Abstract:
Calpains are cysteine proteases showing a unique dependency on Ca2+ for activity. The µ- and m- isoforms are activated in vitro by micro- and millimolar Ca2+ concentrations, respectively. Based on the crystal structure of m-calpain, a negatively charged loop in domain III was proposed to be a Ca2+-sensitive switch regulating activation through electrostatic interactions with the catalytic domain and with cellular membranes. In order to study the role of this structural motif, several acidic residues of the loop were substituted by alanine in µ-calpain. Ca2+-dependent activation and membrane binding properties of the mutants were determined by means of enzyme kinetic, fluorescence- and SPR-spectroscopy measurements. Concomitantly, the effect of domain V autoproteolysis on both events was investigated. Based on these studies, a model for activation and membrane binding of calpain was proposed.