Intracellular AAA+ proteases remove toxic, damaged, and unnecessary proteins and participate in cellular processes that promote health and prevent disease. Because protein synthesis is energetically expensive and proteolysis is irreversible, protein degradation must be carefully regulated to conserve cellular resources. Building on our extensive experience in structure-function studies of AAA+ proteases, we will use a combination of structural biology, biochemistry/biophysics, protein engineering, and molecular genetics to determine how the proteolytic activity of three different AAA+ proteases is mediated and regulated. For example, for the AAA+ HslUV protease, we will determine how temperature and substrate binding control autoinhibition by a novel I-domain structure that blocks the axial pore. For the double-ring ClpAP protease, we will probe the roles of the AAA+ D1 and D2 rings in protein substrate unfolding and translocation in single-molecule optical trapping experiments. For FtsH, an essential membrane-bound bacterial AAA+ protease, we will determine the mechanisms that limit degradation of cytosolic proteins, mediate turnover of an important biosynthetic enzyme, and allow the HflK/HflC membrane partners to control FtsH activity. The unifying theme of this proposal is understanding how the common and unique structural features of different AAA+ proteases contribute to their specialized biological roles. !

Public Health Relevance

. Understanding how cellular proteases eliminate proteins that are damaged, dangerous, or no longer needed is an important goal of basic research, with applications in medicine and biotechnology. AAA+ proteases use the energy of ATP hydrolysis to clear cells of toxic proteins and play important roles in other cellular processes needed to promote health and prevent disease. Our studies will determine the molecular mechanisms that allow different members of this important enzyme family to perform their unique activities and help to maintain cellular and organismal health.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI016892-39
Application #
9531591
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Ernst, Nancy L
Project Start
1980-04-01
Project End
2023-03-31
Budget Start
2018-04-01
Budget End
2019-03-31
Support Year
39
Fiscal Year
2018
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
Hari, Sanjay B; Grant, Robert A; Sauer, Robert T (2018) Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase. Structure 26:1251-1258.e3
Brown, Breann L; Kardon, Julia R; Sauer, Robert T et al. (2018) Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme. Structure 26:580-589.e4
Amberg-Johnson, Katherine; Hari, Sanjay B; Ganesan, Suresh M et al. (2017) Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens. Elife 6:
Totaro, Kyle A; Barthelme, Dominik; Simpson, Peter T et al. (2017) Rational Design of Selective and Bioactive Inhibitors of the Mycobacterium tuberculosis Proteasome. ACS Infect Dis 3:176-181
Baytshtok, Vladimir; Chen, Jiejin; Glynn, Steven E et al. (2017) Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation. J Biol Chem 292:5695-5704
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
Hari, Sanjay B; Sauer, Robert T (2016) The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli. Biochemistry 55:5649-5652
Stein, Benjamin J; Grant, Robert A; Sauer, Robert T et al. (2016) Structural Basis of an N-Degron Adaptor with More Stringent Specificity. Structure 24:232-42
Baytshtok, Vladimir; Fei, Xue; Grant, Robert A et al. (2016) A Structurally Dynamic Region of the HslU Intermediate Domain Controls Protein Degradation and ATP Hydrolysis. Structure 24:1766-1777
Barthelme, Dominik; Sauer, Robert T (2016) Origin and Functional Evolution of the Cdc48/p97/VCP AAA+ Protein Unfolding and Remodeling Machine. J Mol Biol 428:1861-9

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