Researchers investigate a fragile but powerful enzyme in the cell membrane.

The blueprint for rhomboid protease reveals the enzyme is unstable because it is held together mostly by weak bonds.

Imagine walking inside a building so flimsy that it shakes with every step, making you wonder what keeps it standing. HHMI early career scientist Sin Urban has been asking the same question about an unusual class of enzymes, the rhomboid proteases.

These enzymes exist in the cell membranes of most living things, performing essential biological jobs. They let cells converse with each other, allow fruit fly embryos to develop normally, and lend a hand to malaria parasites as they invade red blood cells. Unfortunately, much about how rhomboid proteases work is mysterious, in part because they look so different from other enzymes.

		Water-Powered Scissors Rhomboid protease lives within the cell membrane for a reason. Read More

Urban made a step toward solving this mystery by mutating a rhomboid bit by bit. He learned that the enzyme is unstable because it is held together mostly by weak bonds; he also uncovered a surprising way rhomboid ensnares the water it needs to function.

The first clue about the chemical role of rhomboid proteases came in 2001, when Urban, at the University of Cambridge, discovered that they catalyze the same chemical reaction as a well-understood class of enzymes, the serine proteases. Five years later, Urban and other researchers solved the three-dimensional structure of a rhomboid. But exactly how rhomboid proteases work remained unclear because they don’t resemble serine proteases in shape or amino acid sequence. What’s more, their job—cutting proteins in select places—requires water, which is scarce in the cell membrane where rhomboid proteases are found.

The Rhomboid Protease Molecule
The membrane-embedded rhomboid protease molecule has room for a few water molecules (blue) to help it perform its cellular job.
Credit: Y. Zhou, Y. Zhang and S. Urban.

On top of that, rhomboid proteases are almost impossible to study outside the cell membrane; they break apart if their surroundings go awry. So Urban, now at Johns Hopkins University, and research associate Rosanna P. Baker decided to mimic the cell membrane in the lab and then remove, one at a time, each component of a rhomboid that might be keeping the enzyme’s structure intact. Using protein purified from bacteria, they changed a rhomboid’s amino acid sequence in different ways to create 151 variants, or mutants. By studying how stable each mutant was, the researchers pinpointed the amino acids most important to maintaining the enzyme’s framework.

“We essentially took an Uzi machine gun and poked holes in every side of this molecule,” Urban says. “We think it allowed us to get an unbiased yet quantitative view of the entire architecture.”

They tested each mutant’s stability with an innovative technique called differential static light scattering. In their experiment, an instrument gradually heated up many rows of enzyme samples at the same time. The rising temperature caused various mutants to break apart at different rates—less stable enzymes unraveled at lower temperatures. The unfolded enzymes scattered light, which a camera detected. The team determined the “breaking points” of the 151 variants and combined the results to create a picture, like an architect’s drawing, of the bonds that support a rhomboid’s three-dimensional structure.

Illustration: Rosanna Baker and Sinisa Urban

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