I have been thinking a lot recently about studies in which people have determined the bound conformation of a ligand by transfer-NOESY experiments, essentially by transferring magnetization off another ligand to the protein and then back to the ligand of interest. With the known bound conformation of the first ligand, one can apparently locate the conformation of the second one. Many such unknown protein-bound conformations have been worked out. In my field of research, the ones which are relevant are of agents that bind to tubulin, especially discodermolide. In this case, the conformation of discodermolide was deduced via competition transfer-NOESY experiments with epothilone. These experiments are non-trivial to carry out and, as is the case for other biomolecular NMR studies, should be interpreted carefully. But in the end they look like nifty techniques that can shed light on unknown bioactive conformations, something that's very valuable for drug design.
Essentially it's again a problem of fitting the bound conformation NMR data to a single conformation. In solution we know for sure that this is a fallacious step. The (not so) obvious assumption in doing this for bound conformations is that there's got to be only one conformation in the active site too. But I have always wondered if a ligand in a protein active site could also have multiple conformations. MJ's comments on a past post and the discussion there makes me think that even in a protein active site, there could possibly be multiple conformations of a ligand, something that runs counter to what we conventionally think. How diverse those conformations might be is a different question; one would probably not expect large conformational changes. But even 'small' conformational changes could be significant enough to distinguish between different conformations in the active site. It's a problem worth thinking about.
_(cropped).jpg)
3 weeks ago in Genomics, Medicine, and Pseudoscience
It's a tough question. Certainly molecules with promiscuous binding activity (PXR comes to mind) will tend to allow a great deal of ligand flexibility. Most enzymes and receptors have more restrictive binding pockets than PXR; on the other hand, the protein itself is usually dynamic to some degree. In general it would be best to corroborate conformational singularity in some way. Barring that, as you said, you just have to intepret data conservatively, especially for less intense peaks that may reflect conformational averaging rather than genuine distance information.
ReplyDeleteA few points -
ReplyDelete1.) To be fair, my example in the earlier post was one of the most promiscuous systems out there. Cytochromes P450 can bind an amazing number of organic compounds (some more than others), and given its role in fatty acid metabolism, the notion that a long-chain hydrocarbon might exhibit an unusual amount of flexibility should not be too horribly surprising. I would suggest, without any sense of being rigorous or thorough, if one can estimate the volume of the active site pocket, and you know the sizes of its preferred substrates, you can determine whether you can expect very interesting but challenging-to-assess behavior (a la P450s) or more textbook behavior.
2.) Having said that, I'm sure there are a decent number of exceptions where analogous behavior might occur. I think it's chorismate mutase where the substrate is found predominately in one conformer in solution but the reaction seems to have a transition state similar to another conformer - it was proposed that the enzyme binds the predominate conformer in solution and can do, once bound to the protein, the necessary molecular gymnastics for catalysis to occur. I don't know if this has held up with time, though.
3.) Here's another provocative thought - there might be cases where substrate motion actually either explains strange biochemical results (for instance,non-Michaelis-Menton kinetics) or - in contrast - isn't of much relevance since the motion doesn't hinder catalysis since the bond of interest doesn't stray too far from the necessary residues or metal center where catalysis occurs. I can't think of any examples at the moment, but I wouldn't bet that there aren't any.
I generally agree with the idea that NMR data should be interpreted conservatively - after all, why try to mask the great advantage of NMR in being able to study motion and dynamics?
I should probably get a blog for the occasional rambles on topics such as these....
- MJ
How do you know whether a peak reflects conformational averaging or real distance data? A lot of times it seems that people look at line broadening and conclude a single conformation in the absence of this. But maybe it's just fast averaging then. In any case, you can always find conformations that fit the bound NMR data better than a single deduced conformation. As you mentioned, it's a tough question to decide then whether this is a real event or not, something that would need to be done on a case-by-case basis.
ReplyDeleteMJ, you should indeed get your own blog. It would be valuable resource for these thoughts. I should look up the chorismate mutase example, and although I am not enough of a biochemist to know what kind of effect conformational averaging will have on Michaelis-Menten kinetics, I can guess that any such effects if distinct will provide a valuable probe into such phenomena.
It's been a while since I thought about the chorismate mutase example (it came up early in my grad school career in a class), so I'm not up to date on the system, but it's one of those systems which has gotten a fair amount of attention from the Karplus, Knowles, and Lipscomb groups at Harvard, should be so inclined to do a lit search at any time on the topic.
ReplyDeleteI want to say something smart about how I have seen some neat things about determining homogenous and inhomogenous contributions to linewidths based on lineshape analyses in solid state NMR in the past, but it's been a while, so I will refrain from doing so at this moment. I'm not sure if it's one of those "doesn't quite have a good counterpart in solution NMR" issues, so I'll avoid saying something silly at the moment. Maybe a topic for whenever I get a blog up and running....
- MJ