in. In case you do not want to see the splash screen at start up again, uncheck this check box. You can also uncheck the checkbox in case you are not interested in alert, when unknown groups are treated as HETATM upon loading a structure.
HETATM are atoms in the structure which are not component of the protein but atoms of an external group such as substrate, product, ligand, metal ion or a cofactor. You can also uncheck the checkbox in case you are not interested in alert, when some side chain atoms are missing in the PDB structure file. You can also uncheck the checkbox in case you are not interested in reconstruction of 3D coordinates for these missing atoms. You can also uncheck the checkbox in case you are not interested in Log of activities upon loading a structure. However, for now, you leave the choices as default and click OK button of the “General Preferences” Dialog Box. Now from the file menu, select first Command “Open PDB File...”.
In the open dialog box select, 1FHV PDB file and click open. Since the checkbox was left checked for receiving alert when unknown groups are treated as HETATM upon loading a structure, the General
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communication that some HETATM CONNECT request were ignored because of probably linked to solvent, i.e. water, molecule.
Since the checkbox for receiving alert “when some side chain atoms are missing upon loading a structure ” was also left checked, the General communication that some side chain atoms are missing upon loading a structure is received. You can also uncheck the checkbox in case you not interested in reconstruction of 3D coordinates for these missing atoms. However, when the missing atoms in side chains are constructed, these are shown in pink color in control panel (to be introduced in a short while).
Since the checkbox for receiving Log of activities upon loading a structure was left checked, this log of activities is displayed in a separate window.
Open the control panel window from Menu “Wind” by selecting command ‘Control Panel’.
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The same can also be displayed by clicking in its visible title bar. This will open the control panel window on the right side of the screen, but will cover the log file window. Adjust the size of Log File Window to display both Control panel and Log file windows simultaneously, with blue colored molecule display window in the background of Log File window.
In the last column of control panel, a box for Asp21 is highlighted in pink color. The log window shows first three black lines for missing atoms in Aspartate 21 of the chain A in the OSBS enzyme. The black colour
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lines in the log window are clickable. Click on line displaying missing atom for “ASP 21 of chain A”. This will turn the colour of the message line to red and also select ASP21 in the control panel, highlighted with red color in the first column of control panel.
Do not close the log activities text window as the same may be accessed later-on from ‘Wind’ menu’s last command. Clicking line displaying missing atom for “ASP 21 of chain A”, will display six amino acids, three on N-terminal side of ASP21 and, two on C-terminal side of ASP21. However, to see these residues, Click in the dark blue display window to view six residues.
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Adjust the window positions by clicking in the title bar and dragging the window, to position them as shown above for SwissPDBViewer main window, dark blue display panel window and control panel window. The main window has a Menu bar with various menus. Below menu bar is the toolbar for frequently used commands.
Below toolbar is a message bar with clickable buttons and showing current massage in red color. The message displayed is “Move All”. This means that when user move residues with mouse, all residues in this enzyme will move. On the left of this message, there are two clickable buttons.
Click the first button “Move All” of this bar to toggle to “Move selection”. This means when user move residues with mouse, only selected residues (shown in red colour for their labels in control panel) of the enzyme will move .This will move only selected residues upon using translation (move) and rotation commands. However, click toggle button again to “move all”
residues.
Using, Text command of the Wind menu,
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switch to Log file and click in the line containing message for Missing atom of residue GLU127 of chain A.
Click anywhere in dark blue display window. We see that the display window show the selected GLU127 with some other residues.
Click the first button in tool bar to centre the displayed residues in the dark blue display window.
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The display window shows eight amino acid residues with GLU127 shown in pink. There are six columns in the control panel. In the first column of control panel, the label GLU127 is shown in red, which conveys that GLU127 is a selected residue. Next to label GLU127 in red (In the first column itself), is a small v, which conveys that backbone of the GLU127 is to be displayed in display window. Click on small ‘v’ to erase it.
This will hide the GLU127 in display window. To display it, click next to GLU127 to mark with small ‘v’.
Three residues above GLU127 and two residues below GLU127 are also marked with small v in first column to convey that the backbones of these residues are also to be displayed. But labels of these residues in first column are shown in black; therefore these residues are not selected for performing operations on selected residues. In the next column, a small v is marked for all resides. This means that side chains are also to be shown. However, side chains of only those residues are to be shown for which backbone has been marked with small v for display. Since backbone for only eight residues is marked with small v, therefore, only side chains for these backbones are displayed. The third column is to display the label of amino acid residues in the display window. However, no residue is marked with v. To display labe l for GLU127 in display window, click in the row for GLU127 in the third column. This will mark the residue for display of label and display the residue label in the display window.
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To display label for all residues, press Shift key on keyboard and click anywhere, in the third column, except already marked. Therefore, combination of pressing shift key with a click is used to display or hide any feature such as display backbones or display side chains or display label s, for all the residues.
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This will display the labels for all the displayed residues. Press shift key and click in the third column. It will hide all the labels. In the fourth column Press shift key with a click in fourth column. Open display menu from the menu bar and Select second last command “Render in Solid 3D”.
This will display the atoms as spheres.
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Press shift key with a click in fourth column to display the residues in solid sticks.
Press shift key with a click in first column to unmark so as to hide all the residues. Now no residue is displayed. However, at this stage we can display the whole molecule in ribbon form by pressing shift key with a click in fifth column.
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However, all the ribbons appear in white color. Users can se t the preferences for ribbon colours from
“Prefs” menu with selecting “Ribbons” command.
This will open the following dialog box:
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In this dialog box, just change the colours for top, side and bottom of Helices, Sheets and Coils. Click on Color Button of each to set the color and then select the color from the color dialog box. For colouring helices use three different Green colours with light green for the top and dark green for the bottom and intermediate green for side. For sheets use pink for the bottom red for the top, and intermediate between pink and red for side. These are boxed in the dialog box. For coils, use single yellow for each of top, side and bottom.
After setting for the top of helices, Click OK. Similarly follow for each of the rest of eight settings. After setting colors of coils bottom, this will display the enzyme OSBS as shown next.
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This view is not a real 3D view but only a pseudo3D view. From this image we can make out front and back but not the exact depth. In this image we are not able to see depth of the molecule. Therefore, we are not able to make out whether a particular amino acid is on the front side or on the back side of the molecule.
The actual or real 3D display is viewed in stereovision. Open the Display menu, select “Stereo View” or press ctrl + T keys.
This will display two images of the same molecule.
Left image is for left eye and the right image is for right eye. The two images are taken with approximately 5 degree rotation to present a different view for each of the eye. It requires special training for eyes to view in stereovision. When we see each image with each eye at the same time, our brain can make the depth to see. Simply look at the screen images with two eyes focused at infinity. The distance between 5.5 to 6 cm separated eyes and SwissPdbViewer screen shall be approximately 45-50 cm. Now see through the images without focusing eyes on the images. This is to bring both eyes in parallel, focused at infinity,
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instead of a point focus on the screen. This is achieved when you focus on an imaginary plane farthest away from the object you want to view in stereo. In that case the things between the farthest imaginary plane and your eyes will appear as duplicated. One thing will appear as two things. To check this, focus your eyes at this text and go on reading. Now without stopping reading, simply bring your finger in between screen and your eyes. While still reading, if you realize two transparent fingers (instead of one opaque finger when your two eyes are focused on this finger), it means you focused at farther plane. Now you take away finger and focus at infinity i.e. beyond the plane of this text. The two stereo image structures, shown above, will now appear as four structures and the middle two structures will start coming towards each other and ultimately overlap. If you keep your eyes focused at infinity, i.e. parallel, then this will make the middle image to show the depth of the molecule. With experience, this may take some 30 seconds to a minute.
When you focus two eyes at a nearest point, say at your own nose, both pupils are directed towards nose edge of eye. However when you focus at the farthest point, say moon the both eye pupils are positioned at the middle of eyes. Therefore, try positioning your eye pupils for focus at infinity by bringing your eye pupils in the middle of eyes. If you find it difficult to understand, then visit stereo vision tutorial at http://steipe.biochemistry.utoronto.ca/abc/index.php/Stereo_Vision and practice stereo vision. The other way for stereo viewing includes special stereo mode software to look at Proteins and DNA using anaglyph stereo glasses. One more technique uses special stereo mode hardware with special computer hardware.
Without StereoVision, the analysis of enzyme active site or binding pocket cannot be appreciated in the real sense. This is like watching a 2D photograph without appreciating depth rather than real scene in 3 dimensions.
Concept Map
3.3. Detect Active Site Residues of an Enzyme bound to substrate:
With the previous stereo Vision display, Press Shift key and click in the first column in the control panel , to mark all visible. Again press Shift key and click in the second column in the control panel, to mark all visible. Again press Shift key and click in the fifth column in the control panel to hide ribbons, to mark all invisible. Scroll down to last residue in the control Panel, i.e. residue number OSB789. Select residue OSB789 by clicking its label in first column of control panel. The selection will be conveyed by changing the color of the label (OSB789) to red. This will display as shown.
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Now open ‘Tools’ menu and select ‘Compute H-Bonds’ command.
Now open ‘Display’ menu and select ‘Show Only H-Bonds from Selection’ command. Again open ‘Display’
menu again and select ‘Show Only Groups with Visible H-Bonds’ command.
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However, user can first compute hydrogen bonds of the molecule and then use three commands in order i.e. Select a group or a residue followed by selecting ‘Show Only H-Bonds from Selection’ command of
‘Display’ menu and finally selecting ‘Show Only Groups with Visible H-Bonds’ of ‘Display’ menu. One can select additional residues and repeat these three commands. After opening a file, by default, the selection in the tool bar is for rotation of the molecule, with fourth button on tool bar highlighted.
Therefore, click on the first tool button in the tool Bar to display molecule in center. Press Shift key and click in the third column in the control panel to display labels. Now set the color of OSB789 to cyan by clicking the box in the sixth column for the row OSB789 and selecting the color cyan from the dialog box.
This will display the detected residues in the active site with dashed lines in green color highlighting hydrogen bonding to the bound substrate OSB789.
From the literature information, we know that Few hydrogen bonding or electrostatic interactions are involved in the binding of OSB to the active site; instead, most of the interactions between OSB and the protein were reported either indirect via water molecules or via hydrophobic interactions. However, Four residues ASP161, ASN163, ASP213 and SER262 could be detected by SwissPdbViewer version 4.1.0 to hydrogen bond with bound OSB. This reveals the binding pocket of the OSBS. The binding pocket determines the substrate specificity.
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Concept Map
3.4. Add Active Site Residues using literature information
From the literature information we also know that OSBS has a bound Mg2+. In control panel, mark ‘v’
next to MG790 residue, i.e. second last residue and just click in the fourth column for MG790 to mark ‘v’.
This will display Mg2+ ion as a grey colour sphere.
The literature information also shows that the Mg2+ ion is octahedrally coordinated by ASP161, GLU190, ASP213, two water molecules, and one oxygen of the carboxylate group of OSB. The ASP161, ASP213 and OSB are already displayed. Therefore mark GLU190 to display. The literature information also shows that Lys(133) and Lys(235) are positioned to function as acid/base catalysts in the dehydration reaction of substrate SHCHC to produce OSB. Therefore, mark ‘v’ in the first column of control panel for LYS133 and LYS235 to display these residues. Now center display by clicking first button in toolbar. This will display as shown:
The Lys133, Lys235 and bound Mg2+are the residues for chemical catalysis.
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Concept Map
3.5. Enhancing Graphics to Understand role of active Site Residues
Mark ‘v’ in fourth column for OSB789. Open color menu and select command CPK.
Open ‘Color’ menu again and select command ‘Type’.
This will change the colors of side chains of these residues according to the type of the amino acid residue involved, i.e. acidic (red), basic (blue), polar (yellow) or hydrophobic (grey). This display reveals that four carboxyl groups are coordinating Mg2+ ion and two polar groups are involved in binding OSB substrate. This display also reveals for abstraction of a proton during acid/base catalysis, that one of the Lys is above the plane of OSB and other below the plane. User can set the colours of the type of side chains in the ‘Prefs’
menu with selecting ‘Colors’ command.
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This will open the following dialog box for colours according to type of atoms and amino acid side chains.
Set atoms colors as per CPK types. In chemistry, the CPK colouring is a popular color convention for distinguishing atoms of different chemical elements in molecular models. The scheme is named after the CPK molecular models designed by chemists Robert Corey and Linus Pauling, and improved by Walter Koltun. However, for Carbon instead of black, use white because the background in the present example is black. For nitrogen use blue, oxygen as red and so on. For amino acid side chain, Set as per side chain type, ie. for acidic (red), foe basic (blue), for polar (yellow) and for hydrophobic side chains (grey).
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In addition, user can set the colours for displaying Bonds, Structures such as helices, strands and loops, and this dialog box allows setting color for the background also.
Now press the ‘shift’ key and click in the fifth column of the control panel. This will display the ribbons graphics for the enzyme molecule. This displays the bound substrate with all the active site residues side chains attached to the main chain shown in ribbons form. This helps to understand the complete picture for binding of substrate in the active site of an enzyme. Now appreciate the active site with moving molecule, rotating molecule and zooming molecule. After opening, by default, the selection is for rotation of the molecule, with fourth button on tool bar highlighted. To move protein in X-Y plane i.e. to translate in X-Y plane, select second button in toolbar. To move protein in Z-plane i.e. to zoom in and out, select third button in toolbar. To come back to rotation of molecule mode, click fourth button. Since, we marked the move toggle button to move all; dragging mouse with left mouse button pressed will rotate the whole molecule in three dimensions. However, to rotate the molecule only in X, keep the F5, key pressed. To rotate the molecule only in Y axis, keep F6 key pressed and to rotate the molecule only in Z axis, keep the F7 key pressed.
To appreciate binding of substrate on the whole enzyme, display residues as spheres for both backbone and side chains with color as type color we set and substrate colored cyan and center the display.
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This reveals that most of the surface has hydrophilic residues with red, blue and yellow color and cyan colored substrate is bound within a cleft on the surface between two lobes of the enzyme.
Concept Map 4. Summary
In this module we learnt about analyzing a PDB structure using SwissPDBViewer. The display of the three dimensional structure was appreciated with stereo vision, to see the depth of the molecule. Then we used various commands of SwissPDBViewer to detect the active site residues involved in binding of the substrate. Then we used literature information to add active site residues to complete the picture of active site. Then we enhanced the graphics for appreciating the complete picture of an active site on the enzyme.
In this way students can analyse any enzyme active site. Therefore, students can use literature information as well as substrate bound enzyme to display the active site. I thank you all for visiting ePG Pathshala.
