ATP-dependent AAA+ proteases remove toxic proteins from cells and regulate many other important cellular processes that are required to promote health and prevent disease. Because protein degradation is irreversible, it must be carefully regulated. The architectures of AAA+ proteases and the principles of degradation control are similar in all organisms. 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 unfolds specific target proteins and translocates them into ClpP for degradation. ClpXP is one of the best-characterized AAA+ proteases and is a paradigm for other ATP-dependent proteases and non-proteolytic AAA+ enzymes. These ATP-fueled molecular machines perform mechanical work in the cell, including protein and nucleic-acid remodeling (e.g., helicases), transport of cargo along microtubules, secretion and vesicle recycling, cell-cycle control, viral budding, cytokinesis, activation of apoptosis and the innate immune response, chromosome translocation, viral DNA packaging, peroxisome biogenesis, transcriptional activation, and clamp and helicase loading onto DNA. In humans, mutations in many AAA+ proteins are linked to disease. For example, mutations in the m-AAA mitochondrial protease result in hereditary spastic paraplegia. ClpXP can also promote bacterial pathogenesis and is an antibiotic target. Substantial progress has been made in understanding the general biochemical and structural features of ClpXP and other AAA+ enzymes but important and fundamental questions concerning the molecular mechanisms of these intracellular machines remain unanswered. For instance, in no case, do we understand the ATPase cycle of a AAA+ enzyme and how it is linked to the cycle of conformational changes that power machine function or whether ATP hydrolysis occurs in a sequential or probabilistic fashion. The experiments described in this proposal will answer these questions for ClpXP and provide a conceptual framework and a set of novel tools applicable to studies of the entire superfamily of AAA+ machines. Specifically, we will use biochemical, protein-engineering, and single-molecule approaches to determine how ATP binding, ATP hydrolysis, and the coordination of these reactions among the six subunits of a ClpX ring drive the conformational changes that allow this enzyme to mechanically unfold and translocate protein substrates and to cooperate with ClpP.
Understanding intracellular degradation is a key goal of basic research, with applications in biotechnology and medicine. Enzymes of the AAA+ family clear the cell of toxic proteins and carry out many other ATP-dependent cellular processes needed to promote health and prevent disease. Our studies will illuminate molecular mechanisms employed by a specific AAA+ protease and shed light on the entire enzyme superfamily.
|Amor, Alvaro J; Schmitz, Karl R; Sello, Jason K et al. (2016) Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle. ACS Chem Biol 11:1552-60|
|Olivares, Adrian O; Baker, Tania A; Sauer, Robert T (2016) Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines. Nat Rev Microbiol 14:33-44|
|Carney, Daniel W; Schmitz, Karl R; Scruse, Anthony C et al. (2015) Examination of a Structural Model of Peptidomimicry by Cyclic Acyldepsipeptide Antibiotics in Their Interaction with the ClpP Peptidase. Chembiochem :|
|Iosefson, Ohad; Nager, Andrew R; Baker, Tania A et al. (2015) Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine. Nat Chem Biol 11:201-6|
|Iosefson, Ohad; Olivares, Adrian O; Baker, Tania A et al. (2015) Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine. Cell Rep 12:1032-41|
|Stinson, Benjamin M; Baytshtok, Vladimir; Schmitz, Karl R et al. (2015) Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX. Nat Struct Mol Biol 22:411-6|
|Carney, Daniel W; Compton, Corey L; Schmitz, Karl R et al. (2014) A simple fragment of cyclic acyldepsipeptides is necessary and sufficient for ClpP activation and antibacterial activity. Chembiochem 15:2216-20|
|Schmitz, Karl R; Carney, Daniel W; Sello, Jason K et al. (2014) Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery. Proc Natl Acad Sci U S A 111:E4587-95|
|Cordova, Juan Carlos; Olivares, Adrian O; Shin, Yongdae et al. (2014) Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine. Cell 158:647-58|
|Carney, Daniel W; Schmitz, Karl R; Truong, Jonathan V et al. (2014) Restriction of the conformational dynamics of the cyclic acyldepsipeptide antibiotics improves their antibacterial activity. J Am Chem Soc 136:1922-9|
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