The results, published September 2012 in Nature Chemical Biology, helped answer some of the persistent questions about rhomboid structure.

The drawing revealed why the enzyme is so rickety. Holding the structure together are four close-knit groups of amino acids—four “keystones”—plus a large number of weak connections. As a result, rhomboids sway like a shack held together mostly by loose nails. “The enzyme is so unstable that almost any mutation has an effect,” Urban says. But he believes the enzymes’ unstable nature allows them to respond to changes in their environment. For example, the enzymes may be able to partly break open, allowing the active site to grab onto pieces of proteins that vary in shape and size.

The mutants also held several surprises, which eventually helped the team understand how proteins and water reach a rhomboid’s active site. When the researchers perturbed the amino acids in a certain area of the enzyme, the resulting mutants were just as fragile as the original enzyme but were 15 times more active. These results suggest that this part of the enzyme is the gate to the active site. With their mutations, the team had essentially broken the lock on the gate, letting bits of proteins sneak inside the active site.

Another set of mutants were no more wobbly than the original, but they were much less powerful. Urban and research associate Syed Moin collaborated with Yingkai Zhang’s lab group at New York University to study how the normal rhomboid employs the amino acids that were swapped out in these mutants. Using biochemistry experiments and computer simulations, the team showed that at least two mutations help the enzyme retain water. The researchers reported their findings July 3, 2012, in Structure.

A nagging question had been how a rhomboid gets hold of the water it needs to function inside the water-repellent cell membrane. Researchers had proposed that water funnels in from outside the cell. Now it seems that the enzyme also keeps a few water molecules in store, Urban says. “When water is needed for catalysis, there’s always some in the cup.”

Urban plans to investigate several other puzzling mutants. Perhaps the work will help design a drug that will stabilize the shack, or bring down it for good in pathogens that cause malaria and other diseases.

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