Date of Award
Union College Only
Bachelor of Science
Metacaspase, Protease, Calcium-Binding, Differential Scanning Fluorimetry, Activation Mechanism
Metacaspases are cysteine proteases that initiate programmed cell death, invoke plant immune responses, and coordinate sexual selection in fungi. To function in these pathways metacaspases must first be activated by calcium. Metacaspases from many species have peak in vitro activity in the presence of millimolar levels of calcium, but the molecular mechanism by which calcium activates the metacaspases is unknown. In this thesis I report on the extensive characterization of the calcium-dependency and calcium-binding of metacaspase1 from the fungus Schizophyllum commune (ScMCA-Ia). These experiments have indicated that rearrangements of the active site and active site lid are crucial for metacaspase activation. 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. Sequence alignments indicate that these Asp residues are conserved in ScMCA-Ia and mutagenesis studies reveal that these residues are crucial for activity. We have used differential scanning fluorimetry (DSF) to determine the approximate calcium-binding constants of ScMC-Ia and mutants. Mutating high-affinity calcium-binding Asp residues to Glu renders the enzymes inactive, yet calcium still binds in the µM range, like wildtype. The precise size requirements of the high-affinity site suggest that calcium-binding triggers delicate structural rearrangements. The calcium-binding Asps are found on flexible loops that bear active site Cys and His. As these Asps shift during calcium binding, the loops bearing these residues change position, and the active site is reoriented into a better conformation for catalysis. 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.
Mineo, Charlotte, "Determination of the Calcium-Dependent Activation Mechanism for Metacaspase 1 from the Fungus Schizophyllum commune" (2021). Honors Theses. 2439.