| Calpains are Ca2+-dependent cysteine proteases found in organisms ranging from bacteria to humans. With their endogenous inhibitors they drive and fine-tune Ca2+ signaling pathways through limited, specific proteolysis that modifies the activity of their targets. Calpains regulate processes as common as cell migration and as exclusive as sex determination in nematodes. But how do they respond to Ca2+ signaling when the known Ca2+-binding domains (C2-like and EF-hand) are not even present in all calpain isoforms? The breakthrough in this thesis was the observation that the protease core of calpain (domains I--II) also binds Ca2+. Bacterial expression and biochemical analysis of the cores from both mu- and m-calpain, showed that in isolation they are Ca2+-dependent proteases (minicalpains) with isoform-specific properties reminiscent of their full-length isoform. The protease core is the common element that defines the superfamily. The 2.07A-resolution structure of Ca2+-bound muI--II revealed the conserved Ca2+ switch that aligns the active site. This switch involves cooperative binding of Ca2+ at two non-EF-hand sites, one in each domain, to reposition several loop structures and induce papain-like active site assembly. A mechanism was proposed and tested using mutagenesis and domain swaps. It supported the order and cooperativity of Ca2+ binding, and suggested a hierarchical functional importance for the sequential steps in the mechanism. Accordingly, domain II site assembly is rate-determining, followed in importance by the salt-bridge between sites that provides the basis for cooperativity. Surprisingly, domain I site is more peripheral to activation than originally thought. The 1.95A-resolution structure of mI--II revealed an intrinsic mechanism of silencing of this and similar mini-calpains. A strategically positioned glycine collapses a key alpha-helix leading to rearrangement of the hydrophobic core whereby the conserved tryptophan next to the active site cysteine swings out into the active site cleft and prevents substrate binding. The resistance of non-glycine-containing muI--II to calpastatin inhibition reinforces the patho-physiological consequences of these structure-based mechanisms on calpain signaling. This suggests that active cores produced by autolysis could induce tissue damage during ischemia and neurodegeneration. Minicalpains are, therefore, useful targets for drug screening and design, as shown by the crystal structure of the muI--II-E64 inhibitor complex. |
