Metacaspases are enzymes that transmit cellular signals by cleaving other proteins. These proteases are involved in programmed cell death in plants and fungi and are activated by calcium flux. The existing literature clearly demonstrates that, in vitro, metacaspases require low millimolar concentrations of calcium to function. However, the exact mechanism by which calcium activates the metacaspases is still unclear. Our lab has characterized metacaspase1 from the fungus Schizophyllum commune (ScMCA-Ia) to determine the likely conformational changes needed for metacaspase activation.
Metacaspase structures demonstrate that calcium binds to a pocket of four negatively charged aspartic acid residues. Though the calcium-binding site is far from the active site, the Asp residues are found on the same short flexible loops that bear the active site Cys and His. Sequence alignments demonstrate that the position and identity of these Asp residues is conserved among many metacaspases. Mutating these residues inactivates ScMCA-Ia, highlighting their important role in metacaspase function.
To gain additional insights, we have used differential scanning fluorimetry (DSF) to determine the calcium dissociation constants of ScMC-Ia and several mutants. When calcium-binding Asp residues are replaced with Glu, a longer negative residue, the enzymes are inactive. However, the mutants have nearly the same affinity for calcium as wildtype. Though calcium occupies the binding site, the enzyme is no longer activated by calcium, suggesting that activation occurs through very delicate structural changes. We hypothesize that calcium binding changes the position of the Asp residues which alters the configuration of the loops bearing these groups, and ultimately reorients the active site to promote catalysis.
In addition to the tight binding site discussed above, a second site that binds weakly to calcium is inferred from the high calcium levels needed for metacaspase activity. Kinetics studies suggest that the second 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. This detailed mechanism of metacaspase activation is needed to design highly specific inhibitors to characterize the in vivo pathways involving metacaspases