Scientists at St. Jude Children's Research Hospital used a combination of x-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy to gain in-depth knowledge about how calpain enzymes are regulated in the body. The findings may help scientists better treat an array of disorders because while calpains, which snip apart many target proteins as part of the cell's regulatory machinery, function in many essential cellular processes, defective calpains are associated with muscular dystrophy, type 2 diabetes, gastric cancers, Alzheimer's and Parkinson's diseases, cataracts, and the death of both heart muscle and brain tissue.
Though previous studies had revealed how a few of the molecular parts of the protein calpastatin attach to calpain in the inhibition process and maintain the control necessary to monitor the enzyme, there was no overall picture of calpastatin that revealed how it was so precise in its attachment and potent in its function. Now scientists understand the precision.
“Calpain has multiple domains, and what we saw was that calpastatin wraps itself around pretty much every domain of calpain,” said Tudor Moldoveanu, Ph.D., a postdoctoral fellow in the St. Jude Department of Immunology. 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. It also guarantees that calpastatin will precisely recognize only calpains, rather than mistakenly attach to other similar enzymes in the cell.
The investigators first crystallized a protein to be studied. Then they directed x-rays through the crystal and deduced the protein structure from the diffraction pattern of those x-rays. To overcome the crystallization bottleneck, the team used NMR spectroscopy to tailor the perfect enzyme-inhibitor complex.
Researchers also discovered two other key factors in the equation. First, they found that calpastatin evades being chewed up by calpain, which enables calpastatin to be repeatedly recycled to inhibit the enzyme, making it an even more effective regulator. Second, they showed how calpain itself changes its shape once it is activated by calcium and how this transformation renders it a target of calpastatin attachment and thus inhibition.
The article is published in the November 20 issue of Nature.