Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ATP-dependent proteases are processive, meaning that they degrade full-length proteins into small peptide products without releasing large intermediates along the reaction pathway. In the case of the bacterial ATP-dependent protease ClpAP, ATP hydrolysis by the ClpA component has been proposed to be required for processive proteolysis of full-length protein substrates. We present here data showing that in the absence of the ATPase subunit ClpA, the protease subunit ClpP can degrade full-length protein substrates processively, albeit at a greatly reduced rate. Moreover, the size distribution of peptide products from a ClpP-catalyzed digest is remarkably similar to the size distribution of products from a ClpAP-catalyzed digest. The ClpAP- and ClpP-generated peptide product size distributions are fitted well by a sum of multiple underlying Gaussian peaks with means at integral multiples of approximately 900 Da (7-8 amino acids). Our results are consistent with a mechanism in which ClpP controls product sizes by alternating between translocation in steps of 7-8 (+/-2-3) amino acid residues and proteolysis. On the structural and molecular level, the step size may be controlled by the spacing between the ClpP active sites, and processivity may be achieved by coupling peptide bond hydrolysis to the binding and release of substrate and products in the protease chamber.
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PMID:ClpP hydrolyzes a protein substrate processively in the absence of the ClpA ATPase: mechanistic studies of ATP-independent proteolysis. 1883 65

The ATP-dependent caseinolytic proteases (Clp) are important in resistance against environmental stresses, antibiotic treatments and host immune defences for a number of pathogenic bacteria. ClpP is the proteolytic subunit, whilst ClpA acts both as a chaperone and as an ATPase driving the degradation of damaged or mis-made proteins. The gastric pathogen Helicobacter pylori infects approximately half of the world's population and can cause gastric or duodenal ulcers, gastric malignancies and mucosa-associated lymphoid tissue lymphomas. The conditions of its in vivo environment expose the organism to host immune cells and upon treatment, antibiotics, conditions likely to cause protein damage. We generated isogenic nonpolar mutants in strain SS1 of clpP and clpA and double mutants with both genes inactivated. Such mutants showed increased sensitivity to antibacterials causing protein damage and/or oxidative stress, in addition to a reduced survival in human macrophages. In the mouse infection model the double mutant SS1 clpAP lacked all ability to colonize the murine host. This suggests that the ability to recover from protein damage is of key importance in the pathogenesis of this organism.
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PMID:Helicobacter pylori mutants defective in the clpP ATP-dependant protease and the chaperone clpA display reduced macrophage and murine survival. 1899 3

A common feature of chaperone-proteases is architectural two-fold symmetry across the proteolytic cylinder. Here we investigate the role of symmetry for the function of ClpAP and ClpXP assemblies. We generated asymmetric ClpP particles in which the two rings differ in ClpA and ClpX binding capability and/or in proteolytic activity. Rapid-kinetic fluorescence measurements and steady-state experiments indicate that single 2:1 ClpAP or ClpXP complexes are as efficient in substrate degradation as two 1:1 ClpAP or ClpXP assemblies. This implies that the two chaperone components work independently. However, an asymmetric ClpP particle composed of one active and one inactive ring can stimulate ATPase activity of ClpA regardless of whether ClpA binds to the active ring or to the opposite side of ClpP, across the ring of inactivated protease. Thus, we propose that conformational transitions in ClpP are concerted and allosteric effects are transferred simultaneously to both associated chaperones, leading to synchronized activation.
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PMID:Optimal efficiency of ClpAP and ClpXP chaperone-proteases is achieved by architectural symmetry. 1936 79

The Clp protease ATPase subunit and chaperone ClpX is dispensable in some bacteria, but it is thought to be essential in others, including streptococci and lactococci. We confirm that clpX is essential in the Rx strain of Streptococcus pneumoniae but show that the requirement for clpX can be relieved by point mutations, frame shifts, or deletion of the gene spr1630, which is found in many isolates of S. pneumoniae. Homologs occur frequently in Staphylococcus aureus as well as in a few strains of Listeria monocytogenes, Lactobacillus johnsonii, and Lactobacillus rhamnosus. In each case, the spr1630 homolog is accompanied by a putative transcriptional regulator with an HTH DNA binding motif. In S. pneumoniae, the spr1630-spr1629 gene pair, accompanied by a RUP element, occurs as an island inserted between the trpA and cclA genes in 15 of 22 sequenced genomes.
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PMID:spr1630 is responsible for the lethality of clpX mutations in Streptococcus pneumoniae. 1946 54

The Escherichia coli ATP-dependent protease, ClpAP, is composed of the hexameric ATPase/protein-unfoldase, ClpA, and the tetradecameric proteolytic component, ClpP. ClpP proteolytically degrades folded proteins only when associated with the motor protein ClpA or ClpX, both of which use ATP binding and/or hydrolysis to unfold and translocate proteins into the tetradecameric serine protease ClpP. In addition to ClpA's role in regulating the proteolytic activity of ClpP, ClpA catalyzes protein unfolding of proteins that display target sequences to "remodel" them, in vivo, for regulatory roles beyond proteolytic degradation. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA first requires nucleoside triphosphate binding to assemble into an oligomeric form with protein substrate binding activity. In addition to this nucleotide driven assembly activity, ClpA self-associates in the absence of nucleoside triphosphate binding. An examination of the energetics of the nucleotide driven assembly process cannot be performed without a thermodynamic model of the self-assembly process in the absence of nucleotide cofactor. Here we report an examination of the self-association properties of the E. coli ClpA protein unfoldase through the application of analytical ultracentrifugation and light scattering techniques, including sedimentation velocity, sedimentation equilibrium, and dynamic light scattering approaches. In contrast to published results, application of these approaches reveals that ClpA exists in a monomer-tetramer equilibrium (300 mM NaCl, 10 mM MgCl(2), and 25 mM HEPES, pH 7.5 at 25 degrees C). The implications of these results for the E. coli ClpA self-association and ligand linked association activities are discussed.
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PMID:The Escherichia coli ClpA molecular chaperone self-assembles into tetramers. 1965 Jun 43

ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.
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PMID:Both ATPase domains of ClpA are critical for processing of stable protein structures. 1972 81

Clp proteases are the most widespread energy-dependent proteases in bacteria. Their two-component architecture of protease core and ATPase rings results in an inventory of several Clp protease complexes that often coexist. Here, we present insights into Clp protease function, from their assembly to substrate recruitment and processing, and how this is coupled to the expense of energy.
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PMID:Clp chaperone-proteases: structure and function. 1973 26

Regulated proteolysis by ATP-dependent proteases is universal in all living cells. In Bacillus subtilis, the degradation of the competence transcription factor ComK is mediated by a ternary complex involving the adaptor protein MecA and the ATP-dependent protease ClpCP. Here we demonstrate that a C-terminal, 98-amino acid domain of MecA (residues 121-218) serves as a non-recycling, degradation tag and targets a variety of fusion proteins to the ClpCP protease for degradation. MecA-(121-218) facilitates productive oligomerization of ClpC, stimulates the ATPase activity of ClpC, and allows the activated ClpC complex to stably associate with ClpP. Importantly, the ClpCP protease undergoes dynamic cycles of assembly and disassembly, which are triggered by association with MecA and the degradation of MecA, respectively.
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PMID:Molecular determinants of MecA as a degradation tag for the ClpCP protease. 1976 95

ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.
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PMID:Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine. 1991 67

ClpP is a serine protease whose active sites are sequestered in a cavity enclosed between two heptameric rings of subunits. The ability of ClpP to process folded protein substrates depends on its being partnered by an AAA+ ATPase/unfoldase, ClpA or ClpX. In active complexes, substrates are unfolded and fed along an axial channel to the degradation chamber inside ClpP. We have used cryoelectron microscopy at approximately 11-A resolution to investigate the three-dimensional structure of ClpP complexed with either one or two end-mounted ClpA hexamers. In the absence of ClpA, the apical region of ClpP is sealed; however, it opens on ClpA binding, creating an access channel. This region is occupied by the N-terminal loops (residues 1-17) of ClpP, which tend to be poorly visible in crystal structures, indicative of conformational variability. Nevertheless, we were able to model the closed-to-open transition that accompanies ClpA binding in terms of movements of these loops; in particular, "up" conformations of the loops correlate with the open state. The main part of ClpP, the barrel formed by 14 copies of residues 18-193, is essentially unchanged by the interaction with ClpA. Using difference mapping, we localized the binding site for ClpA to a peripheral pocket between adjacent ClpP subunits. Based on these observations, we propose that access to the ClpP degradation chamber is controlled allosterically by hinged movements of its N-terminal loops, which the symmetry-mismatched binding of ClpA suffices to induce.
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PMID:Binding of the ClpA unfoldase opens the axial gate of ClpP peptidase. 2023 30


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