Affinage

CLPX

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

Length
633 aa
Mass
69.2 kDa
Annotated
2026-04-28
100 papers in source corpus 46 papers cited in narrative 45 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CLPX is a AAA+ ATPase unfoldase that forms asymmetric hexameric rings and uses ATP hydrolysis to mechanically grip, unfold, and translocate polypeptide substrates through conserved aromatic pore-loop residues into the ClpP peptidase chamber for degradation, with substrate selectivity determined by five classes of recognition motifs and modulated by adaptor proteins such as SspB and RssB that dock to the N-terminal zinc-binding domain (ZBD) (PMID:8226770, PMID:12667450, PMID:14536077, PMID:18931677). Independent of ClpP and ATP hydrolysis, CLPX functions as a molecular chaperone that remodels stable protein complexes—disassembling MuA transpososomes by partial unfolding of individual subunits, activating the TrfA replication initiator by monomerization, and inhibiting FtsZ polymerization—demonstrating broad protease-independent remodeling activity (PMID:7557391, PMID:11545746, PMID:9405620, PMID:15948963). In the mitochondrial matrix, CLPX activates 5-aminolevulinate synthase (ALAS) by catalyzing partial unfolding that gates pyridoxal phosphate cofactor incorporation, thereby controlling the committed step of heme biosynthesis; CLPX also regulates ALAS turnover, PPOX activity, and ferrochelatase levels during erythroid differentiation (PMID:25957689, PMID:32091391, PMID:34280433). A dominant loss-of-function mutation (p.Gly298Asp) in the CLPX ATPase active site causes ALAS accumulation and protoporphyrin IX excess, resulting in erythropoietic protoporphyria (PMID:28874591).

Mechanistic history

Synthesis pass · year-by-year structured walk · 16 steps
  1. 1993 High

    Establishing that ClpX is the ATPase specificity subunit of ClpXP resolved how ClpP targets distinct substrate sets, since ClpX directed degradation of lambda O protein independently of ClpA.

    Evidence Biochemical purification and in vitro/in vivo degradation assays in E. coli

    PMID:8226769 PMID:8226770

    Open questions at the time
    • Mechanism of substrate discrimination by ClpX versus ClpA unknown
    • Oligomeric state and ATP coupling mechanism uncharacterized
  2. 1995 High

    Demonstrating that ClpX functions as a ClpP-independent chaperone—protecting lambda O from aggregation, disaggregating aggregates, and disassembling the MuA transpososome—established a dual role for ClpX beyond proteolysis.

    Evidence In vitro chaperone and disaggregation assays (lambda O); reconstituted MuA disassembly with purified components

    PMID:7557391 PMID:7743994

    Open questions at the time
    • Whether chaperone and protease modes use the same substrate-binding surface unknown
    • Structural basis for ATP-binding versus ATP-hydrolysis requirements not resolved
  3. 1997 High

    Identifying a 10-residue C-terminal peptide of MuA as a transferable ClpX-recognition signal, and showing ClpX activates TrfA by monomerization, defined the principle that short sequence motifs direct ClpX to diverse substrates for remodeling or degradation.

    Evidence Peptide-transfer and deletion experiments (MuA); in vitro replication reconstitution (TrfA)

    PMID:9203582 PMID:9405620

    Open questions at the time
    • Full repertoire of ClpX recognition motifs unknown
    • How ZBD versus pore loops contribute to different motif classes not distinguished
  4. 1999 High

    Localizing mammalian CLPX to mitochondria via its N-terminal targeting sequence and demonstrating intrinsic ATPase activity established that CLPX is a conserved mitochondrial matrix AAA+ enzyme.

    Evidence GFP-fusion confocal microscopy with targeting-sequence deletion, in vitro ATPase assays with P-loop mutagenesis (murine and human CLPX)

    PMID:10347188 PMID:11003706

    Open questions at the time
    • Mitochondrial substrates of mammalian CLPXP unidentified
    • Whether mitochondrial CLPX has ClpP-independent chaperone roles unknown
  5. 2001 High

    Showing that ClpX unfolds individual MuA subunits within the intact tetramer—and that the ZBD is dispensable for hexamer formation but essential for substrate-specific recognition—resolved how ClpX couples mechanical unfolding to multisubunit complex disassembly.

    Evidence Biochemical unfolding probes and limited proteolysis with domain deletion analysis

    PMID:11346657 PMID:11545746

    Open questions at the time
    • Number of subunits unfolded per disassembly event not quantified
    • Full structural model of hexameric ClpX lacking
  6. 2003 High

    In vivo substrate trapping identified >50 ClpXP substrates and five classes of recognition signals (two C-terminal, three N-terminal), while crystal and NMR structures of ZBD and H. pylori ClpX revealed the zinc-finger fold, ZBD dimerization, and the IGF-loop/ClpP docking interface, providing a comprehensive substrate-recognition and architectural framework.

    Evidence ClpP-trap mass spectrometry, deletion/fusion/point mutagenesis for motif validation; NMR structure of ZBD dimer; X-ray crystallography of H. pylori ClpX-ADP

    PMID:12667450 PMID:12937164 PMID:14514695 PMID:14525985

    Open questions at the time
    • No full-length hexameric ClpX structure available
    • How different motif classes are prioritized in vivo unclear
  7. 2003 High

    Defining SspB-mediated substrate delivery via the XB tail–ZBD interaction, and RssB-mediated two-step recognition of sigma(S), established that adaptor proteins exploit the ZBD docking site to expand and regulate ClpXP substrate selection.

    Evidence Pulldown assays, point mutagenesis, ternary complex isolation (SspB); deletion analysis and in vivo/in vitro degradation assays (RssB/sigma(S))

    PMID:12445774 PMID:12912910 PMID:14536077

    Open questions at the time
    • Structural basis of the SspB–ZBD interaction at atomic resolution not yet available (resolved later)
    • Whether additional adaptor proteins use the same ZBD interface unknown
  8. 2004 High

    Pore-loop mutagenesis (V154F) demonstrating class-specific substrate recognition within the central channel, and sensor-II arginine mutations linking nucleotide state to ClpP docking, revealed that the pore is a dual-function element for substrate selection and force transduction.

    Evidence Site-directed mutagenesis with in vitro/in vivo degradation assays; biochemical binding and cross-linking assays

    PMID:15004005 PMID:15064753

    Open questions at the time
    • Quantitative contributions of individual pore loops to grip and translocation unknown
    • Allosteric pathway from nucleotide pocket to pore not mapped
  9. 2005 High

    Establishing that ClpX inhibits FtsZ assembly in an ATP-hydrolysis-independent, ClpP-independent manner identified a third functional mode—passive sequestration or capping of a cytoskeletal polymer—linking ClpX to bacterial cell division control.

    Evidence Genetic separation of clpX vs. clpP phenotypes, in vitro FtsZ assembly assays, high-speed AFM visualization

    PMID:15948963 PMID:19136590 PMID:20022957

    Open questions at the time
    • Structural basis of ClpX–FtsZ interaction unresolved
    • Relevance of this mechanism in eukaryotic/mitochondrial division unknown
  10. 2008 High

    Specificity-transplant and crosslinking experiments mapping ssrA-tag contacts to top, middle, and lower channel loops, combined with the finding that a conserved aromatic pore-loop tyrosine grips substrates during translocation, defined the molecular mechanism of substrate engagement and mechanical power stroke.

    Evidence Disulfide crosslinking, specificity-transplant experiments, systematic Tyr→Ala mutagenesis across hexamer subunits

    PMID:18313382 PMID:18931677

    Open questions at the time
    • Exact step size and coordination between subunits during translocation not directly measured at this stage
  11. 2009 High

    Crystal structures of nucleotide-free and nucleotide-bound ClpX hexamers revealing striking asymmetry—with two subunits unable to bind nucleotide—provided the structural basis for the probabilistic firing model of AAA+ ring mechanics.

    Evidence X-ray crystallography of full hexamers in two nucleotide states

    PMID:19914167

    Open questions at the time
    • Dynamic transitions between conformational states not captured by crystallography
    • Correspondence of crystallographic states to functional intermediates not proven
  12. 2011 High

    Single-molecule optical-trap measurements directly quantified ClpX translocation velocity (~80 aa/s), stall force (~20 pN), and fundamental step size (1 nm), establishing ClpXP as a calibrated molecular motor and showing that ClpP binding reduces slippage.

    Evidence Single-molecule optical tweezers force spectroscopy

    PMID:21529717

    Open questions at the time
    • How individual subunit firing events map to observed step sizes not resolved
    • Force-dependent unfolding landscapes for diverse substrates not explored
  13. 2015 High

    Demonstrating that mitochondrial CLPX activates ALAS by catalyzing pyridoxal phosphate cofactor incorporation—conserved from yeast to mammals—connected the AAA+ unfoldase to heme biosynthesis and erythropoiesis, a function entirely distinct from canonical proteolysis.

    Evidence In vitro enzyme reconstitution, yeast genetics/metabolomics, zebrafish knockdown

    PMID:25957689

    Open questions at the time
    • Mechanism of partial unfolding enabling cofactor access not yet characterized
    • Whether CLPX activates other mitochondrial enzymes by cofactor gating unknown
  14. 2017 High

    Identifying a dominant p.Gly298Asp CLPX mutation that poisons hexamer ATPase activity and causes ALAS accumulation and PPIX excess linked CLPX loss-of-function to erythropoietic protoporphyria, establishing a human Mendelian disease mechanism.

    Evidence Patient genetics, in vitro ATPase assays, co-assembly experiments, metabolite measurements

    PMID:28874591

    Open questions at the time
    • Prevalence of CLPX mutations among EPP patients not fully surveyed
    • Whether haploinsufficiency alone is pathogenic or dominant-negative effect is required not resolved
  15. 2020 High

    HDX-MS revealed that CLPX partially unfolds ALAS specifically from the binding site to the active site—not globally—defining a targeted partial-unfolding mechanism that gates cofactor access, mechanistically distinct from canonical processive unfolding.

    Evidence Hydrogen-deuterium exchange mass spectrometry, in vitro reconstitution, ALAS mutagenesis

    PMID:32091391

    Open questions at the time
    • Whether the partial-unfolding mechanism applies to other CLPX chaperone substrates unknown
    • Structural visualization of the CLPX-ALAS intermediate lacking
  16. 2021 High

    Conditional Clpx knockout in erythroid cells showed CLPX controls ALAS2 turnover (not just activation), maintains PPOX activity and FECH levels, and is required for iron utilization during hemoglobin synthesis, broadening CLPX's role to multiple heme pathway enzymes.

    Evidence Conditional knockout cells, enzyme activity measurements, protein stability assays

    PMID:34280433

    Open questions at the time
    • Direct CLPX-PPOX and CLPX-FECH physical interactions not demonstrated
    • Whether CLPX controls these enzymes through CLPXP degradation or chaperone remodeling not distinguished

Open questions

Synthesis pass · forward-looking unresolved questions
  • Major open questions include: (1) the complete mitochondrial substrate repertoire of CLPXP beyond heme pathway enzymes, (2) whether the partial-unfolding cofactor-gating mechanism extends to other mitochondrial enzymes, (3) how CLPX's chaperone versus protease functions are differentially regulated in mammalian mitochondria, and (4) the structural basis of the CLPX–ALAS complex during partial unfolding.
  • No cryo-EM or crystal structure of mitochondrial CLPX-ALAS complex
  • Full mitochondrial CLPXP substrate catalogue not established by trapping approaches
  • Relative contributions of chaperone versus protease modes to mitochondrial homeostasis not quantified

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0044183 protein folding chaperone 6 GO:0140657 ATP-dependent activity 6 GO:0140096 catalytic activity, acting on a protein 5 GO:0098772 molecular function regulator activity 3
Localization
GO:0005739 mitochondrion 4 GO:0005829 cytosol 2
Pathway
R-HSA-392499 Metabolism of proteins 4 R-HSA-1430728 Metabolism 3 R-HSA-1640170 Cell Cycle 3
Partners
ALAS2CLPPFTSZMUARSSBSSPBTFAM
Complex memberships
ClpXAP hybrid complexClpXP protease complex

Evidence

Reading pass · 45 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1993 ClpX is an ATPase subunit that associates with the ClpP protease to form the ClpXP complex, directing ClpP to specific substrates (e.g., lambda O protein) that are not degraded by ClpAP, establishing that substrate selectivity of ClpP is determined by its regulatory ATPase subunit. Biochemical purification, in vitro degradation assays, in vivo genetic analysis The Journal of biological chemistry High 8226769 8226770
1995 ClpX functions as a molecular chaperone independently of ClpP: 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 binding (but not hydrolysis) is required for protection, while hydrolysis is required for disaggregation. In vitro chaperone assays, ELISA-based protein interaction assay, ATPase stimulation assay The EMBO journal High 7743994
1995 ClpX catalyzes ATP-dependent disassembly of the stable MuA transposase tetramer from DNA after recombination without requiring ClpP or any protease; the released MuA is not degraded and retains activity for another round of recombination, demonstrating ClpX as a molecular chaperone that promotes transient conformational change. The C-terminal sequence of MuA is required for ClpX-mediated disassembly and can also target MuA for ClpXP degradation. In vitro reconstitution, purification, transposition assays, deletion analysis Genes & development High 7557391
1997 A 10-amino-acid peptide from the C-terminal domain of MuA transposase is required for recognition by ClpX; this short positively charged peptide is sufficient to convert a heterologous protein into a ClpX substrate. The MuB-binding region of MuA overlaps with the ClpX-recognition region, so MuB inhibits ClpX-mediated disassembly, providing a regulatory mechanism. Deletion analysis, peptide-transfer experiments, in vitro disassembly assays Genes & development High 9203582
1996 ClpX (as a component of MRFalpha) alters the conformation of DNA-bound MuA transposase converting STC1 to a less stable form (STC2), which is a prerequisite for MuA removal and initiation of Mu DNA replication; this demonstrates that ClpX activates MuA for recruitment of host replication factors. In vitro reconstitution with purified replication proteins, biochemical fractionation The EMBO journal High 8631314
2001 The N-terminal domain of ClpX dissociates upon proteolysis but the remaining ClpXΔN retains hexameric assembly, ClpP association, and ATPase/chaperone/proteolytic activity; the conserved IGF/LGF loop region is critical for ClpP binding (cleavage at this loop abolishes ClpP association and activation). Limited proteolysis, deletion analysis, ATPase assays, ClpP binding assays The Journal of biological chemistry High 11346657
2001 ClpX-mediated remodeling of the Mu transpososome involves direct unfolding of MuA subunits (as detected by a biochemical unfolding probe); unfolding of a single transposase subunit is sufficient to destabilize the entire tetrameric complex. Biochemical unfolding probe assay, in vitro remodeling assays Molecular cell High 11545746
2003 The N-terminal zinc binding domain (ZBD) of ClpX is a C4-type zinc-binding domain that forms a constitutive dimer, is essential for degradation of substrates such as lambda O and MuA (but not GFP-ssrA), contains the primary binding site for lambda O and SspB cofactor, and modulates ClpX ATPase responses to ClpP and substrates. Domain deletion/truncation, in vitro degradation assays, zinc-binding characterization The Journal of biological chemistry High 12937164
2003 NMR solution structure of the dimeric ZBD of ClpX reveals a treble-clef zinc finger monomer fold; the dimer interface is unique and a trimer-of-dimers model is proposed to reflect the closed state of the ClpX hexamer. NMR spectroscopy, structural analysis The Journal of biological chemistry High 14525985
2003 Crystal structure of Helicobacter pylori ClpX (lacking the N-terminal Cys cluster region) complexed with ADP reveals two subdomains similar to HslU; the conserved LGF tripeptide resides on a loop that mediates hydrophobic contacts with ClpP heptamer clefts, providing the structural basis for ClpX-ClpP interaction. X-ray crystallography, hexameric modeling The Journal of biological chemistry High 14514695
2003 Mass spectrometry-based in vivo trapping using an inactive ClpP variant identified >50 ClpXP substrates in E. coli; sequence analysis of trapped proteins revealed five classes of ClpX-recognition signals: two C-terminal motifs and three N-terminal motifs. Deletion analysis, fusion proteins, and point mutations confirmed each motif class is sufficient to target proteins for ClpXP degradation. In vivo substrate trapping, mass spectrometry, deletion analysis, fusion protein assays, point mutagenesis Molecular cell High 12667450
2003 The C-terminal SspB tail (XB motif) interacts specifically with the N-terminal zinc-binding domain (N domain) of ClpX to deliver ssrA-tagged substrates; a single point mutation in the SspB C-terminus abolishes SspB-mediated delivery to ClpXP. Pulldown assays, point mutagenesis, in vitro degradation assays Molecular cell High 14536077
2003 Both LexA auto-cleavage fragments are substrates for ClpXP; ClpXP recognizes these fragments using sequence motifs flanking the auto-cleavage site that are dormant (buried) in intact LexA, demonstrating that a protein-processing event can activate latent protease-recognition signals. In vitro and in vivo degradation assays, pulse-chase experiments, mutational analysis Genes & development High 12730132
2003 SspB forms a stable homodimer with two independent ssrA-tag binding sites; SspB-ClpX ternary complex (one SspB dimer, two GFP-ssrA molecules, one ClpX hexamer) was isolated; SspB increases affinity and cooperativity of ssrA-tagged substrate binding to ClpX. Gel filtration, ion-exchange chromatography, ATPase stimulation assays, fluorescence binding assays Chemistry & biology High 12445774
2004 The pore of the ClpX hexamer functions in recognition and catalytic engagement of specific substrate classes: the V154F pore mutation abolishes binding of C-motif 1 substrates and impairs a subsequent engagement step, while leaving other substrate classes unaffected, demonstrating that substrate binding via the pore is class-specific. Site-directed mutagenesis, in vitro and in vivo degradation assays Genes & development High 15004005
2004 ClpX-ClpP affinity varies with the substrate-processing state of ClpX and with ClpP active-site engagement; the IGF loops of ClpX transmit conformational changes driven by ATP hydrolysis to ClpP; a conserved arginine in the sensor II helix of ClpX links nucleotide state to binding of both ClpP and protein substrates. Biochemical binding assays, ATPase assays, mutagenesis, cross-linking Nature structural & molecular biology High 15064753
2004 ClpA and ClpX can bind simultaneously to opposite ends of the ClpP double ring to form functional hybrid ClpXAP complexes, in which each end independently targets its own substrate class; electron microscopy visualized substrate translocation into the chamber by both subunits. Electron microscopy, substrate translocation assays, biochemical reconstitution Journal of structural biology High 15037252
2007 Crystal structures of the ZBD:SspB-XB peptide complex at 1.6 Å resolution show that the XB peptide forms an antiparallel beta-sheet with two ZBD beta-strands in a 1:1 stoichiometry, revealing two independent SspB-tail binding sites per ZBD dimer. X-ray crystallography, biochemical binding analysis Journal of molecular biology High 17258768
2008 The ssrA tag interacts with multiple loops forming the top, middle, and lower portions of the ClpX hexameric central channel; specificity-transplant and disulfide-crosslinking experiments support a two-step binding mechanism: the top loop acts as a specificity filter and the remaining loops bind the tag deep within the pore; nucleotide-dependent conformational changes in these loops drive peptide translocation. Specificity-transplant experiments, disulfide crosslinking, in vitro degradation assays Molecular cell High 18313382
2008 A conserved aromatic-hydrophobic pore-loop motif (tyrosine residue) in ClpX hexamers grips substrates during unfolding and translocation by coupling ATP hydrolysis to mechanical work; removal of the aromatic ring in even a few subunits causes substrate slippage and dramatically increases the energetic cost of unfolding. Site-directed mutagenesis, in vitro unfolding and translocation assays, ATPase assays Nature structural & molecular biology High 18931677
2008 ClpX recognizes the MuA tetramer more tightly than monomeric MuA; residues exposed only in the assembled tetramer enhance recognition via the N domain of ClpX; preferential recognition of the high-priority complex is mediated by contacts exposed upon tetramerization. Binding assays with monomeric vs. tetrameric MuA, N-domain deletion, in vitro disassembly assays Molecular cell High 18406325
2009 Crystal structures of nucleotide-free and nucleotide-bound ClpX hexamers reveal striking asymmetry arising from large rotations between the large and small AAA+ domains of individual subunits; this asymmetry prevents nucleotide binding to two subunits, generates a staggered pore-loop arrangement, and provides a mechanism for coupling ATP-driven conformational changes in one subunit to flexing of the entire ring. X-ray crystallography of ClpX hexamers Cell High 19914167
2010 ClpX binding to ClpP stimulates ClpP cleavage of peptides larger than a few amino acids (requiring ATP binding but not hydrolysis by ClpX) by relieving inhibitory interactions of ClpP channel loop residues; ClpP channel variants alter substrate entry and translocation specificity, supporting a gating model. Biochemical peptide cleavage assays, mutagenesis of ClpP channel residues, active-site modification assays Journal of molecular biology High 20416323
2010 ClpX preferentially unfolds the catalytic-left or catalytic-right (keystone) subunits of the MuA tetramer to destabilize the transpososome; left-end biased Mu replication is not determined by ClpX's intrinsic subunit preference. Altered-specificity MuA proteins with matched DNA sites, in vitro disassembly assays Proceedings of the National Academy of Sciences of the United States of America High 20133746
2011 Single-molecule optical trap experiments directly demonstrated that ClpX generates mechanical force to unfold and translocate polypeptides; translocation velocity reaches ~80 aa/s near-zero force and stalls at ~20 pN; ClpX takes 1, 2, or 3 nm steps with a fundamental step of 1 nm; ClpP binding decreases slip probability and enhances unfolding efficiency; GFP unravels cooperatively via a transient intermediate under ClpXP. Single-molecule optical tweezers/force spectroscopy Cell High 21529717
2012 Human CLPX Walker B mutant (E→A, abolishing ATPase) retains ability to mediate casein degradation by hCLPP (similar to ADEP); most model substrates are recognized by the N-terminal domain of CLPX, but some bypass this and dock directly to the pore-1 motif, demonstrating conserved substrate recognition architecture between bacterial and human CLPXP. Walker B mutagenesis, in vitro degradation assays, domain binding studies Journal of structural biology High 22710082
2012 RNAi-mediated knockdown of ClpX in HeLa cells causes enlarged mtDNA nucleoids, resembling TFAM-knockdown phenotype; ClpX enhances TFAM DNA-binding activity in vitro; the phenotype is rescued by TFAM overexpression but not by ClpP knockdown, indicating that ClpX maintains mtDNA nucleoid distribution through TFAM as a chaperone (not protease) function. RNAi knockdown, confocal microscopy, in vitro DNA-binding assay Experimental cell research Medium 22841477
2013 Deletion of mouse Clpp leads to accumulation of CLPX protein in mitochondria throughout tissues, consistent with CLPX being a primary substrate of the CLPP protease in the mitochondrial matrix. Clpp null mouse model, immunoblotting, proteomics Human molecular genetics Medium 23851121
2015 Mitochondrial ClpX (mtClpX) directly stimulates ALA synthase (ALAS) activity in vitro by catalyzing incorporation of its cofactor pyridoxal phosphate, thereby activating the first step of heme biosynthesis; mtClpX depletion reduces 5-aminolevulinic acid ~5-fold and total heme ~50% in yeast; this activity is conserved in mammalian homologs, and mtClpX depletion impairs vertebrate erythropoiesis. In vitro enzyme reconstitution, metabolomics, yeast genetics, zebrafish knockdown Cell High 25957689
2015 Subunit asymmetry in the ClpX ring is functionally essential: locking one subunit in the nucleotide-unloadable (U) conformation allows the remaining subunits to hydrolyze ATP but alters cooperativity and reduces substrate binding, unfolding, and degradation efficiency, demonstrating that U↔L conformational switching in individual subunits is required for full ClpX function. Covalent hexamer engineering, ATPase assays, substrate degradation assays Nature structural & molecular biology High 25866879
2015 ClpX overexpression in mammalian myoblasts upregulates markers of the mitochondrial unfolded protein response (UPRmt), including the transcription factor CHOP, demonstrating that ClpX stimulates the mammalian UPRmt retrograde signaling pathway. Quantitative proteomics, overexpression, transcription factor analysis Biochimica et biophysica acta Medium 26142927
2017 A dominant p.Gly298Asp mutation in the ATPase active site of human CLPX inactivates ATPase activity; mutant CLPX co-assembles with WT protomers to form a low-activity enzyme; reduced CLPX activity increases posttranslational stability of ALAS (rather than activating it), causing ALAS accumulation and PPIX excess, leading to erythropoietic protoporphyria. Patient genetics, in vitro ATPase assays, co-assembly experiments, metabolite measurements Proceedings of the National Academy of Sciences of the United States of America High 28874591
2019 ATP or ATPγS induces conformational change in the ClpX ring that brings IGF loops closer together enabling multivalent ClpP docking; deletion of one or two IGF loops strongly accelerates ClpXP dissociation and reduces proteolysis processivity; IGF loop length and sequence are critical for ClpX-ClpP interaction kinetics. Single-chain ClpX pseudohexamers, IGF loop deletion mutagenesis, ClpP association/dissociation kinetics, degradation assays Protein science High 30767302
2020 Mitochondrial ClpX activates ALAS (heme biosynthesis enzyme) by partial unfolding limited to a region from the ClpX-binding site to the active site, gating cofactor (pyridoxal phosphate) access; this targeted partial unfolding (not global unfolding) is mechanistically distinct from canonical ClpX substrates. HDX-MS to observe remodeling, in vitro reconstitution, mutagenesis of ALAS structural features eLife High 32091391
2021 In erythroid cells, CLPX primarily regulates ALAS2 by controlling its turnover (stability) rather than its activation; CLPX is also required for PPOX (protoporphyrinogen IX oxidase) activity and FECH (ferrochelatase) level maintenance, and for iron utilization for hemoglobin synthesis during differentiation. Clpx conditional knockout cells, enzyme activity measurements, protein stability assays The Journal of biological chemistry High 34280433
2000 Human CLPX is a 633-amino acid protein containing an N-terminal mitochondrial targeting sequence; expression of full-length CLPX-Myc-His results in import into mitochondria; deletion of the N-terminal targeting sequence abolishes mitochondrial localization. CLPX interacts with ClpP in 293T cells. cDNA cloning, confocal microscopy with GFP fusion, subcellular fractionation, co-immunoprecipitation Mammalian genome High 11003706
1999 Murine ClpX localizes to mitochondria as demonstrated by confocal microscopy of GFP fusions; deletion of the N-terminal mitochondrial targeting sequence abolishes mitochondrial compartmentalization; recombinant murine ClpX displays intrinsic ATPase activity (Km ~25 µM); K300A mutation in the P-loop abolishes both ATP hydrolysis and binding. Confocal microscopy, subcellular fractionation, in vitro ATPase assays, mutagenesis The Journal of biological chemistry High 10347188
2005 ClpX (but not ClpP) inhibits FtsZ assembly in vivo and in vitro through a ClpP-independent mechanism that does not require ATP hydrolysis; ClpX interacts directly with FtsZ in E. coli. Genetic analysis (clpX vs. clpP mutants), in vitro FtsZ assembly assays Molecular microbiology High 15948963
2009 ClpX inhibits FtsZ polymerization in an ATP-independent manner through its N-terminal domain; high-speed AFM directly visualized FtsZ polymer dynamics and showed ClpX disassembles polymers by blocking reassembly; ClpX overexpression in E. coli causes filamentous morphology with abnormal FtsZ localization. In vitro polymerization assays, ATPase-deficient mutant analysis, high-speed atomic force microscopy, in vivo overexpression The Journal of biological chemistry High 20022957
2009 ClpX inhibits FtsZ assembly through a non-canonical mechanism that is independent of its ATP hydrolysis-dependent chaperone activity, as demonstrated by site-directed mutagenesis of ATPase function combined with genetics and biochemistry. Site-directed mutagenesis, genetics (in vivo), biochemical assembly assays Journal of bacteriology High 19136590
1997 ClpX molecular chaperone activates the TrfA replication initiation protein of plasmid RK2 by converting its dimeric (inactive) form to monomers capable of binding the replication origin; this activation is ATP-dependent and is demonstrated in a purified in vitro replication system. In vitro replication assay with purified components, gel filtration to assess monomer/dimer ratio Proceedings of the National Academy of Sciences of the United States of America High 9405620
2021 In CLPP-null mouse embryonal fibroblasts and brains, ClpX co-accumulates with nucleoid component POLDIP2, mRNA granule element LRPPRC, and tRNA processing factor GFM1, establishing that ClpXP primarily acts on assembly of proteins with nucleic acids in the mitochondrial matrix; this pattern is validated in human CLPP-mutant patient fibroblasts. Global proteome profiling of Clpp null mice and patient fibroblasts, subcellular fractionation Cells Medium 34943861
2006 The ZBD of ClpX undergoes large nucleotide-dependent movement towards ClpP in functional ClpXP complexes (switching between capture and feeding conformations); this motion is modulated by the SspB cofactor; an N-terminal extension of ClpX is clipped by bound ClpP, providing evidence for this conformational change. Protease-protection assays, crosslinking, dynamic light scattering, ClpP clipping assay The EMBO journal Medium 16810315
2003 sigma(S) degradation by ClpXP requires sequential recognition of two distinct sites: phosphorylated RssB binds region 2.5 of sigma(S), exposing a second N-terminal region that serves as the ClpX binding site; neither interaction alone is sufficient to commit sigma(S) to degradation. Deletion analysis, in vivo and in vitro degradation assays, reporter fusion assays The EMBO journal High 12912910
2019 ClpX localizes to single foci in close proximity to the division septum in Staphylococcus aureus, supporting a direct role in cell division; ClpX impacts transcription of genes involved in peptidoglycan synthesis, cell division, and type VII secretion independently of ClpP. Fluorescence microscopy (ClpX-GFP localization), transcriptomic analysis of clpX vs. clpXP deletions Scientific reports Medium 31712583

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 472 12667450
2003 Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence. Molecular microbiology 264 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 243 7557391
2011 ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 227 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 225 7743994
2008 Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding. Nature structural & molecular biology 219 18931677
2009 Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine. Cell 211 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 155 23851121
2008 Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates. Molecular cell 132 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 124 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 102 14514695
1997 ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway. Genes & development 101 9203582
2015 Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis. Cell 100 25957689
2003 Targeted delivery of an ssrA-tagged substrate by the adaptor protein SspB to its cognate AAA+ protein ClpX. Molecular cell 97 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
2005 Global virulence regulation in Staphylococcus aureus: pinpointing the roles of ClpP and ClpX in the sar/agr regulatory network. Infection and immunity 88 16299304
2003 Latent ClpX-recognition signals ensure LexA destruction after DNA damage. Genes & development 88 12730132
2002 Characterization of a specificity factor for an AAA+ ATPase: assembly of SspB dimers with ssrA-tagged proteins and the ClpX hexamer. Chemistry & biology 88 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 78 12912910
2005 The ClpX chaperone modulates assembly of the tubulin-like protein FtsZ. Molecular microbiology 76 15948963
2001 Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway. Journal of bacteriology 75 11489852
1999 Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease. The Journal of biological chemistry 75 10318812
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 67 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 66 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 64 10411757
2015 ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells. Biochimica et biophysica acta 61 26142927
2014 Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase. Molecular microbiology 60 24976069
2015 The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1. PloS one 58 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
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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
2004 ClpA and ClpX ATPases bind simultaneously to opposite ends of ClpP peptidase to form active hybrid complexes. Journal of structural biology 48 15037252
1996 Sequence and transcriptional analysis of clpX, a class-III heat-shock gene of Bacillus subtilis. Gene 45 8973311
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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
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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
2010 The chaperone ClpX stimulates expression of Staphylococcus aureus protein A by Rot dependent and independent pathways. PloS one 39 20856878
2000 The ClpX protein of Bacillus subtilis indirectly influences RNA polymerase holoenzyme composition and directly stimulates sigma-dependent transcription. Molecular microbiology 39 10972809
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
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
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.) 36 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 36 31187989
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 34 11003706
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
2015 Reversible Inhibitors Arrest ClpP in a Defined Conformational State that Can Be Revoked by ClpX Association. Angewandte Chemie (International ed. in English) 33 26566002
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
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2008 Unique contacts direct high-priority recognition of the tetrameric Mu transposase-DNA complex by the AAA+ unfoldase ClpX. Molecular cell 31 18406325
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
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
2020 Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding. eLife 29 32091391
2020 The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development. mBio 29 32873765
2019 Roles of the ClpX IGF loops in ClpP association, dissociation, and protein degradation. Protein science : a publication of the Protein Society 28 30767302
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
2019 Role of ClpX and ClpP in Streptococcus suis serotype 2 stress tolerance and virulence. Microbiological research 27 31178057
2020 Global Inventory of ClpP- and ClpX-Regulated Proteins in Staphylococcus aureus. Journal of proteome research 26 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
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
2019 Experiencing community and domestic violence is associated with epigenetic changes in DNA methylation of BDNF and CLPX in adolescents. Psychophysiology 18 31059136
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
2021 ClpX Is Essential and Activated by Single-Strand DNA Binding Protein in Mycobacteria. Journal of bacteriology 13 33229461
2019 Insights to the Assembly of a Functionally Active Leptospiral ClpP1P2 Protease Complex along with Its ATPase Chaperone ClpX. ACS omega 13 31460415
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