CRYSTALLIZATION OF CALPAIN A Ca²⁺ DEPENDENT CYSTEINE PROTEASE. Zongchao Jia, Qilu Ye, Peter L. Davies and John S. Elce. Department of Biochemistry, Queen's University, Kingston, Ontario, K7L 3N6 Canada

The calpains (EC 3.4.22.17) are a family of Ca²⁺ dependent cysteine proteases found in the cytosol of animal cells. Their precise physiological role is uncertain, however it is likely that they are involved in cell signalling and in cytoskeletal modifications. The ubiquitous calpains have a large catalytic subunit (80 kDa) composed of 4 domains, and a small regulatory subunit (30 kDa) composed of 2 domains. The enzymes are activated by Ca²⁺, and then undergo autolysis. In order to understand the structural basis for Ca²⁺ dependent calpain activation and provide a molecular explanation for autolysis, we are interested in determining the structure of calpain II by X-ray crystallography. To avoid autolysis and oxidation which present great problems during recombinant protein production and crystallization, an inactive C105S active-site mutant (80k/21k)has been used. After testing a wide range of crystallization conditions, we obtained crystals of this mutant with size of approximately 0.25x0.17x.03 mm, which were rather fragile and diffracted only to low resolution. Under somewhat different crystallization conditions, the mutant also crystallized in the presence of Ca²⁺, although this was accompanied by heavy Ca²⁺ induced aggregation. Crystallization parameters were varied so that initial nucleation takes place before substantial precipitation, which is difficult to control. We are currently optimizing the conditions to improve crystal quality. (Supported by MRC of Canada and PENCE, Canada)