Project Summary AAA+ proteases remove toxic proteins and regulate many other important cellular processes that promote health and prevent disease. At the same time, protein degradation must be carefully regulated. AAA+ proteases assemble into multi-subunit structures with an internal proteolytic chamber, accessible through narrow channels that exclude natively folded proteins. This mechanism protects most proteins from unintended degradation and requires specific substrates to be recognized, unfolded, and then translocated into the degradation chamber. In the AAA+ ClpXP protease, for example, a ring hexamer of ClpX uses the energy of ATP hydrolysis to unfold specific target proteins and translocate them into ClpP for degradation. ClpXP is one of the best-characterized AAA+ proteases and is a paradigm for other ATP- dependent proteases and AAA+ remodeling machines. These ATP-fueled enzymes perform a wide variety of mechanical remodeling, transport, and regulatory tasks in the cell. In mammals, loss of mitochondrial ClpP results in infertility, hearing loss, and growth defects, whereas mitochondrial ClpX plays an important role in heme biosynthesis. Bacterial ClpXP can promote pathogenesis and is a validated antibiotic target in M. tuberculosis. Substantial progress has been made in understanding the general biochemical and structural features of E. coli ClpXP and other AAA+ enzymes but important and fundamental questions concerning the molecular mechanisms of these machines remain. For example, it is not known how ClpX identifies many classes of N-terminal and C-terminal degrons, whether ClpX rotates with respect to ClpP during normal function, whether proofreading helps ensure degradation specificity, how multiple substrate chains can be simultaneously translocated through the ClpX channel, what the detailed and interacts with polypeptide substrates during mechanical unfolding, whether ATP hydrolysis can occur at multiple positions in the spiral ClpX ring or only at one or a few special positions, and how the detailed ATPase cycle is coupled to mechanical work. The experiments described in this proposal will address these questions and provide a conceptual framework applicable to studies of the entire superfamily of AAA+ machines.