Biochemists ID crucial recognition tag in cellular 'garbage disposal'

Cells must routinely dispose of leftover or waste proteins by breaking them down, but the problem for biochemists studying this fundamental process is that molecules can be toxic garbage in one situation but essential for function in another, says Peter Chien, assistant professor of Biochemistry and Molecular Biology.
Figuring out how bacteria and other cells accurately distinguish waste from useful molecules has been elusive, but his laboratory’s recent progress could offer medical researchers a clue for controlling disease, such as bacterial infections and cancer cell growth. In this week’s early online edition of Structure, Chien and colleagues report a two-part discovery, identifying new players in the protein-degrading system and how they work together at the molecular level.
A major focus of this investigation is phosphodiesterase, or PdeA, and its function in a well-characterized model bacterium, Caulobacter crescentus. The protein PdeA is needed at a particular point in the bacteria’s life-cycle, but is selectively disposed of when no longer needed. It’s known that an energy-dependent protease called ClpXP acts like a garbage disposal to degrade unwanted PdeA, Chien says, but until now it was not understood how ClpXP recognizes and attaches to PdeA, drawing it into the garbage disposal.
Chien says, “We study these proteins and how they interact not only because they’re involved in basic biological function, but because they can help explain how bacteria go virulent, how they become infectious and how we may make them less virulent and less pathogenic. This could lead to developing new antibiotics to fight disease, for example. And, knowing how bacteria control cell cycle progression may help us understand how cancer cells become immortal.”
Using X-ray crystallography, he and colleagues including fellow Biochemistry and Molecular Biology professor Scott Garman were able to build the first detailed structural model of these molecules.
They reveal that the two-part PdeA molecule has a “recognition tag” in one part that is recognized by the ClpXP protease, plus another piece (a type of protein module called a PAS domain). Together, these two parts coordinate the destruction of PdeA. Interestingly, PAS domains are found throughout nature but until now have not been thought to be important in proteolysis, Chien says. Finally, the researchers found that the PdeA protein also has extra recognition tags that only become unmasked when part of the protein is degraded.
Chien says it was a bit of a surprise to learn that one tag in the PAS domain is important and the rest is less so. But structurally, it makes sense that a few small tags in PAS domains should be distributed throughout long protein chains that look like strings of pearls, because as the ClpXP protease finishes degrading one pearl, it might disengage, perceiving its task to be complete. However, with fresh bits of PAS domain and recognition tags cropping up randomly like chocolate chips in a bran muffin, Chien says, full degradation that’s critical to cellular health will continue.   
He adds, “There’s more, it’s not that simple. We found one more player, an adaptor protein called CpdR, which helps the protease recognize its target and tether PdeA to the protease. We call it tethering, but actually we’re not sure whether it’s actively helping the protease pull on the substrate or it’s just holding the protease and substrate together until eventually they touch.”
Taken together, these findings offer molecular biologists a new model of how proteins are degraded, as well as insight into how important the process is for cells, Chien says. “Our results tell us about how particular types of target or substrates might have evolved to prevent undigested or half-digested bits clogging up the works. It appears it’s quite dangerous for a cell to have protein bits floating around.”  
Finally, this new function for the common PAS domain opens up a new possibility for manipulating biological pathways and regulation, plus a target for further research to intervene in that process to reduce bacterial virulence, Chien says.
This work was supported by the National Institute of General Medical Sciences at the National Institutes of Health.