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Gale Rhodes
Chemistry Department
University of Southern Maine

Revised 2006/08/02

Learn how to use Swiss-PdbViewer. Work through sections 1-4 of the Swiss-PdbViewer Tutorial.

Topic: Signal Transduction

G-Protein-Coupled Receptors (GCPRs)

We must resort to a model compound!

Many hormone receptors are GCPRs, but GCPRs are membrane proteins. This means trouble:

  1. They are hard to crystallize, and without crystals, structure determination by X-ray crystallography is not possible.
  2. They do not assume their native conformation in water or organic solvents, so structure determination by NMR is not possible.

Of the few membrane proteins that have yielded crystals and an X-ray model, bovine rhodopsin is a GCPR, but it's a receptor of light, not molecules like hormones. Nevertheless, the overall structure (fold) of rhodopsin is thought to be similar to that of many GCPRs, so we will have to content ourselves with looking at rhodopsin as a model of hormone-recognizing GCPRs.

Rhodopsin is a light receptor in the rods of the retina. Like all GPCRs, it activates a G-protein (see next section) upon absorption of light (hormonal GCPRs activate G-proteins when they bind hormones). In these exercises, you'll explore rhodopsin and discover how it probably resembles and differs from hormone-binding GCPRs.

From the Protein Data Bank, obtain file 1F88, a model of bovine rhodopsin.

  • Open the file in Deep View. It contains two models, labeled A and B, of the protein. Display chain A only. Describe the primary secondary structural features of this protein (display ribbons and color by secondary structure).
  • Color side chains by type and determine the region of the protein surface that is likely to be buried in the cell membrane.
  • Rhodopsin contains a covalently bound ligand, retinal, which occupies a binding site perhaps a little like the hormone-binding site of a GCPR (retinal is probably more deeply buried than a hormone ligand). Display retinal in chain A. Use the Select: Neighbors function of Deep View to find and identify the side chain to which retinal is covalently linked.
  • Study the retinal-binding region. What types of protein-retinal interactions predominate?
  • Proteins destined for incorporation into membranes (like rhodopsin and GCPRs), are synthesized and simultaneoulsy inserted into the ER membrane. Protein synthesis begins at the N-terminus, so the N-terminus usually ends up inside the ER. Will this end be extracellular or intracellular in the finished protein after transfer into the cell membrane? On the basis of your answer, identify the intra- and extra-cellular regions of rhodopsin.

Want a glimpse of the real thing?

You can use the information made available by the Human Genome Project to make a model of a real hormone-binding GCPR. This exercise will show you how to

  1. search the Human Genome for a specific GCPR gene, the beta-1 adrenergic receptor,
  2. learn the function of the receptor as well as diseases associated with defects in its gene,
  3. search for a homologous protein of known structure, and
  4. make a model of the receptor using a known protein as a template,
  5. evaluate the quality of the model.

Click HERE to begin the exercise.

G-Proteins

Upon binding a circulating hormone in its extracellular binding site, a GCPR undergoes conformational change, which triggers activity in a G-protein bound to its intracellular end.

From the Protein Data Bank, obtain file 1GP2, a model of a heterotrimeric G-protein.

  • Open the file in Deep View and display a ribbon-only model. Color ribbon by chain to see the three different subunits. Use the information in your text to identify the alpha, beta, and gamma subunits. According to your text, what are the roles of each subunit?
  • Examine the alpha subunit, which is a GTPase. What secondary structural elements are present? Find a six-strand pleated-sheet structure. Draw a diagram of the sheet using arrows with points at the C-terminus of each strand; your diagram shows you which pairs of strands are parallel and which are antiparallel. (Tip: Color Ribbon: Secondary Structure Succession will help you to trace the direction of the chain.)
  • This subunit binds GDT or GTP. Does it contain anything resembling a nucleotide-binding domain?
  • Examine the beta subunit. What are its principal secondary structural elements?
  • Select the beta subunit and Save: Selected Residues Only. Name the file Gbeta.pdb. Then open this file to create a second layer. Display the Gbeta layer only, and Color Ribbon: Secondary Structure Succession. By eliminating the other subunits, you allow Deep View to assign the full range of colors within just this one subunit.
  • Can you see why this subunit is called a propeller protein? What secondary structural elements are the blades of the propeller? How many blades are there? In each blade, are strands parallel or antiparallel? Find one blade that is NOT composed of contiguous strands. How might this blade help to stabilize the whole structure?
  • Return to the 1GP2 layer, which contains all three subunits. Which subunits interact with each other?
  • Explore the interactions between subunits. Color backbones by chain and sidechains by type. Compute H-bonds. To look at interactions involving subunit alpha, select it (chain A in 1GP2) and Display: Show Only H-bonds From Selection. Now the only H-bonds shown are those involving alpha -- those within alpha or between alpha and other subunits. This makes it easy to find intersubunit H-bonds. Look for H-bonds between groups with different colored backbones. In the same regions, look for hydrophobic interactions -- look for nearnes of gray side chains with different backbone colors. In you estimation, are there more hydrophobin interactions, or more H-bonds, between subunits alpha (yellow backbone) and beta (blue backbone)?
  • Explore the other subunit interfaces in the same manner.

Other Signaling Proteins

In addition to G-proteins and their coupled receptors, your biochemistry text may discuss other signaling proteins. Explore any of these models that are covered in your text:

  • Complex of G-protein alpha (GTP-binding) subunit with catalytic dimer of adenylate cyclase. PDB file 1CJK is one of a series of models of this complex with various GTP analogs bound to the alpha subunit. I can supply you with a paper on this series of structures, which reveal some details of how G-alpha activates adenylate cyclase.
  • Human growth-hormone receptor as a model of receptor tyrosine kinases. See PDB file 3HHR for a model of human growth hormone and its receptor.
  • Src-homology 2 (SH2) domains. See PDB file 1SPS for an SH2 model with bound target peptide, and 1SPR for the peptide-free model.
  • Insulin: 1TRZ
  • Insulin receptor: PDB 1IRK.

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