Light- or drug-dependent switches enable precise control over signaling pathways in living cells.
Cellular signaling pathways act rapidly and are sensitive to subtle changes in the level of activation, making them difficult to manipulate with precision. Klaus Hahn and Nikolay Dokholyan from the University of North Carolina at Chapel Hill and their teams recently discovered an approach to this problem by modulating target proteins through the insertion of light- or drug-sensitive domains to control structural disorder in these proteins.
This work was driven by PhD student Onur Dagliyan, who realized that he could generalize approaches for controlling protein activity used previously in the lab. The team initially found that they could confer rapamycin sensitivity to a kinase by inserting a rapamycin-binding module, or 'knob', into a loop at the back of a kinase away from the catalytic site. Hahn says that the knob they inserted “was very floppy, and the molecular motion killed the kinase.” Rapamycin addition would stabilize the knob and thereby activate the protein. The team then realized that the termini of the light-sensitive LOV domain had a spacing similar to that of the rapamycin-binding module, so they inserted this domain at the same spot in the kinase. In this case, illumination introduced structural disorder into the protein, leading to inhibition of the kinase.
Hahn credits his student with the insight that “this is a mechanism that you may be able to use for many different proteins.” Hahn notes that many proteins are covered with these small loops of an appropriate size to insert a similar knob. Dagliyan developed a simple algorithm for determining the best target sites. From the loops at the protein surface, he eliminated evolutionarily conserved ones from the analysis and then looked for sites that were connected to the protein's active site via secondary structures. The team has applied this approach to activate or inhibit a variety of different proteins in three families: kinases, GTPases and GEFs.
In addition to addressing the biological questions that motivated Hahn and his team to embark on this project, the researchers are also expanding the toolkit. “We are trying to design more knobs [...] and extending it to other proteins,” says Hahn. Along these lines, he is currently thinking about combinatorial switches. He is also eager to help others design such tools or use existing ones.
Dagliyan, O. et al. Engineering extrinsic disorder to control protein activity in living cells. Science 354, 1441–1444 (2016).