Structure-function studies on recombinant calpain

Expression and purification of rat m-calpaln has been developed to produce 5-10 mg of pure enzyme in 3-4 days from 4 L of E. coli. Using a variety of native and mutated calpain subunits in this expression system, a range of structural studies is under way. 1) Site-directed mutagenesis has confirmed that large subunit (80k) residues C105, H262, and N286 constitute the active site triad. 2) Autolysis of calpain small subunit from 28k to 21k was complete within 1 mira It can occur rapidly by intermolecular reaction, as shown by incubation of the inactive mutant C105S-m-80k/28k with active m-80k/21k. 3) A series of deletions has shown that small subunit residues 94116 are not directly involved in catalysis, but are important in stabilization of the heterodimer. Removal of 25 C-terminal residues from the small subunit abolished activity and heterodimer formation. 4) Autolysis of the m-calpain large subunit at A9-K10 was shown to be responsible for the fall in calcium dependency that is characteristic of m-calpaln autolysis. 5) The crystal structures of a homodimer of domain VI (the C-terminal domain of the small subunit), both with and without bound calcium, have been determined. Calcium binds at 3 of the 5 potential EF-hands. One major subunit interaction in the homodimer involves 30 C-terminal residues, and may provide a model of the binding between large and small subunits. This work is funded by MRC of Canada, PENCE, and Queen's University.