PROJECT SUMMARY Integrated quality control (QC) systems are required to sense and manage mitochondrial stress, and their dysregulation is associated with neurodegenerative disease, aging, and cancer. The human ATP-dependent AAA+ protease, LONP1, has emerged as a master regulator of mitochondrial functions and is an integral component of mitochondrial QC systems. Deletion of LONP1 in mice is embryonically lethal and altered LONP1 activity is associated with mitochondrial diseases, aging, and cancer. LONP1 classically functions by degrading oxidatively damaged and unfolded matrix proteins, but also regulates diverse aspects of mitochondrial biology by specifically targeting and degrading folded proteolytic targets such as transcription factor A (TFAM) and cytochrome C oxidase subunit IV (COXIV). Additionally, LONP1 is a single stranded DNA binding protein that localizes to the non-coding control region of mitochondrial DNA, which is important for transcription and genome replication. LONP1 assembles as a 600 kDa hexamer composed of an N-terminal substrate binding domain (NTD), a AAA+ ATPase domain, and a C-terminal protease domain and can function as a protease, a chaperone, or a DNA binding protein. Despite these diverse critical functions, we lack detailed molecular mechanisms describing these activities and their regulation, limiting the fields capacity to determine their specific roles in mitochondrial homeostasis or disease pathogenesis. Recent cryo-electron microscopy (cryo-EM) studies have provided crucial insights into LONP1's conserved, AAA+-mediated hand-over-hand substrate translocation mechanism required to processively engage, unfold, and degrade proteolytic substrates. Strikingly, in these structures, the C-terminal protease domains remain in an inactive conformation even with substrate bound to LONP1's AAA+ domain. These findings contrast recent work on the evolutionarily related bacterial Lon protease and suggest that LONP1 has evolved additional levels of regulation to control or tune proteolytic activity to meet cellular needs. Moreover, these results raise important questions regarding proteolytic regulation and its relationship to other reported cellular functions, including DNA binding. Therefore, the long-term goal of this proposal will be to establish mechanistic models for LONP1's manifold functions and their allosteric regulation in order to define their role in mitochondrial maintenance and disease. I hypothesize that LONP1 adopts distinct structural conformations allosterically regulated by nucleotide state, substrate binding, and/or posttranslational modifications to shift between operational modes to modulate proteolytic and mtDNA binding activities. I will integrate structural studies using cryo-EM with biochemical assays to identify allosteric mechanisms involved in regulating LONP1's proteolytic activity (Aim 1) and elucidate the molecular mechanism and conformational state required for LONP1's mtDNA binding activity...