Assignment 6 |
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Links: http://www.sciencemag.org/cgi/content/abstract/322/5905/1211
http://www.sciencedaily.com/releases/2008/10/081006102607.htm
http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb100_1.html
http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb58_1.html
First of all, some explanation. I read the full article in my most recent Science magazine, which typically arrives 1-2 weeks after the publication date. Only the abstract is available online, and that's the first link. While checking out additional resources, I found the second link, which provides additional information. Note that the second link actually was posted in early October, preceding publication of the article. (When I checked to make sure all the links work, I got an error page with the second link. Do a search for ZM241385 in Science Daily News, and you should immediately find the page.) The remaining two links are PDB Molecule of the Month articles, one recent and one a few years old but related. Both allow you to review GPCR's and their function.
This diagram shows ZM241385, adenosine, and caffeine, all with the same orientation of the purine ring. This orientation is the same that ZM241385 has in the A2A receptor.
1. ZM241385 has many interactions with the A2A receptor, specifically with Glu169, His250, and Asn253 residues on the right side of the diagram, as drawn. Mutations of those residues disrupt binding of ZM241385 and agonists within the ligand pocket. What type of weak interaction(s) would you expect to occur between those residues and ZM241385? Would adenosine have the same interactions?
2. How is the orientation of ZM241385 within the receptor unexpected? Why do the authors (and editors of Science) think this is newsworthy?
3. G protein coupled receptors (GPCR's) are thought to have a key tryptophan residue whose rotation is linked to activation. The W in the diagram indicates the approximate location of the tryptophan in the A2A receptor, and the article states that ZM241385 has a 14 Â2 contact area with the tryptophan. Based upon that, would you expect caffeine to be an adenosine antagonist or agonist? Explain.
4. The Science Daily article refers to the structure of the adrenergic GPCR, which was the PDB molecule of the month for April. Use the 3rd link to locate the PDB article. Why are these receptors hard to study? What did the researchers do to overcome the problem?
5. Finally, for a review of G proteins, check "Exploring the Structure" in the G protein article. How does the change from GTP to GDP affect the structure of the α subunit? What does that imply about the association of βγ with the GTP-α complex?
This is due at the beginning of class on Mon., Dec. 8. Answers must be turned in individually.
Answers:
1. Primarily hydrogen bonds, and adenosine would have similar interactions, because it also has N's in similar locations.
2. It's both extended and perpendicular to the plan of the membrane, which is unusual. Other adenosine receptors have different ligand pockets, with the implication that ZM241385 does not bind to them. This means that ZM241385 is a selective antagonist; since it binds only one specific GPCR it is a better drug (treatment) candidate than less specific ligands.
3. An antagonist, because it doesn't have any group likely to cause tryptophan rotation, and it could still fit into the ligand pocket.
4. Because the proteins are membrane proteins, it's very hard to prepare crystals. The researchers created receptor-lysozyme or receptor-antibody chimeras (combinations) that more readily crystallized.
This question is specifically about the β-adrenergic receptor; although the Molecule of the Month article describes the similarities of G-protein receptors to rhodopsin, the adrenergic receptor abstract makes it clear that the structure was obtained using chimeras.
Anyone who mentioned that had done additional work and received a bonus 0.5 point.