Crystal Structure Reveals Secrets of Enzyme Inhibition

Researchers have used advanced X-ray crystallography techniques to explain how the enzyme inhibitor calpastatin binds to and blocks the enzyme calpain once it has been activated by calcium.

Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals and thereby control cellular functions such as cytoskeletal remodeling, cell cycle progression, gene expression, and apoptotic cell death. Following heart attack or stroke, the influx of blood into the heart muscle causes drastic increases in calcium levels and a burst of calpain activity, which causes significant damage to tissues.

Normally, the activity of calpains is tightly controlled by the endogenous inhibitor calpastatin, which is an intrinsically unstructured protein capable of reversibly binding and inhibiting four molecules of calpain, but only in the presence of calcium. It was not clear how this unstructured protein inhibits calpains without being cleaved itself, nor was it known how calcium induced changes that facilitated the binding of calpastatin to calpain.

Now, in a paper published in the November 20, 2008, issue of the journal Nature investigators at Queen's University (Kingston, ON, Canada) reported that they had obtained the 2.4-angstrom-resolution crystal structure of calcium-bound calpain bound by one of the four inhibitory domains of calpastatin. Calpastatin was seen to inhibit calpain by occupying both sides of the active site cleft. Although the inhibitor passed through the active site cleft, it escaped cleavage in a novel manner by looping out and around the active site cysteine. The inhibitory domain of calpastatin recognized multiple lower affinity sites present only in the calcium-bound form of the enzyme, resulting in an interaction that was tight, specific, and calcium dependent. This crystal structure, and that of a related complex, also revealed the conformational changes that calpain underwent on binding calcium, which included opening of the active site cleft and movement of the domains relative to each other to produce a more compact enzyme.

"This is particularly exciting because the enzyme structure we were seeking – and the way its inhibitor blocks activity without itself being damaged – have proved so elusive until now,” said senior author Dr. Peter Davies, professor of biochemistry at Queen's University.

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