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)

The Escherichia coli GroEL subunit consists of three domains with distinct functional roles. To understand the role of each of the three domains, the effects of mutating a single residue in each domain (Y203C at the apical, T89W at the equatorial, and C138W at the intermediate domain) were studied in detail, using three different enzymes (enolase, lactate dehydrogenase, and rhodanese) as refolding substrates. By analyzing the effects of each mutation, a transfer of signals was detected between the apical domain and the equatorial domain. A signal initiated by the equatorial domain triggers the release of polypeptide from the apical domain. This trigger was independent of nucleotide hydrolysis, as demonstrated using an ATPase-deficient mutant, and, also, the conditions for successful release of polypeptide could be modified by a mutation in the apical domain, suggesting that the polypeptide release mechanism of GroEL is governed by chaperonin-target affinities. Interestingly, a reciprocal signal from the apical domain was suggested to occur, which triggered nucleotide hydrolysis in the equatorial domain. This signal was disrupted by a mutation in the intermediate domain to create a novel ternary complex in which GroES and refolding protein are simultaneously bound in a stable ternary complex devoid of ATPase activity. These results point to a multitude of signals which govern the overall chaperonin mechanism.
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PMID:Functional communications between the apical and equatorial domains of GroEL through the intermediate domain. 1062 39

IscU, a NifU-like Fe/S-escort protein, binds to and stimulates the ATPase activity of Hsc66, a hsp70-type molecular chaperone. We present evidence that stimulation arises from interactions of IscU with the substrate-binding site of Hsc66. IscU inhibited the ability of Hsc66 to suppress the aggregation of the denatured model substrate proteins rhodanese and citrate synthase, and calorimetric and surface plasmon resonance measurements showed that ATP destabilizes Hsc66.IscU complexes in a manner expected for hsp70-substrate complexes. Studies on the interaction of IscU with Hsc66 truncation mutants further showed that IscU does not bind the isolated ATPase domain of Hsc66 but does bind and stimulate a mutant containing the ATPase domain and substrate binding beta-sandwich subdomain. These results support a role for IscU as a substrate for Hsc66 and suggest a specialized function for Hsc66 in the assembly, stabilization, or transfer of Fe/S clusters formed on IscU.
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PMID:The Fe/S assembly protein IscU behaves as a substrate for the molecular chaperone Hsc66 from Escherichia coli. 1105 47

The chaperonins GroEL and GroES were shown to facilitate the refolding of urea-unfolded rhodanese in an ATP-dependent process at 25 or 37 degrees C. A diminished chaperonin activity was observed at 10 degrees C, however. At low temperature, GroEL retains its ability to form a complex with urea-unfolded rhodanese or with GroES. GroEL is also able to bind ATP at 10 degrees C. Interestingly, the ATPase activity of GroEL was highly decreased at low temperatures. Hydrolysis of ATP by GroEL was 60% less at 10 degrees C than at 25 degrees C. We conclude that the reduced hydrolysis of ATP by GroEL is a major but perhaps not the only factor responsible for the diminished chaperonin activity at 10 degrees C. GroEL may function primarily at higher temperatures in which the ability of GroEL to hydrolyze ATP is not compromised.
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PMID:The lower hydrolysis of ATP by the stress protein GroEL is a major factor responsible for the diminished chaperonin activity at low temperature. 1122 29

The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches. HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli. NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains. After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively. Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape. Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation. Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity. Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding.
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PMID:Domain structure of the HSC70 cochaperone, HIP. 1168 74

GroELx and GroESx proteins of symbiotic X-bacteria from Amoeba proteus were overproduced in Escherichia coli transformed with pAJX91 and pUXGPRM, respectively, and their chaperonin functions were assayed. We utilized sigma(70)-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37 degrees C to minimize coexpression of host groE of E. coli. For purifying the proteins, we applied the principle of heat stability for GroELx and pI difference for GroESx to minimize copurification with the hosts GroEL and GroES, respectively. After ultracentrifugation in a sucrose density gradient, the yield and purity of GroELx were 56 and 89%, respectively. The yield and purity of GroESx after anion-exchange chromatography were 62 and 91%, respectively. Purified GroELx had an ATPase activity of 53.2 nmol Pi released/min/mg protein at 37 degrees C. The GroESx protein inhibited ATPase activity of GroELx to 60% of the control at a ratio of 1 for GroESx-7mer/GroELx-14mer. GroESLx helped refolding of urea-unfolded rhodanese up to 80% of the native activity at 37 degrees C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx(7) and GroELx(14) in two stacks of GroELx(7). In this study, we developed a method for the purification of GroESLx and demonstrated that their chaperonin function is homologous to GroESL of E. coli.
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PMID:Purification and characterization of the GroESLx chaperonins from the symbiotic X-bacteria in Amoeba proteus. 1172 84

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.
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PMID:Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU. 1199 2

The ATPase Cdc48 is required for membrane fusion and protein degradation. Recently it has been suggested that Cdc48 in a complex with Ufd1 and Npl4 acts as an ubiquitin-dependent chaperone. Here it is shown that recombinant Cdc48 alone can distinguish between the native and the non-native conformation of model substrates. First, Cdc48 prevents luciferase from aggregating following a heat shock. Second, it inhibits the aggregation of rhodanese upon dilution. Third, Cdc48 binds specifically to heat-denatured luciferase. These chaperone-like functions seem to be independent of ATPase activity. Furthermore, Cdc48 can act as a co-chaperone in the Hsc70-Hsp40 chaperone system. These results show that Cdc48 possesses chaperone-like activities and can functionally interact with Hsc70. Cdc48's ability to recognise denatured proteins can also be a source of unspecific binding in biochemical interaction experiments.
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PMID:Cdc48 can distinguish between native and non-native proteins in the absence of cofactors. 1204 80

We report the characterization of the first chaperonin (Mm-cpn) from a mesophilic archaeon, Methanococcus maripaludis. The single gene was cloned from genomic DNA and expressed in Escherichia coli to produce a recombinant protein of 543 amino acids. In contrast with other known archaeal chaperonins, Mm-cpn is fully functional in all respects under physiological conditions of 37 degrees C. The complex has Mg(2+)-dependent ATPase activity and can prevent the aggregation of citrate synthase. It promotes a high-yield refolding of guanidinium-chloride-denatured rhodanese in a nucleotide-dependent manner. ATP binding is sufficient to effect folding, but ATP hydrolysis is not essential.
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PMID:Nucleotide-dependent protein folding in the type II chaperonin from the mesophilic archaeon Methanococcus maripaludis. 1262

In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins. As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol. Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec). We find that the groESgroEL operon of M. mazei is unable to functionally replace groESgroEL in E. coli. However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo. The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system. This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium. Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction. These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea. Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.
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PMID:Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation. 1457 49

The studies of GroEL, almost exclusively, have been concerned with the function of the chaperonin under non-stress conditions, and little is known about the role of GroEL during heat shock. Being a heat shock protein, GroEL deserves to be studied under heat shock temperature. As a model for heat shock in vitro, we have investigated the interaction of GroEL with the enzyme rhodanese undergoing thermal unfolding at 43 degrees C. GroEL interacted strongly with the unfolding enzyme forming a binary complex. Active rhodanese (82%) could be recovered by releasing the enzyme from GroEL after the addition of several components, e.g. ATP and the co-chaperonin GroES. After evaluating the stability of the GroEL-rhodanese complex, as a function of the percentage of active rhodanese that could be released from GroEL with time, we found that the complex had a half-life of only one and half-hours at 43 degrees C; while, it remained stable at 25 degrees C for more than 2 weeks. Interestingly, the GroEL-rhodanese complex remained intact and only 13% of its ATPase activity was lost during its incubation at 43 degrees C. Further, rhodanese underwent a conformational change over time while it was bound to GroEL at 43 degrees C. Overall, our results indicated that the inability to recover active enzyme at 43 degrees C from the GroEL-rhodanese complex was not due to the disruption of the complex or aggregation of rhodanese, but rather to the partial loss of its ATPase activity and/or to the inability of rhodanese to be released from GroEL due to a conformational change.
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PMID:On the chaperonin activity of GroEL at heat-shock temperature. 1583 70


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