Abstract
Signal transduction is a complicated process that may involve several steps mediated by specific intermolecular interactions. Recently, three-dimensional structures of proteins involved in signal transduction have been obtained and have greatly aided in our understanding of these processes at the molecular level.1,2 In this paper, three-dimensional structures of three proteins that are involved in signal transduction will be described, including (1) a protein tyrosine phosphatase, (2) a pleckstrin homology (PH) domain, and (3) the DNA-binding domain of a member of the ets family of transcription factors, Fli-1, in the DNA-bound form.
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Discussion
David Cistola - I was particularly intrigued with the pleckstrin homology domain work where you mentioned that the overall topology of the pH domain was a ß-barrel-like protein that resembled serum-retinol binding protein and that family of lipid binding proteins. Of course in those proteins, the lipid binding is in the interior between the two ß-sheets and is usually a single acyl chain or something equivalent to a single acyl chain. I am wondering in the pleckstrin homology domain whether you considered the possibility that phospholipids with a single acyl chain might be able to bind with the acyl chain inside of the ß-barrel such as lysophosphotidylcholine and perhaps acyl coenzyme A. Have you screened those lipids as well?
Fesik - We didn’t screen those that you mentioned. However, at first we thought that pH domains may be binding to farnesol or a single lipid, since many proteins that contain pleckstrin homology domains are involved in regulating RAS. For isoprenyl or farnesyl derivatives, however, we didn’t see any binding. It is important to note differences between retinol binding proteins and pH domains. If you look at the apo form of retinol binding protein, there’s a cavity lined with waters. This is a hydrophobic cavity where the lipid binds. If you look at the structure of the pleckstrin homology domain, that cavity is not preformed. The interior of the protein has a nice hydrophobic packing arrangement. Nonetheless, it is possible that a lipid molecule binds in that cavity. However, the experimental evidence to date suggests that the interaction between pH domains and PIP2 consists primarily of a charge-charge interaction and that the fatty acid portion of the lipid or fatty acids in general don’t greatly contribute to the binding to the pleckstrin homology domains.
Cistola - Thank you.
David Gorenstein - Have you considered that the arginines of Fli-1 are binding to the phosphates? If you looked in your model of the Fli-1/DNA complex, are they far enough away? Is it the dynamics of the phosphates and the arginines that’s causing the broadening or something else?
Fesik - In the future, we plan to investigate that further. Today, we can say that we observe the broadening. However, at the present time, we are unsure what is contributing to the line broadening. Since it’s a tight complex, you wouldn’t think that it is exchange broadening. What we think is going on is that the arginine might be experiencing more than one state and that the motion is on the order of the chemical shift difference between these two states. Now this has been observed before by Kurt Wüthrich in other systems and during this meeting we’ve heard other examples where this sort of phenomena could be occurring. For example, Julie Forman-Kay spoke about SH2/phosphopeptide interactions that may be a related phenomenon in which an arginine is experiencing two states and that the motion is of the order of chemical shift difference between the two states.
Gorenstein - We’ve seen that as well in some proteins both from modeling and from phosphorus NMR. I think it is very important. This may be an important component of recognition certainly that the dynamics of the arginine is matching some of the dynamics of the backbone.
Fesik - It may not be as the crystal structures would have you believe in which the side chains were all defined in one location in protein/DNA complexes. In contrast, there might be mobility. In future studies, we plan to try a lot of different sample conditions in an attempt to pin down what’s going on with this Fli-1/DNA complex, especially in regards to the recognition helix containing the two arginines that are required for binding to DNA.
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Fesik, S.W. et al. (1996). NMR Structures of Proteins Involved in Signal Transduction. In: Rao, B.D.N., Kemple, M.D. (eds) NMR as a Structural Tool for Macromolecules. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-0387-9_17
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