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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
9R01GM101988-34
Application #
8292653
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gerratana, Barbara
Project Start
1979-05-01
Project End
2016-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
34
Fiscal Year
2012
Total Cost
$455,820
Indirect Cost
$160,437
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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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
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