Affinage

CLPX

ATP-dependent clpX-like chaperone, mitochondrial · UniProt O76031

Length
633 aa
Mass
69.2 kDa
Annotated
2026-06-09
100 papers in source corpus 46 papers cited in narrative 43 extracted findings
Cross-family judge vs UniProt: tie faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ClpX is an ATP-driven AAA+ unfoldase that powers both regulated protein degradation and ATP-dependent protein remodeling (PMID:8226769, PMID:8226770, PMID:7557391, PMID:21529717). As the substrate-selection subunit of the ClpXP protease, ClpX recognizes degradation-tag classes—two C-terminal and three N-terminal motifs catalogued across more than fifty E. coli substrates (PMID:12667450)—and directs ClpP toward substrates distinct from those of ClpA, with selectivity set by which ATPase associates with ClpP (PMID:8226769, PMID:8226770). Recognition is bipartite: the N-terminal C4-type zinc-binding domain (ZBD) forms a Zn-dependent dimer that binds substrates such as lambda O and the adaptors SspB and RssB, while an aromatic pore loop bearing a critical tyrosine grips the polypeptide in a nucleotide-dependent manner for translocation (PMID:12937164, PMID:18931677, PMID:14536077, PMID:12912910). The hexameric ring is intrinsically asymmetric, with subunits switching between nucleotide-loadable and unloadable states; this asymmetry creates staggered pore loops and couples ATP hydrolysis in one subunit to flexing of the whole ring, generating mechanical force (~20 pN stall) that unfolds substrates in ~1 nm steps (PMID:19914167, PMID:25866879, PMID:18931677, PMID:21529717). IGF/LGF loops on the ring dock multivalently into ClpP clefts, and ATP binding—not hydrolysis—reconfigures these loops to tune ClpX–ClpP affinity, open the ClpP substrate gate, and sustain processive degradation (PMID:11346657, PMID:15064753, PMID:30767302, PMID:20416323). Independently of ClpP, ClpX is an autonomous chaperone that disassembles the MuA transpososome by selectively unfolding keystone subunits, monomerizes the TrfA replication initiator, protects and disaggregates lambda O, and inhibits FtsZ assembly (PMID:7557391, PMID:11545746, PMID:20133746, PMID:9405620, PMID:7743994, PMID:20022957, PMID:15948963, PMID:19136590). The mammalian mitochondrial ortholog is imported via an N-terminal transit peptide into the matrix (PMID:11003706, PMID:10347188) and acts largely non-proteolytically in heme biosynthesis: it activates δ-aminolevulinate synthase (ALAS) by limited, targeted partial unfolding that permits pyridoxal-phosphate cofactor loading, while also controlling ALAS turnover, PPOX activity, FECH levels, and iron utilization during erythropoiesis (PMID:25957689, PMID:32091391, PMID:34280433), and it maintains mtDNA nucleoid distribution by enhancing TFAM DNA binding (PMID:22841477). A dominant ATPase-inactivating CLPX mutation (p.Gly298Asp) stabilizes ALAS, causing protoporphyrin accumulation and erythropoietic protoporphyria (PMID:28874591).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 1993 High

    Established ClpX as a distinct regulatory ATPase that confers substrate selectivity on the ClpP peptidase, defining the two-component architecture of energy-dependent proteolysis.

    Evidence Protein purification, in vitro proteolysis, and clpX/clpP deletion genetics in E. coli

    PMID:8226769 PMID:8226770

    Open questions at the time
    • Did not define the structural basis of substrate recognition
    • Mechanism of ClpX–ClpP coupling not resolved
  2. 1995 High

    Showed that ClpX is not only a protease component but an autonomous, ATP-dependent chaperone able to remodel stable protein–DNA complexes and counter aggregation, separating its unfoldase activity from degradation.

    Evidence In vitro MuA transpososome disassembly and lambda O aggregation/disaggregation assays with purified ClpX

    PMID:7557391 PMID:7743994

    Open questions at the time
    • Did not identify the substrate recognition determinants
    • Distinction between ATP binding vs hydrolysis only partially dissected
  3. 1997 High

    Defined transferable peptide recognition signals, showing a short C-terminal motif is sufficient to make a protein a ClpX substrate and that recognition sites overlap with functional partner-binding regions.

    Evidence Peptide deletion/competition and in vitro disassembly with MuA and TrfA

    PMID:9203582 PMID:9405620

    Open questions at the time
    • Full repertoire of recognition signals not yet catalogued
    • Structural element of ClpX reading these signals not identified
  4. 2000 Medium

    Identified mammalian CLPX as a mitochondrially targeted ortholog, extending ClpX biology from bacteria to a defined organellar compartment.

    Evidence cDNA cloning, GFP-tagged localization with N-terminal deletion controls, FISH mapping; murine ATPase characterization

    PMID:10347188 PMID:11003706

    Open questions at the time
    • Mitochondrial substrates and physiological role undefined
    • Functional interaction with mitochondrial ClpP not established at this stage
  5. 2001 High

    Mapped the functional architecture of ClpX: the IGF/LGF loop mediates ClpP binding, the N-domain contributes to substrate binding, and the Zn-coordinating C4 finger is required for oligomerization and all downstream activities.

    Evidence Limited proteolysis, deletion analysis, zinc-release experiments, and crystallography of H. pylori ClpX

    PMID:11278349 PMID:11346657 PMID:14514695

    Open questions at the time
    • Nucleotide-dependent dynamics of these elements not yet resolved
    • How asymmetry across subunits is organized unknown
  6. 2003 High

    Systematized substrate recognition by cataloguing five degradation-tag classes and defining the ZBD as the binding hub for substrates and the SspB/RssB adaptors, while NMR/structural work fixed the ZBD as a treble-clef zinc-finger dimer.

    Evidence In vivo substrate trapping with mass spectrometry, ZBD deletion/binding assays, adaptor mutagenesis, NMR structure

    PMID:12667450 PMID:12730132 PMID:12912910 PMID:12937164 PMID:14525985 PMID:14536077

    Open questions at the time
    • RssB's second non-targeting role mechanistically unresolved (Medium-confidence)
    • How distinct tag classes route to pore vs ZBD not fully separated
  7. 2004 High

    Demonstrated that the central pore performs both recognition and catalytic engagement of specific substrate classes, and that ClpX–ClpP affinity is dynamically tuned by the ATPase cycle through IGF loops and a sensor-II arginine.

    Evidence Pore (V154F) and sensor-II/IGF mutagenesis with in vitro/in vivo degradation; EM of hybrid ClpXAP complexes

    PMID:15004005 PMID:15037252 PMID:15064753

    Open questions at the time
    • Atomic basis of pore-loop staggering not yet visualized
    • Hybrid complex prevalence in vivo only inferred from stoichiometry
  8. 2008 High

    Resolved the mechanics of substrate gripping, showing an aromatic pore-loop tyrosine grips polypeptide in a nucleotide-dependent way and that ssrA engagement is a multi-loop, two-step process.

    Evidence Single-subunit pore-loop mutagenesis, specificity-transplant and disulfide-crosslinking unfolding assays; enhanced tetramer recognition via N domain

    PMID:18313382 PMID:18406325 PMID:18931677

    Open questions at the time
    • Conformational trajectory of loops during a power stroke inferred indirectly
    • Coordination between adjacent subunit loops not directly observed
  9. 2009 High

    Provided the structural framework for asymmetric ATPase action, showing inter-domain rotations produce ring asymmetry that excludes nucleotide from some subunits and staggers pore loops.

    Evidence Crystal structures of nucleotide-free and nucleotide-bound ClpX hexamers

    PMID:19914167

    Open questions at the time
    • Static structures do not capture the dynamic hydrolysis cycle
    • Force-generation steps not directly visualized
  10. 2011 High

    Directly measured ClpX as a mechanochemical motor, quantifying force, step size, velocity, and the role of ClpP in reducing slippage.

    Evidence Single-molecule optical tweezers force-extension measurements

    PMID:21529717

    Open questions at the time
    • Coupling of individual ATP hydrolysis events to discrete steps not fully assigned
    • Behavior on structurally diverse substrates limited
  11. 2015 High

    Established that mitochondrial ClpX activates heme biosynthesis by loading the PLP cofactor onto ALAS, a non-canonical function distinct from degradation, and that subunit conformational switching (L/U states) governs cooperative ring activity.

    Evidence Yeast genetics/metabolomics, in vitro PLP-incorporation reconstitution, vertebrate depletion; covalent single-chain pseudohexamers with locked subunit

    PMID:25866879 PMID:25957689

    Open questions at the time
    • Mechanism of cofactor delivery not yet defined at this stage
    • Generality across ALAS isoforms unaddressed
  12. 2017 High

    Linked CLPX to human disease, showing a dominant ATPase-inactivating mutation stabilizes ALAS and causes erythropoietic protoporphyria, revealing CLPX dually controls ALAS activation and turnover.

    Evidence Patient genetics, mutant/WT coassembly biochemistry, ALAS stability and PPIX quantification

    PMID:28874591

    Open questions at the time
    • Physiological balance between activation and turnover in normal cells not quantified
    • Whether ClpP participates in ALAS turnover unresolved
  13. 2020 High

    Defined the structural mechanism of ALAS activation as targeted partial unfolding from the binding site to the active site, contrasting with canonical global unfolding.

    Evidence HDX-MS unfolding mapping plus ALAS mutagenesis and cofactor-incorporation assays

    PMID:32091391

    Open questions at the time
    • How ClpX limits unfolding to a defined region mechanistically unclear
    • Energetic cost of partial vs global unfolding not compared
  14. 2021 High

    Expanded mitochondrial CLPX function in erythropoiesis, showing it controls ALAS2 stability, PPOX and FECH activity, and iron utilization, and that its principal physiological substrates are nucleic-acid-associated proteins.

    Evidence Conditional Clpx knockout mice with pathway enzyme assays; comparative proteomics of ClpP-null mouse and human fibroblasts

    PMID:34280433 PMID:34943861

    Open questions at the time
    • Direct substrate engagement for PPOX/FECH regulation not reconstituted
    • Mechanism distinguishing chaperone vs proteolytic roles in vivo not fully separated

Open questions

Synthesis pass · forward-looking unresolved questions
  • How ClpX selects between proteolytic destruction, productive remodeling, and cofactor-loading partial unfolding on a given substrate—and what dictates this branching in the mitochondrial matrix—remains unresolved.
  • No unified model linking substrate features to outcome (degrade vs remodel vs activate)
  • Full mammalian mitochondrial substrate set incompletely defined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0044183 protein folding chaperone 5 GO:0140096 catalytic activity, acting on a protein 5 GO:0140657 ATP-dependent activity 5 GO:0003677 DNA binding 1
Localization
GO:0005739 mitochondrion 4
Pathway
R-HSA-1430728 Metabolism 2 R-HSA-392499 Metabolism of proteins 2
Partners
ALASCLPPFTSZMUARSSBSSPBTFAMTRFA
Complex memberships
ClpXP protease

Evidence

Reading pass · 43 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1993 ClpX was identified as an alternative ATPase subunit that associates with ClpP to form the ClpXP protease, directing ClpP toward specific substrates (e.g., lambda O protein) distinct from those of ClpAP; selectivity of degradation by ClpP is determined by which regulatory ATPase (ClpA vs ClpX) associates with it. Protein purification, in vitro proteolysis assay, in vivo genetic analysis (clpX/clpP deletion mutants) The Journal of biological chemistry High 8226769 8226770
1995 ClpX, in an ATP-dependent manner, disassembles the stable MuA transposase tetramer from post-recombination DNA without degrading MuA; this activity is independent of ClpP and identifies ClpX as a molecular chaperone capable of remodeling stable protein-DNA complexes. In vitro disassembly assay with purified ClpX, MuA, and DNA; demonstration that released MuA is undegraded and functional; deletion analysis mapping C-terminal MuA recognition sequence Genes & development High 7557391
1995 ClpX alone (without ClpP) acts as a molecular chaperone: it protects lambda O protein from heat-induced aggregation, disaggregates preformed lambda O aggregates, and promotes lambda O binding to its DNA recognition sequence. ATP (but not its hydrolysis) is required for protection from aggregation, whereas disaggregation requires ATP hydrolysis. In vitro aggregation protection assay, disaggregation assay, DNA-binding assay, modified ELISA, ATPase stimulation measurement The EMBO journal High 7743994
1997 A 10-amino-acid peptide at the C-terminal domain of MuA transposase is required for recognition by ClpX and is sufficient to convert a heterologous protein into a ClpX substrate. The MuB protein-interaction region of MuA overlaps with the ClpX-recognition site, such that MuB inhibits ClpX-mediated disassembly. Deletion analysis, peptide competition assay, in vitro disassembly assay with purified components Genes & development High 9203582
1997 ClpX (molecular chaperone activity, independent of ClpP) activates the TrfA replication initiator protein of plasmid RK2 by converting TrfA dimers into monomers in an ATP-dependent reaction, thereby enabling TrfA to bind origin iterons and initiate replication. In vitro replication reconstitution from purified components; gel-filtration to assess TrfA oligomeric state; iteron-binding assay Proceedings of the National Academy of Sciences of the United States of America High 9405620
2001 The IGF/LGF loop of ClpX (identified by limited proteolysis) is the structural element that mediates ClpX-ClpP interaction; cleavage within this loop abolishes ClpP binding and activation. The N-terminal domain of ClpX (which dissociates upon cleavage) is dispensable for ATPase, chaperone, and proteolytic activity but lies on the outside ring surface where it contributes to substrate binding. Limited proteolysis with lysylendopeptidase C, deletion analysis, ATPase assay, ClpP-binding assay, proteolysis assay The Journal of biological chemistry High 11346657
2001 ClpX selectively unfolds individual MuA subunits within the transpososome; direct recognition and unfolding of a single transposase subunit is sufficient to destabilize the entire tetrameric complex, explaining how ClpX remodels without fully disassembling the complex. Biochemical unfolding probe assay, in vitro remodeling assay with purified proteins, differential ClpX engagement analysis Molecular cell High 11545746
2003 The N-terminal zinc-binding domain (ZBD) of ClpX forms a stable C4-type zinc-dependent dimer that is required for degradation of substrates such as lambda O and MuA but not GFP-ssrA; ZBD contains the primary binding site for lambda O and the SspB cofactor; removal of ZBD renders ClpX ATPase activity insensitive to ClpP, substrates, or SspB. Deletion analysis, binding assays (ELISA, pull-down), ATPase assay, proteolysis assay with ClpXP The Journal of biological chemistry High 12937164
2003 More than 50 ClpXP substrates were identified in E. coli, and analysis of their sequences established five recurring degradation-tag motif classes (two C-terminal, three N-terminal). Deletion, fusion, and point-mutation experiments confirmed that each motif class is sufficient to target proteins for ClpXP degradation. In vivo substrate trapping with inactive ClpP variant, mass spectrometry identification, deletion/fusion/point-mutation validation Molecular cell High 12667450
2003 The SspB adaptor protein delivers ssrA-tagged substrates to ClpX via a bipartite mechanism: the N-terminal region of SspB binds ssrA-tagged substrates, while a conserved C-terminal motif (XB) of SspB docks specifically to the N-terminal domain (ZBD) of ClpX, positioning substrates for degradation. A single point mutation in the XB region abolishes SspB-stimulated degradation. Binding assays, point mutation analysis, in vitro degradation assay Molecular cell High 14536077
2003 The NMR solution structure of the ClpX N-terminal zinc-binding domain (ZBD) dimer was determined; the monomer fold belongs to the treble clef zinc finger family. The dimeric ZBD structure is unique and a trimer-of-dimers model was proposed to reflect the closed-state ClpX hexamer. NMR spectroscopy, structure determination of ZBD dimer The Journal of biological chemistry High 14525985
2003 Crystal structure of H. pylori ClpX (lacking the N-terminal Cys-cluster region) in complex with ADP revealed two subdomains similar to HslU; the conserved LGF tripeptide is located on the tip of the ClpP-binding loop; a hexameric model suggests six LGF tripeptides contact the hydrophobic clefts of ClpP asymmetrically. X-ray crystallography, structural modeling The Journal of biological chemistry High 14514695
2003 ClpX recognizes latent degradation signals in both cleavage fragments of LexA that flank the auto-cleavage site but are cryptic in intact LexA; ClpXP degrades both LexA fragments after DNA-damage-triggered auto-cleavage, and this degradation is important for cell survival after DNA damage. In vitro degradation assay, in vivo stability assay, deletion/mutation analysis of LexA sequences Genes & development High 12730132
2003 sigma(S) proteolysis by ClpXP requires two distinct regions in sigma(S): region 2.5 (a long alpha-helix) binds phosphorylated RssB, and an N-terminal region exposed only upon RssB binding serves as the ClpX-binding site. Binding of ClpX alone is not sufficient for degradation; RssB plays a second role beyond substrate targeting. In vitro binding assay, fusion protein analysis, in vivo degradation assay The EMBO journal Medium 12912910
2004 The V154F pore mutation in ClpX severely impairs binding and engagement of substrates carrying C-motif 1 degradation signals, while efficiently processing substrates with other recognition signal classes. This establishes that the ClpX pore functions in both recognition and catalytic engagement of specific substrate classes. Site-directed mutagenesis, in vitro and in vivo substrate degradation assays Genes & development High 15004005
2004 ClpX-ClpP affinity varies with the protein-processing task of ClpX and with catalytic engagement of ClpP active sites; functional communication between the symmetry-mismatched rings depends on ClpX ATPase activity and is transmitted through conformational changes in the IGF loops; a conserved arginine in the sensor II helix of ClpX links its nucleotide state to ClpP binding and substrate processing. Biochemical binding assays, ATPase assays, mutagenesis of IGF loops and sensor II helix, in vitro degradation assays Nature structural & molecular biology High 15064753
2004 ClpA and ClpX hexamers can simultaneously bind opposite ends of the same ClpP double ring, forming hybrid ClpXAP complexes. These hybrid complexes independently translocate their respective substrates without redistribution of the ATPases; stoichiometry data suggest heteromeric complexes predominate in growing cells. Electron microscopy, biochemical reconstitution, substrate translocation assay, in vivo stoichiometry analysis Journal of structural biology High 15037252
2006 The N-terminal ZBD of ClpX undergoes large nucleotide-dependent movement: in functional ClpXP complexes, the ZBD moves toward ClpP and into the AAA+ ring ('capture' to 'feeding' conformation). This motion is modulated by the cofactor SspB. Evidence includes ClpP-mediated clipping of an N-terminal extension and protease-protection, crosslinking, and light scattering experiments. Protease protection assay, chemical crosslinking, dynamic light scattering, proteolysis by ClpP of N-terminal extension The EMBO journal Medium 16810315
2007 Crystal structure of the ClpX ZBD in complex with the SspB C-terminal XB peptide at 1.6 Å resolution revealed that XB forms an antiparallel beta-sheet with two beta-strands of ZBD in a 1:1 stoichiometry, indicating two independent SspB-tail binding sites per ZBD dimer; biochemical analysis confirmed key determinants of SspB recognition. X-ray crystallography, biochemical binding assays, mutagenesis Journal of molecular biology High 17258768
2007 The zinc-binding domain (ZBD) of ClpX is required for Spx proteolysis by ClpXP in B. subtilis; disulfide stress (diamide) releases Zn from ClpX ZBD and inhibits ClpXP activity; Cys-to-Ser mutations at Zn-coordinating residues of ZBD reduce ClpXP-mediated Spx degradation both in vitro and in vivo. In vitro proteolysis assay, in vivo stability assay, site-directed mutagenesis of ZBD cysteines, Zn-release measurement by electrophoresis Journal of bacteriology Medium 17827297
2008 An aromatic-hydrophobic pore loop (containing a critical tyrosine residue) in each ClpX subunit grips substrate polypeptides during translocation and unfolding; removal of the aromatic ring in even a few subunits causes substrate slippage, frequent unfolding failure, and enormously increased energetic cost. The tyrosine's effect depends on the nucleotide state of the resident subunit, supporting nucleotide-dependent pore-loop conformational changes as the driver of translocation. Site-directed mutagenesis of pore-loop residues in individual subunits, in vitro unfolding/translocation assays, ATPase measurements Nature structural & molecular biology High 18931677
2008 The ssrA tag binds to distinct loops at the top, middle, and lower portions of the ClpX hexamer central channel via a two-step mechanism: a top loop acts as a specificity filter and remaining loops form a binding site deep in the pore. Crosslinking reveals a staggered arrangement of pore loops and nucleotide-dependent changes in their conformations. Specificity-transplant experiments, disulfide-crosslinking, in vitro degradation assays Molecular cell High 18313382
2008 ClpX recognizes and preferentially engages the tetrameric MuA-DNA complex over monomeric MuA; residues exposed only in the tetramer enhance recognition via the ClpX N domain, and this enhanced recognition is required for high-priority disassembly. Altered-specificity MuA/DNA binding experiments, in vitro disassembly assays, N-domain deletion analysis Molecular cell High 18406325
2009 Crystal structures of nucleotide-free and nucleotide-bound ClpX hexamers revealed striking ring asymmetry arising from large rotations between large and small AAA+ domains of individual subunits; this asymmetry prevents nucleotide binding to two subunits, creates a staggered pore-loop arrangement, and provides a mechanism for coupling ATP binding/hydrolysis in one subunit to flexing motions of the entire ring. X-ray crystallography of ClpX hexamers in two nucleotide states Cell High 19914167
2009 ClpX inhibits FtsZ assembly in B. subtilis and E. coli through a mechanism that does not require ATP hydrolysis and is independent of ClpP; the N-terminal domain of ClpX is required for inhibition of FtsZ polymerization; ClpX disassembles FtsZ polymers by blocking reassembly rather than severing filaments. In vitro FtsZ polymerization assay, ATPase-dead ClpX mutant analysis, N-terminal domain deletion, high-speed AFM single-molecule analysis, in vivo genetic analysis The Journal of biological chemistry / Molecular microbiology / Journal of bacteriology High 15948963 19136590 20022957
2010 ClpX binding to ClpP stimulates ClpP cleavage of peptides larger than a few amino acids and enhances ClpP active-site reactivity; stimulation requires ATP binding but not hydrolysis by ClpX. ClpP channel-loop residues normally exclude substrates larger than small peptides; ClpX binding relieves these inhibitory interactions to open a gate for substrate entry. Peptide cleavage assays, active-site modification assays, alanine substitution of ClpP channel residues, ClpXP translocation assay Journal of molecular biology High 20416323
2010 ClpX unfolds the 'keystone' catalytic-left or catalytic-right subunit of the MuA tetramer (those making extensive intersubunit contacts) to destabilize the entire transpososome; the other two subunits are recognized much less efficiently, and their extraction does not destabilize the complex. Altered-specificity MuA proteins/DNA sites, in vitro disassembly assay, transposase subunit-specific analysis Proceedings of the National Academy of Sciences of the United States of America High 20133746
2011 Single-molecule optical tweezers experiments directly demonstrated that ClpX generates mechanical force (up to ~20 pN stall force) to unfold and translocate polypeptides through its central pore; translocation velocity is force-dependent (maximum ~80 aa/s near-zero force); ClpX takes 1, 2, or 3 nm steps (fundamental step ~1 nm); ClpP binding decreases substrate slip probability and enhances unfolding efficiency; GFP unravels cooperatively via a transient intermediate under ClpXP. Single-molecule optical tweezers (force-extension measurements) Cell High 21529717
2012 Human mitochondrial CLPX localizes to the mitochondrial matrix, and its Walker B (ATPase-inactive) mutant retains the ability to mediate casein degradation by hCLPP in a manner similar to the small-molecule ClpP activator ADEP; most model substrates are recognized by the N-terminal domain of hCLPX, while some bypass it and dock directly to the pore-1 motif. Walker B mutagenesis, in vitro binding assays with casein and physiological substrates, proteolysis assay with hCLPP Journal of structural biology Medium 22710082
2012 Human ClpX (mtClpX) maintains mtDNA nucleoid distribution by enhancing TFAM DNA-binding activity, functioning as a chaperone rather than as a protease; ClpX knockdown causes enlarged mtDNA nucleoids; ClpX and TFAM colocalize in mitochondria; this phenotype is not reproduced by ClpP knockdown. RNAi knockdown in HeLa cells, fluorescence imaging, in vitro TFAM DNA-binding assay with ClpX, ClpP knockdown as negative control Experimental cell research Medium 22841477
2015 Mitochondrial ClpX (mtClpX) directly activates 5-aminolevulinate synthase (ALAS), the first enzyme of heme biosynthesis, by catalyzing incorporation of its cofactor pyridoxal phosphate (PLP) into ALAS. This activity is conserved in mammalian homologs; mtClpX depletion reduces ALA and heme levels, and impairs vertebrate erythropoiesis, establishing ClpX as a stimulator of heme biosynthesis via a mechanism distinct from canonical protein unfolding. Yeast genetic interaction and metabolomic analysis, in vitro reconstitution of PLP incorporation into ALAS by ClpX, mtClpX depletion in vertebrates (erythropoiesis phenotype) Cell High 25957689
2015 Conformational switching of ClpX subunits between nucleotide-loadable (L) and nucleotide-unloadable (U) states is required for efficient substrate binding, unfolding, and degradation; locking one subunit in the U conformation reduces cooperativity of ATP hydrolysis and substrate processing efficiency, supporting an asymmetric and probabilistic model of AAA+ ring activity. Covalent single-chain ClpX pseudohexamers with one subunit locked in U conformation, ATPase assays, substrate unfolding/degradation assays Nature structural & molecular biology High 25866879
2017 A dominant gain-of-function mutation in the ATPase active site of human CLPX (p.Gly298Asp) inactivates ATPase activity; coassembly of mutant and WT CLPX protomers produces a low-activity enzyme that increases posttranslational stability of ALAS (by reducing CLPX-mediated turnover), causing PPIX accumulation and erythropoietic protoporphyria. This establishes that CLPX controls heme biosynthesis by both activating ALAS (cofactor loading) and mediating its turnover. Patient genetic analysis, ATPase assay of mutant CLPX, coassembly experiments, ALAS stability assay, metabolite quantification (PPIX) Proceedings of the National Academy of Sciences of the United States of America High 28874591
2019 IGF-motif loops of ClpX require ATP binding (not hydrolysis) to rearrange the ClpX ring for efficient multivalent docking to ClpP; deletion of one or two IGF loops modestly slows ClpXP association but strongly accelerates dissociation, and reduces the processivity of ATP-dependent proteolysis. Single-chain ClpX pseudohexamers with IGF loop deletions, kinetic binding assays, degradation processivity assays Protein science High 30767302
2020 Mitochondrial ClpX activates ALAS through partial, targeted unfolding of a region extending from the ClpX-binding site to the active site, rather than global unfolding. Specific sequence and structural features within ALAS position mtClpX and provide a grip for this limited remodeling, which is required for cofactor (PLP) binding to ALAS. This contrasts with the global unfolding canonically performed by ClpX homologs. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map unfolding, mutagenesis of ALAS binding/structural elements, in vitro cofactor-incorporation assay eLife High 32091391
2021 In erythroid cells, CLPX regulates ALAS2 primarily by controlling its turnover (stability) rather than activating it; CLPX is also required for PPOX activity and maintenance of FECH levels (terminal heme synthesis enzymes), and for iron utilization during erythroid differentiation, indicating multiple distinct mechanisms by which CLPX controls the heme synthesis pathway. Clpx conditional knockout mice, enzyme activity assays for ALAS2, PPOX, FECH, protein stability (degradation) assays, iron utilization assay during erythroid differentiation The Journal of biological chemistry High 34280433
2000 Human and mouse CLPX encode mitochondrially targeted orthologs of bacterial ClpX; the protein contains an N-terminal mitochondrial transit peptide and a C4 zinc finger motif. Expression of a C-terminally tagged full-length CLPX cDNA confirmed mitochondrial import; deletion of the N-terminal targeting sequence abolished mitochondrial localization. cDNA cloning, confocal microscopy of GFP-tagged CLPX, N-terminal deletion analysis, FISH chromosomal mapping Mammalian genome Medium 11003706
1999 Murine ClpX has intrinsic ATPase activity (Km ~25 µM, Vmax ~660 pmol/min/µg); substitution of lysine 300 (P-loop) with alanine abolishes both ATP hydrolysis and binding; ClpX-GFP fusions localize to mitochondria via an N-terminal targeting sequence; deletion of the targeting sequence abolishes mitochondrial localization; ClpX co-immunoprecipitates with ClpP in overexpression experiments. Recombinant protein expression, ATPase assay, P-loop mutagenesis, confocal microscopy, co-IP The Journal of biological chemistry Medium 10347188
2001 The Zn(II) ion bound to the C4-type zinc finger motif of E. coli ClpX is required for its oligomerization, ATP binding, ClpP binding, and ClpXP-dependent proteolysis; release of Zn(II) prevents all of these activities. The ClpXDeltaCys mutant (all four Cys replaced by Ser) phenocopies Zn-free ClpX. Zinc release experiments, ATPase assay, ClpP-binding assay, proteolysis assay, native PAGE for oligomerization The Journal of biological chemistry High 11278349
2006 The ClpX N-terminal ZBD binds hydrophobic residues with sequence preferences different from the AAA+ domain; the SspB2 C-terminus interacts with a hydrophobic patch on the surface of ZBD as determined by NMR and mutagenesis; SspB2 binds ZBD2 with defined affinity and geometry measured by dual polarization interferometry. NMR spectroscopy, site-directed mutagenesis, dual polarization interferometry Proceedings of the National Academy of Sciences of the United States of America Medium 17090685
1998 The yeast Saccharomyces cerevisiae mitochondrial ClpX homolog Mcx1p localizes to the mitochondrial matrix space and is peripherally associated with the inner membrane; no ClpP homolog was identified in the yeast genome, suggesting Mcx1p performs non-proteolytic chaperone function in mitochondria. Yeast genome analysis, subcellular fractionation, membrane association assay FEBS letters Medium 9827555
2015 In S. aureus, ClpX controls Protein A expression through two mechanisms: (1) by stimulating translation of Rot (an activator of spa transcription), thereby maintaining Rot above a threshold level required for spa transcription; and (2) by independently stimulating translation of the spa mRNA itself. Rot antibody Western blot quantification, inducible Rot expression experiments, clpX deletion strain analysis, in vivo translation/stability assays PloS one Medium 20856878
2021 In ClpP-null mouse and human fibroblast cells, the primary consequence of ClpP absence is accumulation of ClpX together with nucleoid-associated proteins (POLDIP2, LRPPRC, GFM1/GRSF1 in mouse), revealing that ClpXP primarily acts on proteins associated with nucleic acids; mitoribosomal accumulation was minor. Global proteomics of ClpP-null mouse fibroblasts and brain plus ClpP-mutant human patient fibroblasts; comparative analysis across two species Cells Medium 34943861

Source papers

Stage 0 corpus · 100 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2003 Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Molecular cell 475 12667450
2003 Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence. Molecular microbiology 266 12791139
1993 ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. Sequence and in vivo activities. The Journal of biological chemistry 263 8226770
1995 Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes & development 245 7557391
2011 ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 231 21529717
1995 The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone. The EMBO journal 227 7743994
2008 Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding. Nature structural & molecular biology 221 18931677
2009 Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine. Cell 214 19914167
1993 Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli. The Journal of biological chemistry 209 8226769
1998 Stress induction of the Bacillus subtilis clpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance. Molecular microbiology 160 9643546
2006 In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted. Molecular microbiology 157 16803592
2013 Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors. Human molecular genetics 156 23851121
2008 Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates. Molecular cell 133 18313382
2004 Role of the processing pore of the ClpX AAA+ ATPase in the recognition and engagement of specific protein substrates. Genes & development 130 15004005
2004 Communication between ClpX and ClpP during substrate processing and degradation. Nature structural & molecular biology 126 15064753
1994 A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein. Molecular microbiology 115 8022280
2001 Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis. The Journal of biological chemistry 111 11346657
1996 ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis. The EMBO journal 106 8631314
2003 Crystal structure of ClpX molecular chaperone from Helicobacter pylori. The Journal of biological chemistry 103 14514695
2015 Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis. Cell 101 25957689
1997 ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway. Genes & development 101 9203582
2003 Targeted delivery of an ssrA-tagged substrate by the adaptor protein SspB to its cognate AAA+ protein ClpX. Molecular cell 98 14536077
2003 The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function. The Journal of biological chemistry 96 12937164
2003 Latent ClpX-recognition signals ensure LexA destruction after DNA damage. Genes & development 90 12730132
2005 Global virulence regulation in Staphylococcus aureus: pinpointing the roles of ClpP and ClpX in the sar/agr regulatory network. Infection and immunity 89 16299304
2002 Characterization of a specificity factor for an AAA+ ATPase: assembly of SspB dimers with ssrA-tagged proteins and the ClpX hexamer. Chemistry & biology 89 12445774
2001 Loss-of-function mutations in yjbD result in ClpX- and ClpP-independent competence development of Bacillus subtilis. Molecular microbiology 81 11703662
2003 Sequential recognition of two distinct sites in sigma(S) by the proteolytic targeting factor RssB and ClpX. The EMBO journal 79 12912910
2005 The ClpX chaperone modulates assembly of the tubulin-like protein FtsZ. Molecular microbiology 76 15948963
1999 Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease. The Journal of biological chemistry 76 10318812
2001 Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway. Journal of bacteriology 75 11489852
2017 Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria. Proceedings of the National Academy of Sciences of the United States of America 70 28874591
2015 Stepwise decrease in daptomycin susceptibility in clinical Staphylococcus aureus isolates associated with an initial mutation in rpoB and a compensatory inactivation of the clpX gene. Antimicrobial agents and chemotherapy 67 26324273
2010 Control of substrate gating and translocation into ClpP by channel residues and ClpX binding. Journal of molecular biology 67 20416323
1999 Role of lon and ClpX in the post-translational regulation of a sigma subunit of RNA polymerase required for cellular differentiation in Bacillus subtilis. Molecular microbiology 66 10411757
2015 ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells. Biochimica et biophysica acta 62 26142927
2014 Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase. Molecular microbiology 61 24976069
2015 The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1. PloS one 59 25933022
2010 Mycobacterium tuberculosis ClpX interacts with FtsZ and interferes with FtsZ assembly. PloS one 54 20625433
1997 The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone. Proceedings of the National Academy of Sciences of the United States of America 52 9405620
2003 Energy-dependent degradation: Linkage between ClpX-catalyzed nucleotide hydrolysis and protein-substrate processing. Protein science : a publication of the Protein Society 51 12717012
2004 ClpA and ClpX ATPases bind simultaneously to opposite ends of ClpP peptidase to form active hybrid complexes. Journal of structural biology 49 15037252
2000 Mutations conferring amino acid residue substitutions in the carboxy-terminal domain of RNA polymerase alpha can suppress clpX and clpP with respect to developmentally regulated transcription in Bacillus subtilis. Molecular microbiology 49 10972808
1996 Sequence and transcriptional analysis of clpX, a class-III heat-shock gene of Bacillus subtilis. Gene 45 8973311
2016 The Cell Wall Polymer Lipoteichoic Acid Becomes Nonessential in Staphylococcus aureus Cells Lacking the ClpX Chaperone. mBio 43 27507828
2012 Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX. Journal of structural biology 43 22710082
2007 Structural basis of SspB-tail recognition by the zinc binding domain of ClpX. Journal of molecular biology 43 17258768
2007 Requirement of the zinc-binding domain of ClpX for Spx proteolysis in Bacillus subtilis and effects of disulfide stress on ClpXP activity. Journal of bacteriology 43 17827297
1998 ClpX and ClpP are essential for the efficient acquisition of genes specifying type IA and IB restriction systems. Molecular microbiology 43 9593294
1998 Mcx1p, a ClpX homologue in mitochondria of Saccharomyces cerevisiae. FEBS letters 42 9827555
2012 Maintenance of mitochondrial genome distribution by mitochondrial AAA+ protein ClpX. Experimental cell research 41 22841477
2001 ClpX-mediated remodeling of mu transpososomes: selective unfolding of subunits destabilizes the entire complex. Molecular cell 41 11545746
2009 AAA+ chaperone ClpX regulates dynamics of prokaryotic cytoskeletal protein FtsZ. The Journal of biological chemistry 40 20022957
2003 Solution structure of the dimeric zinc binding domain of the chaperone ClpX. The Journal of biological chemistry 40 14525985
2000 The ClpX protein of Bacillus subtilis indirectly influences RNA polymerase holoenzyme composition and directly stimulates sigma-dependent transcription. Molecular microbiology 40 10972809
2010 The chaperone ClpX stimulates expression of Staphylococcus aureus protein A by Rot dependent and independent pathways. PloS one 39 20856878
2019 The ClpX chaperone controls autolytic splitting of Staphylococcus aureus daughter cells, but is bypassed by β-lactam antibiotics or inhibitors of WTA biosynthesis. PLoS pathogens 38 31518377
2021 EZH2i EPZ-6438 and HDACi vorinostat synergize with ONC201/TIC10 to activate integrated stress response, DR5, reduce H3K27 methylation, ClpX and promote apoptosis of multiple tumor types including DIPG. Neoplasia (New York, N.Y.) 37 34246076
2019 De Novo Design of Boron-Based Peptidomimetics as Potent Inhibitors of Human ClpP in the Presence of Human ClpX. Journal of medicinal chemistry 37 31187989
2009 ClpX inhibits FtsZ assembly in a manner that does not require its ATP hydrolysis-dependent chaperone activity. Journal of bacteriology 37 19136590
2003 Essentiality of clpX, but not clpP, clpL, clpC, or clpE, in Streptococcus pneumoniae R6. Journal of bacteriology 37 12700276
2017 The Protein Chaperone ClpX Targets Native and Non-native Aggregated Substrates for Remodeling, Disassembly, and Degradation with ClpP. Frontiers in molecular biosciences 35 28523271
2005 Remodeling protein complexes: insights from the AAA+ unfoldase ClpX and Mu transposase. Protein science : a publication of the Protein Society 35 16046622
2001 Structure-function analysis of the zinc-binding region of the Clpx molecular chaperone. The Journal of biological chemistry 35 11278349
2000 Regulation of sigma S degradation in Salmonella enterica var typhimurium: in vivo interactions between sigma S, the response regulator MviA(RssB) and ClpX. Journal of molecular microbiology and biotechnology 35 10939250
2000 Human and mouse mitochondrial orthologs of bacterial ClpX. Mammalian genome : official journal of the International Mammalian Genome Society 35 11003706
2015 Reversible Inhibitors Arrest ClpP in a Defined Conformational State that Can Be Revoked by ClpX Association. Angewandte Chemie (International ed. in English) 34 26566002
2017 A Chemical Disruptor of the ClpX Chaperone Complex Attenuates the Virulence of Multidrug-Resistant Staphylococcus aureus. Angewandte Chemie (International ed. in English) 33 28906057
2006 Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX. Proceedings of the National Academy of Sciences of the United States of America 33 17090685
2015 Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX. Nature structural & molecular biology 32 25866879
2008 Unique contacts direct high-priority recognition of the tetrameric Mu transposase-DNA complex by the AAA+ unfoldase ClpX. Molecular cell 32 18406325
2020 The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development. mBio 31 32873765
1998 Formation of the preprimosome protects lambda O from RNA transcription-dependent proteolysis by ClpP/ClpX. Proceedings of the National Academy of Sciences of the United States of America 31 9860956
2020 Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding. eLife 30 32091391
2019 Roles of the ClpX IGF loops in ClpP association, dissociation, and protein degradation. Protein science : a publication of the Protein Society 30 30767302
1999 Molecular cloning and characterization of a mouse homolog of bacterial ClpX, a novel mammalian class II member of the Hsp100/Clp chaperone family. The Journal of biological chemistry 30 10347188
2019 Role of ClpX and ClpP in Streptococcus suis serotype 2 stress tolerance and virulence. Microbiological research 28 31178057
2002 Degradation of a Caulobacter soluble cytoplasmic chemoreceptor is ClpX dependent. Journal of bacteriology 28 12426352
2021 The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover. The Journal of biological chemistry 27 34280433
2020 Global Inventory of ClpP- and ClpX-Regulated Proteins in Staphylococcus aureus. Journal of proteome research 27 33210542
2006 Large nucleotide-dependent movement of the N-terminal domain of the ClpX chaperone. The EMBO journal 25 16810315
1999 The Oenococcus oeni clpX homologue is a heat shock gene preferentially expressed in exponential growth phase. Journal of bacteriology 24 10542163
2019 The clpX gene plays an important role in bacterial attachment, stress tolerance, and virulence in Xanthomonas campestris pv. campestris. Archives of microbiology 21 31741013
2000 ClpP/ClpX-mediated degradation of the bacteriophage lambda O protein and regulation of lambda phage and lambda plasmid replication. Archives of microbiology 21 10985747
2003 Mu transpososome architecture ensures that unfolding by ClpX or proteolysis by ClpXP remodels but does not destroy the complex. Chemistry & biology 20 12770828
2019 Experiencing community and domestic violence is associated with epigenetic changes in DNA methylation of BDNF and CLPX in adolescents. Psychophysiology 19 31059136
2001 Expression of clpX, an ATPase subunit of the Clp protease, is heat and cold shock inducible in Lactococcus lactis. Journal of dairy science 19 11518300
2018 The role of ClpX in erythropoietic protoporphyria. Hematology, transfusion and cell therapy 18 30057992
2010 The AAA+ ClpX machine unfolds a keystone subunit to remodel the Mu transpososome. Proceedings of the National Academy of Sciences of the United States of America 18 20133746
2010 Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species. Protein science : a publication of the Protein Society 16 20014030
2003 Involvement of ClpX protein in the post-transcriptional regulation of a competence specific transcription factor, ComK protein, of Bacillus subtilis. Journal of biochemistry 16 12761164
2023 ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis. Communications biology 15 37798322
2021 Potent preclinical sensitivity to imipridone-based combination therapies in oncohistone H3K27M-mutant diffuse intrinsic pontine glioma is associated with induction of the integrated stress response, TRAIL death receptor DR5, reduced ClpX and apoptosis. American journal of cancer research 15 34659909
2013 Deletion in the C-terminal domain of ClpX delayed entry of Salmonella enterica into a viable but non-culturable state. Research in microbiology 15 23385142
2003 The ATPase ClpX is conditionally involved in the morphological differentiation of Streptomyces lividans. Molecular genetics and genomics : MGG 15 12589431
2021 Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA. Cells 14 34943861
2019 Staphylococcus aureus ClpX localizes at the division septum and impacts transcription of genes involved in cell division, T7-secretion, and SaPI5-excision. Scientific reports 14 31712583
2022 Tag-Dependent Substrate Selection of ClpX Underlies Secondary Differentiation of Chlamydia trachomatis. mBio 13 36154190
2021 ClpX Is Essential and Activated by Single-Strand DNA Binding Protein in Mycobacteria. Journal of bacteriology 13 33229461
2012 Role of the N-terminal domain of the chaperone ClpX in the recognition and degradation of lambda phage protein O. The journal of physical chemistry. B 13 22360725

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