AAA+ proteases remove toxic proteins from cells and regulate many other important cellular processes that are required to promote health and prevent disease. As 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 uses the energy of ATP hydrolysis to unfold 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 AAA+ remodeling machines. These ATP-fueled enzymes perform a wide variety of remodeling, transport, and regulatory tasks in the cell, all of which require mechanical work. In mammals, loss of mitochondrial ClpP results in infertility, hearing loss, and a growth defect, 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 ClpXP and other AAA+ enzymes but important and fundamental questions concerning the molecular mechanisms of these machines remain unanswered. For example, it is not known how ClpX grips and interacts with polypeptide substrates during mechanical unfolding and translocation, how the hexameric ClpX ring engages protein substrates and coordinates ATP binding and hydrolysis with conformational switching between nucleotide loadable and unloadable subunits during function, or how ClpX binds and collaborates with ClpP during degradation. The experiments described in this proposal will answer these questions and provide a conceptual framework and a set of novel tools applicable to studies of the entire superfamily of AAA+ machines.

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 #
5R01GM101988-39
Application #
9266792
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Barski, Oleg
Project Start
1979-05-01
Project End
2020-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
39
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Olivares, Adrian O; Kotamarthi, Hema Chandra; Stein, Benjamin J et al. (2017) Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines. Proc Natl Acad Sci U S A :
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
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 11:1552-1560
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
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
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 :
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; 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
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
Schmitz, Karl R; Sauer, Robert T (2014) Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase. Mol Microbiol 93:617-28

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