Determination of the Calcium‐Dependent Activation Mechanism for Metacaspase 1 from Schizophyllum commune

Abstract

Metacaspases are cysteine proteases implicated in initiating programmed cell death in plants and fungi, and have recently been shown to play a key role in the plant immune response. These proteins are distantly related to caspases, the enzymes that induce apoptosis in animals. Notably, metacaspases require calcium binding for activity, whereas caspases are instead activated by upstream signalling pathways that utilize calcium as a signal transducer. Metacaspase activity is greatest in the presence of millimolar levels of calcium. However, the exact mechanism by which calcium activates the metacaspases is still unclear. Our lab has performed an extensive characterization of metacaspase1 from the fungus Schizophyllum commune (ScMCA-Ia) to determine the likely conformational changes needed for metacaspase activation. The available metacaspase crystal structures from yeast and T. brucei demonstrate calcium-binding to a conserved site of four aspartic acid residues outside of the active site. While calcium itself binds to this allosteric site, the calcium-binding Asps are found on flexible loops and are only a few residues away from the active site. Sequence alignments demonstrate that these Asp residues are conserved in ScMCA-Ia and mutagenesis studies that exchange Asp for Ala reveal that this site is crucial for activity on small peptide substrates. We have used differential scanning fluorimetry to determine the calcium-binding constants of ScMC-Ia and associated mutants. Mutating calcium-binding Asp residues to Glu renders the enzymes inactive, however differential scanning fluorimetry indicates that the mutants still bind to calcium with Kd values similar to wildtype, in the µM range. This suggests that calcium-binding to this high-affinity site changes the position of calcium binding residues, altering the positions of the loops bearing these residues, and ultimately changing the orientation of the active site. Even slight changes to the calcium binding site (Asp to Glu) cannot be tolerated. In addition to the micromolar binding site, a second site that binds calcium in the mM range has been inferred from the low millimolar calcium levels needed for optimal metacaspase activity. Kinetics experiments suggest that the low-affinity calcium binding site is important for substrate binding. Together, these findings comprise a complete model for metacaspase activation in which high-affinity calcium binding reorients the active site for catalysis and low-affinity binding alters the substrate binding pocket to enhance substrate binding and catalysis. This mechanistic understanding of metacaspase activation and conformational changes is needed to design metacaspase-specific inhibitors to treat fungal diseases.