Open and compressed conformations of Francisella tularensis ClpP

Proc. Natl. Acad. Sci. U.S.A.

Juravsky

et al. 2020

The 300-kDa ClpP1P2 protease fromMycobacterium tuberculosiscollaborates 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 targetM. tuberculosis.

Section: Discussionmentioning

confidence: 99%

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

Proc. Natl. Acad. Sci. U.S.A.

Juravsky

et al. 2020

“…There is significant evidence from a variety of different biophysical techniques that ClpP is a highly dynamic molecular complex. For example, ClpP can be crystallized in three distinct conformations (4), including extended (25,26,31), compact (32,33), and compressed (31,34,35) forms that differ in the height of the barrel and in the conformation of the handle region. Further insight comes from NMR studies focusing on a pair of Ile residue probes showing the presence of at least two handle-region conformations in solution (36).…”

Section: Significancementioning

confidence: 99%

Reversible inhibition of the ClpP protease via an N-terminal conformational switch

Proc. Natl. Acad. Sci. U.S.A.

Bonomi

et al. 2018

Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly β-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of and ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.

“…It has been shown that ClpP, while maintaining its tetradecameric architecture, can assume several conformations that mainly differ in the height of its barrel-like structure and in the conformation of the handle domain (11,67). High-resolution ClpP structures from various species include an active extended conformation with a fully formed catalytic triad and handle domain (38,68), as well as compressed/compact forms with kinked/truncated handle helices and unstructured handle β-sheets with misaligned active site residues (69)(70)(71)(72). While it is widely accepted that the extended form is prevalent in solution and is the active state, it is yet to be established whether the compressed/compact forms can be populated under conditions that are distinct from those used for crystallization, where crystal contacts can affect the ClpP structure (11,73).…”

Section: Discussionmentioning

confidence: 99%

An allosteric switch regulatesMycobacterium tuberculosisClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

Juravsky

et al. 2019

Preprint

Short title: NMR and cryo-EM of M. tuberculosis ClpP1P2The 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. the binding of essential AAA+ unfoldases, ClpX or ClpC1, that use the energy of ATP to unfold and translocate substrates into its catalytic chamber for degradation (28). Lassomycin (29), ecumicin (30), and rufomycin (31) are promising antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by binding to ClpC1 and decoupling ATP-dependant protein unfolding from proteolysis. Similarly, ADEPs also kill M. tuberculosis by preventing the binding of AAA+ regulatory unfoldases to MtClpP1P2 (32). MtClpP1P2 reacts with standard inhibitors of serine proteases, such as chloromethyl ketones, which modify the serine and histidine residues at enzyme active sites (33). Peptide boronates have been shown to also directly engage MtClpP1P2 active sites, causing inhibition at low micromolar concentrations and preventing growth of M. tuberculosis (34-36). More recently Cediranib, an anti-cancer drug, was proposed as a novel non-covalent inhibitor of MtClpP1P2 (37).Most bacteria possess a single clpP gene, giving rise to a structure comprising a pair of heptameric rings that are arranged coaxially to form a homotetradecameric barrel-like protein complex enclosing fourteen Asp-His-Ser catalytic triads (11, 12,38). Each of the identical protomers consists of an N-terminal domain that forms gated narrow pores on the apical surface of the barrel, a head domain that generates the main body of the ClpP barrel, and a handle region comprising a helix and a β-sheet that mediate ClpP ring-ring interactions (11,(38)(39)(40). In addition, the handle provides crucial contacts that align the catalytic triad and generates a binding grove for substrate polypeptides (41). Opening of the pores that allow substrate translocation is tightly regulated by AAA+ regulators (42,43). Actinobacteria, the phylum to which mycobacteria such as M. tuberculosis belong, are unique in that they contain two clpP genes, clpP1 and clpP2, that encode for MtClpP1 and MtClpP2, respectively (44,45). Initial structure-function studies concluded that MtClpP1 and SI AppendixAn allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR ...