Graphics Gallery

Gale Rhodes
Chemistry Department
University of Southern Maine

Revised 2006/08/02

Learn how to use Swiss-PdbViewer. See the Swiss-PdbViewer Tutorial.

Topic: Glycogen Metabolism

Examples

Featuring some of the enzymes and regulatory proteins involved in glycogen metabolism.

 

Glycogen Phosphorylase

From the Protein Data Bank, download 1GPA, an x-ray crystallographic model of glycogen phosphorylase a with bound pyridoxal phosphate.

  • Describe the quaternary structure of this enzyme. It helps to Color: Chain.
  • Explore the contacts between subunits: Display only the residues that make contact with adjacent chains (Select: Groups Close to Another Chain, then <return>). Color the model CPK, and then color backbone by chain. This way, you can discern side-chain atoms easily, and the backbone color helps you to distinguish the chains. Zoom in on some of the inter-chain contacts. Can you find inter-chain hydrogen bonds? salt bridges? hydrophobic interactions?
  • Limit the display to chain A. What are the predominant secondary structural elements? Can you distinguish domains within a single chain?
  • Focus on the cofactor pyridoxal phosphate (PLP). The cofactor is covalently linked to the enzyme. What is the chemical nature of the link? Are there additional, noncovalent forces that hold or orient PLP?
  • Focus on the phosphate groups at the subunit interfaces. Zoom in on one of them and study its surroundings. On what residue does the phosphate group reside? With what residues does the phosphate group interact? What is the sequence surrounding this phosphorylated residue? Does the sequence resemble the phosphorylation targets of regulatory protein phosphorylases?

From the Protein Data Bank, download 1GPB, an x-ray crystallographic model of gycogen phosphorylase b with bound pyridoxal phosphate. This file contains only one subunit of the enzyme.

  • Superimpose 1GPB onto the A chain of 1 GPA. The simplest way is to select and save chain A of 1GPA, close 1GPA, and then reload your saved chain A. Then use Fit: Magic Fit followed by Fit: Generate Structural Alignment to superimpose them. Blink the two models to see the differences between their conformations.
  • Limit the display to PLP and its neighbors in both layers. Blink the models. Can you discern any differences that might affect the accessibility or reactivity of the PLP phosphate group?
  • Add ribbons for all residues not currently displayed. Blink. Can you discern any differences that might affect the accessibility or reactivity of the PLP phosphate group?
  • You have probably noticed by now that in 1GPA (phosphorylase a), PLP has a neighbor that is not present in 1GPB (phosphorylase b). Identify the neighbor. What substance might bind to this same site when the enzyme is functioning?

 

Cyclic-AMP-Dependent Protein Kinase (cAPK)

From the Protein Data Bank, download 1ATP, a model of cAPK with bound ATP and an inhibitor peptide that mimics cAPK's phosphorylation target on the alpha or beta regulatory subunits of phosphorylase kinase B and other phosphorylation targets of cAPK.

  • Display the model in ribbon form, with ribbons colored by chain. The enzyme is yellow, the target peptide is blue.
  • Add ATP and its associated Mg2+ ions, with dotted surfaces.
  • In the target peptide, cAPK phosphorylates residue 21, a serine or threonine. In this inhibitor peptide (chain I), residue 21 is ALA. Suggest why this peptide inhibits cAPK. Predict the type of inhibition (competitive, uncompetitive, etc).
  • Use SPV to change residue 21 to THR. Measure the distance from the threonine -OH to the ATP phosphate.
  • Compare the expected affinity of target peptide for cAPK before and after phosphorylation.

 

Phosphorylase Kinase

From the Protein Data Bank, download 2PHK, a model of phosphorylase kinase complexed with a substrate or target peptide like the one it would phosphorylate.

  • Display ribbon only, and color by chain to distinguish the enzyme from its substrate/target.
  • Explore the binding to the target to the enzyme. Here's a quick way to focus on hydrogen bonding to the target: Select chain B, the target. Tools: Compute H bonds, then Display: Show only H-bonds from selection, and then Display: Show only groups with visible H bond. Now you should have a ribbon model showing in wireframe only those residues of enzyme and target that are involved in hydrogen bonding.
  • Here's a quick way to focus on other types of interactions between enzyme and substrate/target: Select chain B. Select the neighbors of B within 4 angstroms. Then color side chains by type and backbone by chain.

 

Calmodulin

From the Protein Data Bank, download 2BBM, a model of calmodulin (from fruit fly) in complex with a target peptide.

This model was derived from NMR data. 2BBM is the the averaged, energy-minimized model computed from the ensemble of 21 models that were obtained from NMR analysis. (See this Technical Note about structure determination by NMR.)

  • Read the file header to find out what organisms the calmodulin and target peptide are from.
  • The target is a synthetic peptide. Should this make any difference in the way the peptide binds to calmodulin?
  • Display model as ribbons and color by chain to distinguish calmodulin from the target.
  • Display all atoms. Remove the hydrogens. Select and display the target peptide (chain B). Add its neighbors to 4 angstroms. Color backbone by chain and sidechains by type. What kinds of interactions bind calmodulin to its target?
  • Identify the ligands of the four Ca2+ ions. Are the calcium ions or their ligands involved in binding the target peptide? How would you describe the role of the calcium ions?
  • Read in your textbook about the EF hand structural motif. Display ribbons and color them by secondary structure succession. Identify EF hand motifs in calmodulin.
  • To see how well the 21 models in the ensemble agree with each other, color the model by B-factor. In the PDB file of an NMR model, the B-factor column for each atom contains a measure of how much that atom position varies throughout the models in the ensemble. If the command Color: B-Factor colors the entire model blue, use Prefs: General to turn on Scale B-factors colors so that min = dark blue and max = red. Then color by B-factor again. Do the 21 models differ more in mainchain or sidechain atoms? in buried or surface atoms?

Now let's compare the conformations of calmodulin with and without the target peptide. Download 4CLN, an averaged NMR model of fruit fly calmodulin alone (without the target peptide). Open 4CLN and then 2BBM in Deep View.

  • Display both models in single-strand ribbon, and color the ribbon by secondary structure succession. The N-terminal domains will be blue to blue-green in both models. Manually superimpose the N-terminal domains of the two models. You will need to display the Layer Infos window in order to easily turn on and off movement of individual models, so you can move one onto the other. This will give you some practice using the full range of rotation, translation, and zoom tools. To superimpose them well, you'll need to move the model around and check the alignment from all sides.
  • Once you have superimposed the N-terminal domains nicely, blink between the models. As you go from 4CLN to 2BBM, the target peptide pops into view, and the two calmodulin domains appear to gather around it. This comparison shows dramatically the conformational differences between calmodulin when it is free and when it is bound to a target peptide.

The following exploration will give you some insights into NMR models like 1BBM. (See this Technical Note about structure determination by NMR.) You will use two PDB files, 1CFC, which contains an ensemble of 25 calmodulin models obtained from NMR data, and 1CFD, an averaged, energy-minimized model of the ensemble of models in 1CFC.This calmodulin is the calcium-free protein from the African clawed toad.

  • Be sure that you have plenty of memory allocated for Swiss-PdbViewer (12 Mb is enough on my computer). First, download 1CFC. In the SPV dialog that appears, specify 25 models to be loaded.
  • Make a ribbon display, and then color ribbons by secondary structure succession. followed by 1CFD. Blink through all the 1CFC models to see the full range of models that satisfy the NMR data.
  • What parts of the model are well constrained by the data? What parts are not constrained?
  • Do you think that the differences among the models reflect data that is inadequate to constrain the models, or real conformational flexibility in solution?
  • Download 1CFD, the averaged model. Color it by B-factor. For an NMR model, the colors indicate how well atom positions in the averaged model (1CFD) agree across the ensemble of models (1CFC). From what you saw in blinking through the 1CFC models, you would think that coloring 1CFD in this way would make one domain blue and the other red (because one domain is superimposed for all models, and one shows the variation in domain-domain relationship. But the result is quite different. Read the file header to find out why.
  • How could you use these models to get some idea of how calmodulin conformation changes upon binding calcium?

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