Small angle x-ray scattering has been used to monitor calpain structural transitions during the activation process triggered by Ca2+ binding. The scattering pattern of the unliganded enzyme in solution does not display any significant difference with that calculated from the crystal structure. The addition of Ca2+ promotes the formation of large aggregates, indicating the exposure of hydrophobic patches on the surface of the protease. In contrast, Ca2+ addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change with no dissociation of the heterodimer. The resulting conformation appears to be slightly more extended than the unliganded form. From the comparison between ab initio models derived from our data with the crystal structure, the major observable conformational change appears to be localized at level of the L-subunit and in particular seems to confirm the mutual movement already observed by the crystallographic analysis of the dII (dIIb) and the dI (dIIa) domains creating a functional active site. This work not only provides another piece of supporting evidence for the calpain conformational change in the presence of Ca2+, but actually constitutes the first experimental observation of this change for intact heterodimeric calpain in solution.
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