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New insight into the controls on a go-to enzyme
Date:11/19/2008

St. Jude researchers used the analytical technique of X-ray crystallography, with help from nuclear magnetic resonance (NMR) spectroscopy. In this widely used method of determining protein structure, researchers first crystallize a protein to be studied. Then, they direct X-rays through the crystal and deduce the protein structure from the diffraction pattern of those X-rays. To overcome the crystallization bottleneck, a lengthy and unpredictable variable in X-ray crystallography, the investigators used NMR spectroscopy to tailor the perfect enzyme-inhibitor complex.

Tudor Moldoveanu, Ph.D., a postdoctoral fellow in Green's laboratory, performed X-ray structural analysis on such a protein crystal that consisted of a critical part of the calpastatin molecule attached to calpain. The structural picture obtained of the two proteins clutched together clearly revealed why calpastatin so specifically attaches to calpain.

"Calpain has multiple domains, and what we saw was that calpastatin wraps itself around pretty much every domain of calpain," said Moldoveanu, the report's first author. This attachment not only blocks the portion of the enzyme called the active site, where calpain performs its snipping function, but also covers regions away from that site. Such a broad molecular embrace guarantees that calpastatin will potently and rapidly shut down calpain's function, Moldoveanu said. This broad embrace also guarantees that calpastatin will precisely recognize only calpains, rather than mistakenly attach to other similar enzymes in the cell.

Furthermore, the researchers discovered how calpastatin evades being chewed up by calpain. Calpastatin's survival enables it to be repeatedly recycled to inhibit calpain, making it an even more effective regulator.

The researchers' structural information also showed how calpain changes its shape once it is activated by calcium and how this transformation renders it a target of calpastatin attac
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Contact: Summer Freeman
summer.freeman@stjude.org
901-595-3061
St. Jude Children's Research Hospital
Source:Eurekalert

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