A bit mysteriously, some proteins manage to multitask even with structures that are unfolded and floppy—“intrinsically disordered.” In this week’s issue of Nature, scientists at The Scripps Research Institute (TSRI) report their discovery of an important trick that a well-known intrinsically disordered protein (IDP) uses to expand and control its functionality.
Links to Disease
E1A is worth studying not just because it facilitates adenovirus infections, but also because it’s a prime example of an IDP. Such proteins frequently play outsized roles in cells, as crucial “molecular hubs” within very large protein-interaction networks. IDPs also include proteins that are linked to major diseases, including the tumor suppressor protein p53, the alpha synuclein protein of Parkinson’s disease, and the amyloid beta and tau proteins of Alzheimer’s disease.
The simple, flexible structures of IDPs are often promiscuously “sticky,” which in principle explains why they would have multiple molecular partners. But IDPs don’t connect willy-nilly with other proteins, and scientists have wondered how they regulate their diverse interactions.
Wright’s laboratory and others have been studying these interactions using a technique called nuclear magnetic resonance (NMR) spectroscopy. However, E1A’s intrinsic stickiness means that it tends to aggregate at NMR-friendly concentrations, rendering this method of analysis problematic. (Most proteins, by folding up into complex shapes, effectively cloak their stickier bits.)
A Sensitive Technique
For the new study, Wright and his colleagues turned to Deniz, whose laboratory specializes in the use of sensitive, cutting-edge techniques to study the dynamics of disordered proteins and other biological molecules. One of these techniques, a quantum optics method known as single-molecule FRET, uses a tiny fluorescent beacon system to register distances between selected parts of a protein. In effect, this allows investigators to monitor in real time the shape-changes of E1A—characterized by Wright’s laboratory in earlier work—which mark its rapid couplings and uncouplings with other proteins.
“The technique is sensitive enough that we can use it at extremely low protein concentrations, even focusing on single E1A proteins to avoid the loss of information that comes from the usual averaging of results over multiple proteins,” Deniz said.
Postdoctoral fellows Allan Chris M. Ferreon and Josephine C. Ferreon, in the Deniz and Wright laboratories, respectively, used the single-molecule FRET method to detail the strengths (“affinities”) with which E1A binds to two of its most important protein partners. By mapping how these binding affinities change under different conditions, they were able to obtain key insights into how E1A manages its multiple interactions.
First, like many folded proteins, E1A turns out to employ a basic regulatory mechanism called allostery: when one protein partner binds at one part of the E1A structure, it changes the ability of the other major binding site on E1A to bind other partners.
For most proteins that use allostery, this change makes partner-binding at the other site more likely (“positive cooperativity”). For a minority, it makes partner-binding at the other site less likely (“negative cooperativity”). But E1A turns out to have the capacity for either positive cooperativity or negative cooperativity between its two major binding regions—depending on whether a third part of the protein is occupied. “Allostery itself is a mechanism for modulating a protein’s functions, and here we show that E1A takes it to another level by modulating allostery—modulating the modulation, in effect,” said Josephine Ferreon.
The finding helps explain how E1A generates and manages its functional complexity—a complexity that for viral proteins seems particularly necessary, considering how tiny viral genomes are in comparison to those of their animal hosts. Moreover, some of E1A’s key binding partners in infected cells are themselves hub-type IDPs. “So now you multiply the complexity—and you can see how proteins such as E1A manage to achieve so much so quickly within a cell,” said Allan Ferreon.
Wright regards the study as the start of a rewarding line of investigation using sensitive techniques such as single-molecule FRET. “The fact that we can get around the usual technical obstacles relating to IDPs and do these single-molecule experiments really opens up the study of IDP hub interactions,” he said.
Deniz concludes, “We’re definitely going to be studying more of these hub proteins, and I think we’re going to discover other fundamental principles by which they achieve complex layers of biological regulation and function.”
The study, “Modulation of allostery by protein intrinsic disorder” was funded by the National Institutes of Health (grants GM066833 and CA96865) and by the Skaggs Institute for Chemical Biology at TSRI.
About The Scripps Research Institute
The Scripps Research Institute (TSRI) is one of the world's largest independent, not-for-profit organizations focusing on research in the biomedical sciences. TSRI is internationally recognized for its contributions to science and health, including its role in laying the foundation for new treatments for cancer, rheumatoid arthritis, hemophilia, and other diseases. An institution that evolved from the Scripps Metabolic Clinic founded by philanthropist Ellen Browning Scripps in 1924, the institute now employs about 3,000 people on its campuses in La Jolla, CA, and Jupiter, FL, where its renowned scientists—including three Nobel laureates—work toward their next discoveries. The institute's graduate program, which awards PhD degrees in biology and chemistry, ranks among the top ten of its kind in the nation. For more information, see www.scripps.edu.
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