PROJECT SUMMARY Systems to identify, endure, and respond to stress are critical for longevity and cellular function. As the hub of bioenergetic processes, mitochondria have an array of stress tolerance systems, one of which is comprised of proteases localized to the inner mitochondrial membrane (IMM). IMM proteases acutely manage stress, but chronic imbalances in the activity of IMM proteases contribute to mitochondrial dysfunction associated with ageing and etiologically-diverse age-associated diseases including many neurodegenerative disorders and cardiomyopathy. Remarkably, interventions to rescue and restore balance to IMM protease activity have demonstrated therapeutic benefit for mitigating pathologic mitochondrial dysfunction implicated in both acute and chronic age-associated diseases as well as lifespan extension. This multimodal therapeutic value has led to significant interest in defining the molecular mechanisms that drive IMM protease function. One central stress responsive IMM protease that remains understudied is the ATP-independent zinc metalloprotease OMA1. Basally inactive, OMA1 is activated in response to mitochondrial insults through a poorly defined mechanism. Active OMA1 site-specifically cleaves select IM substrates to protect mitochondria from acute mitochondrial insults through modulation of morphology and the activation of stress-responsive transcription factors. Despite the central role of OMA1 in mounting a mitochondrial stress response and its high therapeutic value, no structural information of OMA1 activation or proteolytic activity has been defined. Here, we integrate cryo-EM structure determination (Aim 1) and deep mutational scanning (Aim 2) to establish the first structure-function relationship for OMA1 activation and proteolytic activity. Through these efforts, we will define the molecular mechanism of OMA1 activation and proteolysis. This will reveal new insights into OMA1-dependent regulation of mitochondrial proteostasis and function and establish a structural basis to develop new strategies to therapeutically target OMA1 to mitigate pathologic mitochondrial dysfunction associated with ageing and ageing-associated neurodegeneration and cardiomyopathy.