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Key Points In ATP-dependent proteases, a ring-shaped AAA+ machine harnesses the chemical energy of ATP binding and hydrolysis to mechanically unfold target proteins by translocating them through an axial pore and into the degradation chamber of a self-compartmentalized peptidase Recognition of 'degron' sequences in specific target proteins involves the direct binding of amino-acid sequences to the axial pore of the AAA+ ring, binding of sequences to auxiliary domains and/or binding mediated by adaptor proteins. Degron sequences can be revealed or added to substrates by protein-modification reactions Novel antibiotics kill some bacteria by binding to the ClpP peptidase and transforming it into a rogue enzyme that indiscriminately degrades nascent polypeptides and unstructured cellular proteins Single-molecule optical trapping has directly visualized the unfolding and translocation activities of the ClpXP and ClpAP AAA+ proteases. These experiments and solution studies support a probabilistic model of AAA+ ring function and show that each power stroke has a constant — and typically low — probability of unfolding a stable protein domain Although protein degradation by AAA+ proteases is typically highly processive, multidomain substrates are sometimes only partially proteolysed, with the released products having new biological functions AAA+ enzymes can function independently of peptidases to solubilize aggregated proteins, to disassemble macromolecular complexes or to catalyse the incorporation of cofactors into enzymes AAA+ proteolytic machines unfold and degrade damaged and unneeded proteins in all domains of life. In this Review, Sauer and colleagues discuss the molecular mechanisms and structures of bacterial AAA+ machines, focusing on recent studies of ClpXP as a paradigm. To maintain protein homeostasis, AAA+ proteolytic machines degrade damaged and unneeded proteins in bacteria, archaea and eukaryotes. This process involves the ATP-dependent unfolding of a target protein and its subsequent translocation into a self-compartmentalized proteolytic chamber. Related AAA+ enzymes also disaggregate and remodel proteins. Recent structural and biochemical studies, in combination with direct visualization of unfolding and translocation in single-molecule experiments, have illuminated the molecular mechanisms behind these processes and suggest how remodelling of macromolecular complexes by AAA+ enzymes could occur without global denaturation. In this Review, we discuss the structural and mechanistic features of AAA+ proteases and remodelling machines, focusing on the bacterial ClpXP and ClpX as paradigms. We also consider the potential of these enzymes as antibacterial targets and outline future challenges for the field.

ClpP4 is a nuclear-encoded plastid protein that functions as a proteolytic subunit of the ATP-dependent Clp protease of higher plants. Given the lack of viable clpP4 knockout mutants, antisense clpP4 repression lines were prepared to study the functional importance of ClpP4 in Arabidopsis thaliana. Screening of transformants revealed viable lines with up to 90% loss of wild type levels of ClpP4 protein, while those with > 90% were severely bleached and strongly retarded in vegetative growth, failing to reach reproductive maturity. Of the viable antisense plants, repression of clpP4 expression produced a pleiotropic phenotype, of which slow growth and leaf variegation were most prominent. Chlorosis was most severe in younger leaves, with the affected regions localized around the mid-vein and exhibiting impaired chloroplast development and mesophyll cell differentiation. Chlorosis lessened during leaf expansion until all had regained the wild type appearance upon maturity. This change in phenotype correlated with the developmental expression of ClpP4 in the wild type, in which ClpP4 was less abundant in mature leaves due to post-transcriptional/translational regulation. Repression of ClpP4 caused a concomitant down-regulation of other nuclear-encoded ClpP paralogs in the antisense lines, but no change in other chloroplast-localized Clp proteins. Greening of the young chlorotic antisense plants upon maturation was accelerated by increased light, either by longer photoperiod or by higher growth irradiance; conditions that both raised levels of ClpP4 in wild type leaves. In contrast, shift to low growth irradiance decreased the relative amount of ClpP4 in wild type leaves, and caused newly developed leaves of fully greened antisense lines to regain the chlorotic phenotype.

Visualizing biomolecular and cellular processes inside intact living organisms is a major goal of chemical biology. However, existing molecular biosensors, based primarily on fluorescent emission, have limited utility in this context due to the scattering of light by tissue. In contrast, ultrasound can easily image deep tissue with high spatiotemporal resolution, but lacks the biosensors needed to connect its contrast to the activity of specific biomolecules such as enzymes. To overcome this limitation, we introduce the first genetically encodable acoustic biosensors—molecules that ‘light up’ in ultrasound imaging in response to protease activity. These biosensors are based on a unique class of air-filled protein nanostructures called gas vesicles, which we engineered to produce nonlinear ultrasound signals in response to the activity of three different protease enzymes. We demonstrate the ability of these biosensors to be imaged in vitro, inside engineered probiotic bacteria, and in vivo in the mouse gastrointestinal tract. Engineering cleavage sites into gas vesicle proteins enables protease-responsive regulation of gas vesicle mechanics and activates them as ultrasound contrast agents for imaging applications in cells and living mice.

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC–ClpP proteolytic complex from Bacillus subtilis . Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC–ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC–ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin–proteasome system. In Gram-positive bacteria, arginine phosphorylation by the McsB kinase functions as a general post-translational marker for Clp-mediated proteolysis. Degradation signal for the ClpC–ClpP proteolytic complex Protein ubiquitination can mark proteins for degradation by the proteasome in eukaryotic cells, but it is unknown whether such a tagging system for the equivalent bacterial Clp proteases exists. Here, Tim Clausen and colleagues report that arginine phosphorylation by the McsB kinase functions as a general post-translational marker for proteasomal degradation in Gram-positive bacteria, tagging at least 25% of all proteins degraded by Clp. The phosphoarginine degradation pathway is essential to cope with proteotoxic stress in vivo .

The caseinolytic protease (ClpP) is an emerging antibacterial target. Pseudomonas plecoglossicida ( Pp ), a pathogen causing visceral white spot disease in Larimichthys crocea , encodes two ClpP paralogs, Pp ClpP1 and Pp ClpP2. This study characterizes their distinct structural and functional properties. Phylogenetic and biochemical analysis revealed that Pp ClpP2 functions as a canonical serine protease with high peptidase activity, while Pp ClpP1 is evolutionarily divergent, exhibiting low inherent activity due to an unconventional Ser-His-Pro catalytic triad and a truncated N-terminal domain. Cryo-EM structure determination of Pp ClpP1 confirmed a homotetradecameric assembly with a dilated axial pore and a non-canonical catalytic geometry. In contrast, AlphaFold-predicted Pp ClpP2 displayed a compact structure with a canonical Ser-His-Asp triad. The subunits formed a stable heterotetradecamer ( Pp ClpP1P2) with enhanced proteolytic activity compared to individual homotetradecameric. Pull-down assays demonstrated that Pp ClpP2, but not Pp ClpP1, specifically interacts with the unfoldase Pp ClpX, and the Pp ClpP1P2 heterotetradecamer further augmented Pp ClpX-mediated degradation of model substrates. Notably, the proteasome inhibitor bortezomib (BTZ) selectively inhibited Pp ClpP1 by binding to a unique pocket near the active site without engaging the catalytic serine, thereby suppressing bacterial growth in a Pp ClpP1-dependent manner. This study elucidates the structural basis of functional divergence between Pp ClpP paralogs, highlights their synergistic interplay in proteolysis, and identifies Pp ClpP1 as a druggable target for antibacterial development.

The caseinolytic protease (Clp) system has recently emerged as a promising anti-tuberculosis target. The anti-cancer drug bortezomib exhibits potent anti-mycobacterial activity and binds to Mycobacterium tuberculosis ( Mtb ) Clp protease complexes. We determine cryo-EM structures of Mtb ClpP1P2, ClpC1P1P2 and ClpXP1P2 complexes bound to bortezomib in different conformations. Structural and biochemical data indicate that sub-stoichiometric binding by bortezomib to the protease active sites orthosterically activates the Mtb ClpP1P2 complex. Bortezomib activation of Mtb ClpP1P2 induces structural changes promoting the recruitment of the chaperone-unfoldases, Mtb ClpC1 or Mtb ClpX, facilitating holoenzyme formation. The structures of the Mtb ClpC1P1P2 holoenzyme indicate that Mtb ClpC1 motion, induced by ATP rebinding at the Mtb ClpC1 spiral seam, translocates the substrate. In the Mtb ClpXP1P2 holoenzyme structure, we identify a specialized substrate channel gating mechanism involving the Mtb ClpX pore-2 loop and Mtb ClpP2 N-terminal domains. Our results provide insights into the intricate regulation of the Mtb Clp system and suggest that bortezomib can disrupt this regulation by sub-stoichiometric binding at the Mtb Clp protease sites. The study reveals how bortezomib activates Mycobacterium tuberculosis Clp protease complexes. Cryo-EM structures reveal how sub-stoichiometric bortezomib binding triggers structural changes, mediates holoenzyme formation and disrupts Clp system regulation.

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability. Maintenance and quality control of the mitochondrial respiratory chain complexes responsible for bulk energy production are unclear. Here, the authors show that the mitochondrial protease ClpXP is required for the rapid turnover of the core N-module of respiratory complex I, which happens independently of other modules in the complex.

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 human ClpXP complex (hClpXP) orchestrates mitochondrial protein quality control through targeted degradation of misfolded and unnecessary proteins. While bacterial ClpXP systems are well characterized, the assembly and regulation of human ClpXP remain poorly understood. In this study, we elucidate the complete assembly pathway of hClpXP through high-resolution cryo-electron microscopy (cryo-EM) structures. Our findings confirm that hClpP exists as a single-ring heptamer in isolation and reveal a previously undocumented initial assembly complex in which hexameric hClpX first engages with heptameric hClpP. We further demonstrate how this interaction drives substantial conformational rearrangements that facilitate the formation of tetradecameric hClpP within the fully assembled complex. Notably, we characterize a unique eukaryotic sequence in hClpX, termed the E-loop, which plays a critical role in stabilizing hexamer assembly and maintaining ATPase activity. Additionally, we show that peptide binding at the hClpP active site triggers further structural changes essential for achieving full proteolytic competence. Together, these structures provide unprecedented mechanistic insights into the stepwise assembly and activation of hClpXP, significantly advancing our understanding of this essential mitochondrial protein degradation machinery. Mitochondrial ClpXP maintains protein quality through targeted degradation. Here, the authors use cryo-EM to define the stepwise assembly of human ClpXP, identifying key intermediates and a unique E-loop element that regulates complex formation and proteolytic activation.

The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop–substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis.Cryo-EM structures of Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis reveal contacts that drive substrate translocation and a dynamic switch mechanism at the ClpA–ClpP interface.