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Title: Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

Journal Article · · Journal of Biological Chemistry
 [1];  [2];  [2];  [3];  [1];  [4];  [2]
  1. Frederick National Lab., MD (United States)
  2. Harvard Medical School, Boston, MA (United States)
  3. National Inst. of Health (NIH), Bethesda, MD (United States)
  4. National Inst. of Health (NIH), Bethesda, MD (United States); NCI, Bethesda, MD (United States)
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The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3–4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity of Mtb ClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2–3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Inst. of Health; NCI
Grant/Contract Number:
W-31-109-Eng-38; RO-051923-170; HHSN261200800001E
OSTI ID:
1248396
Journal Information:
Journal of Biological Chemistry, Vol. 291, Issue 14; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 33 works
Citation information provided by
Web of Science

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Cited By (11)

Insights into ClpXP proteolysis: heterooligomerization and partial deactivation enhance chaperone affinity and substrate turnover in Listeria monocytogenes journal January 2017
Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease: ADEPs target ClpP1P2 of M. tuberculosis journal April 2016
AAA+ Machines of Protein Destruction in Mycobacteria journal July 2017
Towards Selective Mycobacterial ClpP1P2 Inhibitors with Reduced Activity against the Human Proteasome journal February 2017
Inhibiting Mycobacterium tuberculosis ClpP1P2 by addressing the equatorial handle domain of ClpP1 subunit preprint July 2019
A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery posted_content December 2019
Reversible inhibition of the ClpP protease via an N-terminal conformational switch journal June 2018
An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR journal March 2020
Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors journal September 2019
Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors posted_content March 2019
A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery journal January 2020

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
enzyme mechanism
protease
protein complex
protein degradation
x-ray crystallography