Nucleotide-induced ClpC oligomerization and its non-preferential association with ClpP isoforms of pathogenic Leptospira.

Bacterial caseinolytic protease-chaperone complexes participate in the elimination of misfolded and aggregated protein substrates. The spirochete Leptospira interrogans possess a set of Clp-chaperones (ClpX, ClpA, and ClpC), which may associate functionally with two different isoforms of LinClpP (ClpP1 and ClpP2). The L. interrogans ClpC (LinClpC) belongs to class-I chaperone with two active ATPase domains separated by a middle domain. Using the size exclusion chromatography, ANS dye binding, and dynamic light scattering analysis, the LinClpC is suggested to undergo nucleotide-induced oligomerization. LinClpC associates with either pure LinClpP1 or LinClpP2 isoforms non-preferentially and with equal affinity. Regardless, pure LinClpP isoforms cannot constitute an active protease complex with LinClpC. Interestingly, the heterocomplex LinClpP1P2 in association with LinClpC forms a functional proteolytic machinery and degrade ß-casein or FITC-casein in an energy-independent manner. Adding either ATP or ATPγS further fosters the LinClpCP1P2 complex protease activity by nurturing the functional oligomerization of LinClpC. The antibiotic, acyldepsipeptides (ADEP1) display a higher activatory role on LinClpP1P2 protease activity than LinClpC. Altogether, this work illustrates an in-depth study of hetero-tetradecamer LinClpP1P2 association with its cognate ATPase and unveils a new insight into the structural reorganization of LinClpP1P2 in the presence of chaperone, LinClpC to gain protease activity.

ClpP Peptidase as a Plausible Target for the Discovery of Novel Antibiotics.

Antimicrobial resistance (AMR) to currently available antibiotics/drugs is a global threat. It is desirable to develop new drugs that work through a novel target(s) to avoid drug resistance. This review discusses the potential of the caseinolytic protease P (ClpP) peptidase complex as a novel target for finding novel antibiotics, emphasising the ClpP's structure and function. ClpP contributes to the survival of bacteria via its ability to destroy misfolded or aggregated proteins. In consequence, its inhibition may lead to microbial death. Drugs inhibiting ClpP activity are currently being tested, but no drug against this target has been approved yet. It was demonstrated that Nblocked dipeptides are essential for activating ClpP's proteolytic activity. Hence, compounds mimicking these dipeptides could act as inhibitors of the formation of an active ClpP complex. Drugs, including Bortezomib, Cisplatin, Cefmetazole, and Ixazomib, inhibit ClpP activation. However, they were not approved as drugs against the target because of their high toxicity, likely due to the presence of strong electrophiles in their warheads. The modifications of these warheads could be a good strategy to reduce the toxicity of these molecules. For instance, a boronate warhead was replaced by a chloromethyl ketone, and this new molecule was shown to exhibit selectivity for prokaryotic ClpP. A better understanding of the structure and function of the ClpP complex would benefit the search for compounds mimicking N-blocked dipeptides that would inhibit ClpP complex activity and cause bacterial death.

Structure of phosphorylated-like RssB, the adaptor delivering σs to the ClpXP proteolytic machinery, reveals an interface switch for activation.

In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-ß5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.

An NMR Study of a 300-kDa AAA+ Unfoldase.

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.

Assessment of the structure-activity relationship and antileukemic activity of diacylpyramide compounds as human ClpP agonists.

Human caseinolytic protease P (ClpP) is required for the regulatory hydrolysis of mitochondrial proteins. Allosteric ClpP agonists dysfunctionally activate mitochondrial ClpP in antileukemic therapies. We previously developed ZG111, a potent ClpP agonist derived from ICG-001, inhibits the proliferation of pancreatic ductal adenocarcinoma cell lines in vitro and in vivo by degrading respiratory chain complex proteins. Herein, we studied the structure-activity relationships of ICG-001 analogs as antileukemia agents. Compound ZG36 exhibited improved stabilization effects on the thermal stability of ClpP in acute myeloid leukemia (AML) cell lines compared with the stabilization effects of ZG111, indicating a direct binding between ZG36 and ClpP. Indeed, the resolved ZG36/ClpP structural complex reveals the mode of action of ZG36 during ClpP binding. Compound ZG36 nonselectively degrades respiratory chain complexes and decreases the mitochondrial DNA, eventually leading to the collapse of mitochondrial function and leukemic cell death. Finally, ZG36 treatment inhibited 3-D cell growth in vitro and suppressed the tumorigenesis of AML cells in xenografted mice models. Collectively, we developed a new class of human ClpP agonists that can be used as potential antileukemic therapies.

Molecular determinants of protein half-life in chloroplasts with focus on the Clp protease system.

Proteolysis is an essential process to maintain cellular homeostasis. One pathway that mediates selective protein degradation and which is in principle conserved throughout the kingdoms of life is the N-degron pathway, formerly called the 'N-end rule'. In the cytosol of eukaryotes and prokaryotes, N-terminal residues can be major determinants of protein stability. While the eukaryotic N-degron pathway depends on the ubiquitin proteasome system, the prokaryotic counterpart is driven by the Clp protease system. Plant chloroplasts also contain such a protease network, which suggests that they might harbor an organelle specific N-degron pathway similar to the prokaryotic one. Recent discoveries indicate that the N-terminal region of proteins affects their stability in chloroplasts and provides support for a Clp-mediated entry point in an N-degron pathway in plastids. This review discusses structure, function and specificity of the chloroplast Clp system, outlines experimental approaches to test for an N-degron pathway in chloroplasts, relates these aspects into general plastid proteostasis and highlights the importance of an understanding of plastid protein turnover.

Comprehensive structural characterization of the human AAA+ disaggregase CLPB in the apo- and substrate-bound states reveals a unique mode of action driven by oligomerization.

The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space (IMS) and functions to promote the solubilization of various mitochondrial proteins. Loss-of-function CLPB mutations are associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. How CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo-tetradecamers in apo-state and is remodeled into homo-dodecamers upon substrate binding. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of 2 amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.

The structure of caseinolytic protease subunit ClpP2 reveals a functional model of the caseinolytic protease system from Chlamydia trachomatis.

Chlamydia trachomatis (ct) is the most reported bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Caseinolytic proteases (ClpP) from pathogenic bacteria are attractive antibiotic targets, particularly for bacterial species that form persister colonies with phenotypic resistance against common antibiotics. ClpP functions as a multisubunit proteolytic complex, and bacteria are eradicated when ClpP is disrupted. Although crucial for chlamydial development and the design of agents to treat chlamydia, the structures of ctClpP1 and ctClpP2 have yet to be solved. Here, we report the first crystal structure of full-length ClpP2 as an inactive homotetradecamer in a complex with a candidate antibiotic at 2.66 Å resolution. The structure details the functional domains of the ClpP2 protein subunit and includes the handle domain, which is integral to proteolytic activation. In addition, hydrogen-deuterium exchange mass spectroscopy probed the dynamics of ClpP2, and molecular modeling of ClpP1 predicted an assembly with ClpP2. By leveraging previous enzymatic experiments, we constructed a model of ClpP2 activation and its interaction with the protease subunits ClpP1 and ClpX. The structural information presented will be relevant for future rational drug design against these targets and will lead to a better understanding of ClpP complex formation and activation within this important human pathogen.

Acyldepsipeptide Antibiotics and a Bioactive Fragment Thereof Differentially Perturb Mycobacterium tuberculosis ClpXP1P2 Activity in Vitro.

Proteolytic complexes in Mycobacterium tuberculosis (Mtb), the deadliest bacterial pathogen, are major foci in tuberculosis drug development programs. The Clp proteases, which are essential for Mtb viability, are high-priority targets. These proteases function through the collaboration of ClpP1P2, a barrel-shaped heteromeric peptidase, with associated ATP-dependent chaperones like ClpX and ClpC1 that recognize and unfold specific substrates in an ATP-dependent fashion. The critical interaction of the peptidase and its unfoldase partners is blocked by the competitive binding of acyldepsipeptide antibiotics (ADEPs) to the interfaces of the ClpP2 subunits. The resulting inhibition of Clp protease activity is lethal to Mtb. Here, we report the surprising discovery that a fragment of the ADEPs retains anti-Mtb activity yet stimulates rather than inhibits the ClpXP1P2-catalyzed degradation of proteins. Our data further suggest that the fragment stabilizes the ClpXP1P2 complex and binds ClpP1P2 in a fashion distinct from that of the intact ADEPs. A structure-activity relationship study of the bioactive fragment defines the pharmacophore and points the way toward the development of new drug leads for the treatment of tuberculosis.

AAA+ protease-adaptor structures reveal altered conformations and ring specialization.

ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here we present high-resolution cryo-EM structures of Escherichia coli ClpAPS complexes, showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze the protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated, tucked away from the channel and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies show additional structures and functions for key AAA+ elements. Pore-loop tucking may be used broadly by AAA+ unfoldases, for example, during enzyme pausing/unloading.

ATP hydrolysis tunes specificity of a AAA+ protease.

In bacteria, AAA+ proteases such as Lon and ClpXP degrade substrates with exquisite specificity. These machines capture the energy of ATP hydrolysis to power unfolding and degradation of target substrates. Here, we show that a mutation in the ATP binding site of ClpX shifts protease specificity to promote degradation of normally Lon-restricted substrates. However, this ClpX mutant is worse at degrading ClpXP targets, suggesting an optimal balance in substrate preference for a given protease that is easy to alter. In vitro, wild-type ClpXP also degrades Lon-restricted substrates more readily when ATP levels are reduced, similar to the shifted specificity of mutant ClpXP, which has altered ATP hydrolysis kinetics. Based on these results, we suggest that the rates of ATP hydrolysis not only power substrate unfolding and degradation, but also tune protease specificity. We consider various models for this effect based on emerging structures of AAA+ machines showing conformationally distinct states.

The Bacterial ClpXP-ClpB Family Is Enriched with RNA-Binding Protein Complexes.

In the matrix of bacteria/mitochondria/chloroplasts, Lon acts as the degradation machine for soluble proteins. In stress periods, however, proteostasis and survival depend on the strongly conserved Clp/Hsp100 family. Currently, the targets of ATP-powered unfoldases/disaggregases ClpB and ClpX and of peptidase ClpP heptameric rings are still unclear. Trapping experiments and proteome profiling in multiple organisms triggered confusion, so we analyzed the consistency of ClpP-trap targets in bacteria. We also provide meta-analyses of protein interactions in humans, to elucidate where Clp family members are enriched. Furthermore, meta-analyses of mouse complexomics are provided. Genotype-phenotype correlations confirmed our concept. Trapping, proteome, and complexome data retrieved consistent coaccumulation of CLPXP with GFM1 and TUFM orthologs. CLPX shows broad interaction selectivity encompassing mitochondrial translation elongation, RNA granules, and nucleoids. CLPB preferentially attaches to mitochondrial RNA granules and translation initiation components; CLPP is enriched with them all and associates with release/recycling factors. Mutations in CLPP cause Perrault syndrome, with phenotypes similar to defects in mtDNA/mtRNA. Thus, we propose that CLPB and CLPXP are crucial to counteract misfolded insoluble protein assemblies that contain nucleotides. This insight is relevant to improve ClpP-modulating drugs that block bacterial growth and for the treatment of human infertility, deafness, and neurodegeneration.

Characterization of TR-107, a novel chemical activator of the human mitochondrial protease ClpP.

We recently described the identification of a new class of small-molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small-molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. We selected one compound (TR-107) with excellent potency, specificity, and drug-like properties for further evaluation. TR-107 showed ClpP-dependent growth inhibition in the low nanomolar range that was equipotent to paclitaxel in triple-negative breast cancer (TNBC) cell models. TR-107 also reduced specific mitochondrial proteins, including OXPHOS and TCA cycle components, in a time-, dose-, and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed the inactivation of OXPHOS and increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared with other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. Using human TNBC MDA-MB-231 xenografts, the antitumor response to TR-107 was investigated. Oral administration of TR-107 resulted in a reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

Antibacterial peptide CyclomarinA creates toxicity by deregulating the Mycobacterium tuberculosis ClpC1-ClpP1P2 protease.

The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1-ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.

ClpP inhibitors are produced by a widespread family of bacterial gene clusters.

The caseinolytic protease (ClpP) is part of a highly conserved proteolytic complex whose disruption can lead to antibacterial activity but for which few specific inhibitors have been discovered. Specialized metabolites produced by bacteria have been shaped by evolution for specific functions, making them a potential source of selective ClpP inhibitors. Here, we describe a target-directed genome mining strategy for discovering ClpP-interacting compounds by searching for biosynthetic gene clusters that contain duplicated copies of ClpP as putative antibiotic resistance genes. We identify a widespread family of ClpP-associated clusters that are known to produce pyrrolizidine alkaloids but whose connection to ClpP has never been made. We show that previously characterized molecules do not affect ClpP function but are shunt metabolites derived from the genuine product of these gene clusters, a reactive covalent ClpP inhibitor. Focusing on one such cryptic gene cluster from Streptomyces cattleya, we identify the relevant inhibitor, which we name clipibicyclene, and show that it potently and selectively inactivates ClpP. Finally, we solve the crystal structure of clipibicyclene-modified Escherichia coli ClpP. Clipibicyclene's discovery reveals the authentic function of a family of natural products whose specificity for ClpP and abundance in nature illuminate the role of eco-evolutionary forces during bacterial competition.

Recent structural insights into the mechanism of ClpP protease regulation by AAA+ chaperones and small molecules.

ClpP is a highly conserved serine protease that is a critical enzyme in maintaining protein homeostasis and is an important drug target in pathogenic bacteria and various cancers. In its functional form, ClpP is a self-compartmentalizing protease composed of two stacked heptameric rings that allow protein degradation to occur within the catalytic chamber. ATPase chaperones such as ClpX and ClpA are hexameric ATPases that form larger complexes with ClpP and are responsible for the selection and unfolding of protein substrates prior to their degradation by ClpP. Although individual structures of ClpP and ATPase chaperones have offered mechanistic insights into their function and regulation, their structures together as a complex have only been recently determined to high resolution. Here, we discuss the cryoelectron microscopy structures of ClpP-ATPase complexes and describe findings previously inaccessible from individual Clp structures, including how a hexameric ATPase and a tetradecameric ClpP protease work together in a functional complex. We then discuss the consensus mechanism for substrate unfolding and translocation derived from these structures, consider alternative mechanisms, and present their strengths and limitations. Finally, new insights into the allosteric control of ClpP gained from studies using small molecules and gain or loss-of-function mutations are explored. Overall, this review aims to underscore the multilayered regulation of ClpP that may present novel ideas for structure-based drug design.

Heterozygous variants of CLPB are a cause of severe congenital neutropenia.

Severe congenital neutropenia is an inborn disorder of granulopoiesis. Approximately one third of cases do not have a known genetic cause. Exome sequencing of 104 persons with congenital neutropenia identified heterozygous missense variants of CLPB (caseinolytic peptidase B) in 5 severe congenital neutropenia cases, with 5 more cases identified through additional sequencing efforts or clinical sequencing. CLPB encodes an adenosine triphosphatase that is implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, 3-methylglutaconic aciduria was not observed and, other than neutropenia, these clinical features were uncommon in our series. Moreover, the CLPB variants are distinct, consisting of heterozygous variants that cluster near the adenosine triphosphate-binding pocket. Both genetic loss of CLPB and expression of CLPB variants result in impaired granulocytic differentiation of human hematopoietic progenitor cells and increased apoptosis. These CLPB variants associate with wild-type CLPB and inhibit its adenosine triphosphatase and disaggregase activity in a dominant-negative fashion. Finally, expression of CLPB variants is associated with impaired mitochondrial function but does not render cells more sensitive to endoplasmic reticulum stress. Together, these data show that heterozygous CLPB variants are a new and relatively common cause of congenital neutropenia and should be considered in the evaluation of patients with congenital neutropenia.

Substrates and interactors of the ClpP protease in the mitochondria.

The ClpP protease is found across eukaryotic and prokaryotic organisms. It is well-characterized in bacteria where its function is important in maintaining protein homeostasis. Along with its ATPase partners, it has been shown to play critical roles in the regulation of enzymes involved in important cellular pathways. In eukaryotes, ClpP is found within cellular organelles. Proteomic studies have begun to characterize the role of this protease in the mitochondria through its interactions. Here, we discuss the proteomic techniques used to identify its interactors and present an atlas of mitochondrial ClpP substrates. The ClpP substrate pool is extensive and consists of proteins involved in essential mitochondrial processes such as the Krebs cycle, oxidative phosphorylation, translation, fatty acid metabolism, and amino acid metabolism. Discoveries of these associations have begun to illustrate the functional significance of ClpP in human health and disease.

Structure and function of ClpXP, a AAA+ proteolytic machine powered by probabilistic ATP hydrolysis.

ClpXP is an archetypical AAA+ protease, consisting of ClpX and ClpP. ClpX is an ATP-dependent protein unfoldase and polypeptide translocase, whereas ClpP is a self-compartmentalized peptidase. ClpXP is currently the only AAA+ protease for which high-resolution structures exist, the molecular basis of recognition for a protein substrate is understood, extensive biochemical and genetic analysis have been performed, and single-molecule optical trapping has allowed direct visualization of the kinetics of substrate unfolding and translocation. In this review, we discuss our current understanding of ClpXP structure and function, evaluate competing sequential and probabilistic mechanisms of ATP hydrolysis, and highlight open questions for future exploration.

Substrate Profiling of Mitochondrial Caseinolytic Protease P via a Site-Specific Photocrosslinking Approach.

Approaches for profiling protease substrates are critical for defining protease functions, but remain challenging tasks. We combine genetic code expansion, photocrosslinking and proteomics to identify substrates of the mitochondrial (mt) human caseinolytic protease P (hClpP). Site-specific incorporation of the diazirine-bearing amino acid DiazK into the inner proteolytic chamber of hClpP, followed by UV-irradiation of cells, allows to covalently trap substrate proteins of hClpP and to substantiate hClpP's major involvement in maintaining overall mt homeostasis. In addition to confirming many of the previously annotated hClpP substrates, our approach adds a diverse set of new proteins to the hClpP interactome. Importantly, our workflow allows identifying substrate dynamics upon application of external cues in an unbiased manner. Identification of unique hClpP-substrate proteins upon induction of mt oxidative stress, suggests that hClpP counteracts oxidative stress by processing of proteins that are involved in respiratory chain complex synthesis and maturation as well as in catabolic pathways.