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Abstract
Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.
Highlights
Known, but within this report, the word calpain refers only to the - and m-calpains
The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit
The structure of calpain in the absence of Ca2ϩ [12, 13] showed that the active site was not assembled to a catalytically active conformation, so that it was clearly of interest to solve the structure in the presence of Ca2ϩ to understand the mechanism of calpain activation
Summary
Known, but within this report, the word calpain refers only to the - and m-calpains. Whereas the exact physiological roles of calpains remain to be defined, many studies suggest that they have important cellular roles They have been implicated in several important cellular functions, including signal transduction, apoptosis, cell cycle regulation, and cytoskeletal reorganization [1,2,3,4]. The initial effects of Ca2ϩ binding to calpain include a conformational change that is essential to assemble the active site and some limited autolysis of both subunits. Several structural features have been identified that maintain the active site in an inactive conformation These involve on one side contacts between the large subunit N-terminal peptide of domain I and domain VI of the small subunit and on the other side contacts between domains II and III of the large subunit.
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