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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 11; pp. 5895 - 5906
• Main Authors: Vahidi, Siavash, Ripstein, Zev A., Juravsky, Jordan B., Rennella, Enrico, Goldberg, Alfred L., Mittermaier, Anthony K., Rubinstein, John L., Kay, Lewis E.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 17.03.2020
• Subjects: Allosteric properties; Antibiotics; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biological Sciences; Cryoelectron Microscopy - methods; Crystallography, X-Ray; Dynamic structural analysis; Endopeptidase Clp - chemistry; Endopeptidase Clp - metabolism; Escherichia coli; Homeostasis; Inhibitors; Leucine; Models, Molecular; Mycobacterium tuberculosis; Mycobacterium tuberculosis - metabolism; NMR; Nuclear magnetic resonance; Peptides; Protease; Protein Conformation; Protein Interaction Domains and Motifs; Proteinase; Proteolysis; Serine Endopeptidases - chemistry; Serine Endopeptidases - metabolism; Spectroscopy; Structure-function relationships; Substrates; Tuberculosis; Virulence; ClpP handle region; intra-/interring cooperativity; protein homeostasis; allostery
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1921630117

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 of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as 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 antituberculosis (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. Electron cryomicroscopy (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, on 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 interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis.