ProteinShader Tutorial: 1A05

3-Isopropylmalate Dehydrogenase: Three-Layer (Alpha-Beta-Alpha) Sandwich [PDB:1A05]

The 3-isopropylmalate dehydrogenase enzyme catalyzes dehydrogenation and decarboxylation reactions that are important in the biosynthetic pathway for the amino acid leucine [1]. The structure of this bifunctional dimeric enzyme from Thiobacillus ferrooxidans has been solved by x-ray crystallography [1, 2], and can be loaded into the ProteinShader program by selecting Open from the File menu and then using the file chooser box to select the 1A05.pdb file.

Cartoon-style displays with halftoning

When the enzyme is first loaded, it will be displayed as a pen-and-ink style drawing of ribbons and tubes. To obtain the orientation shown in Figure 1A, use the Orientation menu above the canvas to select Bottom. It may also be helpful to zoom in a little by using the middle mouse button or scroll-wheel (see mouse motion control for a summary of the mouse controls). Alternatively, if the canvas has the focus (because it was clicked on), pressing the "i" key once will zoom the camera in by 5 angstroms, while the "o" key will zoom the camera out by 5 ansgstroms.

By default, alpha-helices are shown as tubes, while beta-strands are shown as wide ribbons, and general loop regions are shown as thin ribbons. To place more emphasis on the beta-strand ribbons, click on the β-Strands radio button of the Decorations subpanel, and then select Vertical Bars from the Halftone Texture menu and None from the BendTexture menu. To de-emphasize the loop regions, switch from the Decorations subpanel to the Cartoon Visibility subpanel and click on the Loops radio button before pressing the Translucent button. The degree of translucency can be adjusted with the slider control below the button.

With the loop regions faded out and the orientation as in Figure 1B, it should be more obvious that the protein has two pockets, one above and a little to the right of the large central domain, and one below and a little to the left. Each pocket serves as a binding site for the enzyme's substrate, 3-isopropylmalate. By default, the ProteinShader program does not show heterogens when a new structure is loaded, so go the Visibility menu above the canvas and select the Heterogens checkbox. The 3-isopropylmalate molecules (one in each pocket) should appear as in Figure 1C, with spheres colored according to atom type (gray for carbon and red for oxygen). A small green sphere that is mostly obscured by the 3-isopropymalate is a magnesium ion.

The Visiblity menu above the canvas is intended as a quick and convenient way to show or hide all amino acids, heterogens, or waters. For more fine grained control, there is a Cartoon Visibility subpanel (for tube and ribbon segments) and an Atom Visibility subpanel (for space filling, balls and sticks, and sticks style displays). In a cartoon-style display, heterogens are currently shown only as space filling models, so manipulating the visibility of individual heterogens requires the use of the Atom Visibility subpanel. For example, to get a better look at the magnesium ions while still in the Figure 1C view, make sure that the Chain menu near the top left of the Control Panel is showing chain A, and then select Heterogens from the menu right below the Chain menu. The list of residues right below the menus should now show four heterogens: two magnesium ions and two isopropylmalates.

To select the two isopropylmalates, click on HET_IPM 401 and, while holding down the keyboard Ctrl button, click on HET_IPM 402. Now use the menu at the top right of the Control Panel to change from the Decorations subpanel to the Atom Visibility subpanel. Click on the Selected radio button near the top of the subpanel and make sure that Residues is showing in the menu below the radio button. Click the Invisible button to hide the isopropylmalates. The two magnesium ions should now be easy to see. Click the Opaque button to set the isopropylmalates visible again.

Figure 1. Ribbons and tubes style display of the enzyme 3-isopropylmalate dehydrogenase. (A) Pen-and-ink style rendering with the default settings for halftoning. (B) Same as part A after adding a vertical bars pattern to the beta-strand ribbons and fading out the loop regions. (C) Same as part B, but two molecules of the 3-isopropylmalate substrate are shown in their binding sites. The images were captured as described in the Saving Images section of the tutorials main page.

Cartoon-style displays in color

The three-layer alpha-beta-alpha sandwich structure of 3-isopropylmalate dehydrogenase may be a little easier to resolve using color as shown in Figure 2. Starting from the image shown in Figure 1C, go back to the Decorations subpanel and, after choosing Model from the menu under the Selected radio button, click on the Plain button at the very bottom of the subpanel. In this context, plain means color images with no special patterns mapped onto the surfaces of the ribbons and tubes. By default, the beta-strands should be green, the alpha-helices red, and the loop regions gray.

To emphasize the beta-strands, click on the β-Strands radio button to select all beta-strands. Then use the Patterns button to apply Vertical Bars to the beta-strands. The image should now appear as in Figure 2A. To rotate the enzyme about its current vertical axis, hold down the shift key and right-click while dragging the mouse horizontally across the canvas. Dragging about halfway across the canvas should produce a rotation of about 180 degrees, which should be close to the view shown in Figure 2B. Simply selecting Top from the Orientation menu will produce exactly the same orientation as in Figure 2B, but slowly rotating the protein around will studying the secondary structure will give a better sense of why the protein is referred to as an alpha-beta-alpha sandwich.

Figure 2. The three-layer alpha-beta-alpha structure of 3-isopropylmalate dehydrogenase with alpha-helices in red and beta-strands in green. (A) The view with Bottom selected from the Orientation menu. (B) Same as part A, except that the enzyme has been rotated 180 degrees by selecting Top from the Orientation menu. The images were captured as described in the Saving Images section of the tutorials main page.

Visualizing side chains in a binding site

The 3-isopropylmalate substrate binding site includes a hydrophobic pocket that recognizes the substrate's gamma-isopropyl group [1]. Visualizing the amino acid side chains that form this pocket is a little easier if ribbons and tubes are shown as a pen-and-ink style display, while the space filling side chains are shown in color as in Figure 3. Starting from the image in Figure 2B, use the Decorations subpanel to select the entire Model from the menu under the Selected radio button, and then apply Noise from the Halftone Texture menu and Diagonals 1 from the Bend Texture menu. Next, click on the β-Strands radio button to select all beta-strands, and apply Vertical Bars from the Halftone Texture menu and None from the Bend Texture menu. The ribbons and tubes should now look similar to Figure 3A, but to get a better view of the 3-isopropylmalate substrate molecule in the lower half of figure, hold down the shift key and right-click while dragging the mouse a short distance horizontally across the canvas.

To set space filling amino acid side chains visible in a ribbons and tubes style display, use the menu at the top right of the Control Panel to switch to the Cartoon Side Chains subpanel. Then go to the Chain menu near the top left of the Control Panel and select chain B. With Amino Acids selected in the menu right below the Chain menu, a list of all the amino acids in chain B should be displayed. Scroll down the list and select VAL 193. Then make sure that Residues is showing in the menu below the Selected radio button before clicking on the Space Filling button of the Cartoon Side Chains subpanel. By default, the spheres are colored by atom type, which will be gray for carbon. To color all side chains by amino acid type, switch to the Atom Color subpanel, and then select the entire Model from the menu below the Selected radio button before clicking on the Amino Acid button at the bottom of the subpanel. The VAL 193 side chain near the gamma-isopropyl group of the substrate in the lower half of Figure 3A should now be green.

To see the other amino acid side chains that form the hydrophobic pocket, go back to the Cartoon Side Chains subpanel and switch to chain A before selecting GLU 88, LEU 91, and LEU 92 and clicking on the space filling button (multiple residues can be selected at the same time by holding down the Ctrl key while left-clicking on residues). For a close up view of these residues as in Figure 3B, the mouse can be used to zoom in or translate the image up or down or left to right as described in the Mouse Motion Control section. To see the equivalent side chains near the gamma-isopropyl group in the top half of Figure 3A, repeat the above procedure with VAL 193 on chain A and GLU 88, LEU 91, and LEU 92 on chain B (chains A and B of this dimeric protein are identical).

Figure 3. Visualization of a hydrophobic pocket that recognizes an isopropyl group. (A) The 3-isopropylmalate dehydrogenase enzyme is shown as tubes and ribbons using halftoning for a pen-and-ink style appearance, while two substrate molecules and subset of the side chains that recognize them are shown as a space filling style display. (B) A closeup view of the lower portion of part A. The 3-isopropylmalate substrate is colored by atom type, while side chains are colored by amino acid type (red for glutamate and green for leucine and valine). One of the side chains (Val 193) belongs to chain B, while three others (Glu 88, Leu 91, and Leu 92) belong to chain A. For Glu 88 (the only side chain in red), it is a CH2 group that is part of the hydrophobic pocket in close proximity to the gamma-isopropyl group of the 3-isopropylmalate substrate. The images were captured as described in the Saving Images section of the tutorials main page.

References

1. Imada K, Inagaki K, Matsunami H, Kawaguchi H, Tanaka H, Tanaka N, Namba K: Structure of 3-isopropylmalate dehydrogenase in complex with 3-isopropylmalate at 2.0 A resolution: the role of Glu88 in the unique substrate-recognition mechanism. Structure 1998, 6: 971-982.

2. 3-Isopropylmalate Dehydrogenase PDB entry 1A05 [http://www.rcsb.org/pdb/explore/explore.do?structureId=1A05]