scholarly journals Mutation of the ATP-Binding Pocket of SSA1 Indicates That a Functional Interaction Between Ssa1p and Ydj1p Is Required for Post-translational Translocation Into the Yeast Endoplasmic Reticulum

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 501-512 ◽  
Author(s):  
Amie J McClellan ◽  
Jeffrey L Brodsky
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Binding Pocket ◽  
Atp Binding ◽  
Dominant Effect ◽  
Functional Interaction ◽  
Mutant Proteins

Abstract The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.

scholarly journals The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

2006 ◽  
Vol 401 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Fiona L. L. Stratford ◽  
Mohabir Ramjeesingh ◽  
Joanne C. Cheung ◽  
Ling-JUN Huan ◽  
Christine E. Bear
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Vital Role ◽  
Atp Binding ◽  
Primary Role ◽  
Binding Domains ◽  
Membrane Spanning ◽  
Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

scholarly journals Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

2010 ◽  
Vol 21 (6) ◽  
pp. 871-884 ◽  
Author(s):  
Atanas V. Koulov ◽  
Paul LaPointe ◽  
Bingwen Lu ◽  
Abbas Razvi ◽  
Judith Coppinger ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Protein Misfolding ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Inherited Disease ◽  
Hsp90 Chaperone ◽  
Terminal Domains

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

In vitro and in vivo activity of regorafenib (REGO) in drug-resistant gastrointestinal stromal tumors (GIST).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 10510-10510 ◽  
Author(s):  
Cesar Serrano-Garcia ◽  
Michael C. Heinrich ◽  
Meijun Zhu ◽  
Chandrajit P. Raut ◽  
Grant Eilers ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Initial Response ◽  
Binding Pocket ◽  
Atp Binding ◽  
Activation Loop ◽  
Line Therapy ◽  
Exon 11 ◽  
Clinical Trial Results

10510 Background: KIT and PDGFRA mutations (mut) are the crucial transforming events in most GISTs, and tyrosine kinase inhibitors (TKIs) with activity against KIT and PDGFRA, such as imatinib (IM) (front-line therapy) and sunitinib (SU) (second-line therapy), are effective treatments in GIST patients (pts). Resistance to IM and SU is commonly associated with evolution of secondary kinase mut. REGO is a multi-targeted TKI that inhibits KIT, PDGFR, and other oncologic targets and has recently shown benefit in pts with metastatic GIST after progression on standard treatments. We evaluated the in vitro and in vivo activity of REGO compared with IM, SU, and sorafenib (SOR) (a multi-TKI structurally related to REGO). Methods: REGO, IM, SU, and SOR inhibition of viability and KIT phosphorylation was assessed in human GIST cell lines and in Ba/F3 cells transformed by KIT oncoproteins with IM-resistant ATP binding pocket or activation-loop mut. KIT/PDGFRA genotyping was performed in GISTs responding or progressing on REGO in the academic phase II clinical trial. Results: In GISTs with KIT exon 11 mutant oncoproteins, REGO potently inhibited viability, KIT phosphorylation, and downstream effector phosphorylation (AKT, MAPK, S6). IM-resistant activation loop mut were more potently inhibited by REGO than SU, whereas the gatekeeper IM-resistant mut T670I was inhibited by both REGO and SU, and the common ATP-binding pocket mutant V654A was more potently inhibited by SU than REGO. Two GIST metastases progressing in one pt after initial response to REGO contained KIT V654A mut. SOR and REGO demonstrated comparable in vitro overall activity. Representative GIST cell line viability IC50s are shown in the Table (values in bold indicate expected clinical relevance). Conclusions: In vitro studies confirm REGO is a potent inhibitor of KIT exon 11 mut in GIST and appears to have stronger activity than SU against the most common KIT activation-loop mut observed in GIST. Ongoing clinical correlative analyses from REGO-treated study patients will be presented. [Table: see text]

scholarly journals The Sso7d protein ofSulfolobus solfataricus: in vitro relationship among different activities

Archaea ◽  
2002 ◽  
Vol 1 (2) ◽  
pp. 87-93 ◽  
Author(s):  
Annamaria Guagliardi ◽  
Laura Cerchia ◽  
Mosè Rossi
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Physiological Role ◽  
Protein Aggregates ◽  
Dependent Manner ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Negative Supercoiling

The physiological role of the nonspecific DNA-binding protein Sso7d from the crenarchaeonSulfolobus solfataricusis unknown. In vitro studies have shown that Sso7d promotes annealing of complementary DNA strands (Guagliardi et al. 1997), induces negative supercoiling (Lopez-Garcia et al. 1998), and chaperones the disassembly and renaturation of protein aggregates in an ATP hydrolysis-dependent manner (Guagliardi et al. 2000). In this study, we examined the relationships among the binding of Sso7d to double-stranded DNA, its interaction with protein aggregates, and its ATPase activity. Experiments with 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that exposed hydrophobic surfaces in Sso7d are responsible for interactions with protein aggregates and double-stranded DNA, whereas the site of ATPase activity has a non-hydrophobic character. The interactions of Sso7d with double-stranded DNA and with protein aggregates are mutually exclusive events, suggesting that the disassembly activity and the DNA-related activities of Sso7d may be competitive in vivo. In contrast, the hydrolysis of ATP by Sso7d is independent of the binding of Sso7d to double-stranded DNA or protein aggregates.

scholarly journals Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

scholarly journals ATPase activity of the DEAD-box protein Dhh1 controls processing body formation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Christopher Frederick Mugler ◽  
Maria Hondele ◽  
Stephanie Heinrich ◽  
Ruchika Sachdev ◽  
Pascal Vallotton ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Liquid Droplets ◽  
Processing Bodies ◽  
Dead Box ◽  
Posttranscriptional Gene Regulation ◽  
The Dead ◽  
Processing Body

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

scholarly journals Bacillus subtilis Smc condenses chromosomes in a heterologous cell system, which is down-regulated by ScpAB

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Tobias Knust ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Cell System ◽  
Epifluorescence Microscopy ◽  
Live Cells ◽  
Atp Binding ◽  
E Coli ◽  
Negative Effect

Abstract Objective Structural maintenance of chromosomes (SMC) proteins are key players in chromosome dynamics in all types of organisms. The so-called condensin subfamily is essential for chromosome condensation in eukaryotic cells, as is the bacterial SMC complex (called MukBEF in Escherichia coli). We expressed the Bacillus subtilis Smc protein and its two complex partners ScpA and ScpB in E. coli cells, and monitored effects on chromosome compaction by DNA staining of live cells using epifluorescence microscopy. Data description We show that expression of BsSmc leads to strong chromosome compaction, while expression of ScpAB does not show any effect. Chromosome compaction by Smc was also found for mutant versions lacking ATP binding or ability for head engagement, and was counteracted by concomitant expression of ScpAB. Our findings show that the SMC complex can act as autonomous condensation system in a heterologous bacterial host system, for which neither ATP binding nor ATP hydrolysis are required. Our investigation suggests that the negative effect on compaction activity of Smc exerted by ScpAB in vivo does not involve an effect on ATPase activity, but more likely a stabilization of the engagement of head domains, which in turn may affect ATPase activity.

scholarly journals In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis

1998 ◽  
Vol 143 (4) ◽  
pp. 901-910 ◽  
Author(s):  
Wolfgang M.J. Obermann ◽  
Holger Sondermann ◽  
Alicia A. Russo ◽  
Nikola P. Pavletich ◽  
F. Ulrich Hartl
Keyword(s):  
Crystal Structure ◽  
Atpase Activity ◽  
Binding Site ◽  
Atp Hydrolysis ◽  
Specific Inhibitor ◽  
Nucleotide Binding ◽  
Basic Mechanism ◽  
Atp Binding ◽  
Terminal Domain

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis–dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

scholarly journals Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain

2021 ◽  
Author(s):  
Rongde Qiu ◽  
Jun Zhang ◽  
Jeremy D. Rotty ◽  
Xin Xiang
Keyword(s):  
Atp Hydrolysis ◽  
Cytoplasmic Dynein ◽  
The Other ◽  
Atp Binding ◽  
Motor Activation ◽  
Early Endosomes ◽  
Arginine Finger ◽  
Atp Binding And Hydrolysis

SummaryCytoplasmic dynein is activated by dynactin and cargo adapters in vitro, and the activation also needs LIS1 (Lissencephaly 1) in vivo. How this process is regulated remains unclear. Here we found in Aspergillus nidulans that a dynein AAA4 arginine-finger mutation bypasses the requirement of LIS1 for dynein activation driven by the early endosomal adapter HookA. As the AAA4 arginine-finger is implicated in AAA3 ATP hydrolysis, we examined AAA3 mutants defective in ATP binding and hydrolysis respectively. Astonishingly, blocking AAA3 ATP hydrolysis allows dynein activation by dynactin in the absence of LIS1 or HookA. As a consequence, dynein accumulates at microtubule minus ends while early endosomes stay near the plus ends. On the other hand, blocking AAA3 ATP binding abnormally prevents LIS1 from being dissociated from dynein upon motor activation. Thus, the AAA3 ATPase cycle regulates the coordination between dynein activation and cargo binding as well as the dynamic dynein-LIS1 interaction.

scholarly journals Isolation and Characterization of a Second Subunit of Molecular Chaperonin from Pyrococcus kodakaraensis KOD1: Analysis of an ATPase-Deficient Mutant Enzyme

1999 ◽  
Vol 65 (4) ◽  
pp. 1801-1805 ◽  
Author(s):  
Michi Izumi ◽  
Shinsuke Fujiwara ◽  
Masahiro Takagi ◽  
Shigenori Kanaya ◽  
Tadayuki Imanaka
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Deficient Mutant ◽  
Α Subunit ◽  
Mutant Proteins ◽  
E Coli ◽  
Isolation And Characterization ◽  
Insoluble Form ◽  
Cobyric Acid

ABSTRACT The cpkA gene encoding a second (α) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (β subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.

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