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An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

View ORCID ProfileSiavash Vahidi, View ORCID ProfileZev A. Ripstein, Jordan B. Juravsky, View ORCID ProfileEnrico Rennella, View ORCID ProfileAlfred L. Goldberg, View ORCID ProfileAnthony K. Mittermaier, View ORCID ProfileJohn L. Rubinstein, View ORCID ProfileLewis E. Kay
doi: https://doi.org/10.1101/2019.12.11.873281
Siavash Vahidi
1Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
2Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
3Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
4Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada
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Zev A. Ripstein
2Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
4Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada
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Jordan B. Juravsky
4Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada
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Enrico Rennella
1Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
2Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
3Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
4Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada
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Alfred L. Goldberg
5Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02138, USA
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Anthony K. Mittermaier
6Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, H3A 0B8, Canada
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John L. Rubinstein
2Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
4Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada
7Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 1L7, Canada
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Lewis E. Kay
1Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
2Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
3Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
4Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada
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  • For correspondence: kay@pound.med.utoronto.ca
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Abstract

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as novel antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of anti-TB therapeutics that target the Clp system. Here we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Cryo-EM structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that upon binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function, and shows extensive allosteric interactions involving both intra- and inter-ring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis.

Significance Statement The MtClpP1P2 protease is part of the essential protein degradation machinery that helps maintain protein homeostasis in Mycobacterium tuberculosis, the causative agent of TB. Antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by disrupting MtClpP1P2 function have attracted recent attention. Here we characterize a switch that can control MtClpP1P2 activity through binding of small peptides, leading to a concerted conformational change that potentially can be exploited by drug molecules to interfere with MtClpP1P2 function. Overall, this work highlights the power of a combined NMR and cryo-EM approach to provide detailed insights into the structure-dynamics-function relationship of molecular machines critical to human health.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 12, 2019.
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An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR
Siavash Vahidi, Zev A. Ripstein, Jordan B. Juravsky, Enrico Rennella, Alfred L. Goldberg, Anthony K. Mittermaier, John L. Rubinstein, Lewis E. Kay
bioRxiv 2019.12.11.873281; doi: https://doi.org/10.1101/2019.12.11.873281
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An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR
Siavash Vahidi, Zev A. Ripstein, Jordan B. Juravsky, Enrico Rennella, Alfred L. Goldberg, Anthony K. Mittermaier, John L. Rubinstein, Lewis E. Kay
bioRxiv 2019.12.11.873281; doi: https://doi.org/10.1101/2019.12.11.873281

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