Human Growth Hormone: Four Alpha-Helix Bundle [PDB:1HGU]

The core of the hGH (human growth hormone) protein is a four-helix bundle structure comprised of two pairs of parallel alpha-helices that are joined in an antiparallel fashion [1]. X-ray crystallography has been used to determine the three-dimensional structure of the hGH protein by itself [2, 3] and complexed with the extracellular domain of its receptor [4, 5]. To load the hGH protein structure, select Open from the File menu of the ProteinShader program and then use the file chooser box to select the 1HGU.pdb file.

Cartoon-style displays with side chains

When the hGH protein is first loaded, it will be displayed as a pen-and-ink style drawing of ribbons and tubes. To switch to an all tubes style display as shown in Figure 1A, go to the Style menu above the canvas and select Tubes from the Cartoon submenu. The tubes representing alpha-helices have a thicker diameter and are drawn a little more darkly than the loop regions connecting them. The amino-terminus of the polypeptide chain is seen at the top left of the figure, while the carboxyl-terminus is near the top center. What appears to be a chain termination near the bottom right of Figure 1A is in fact a gap in the structure. Amino acid residues 37 to 39 are disordered in the 1HGU crystal structure, presumably because they are part of a flexible loop that is moving around in the solvent that makes up part of the crystal. When data on atom positions is missing from a PDB file, the ProteinShader program does not attempt to fill in missing parts of a tube or ribbon, but rather simply puts end caps on the tube or ribbon before and after the gap.

Amino acid side chains that strongly modulate binding of hGH to its receptor have been identified by a strategy known as alanine-scanning mutagenesis [6]. To illustrate these side chains as shown in Figure 1B, use the menu at the top right of the Control Panel to switch from the Decorations subpanel to the Cartoon Side Chains subpanel. Then go to the list of amino acids on the left side of the Control Panel and left-click with the mouse to select residues number 10, 54, 56, 58, 64, 68, 171, 172, 174, 175, 176, 178, 182, and 185 while holding down the keyboard Ctrl key (command key on Macintosh). When the first of these residues was clicked on, the menu under the Selected radio button near the top of the Control Panel should have automatically switched to Residues, so clicking the Space Filling button should now cause the side chains of the selected residues to appear as spheres. By default, the spheres are colored by atom type. To color by amino acid type, switch from the Cartoon Side Chainssubpanel to the Atom Color subpanel and use the Amino Acid button at the very bottom of the subpanel.

To get a better look at the spatial arrangement of the sides chains that affect receptor binding, use the left-side mouse button to drag the mouse across the canvas and rotate the image to a position similar to Figure 1C (the protein can be returned to its original orientation at any time by using the Orientation menu). For a more schematic view of the side chains, go back to the Cartoon Side Chains subpanel, and, after making sure that Residues is still selected in the menu under the Selected radio button, click on the Balls and Sticks button. To color the balls by atom type, go back to the Atom Color subpanel and use the Atom Type button. To obtain an image similar to Figure 1D, use the Style menu above the canvas to select Ribbons from the Cartoon submenu, and then go back to the Decorations subpanel, choose Model from the menu under the Selected radio button, and select Hatching 1 from the Bend Texture menu.

In addition to rotating the image with the mouse, the center mouse button or scroll-wheel can be used to zoom in and out on the image, while the right-side mouse button can be used for moving the image up and down or side to side (see Mouse Motion Control section for details and for Macintosh single-button mouse options). By using these mouse manipulations, it should be fairly easy to obtain a closeup view of the side chains similar to Figure 2.

A [1HGU.pdb tubes] B [1HGU.pdb tubes-spheres]
C [1HGU.pdb tubes-spheres (left)] D [1HGU.pdb ribbons-spheres]

Figure 1. Illustration of hGH side chains that affect receptor binding. (A) hGH rendered as tubes with halftoning. (B) The same image as part A, except that amino acid side chains implicated in receptor binding are shown as a space filling (spheres) style display with colors based on amino acid type. (C) The same image as part B, except that the protein has been rotated to get a better look at the side chains. (D) The protein is shown in the same orientation as in part C, but using ribbons rather than tubes and with side chains shown as a balls and sticks style display with colors based on atom type. The images were captured as described in the Saving Images section of the tutorials main page.

[1HGU.pdb ribbons-spheres closeup]

Figure 2. Closeup view of amino acid side chains that modulate hGH receptor binding. The image is the same as in Figure 1D, except that mouse controls have been used to zoom in on a region of the protein. The images were captured as described in the Saving Images section of the tutorials main page.

hGH bound to receptor extracellular domains

Binding of hGH to its cognate receptor is required for regulation of normal human growth and development, and this binding appears to activate signal transduction through a mechanism that involves dimerization of receptor molecules [4, 7]. The structure of hGH complexed with two molecules of the extracellular domain of its receptor has been solved by x-ray crystallography [4, 5], and can be loaded into the ProteinShader program by using the File menu above the canvas to open the 3HHR.pdf file.

When the structure is first opened, it will be shown as a ribbons and tubes style display. To obtain an orientation similar to Figure 3A, select Right from the Orientation menu above the canvas, and then drag the mouse vertically downwards a short distance. Beta-strands, which dominate the receptor extracellular domain, are shown as wide ribbons, while alpha-helices, which dominate the hGH structure, are shown as tubes. To place stripes on the beta-strands, go to the Decorations subpanel and click on the β-Strands radio button to select all beta-strands, and then select Vertical Bars from the Halftoning Texture menu and None from the Bend Texture menu.

To fade out the loop regions (thin ribbons) between alpha-helices and beta-strands, go to the menu at the top right of the Control Panel and change it from the Decorations subpanel to the Cartoon Visibility subpanel. Click on the Loops radio button to select all loops, and then click on the Translucent button. The default value for translucency is 75%, but can be adjusted by using the slider control below the Translucent button. To explore the three-dimensional structure, use the Mouse Motion Controls and the Orientation menu above the canvas.

The hGH amino acid side chains illustrated in Figures 1 and 2 define the high affinity receptor binding site, Site1, which binds the receptor first, after which a second lower affinity site on the other side of hGH, Site2, can bind a second copy of the receptor [8]. To see where the Site1 side chains contact the receptor, try using the Cartoon Side Chains subpanel as described above for Figure 1B. It may also be helpful to experiment with visibility status, decorations, and color changes by using the Cartoon Visibility, Decorations, and Cartoon Color subpanels.

A [3HHR.pdb endview] B [3HHR.pdb sideview]

Figure 3. The hGH protein is shown bound to the extracellular domain of its receptor, hGHbp. (A) A ribbons and tubes style display of the hGH protein bound to two molecules of hGHbp. The two beta-strand dominated domains on the left half of the image are one copy of hGHbp, while the two beta-strand dominated domains on the right half are the second copy. The view shows one end of the four-helix bundle that forms the core of the hGH protein. (B) Same image as in part A, but after rotating it approximately 90 degrees to get a side view of the four-helix bundle. The images were captured as described in the Saving Images section of the tutorials main page.

References

1. Brandon C, Tooze J: Alpha-Domain Structures. In Introduction to Protein Structure. 2nd edition. New York: Garland Publishing; 1998: 35-46.

2. Chantalat L, Jones ND, Korber, F, Navaza, J, Pavlovsky AG: The crystal-structure of wild-type growth-hormone at 2.5 angstrom resolution. Protein Pept Lett 1995, 2: 333-340.

3. Human growth hormone PDB entry 1HGU [http://www.rcsb.org/pdb/explore/explore.do?structureId=1HGU]

4. de Vos AM, Ultsch M, Kossiakoff AA: Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 1992, 255: 306-312.

5. Human growth hormone and extracellular domain of its receptor PDB entry 3HHR [http://www.rcsb.org/pdb/explore.do?structureId=3HHR]

6. Cunningham BC, Wells JA: High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 1989, 244: 1081-1085.

7. Brandon C, Tooze J: Signal Transduction. In Introduction to Protein Structure. 2nd edition. New York: Garland Publishing; 1998: 251-281.

8. Kossiakoff AA: The structural basis for biological signaling, regulation, and specificity in the growth hormone-prolactin system of hormones and receptors. Adv Protein Chem 2004, 68:147-169.