Critical role of Asp283 in modulating calcium-dependent autoprocessing of yeast Metacaspase ScMCA-Ia

Metacaspases are Ca 2+ -dependent cysteine proteases essential for programmed cell death. This study elucidates the regulatory mechanisms of Saccharomyces cerevisiae type I metacaspase (ScMCA-IA), focusing on Ca 2+ -dependent autoprocessing and the function of its N-terminal hydrophobic prodomain. Through cloning, heterologous expression, and site-directed mutagenesis, the roles of Asp 236 , Asp 252 , Asp 253 , and Asp 283 in ScMCA-IA autoprocessing were systematically investigated. A truncated variant (ScMCA-IA-ΔN86) was engineered to assess prodomain contribution. Results demonstrate that ΔN86 exhibits gradual autoprocessing and processing patterns consistent with intermolecular cleavage. The N-terminal region critically modulates Ca 2+ affinity and processing kinetics. Mutagenesis revealed distinct aspartate contributions to maturation efficiency: D236A compromised truncated variant stability; D252A attenuated processing efficiency; D253A yielded predominantly unprocessed enzyme; while D283A profoundly impaired autoprocessing, establishing Asp 283 as a critical residue for calcium-dependent autoprocessing and acquisition of the processed conformation. These findings provide mechanistic insights into metacaspase regulation, with Asp 283 playing a key role in the Ca 2+ -dependent proteolytic maturation. However, direct catalytic activity was not measured; thus, the relationship between processing and enzymatic activation remains to be determined.