Structural and biochemical characterization of disease-associated mutations in the human mitochondrial processing...

The mitochondrial processing peptidase (MPP) is an essential heterodimeric protease responsible for cleavage of N-terminal mitochondrial targeting sequences (N-MTSs) from most nuclear-encoded mitochondrial proteins following import into the mitochondrial matrix. Pathogenic variants in the α-subunit of MPP (PMPCA) cause a spectrum of mitochondrial disorders, including spinocerebellar ataxia type 2 (SCAR2), encephalopathy, and severe neurodevelopmental disease; however, the molecular mechanisms underlying these phenotypes remain poorly understood. Here, we combined biochemical and structural approaches to characterize five disease-associated PMPCA variants (S96L, R185W, R268W, A377T, and G515R). Enzymatic analyses revealed variant-specific defects in substrate processing, with most mutations severely impairing cleavage of the rapidly processed substrate malate dehydrogenase 2. In contrast, several variants exhibited only modest impairment in the processing of the slowly cleaved substrate PINK1. Mass photometry demonstrated reduced heterodimerization of all variants, whereas circular dichroism identified varying degrees of structural destabilization, particularly for S96L and G515R. To define the structural basis of these defects, hydrogen-deuterium exchange mass spectrometry was performed on wild type MPP and selected variants. Pathogenic mutations altered substrate-induced conformational changes within previously characterized substrate-recognition elements, including the glycine-rich loop. Furthermore, we identified a previously unrecognized region, termed the INS loop, that exhibited substrate-dependent protection uniquely in the wild type enzyme but was consistently disrupted across pathogenic variants. Together, these findings demonstrate that PMPCA mutations impair MPP function through diverse mechanisms involving protein stability, dimerization, substrate selectivity, and allosteric conformational dynamics. Our work provides a mechanistic framework for understanding genotype-phenotype relationships in PMPCA-associated disease.