Role of Calcium in Proteolytic Activity of a Type I Metacaspase from Schizophyllum commune

Abstract

Metacaspases are members of the C14 class of cysteine proteases that also includes the caspases. They are found in fungi, plants and protozoa, but not in animals. Unlike the caspases, metacaspases cleave after positively charged residues such as Lys and Arg and are activated by calcium ions. The role of metacaspases in vivo is not yet clearly defined. They have been implicated in cell death and protein quality control, and recent work in A. thaliana suggests that they are activated in response to plant wounding as part of a primitive immune system. Our work centers on understanding how the Type I metacaspases of the fungus Schizophyllum commune are activated by calcium ions. Metacaspase 1 (MC1) activity can be reduced by >90% by mutation of conserved aspartic acid residues implicated in calcium binding. A homology model for MC1 based on the structures of Type I metacaspases from S. cerevisiae and T. brucei was refined. Using the model we identified a loop near the active site that also contains the putative calcium binding site and may undergo conformational changes upon calcium binding. Mutation of a residue in the hinge region of this loop from Gly to Ala significantly reduced enzymatic activity. Kinetic solvent viscosity effects suggest that at low calcium concentrations the enzymatic reaction is limited by substrate binding, while at high calcium levels product release becomes rate‐limiting. We will combine these data to present a model for the role of calcium in Type I metacaspase activation.Support or Funding InformationCRM was supported by a Scholarship from the Arnold and Mabel Beckman Foundation