Water-Powered Scissors

Why does rhomboid protease, an enzyme that relies on water to function, reside in the waterless environment of the cell membrane? HHMI early career scientist Sin Urban discovered that the protein’s dwelling allows it to use a one-of-a kind method for connecting with the right target molecules.

Rhomboids are serine proteases—they use water to snip off the anchors of membrane-embedded proteins so they can float away and interact with other molecules outside the cell. “Essentially, they are water-powered scissors,” says Urban. “They are cutting things, and they require water to do so. But the membrane is the one place in the cell that is supposed to be devoid of water.”

Urban knew that rhomboid proteins are rickety structures that rely on the surrounding membrane to hold their shape. But he wondered if there was more to it. Was the membrane just a passive place where a rhomboid could live or did it play an active role in the enzyme’s function?

To learn more, Urban and his Johns Hopkins University colleague Syed M. Moin placed the proteases in little balls of detergent, called micelles, which stabilized the wobbly enzymes enough to make it possible to study their behavior outside the membrane.

When they compared rhomboid’s behavior in the micelles to how it acts in the membrane, they found that two things had changed. “If you put them in detergent, rhomboid proteases lose their ability to discriminate between correct and incorrect targets and they cleave proteins where they’re not supposed to,” says Urban.

Rhomboid proteases are like barrels with lids that allow only certain proteins to enter the enzymes and get clipped. The membrane, Urban and Moin discovered, helps keep the lid shut. Without the membrane, the lid swings wide open, allowing any protein to enter. But when the lid is shut, a protein must squeeze through a narrow entryway within the enzyme’s structure that restricts how far the protein can insert itself and where it can be cut.

Another surprising finding was that, like the rhomboid proteases themselves, the proteins they interact with are intrinsically unstable. They fold into coiled helices in the membrane, but they prefer to be unfurled. As soon as one of these proteins enters a rhomboid’s barrel, it spontaneously unwinds and is clipped. The results were published online on November 13, 2012, in the journal eLife.

“Rhomboid proteases are essentially providing an alternative environment,” explains Urban. “Proteins that like that environment come in and as a consequence get close enough to the active site that they get clipped. Proteins that prefer to be in the membrane never unwind, never fully enter, and are just ejected.”

This mechanism is very different from the way other serine proteases, and most enzymes, go about their business. Soluble serine proteases recognize and clip a specific sequence of amino acids on their targets. Rhomboid proteases, on the other hand, seem to be attracted only to those proteins that are flimsy, regardless of what amino acids they contain. “I’m not aware of any other protease that works this way,” says Urban.

Now that he knows how rhomboid proteases work, Urban wonders if they might do more than simply cleave the anchors off membrane proteins. “One function of rhomboid might be to troll the membrane looking for things that have become unstable,” says Urban. “That’s an area of biology that we haven’t explored, and this biochemical mechanism hints that maybe it’s the underlying function of rhomboid.”

-- Nicole Kresge
HHMI Bulletin, Winter 2013

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