Transcription from heterologous rRNA operon promoters in chloroplasts reveals requirement for specific activating factors

The abundant molecular chaperone Hsp90 is a key regulator of protein structure in the cytosol of eukaryotic cells. Although under physiological conditions a specific subset of proteins is substrate for Hsp90, under stress conditions Hsp90 seems to perform more general functions. However, the underlying mechanism of Hsp90 remained enigmatic. Here, we analyzed the function of conserved Hsp90 domains. We show that Hsp90 possesses two chaperone sites located in the N- and C-terminal fragments, respectively. The C-terminal fragment binds to partially folded proteins in an ATP-independent way potentially regulated by cochaperones. The N-terminal domain contains a peptide binding site that seems to bind preferentially peptides longer than 10 amino acids. Peptide dissociation is induced by ATP binding. Furthermore, the antitumor drug geldanamycin both inhibits the weak ATPase of Hsp90 and stimulates peptide release. We propose that the existence of two functionally different chaperone sites together with a substrate-selecting set of cochaperones allows Hsp90 to guide the folding of a subset of target proteins and, at the same time, to exhibit general chaperone functions.     

Change in the therapist: the role of patient-induced inspiration

BACKGROUND: The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. RESULTS: The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. CONCLUSIONS: The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes in protein structure as a result of calcium binding may facilitate phosphorylation. A small, but significant, movement of metal ions and sidechains could position catalytically important threonine residues for phosphorylation. The second calcium site represents a new calcium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in the active site could be transmitted to the peptide-binding domain.     

Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein sigma1

The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.     

Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.     

Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent

The Hsp90 chaperone is required for the activation of several families of eukaryotic protein kinases and nuclear hormone receptors, many of which are protooncogenic and play a prominent role in cancer. The geldanamycin antibiotic has antiproliferative and antitumor effects, as it binds to Hsp90, inhibits the Hsp90-mediated conformational maturation/refolding reaction, and results in the degradation of Hsp90 substrates. The structure of the geldanamycin-binding domain of Hsp90 (residues 9-232) reveals a pronounced pocket, 15 A deep, that is highly conserved across species. Geldanamycin binds inside this pocket, adopting a compact structure similar to that of a polypeptide chain in a turn conformation. This, and the pocket's similarity to substrate-binding sites, suggest that the pocket binds a portion of the polypeptide substrate and participates in the conformational maturation/refolding reaction.     

Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP

To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5'-(beta, gamma-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization.     

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.     

Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action

Applying stopped-flow fluorescence spectroscopy for measuring conformational changes of the DnaK molecular chaperone (bacterial Hsp70 homologue) and its binding to target peptide, we found that after ATP hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which possesses limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone. In the presence of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted back to the DnaK conformation, and the resulting DnaJ-DnaK(ADP) complex binds to peptide substrates more tightly. Formation of the DnaJ(substrate-DnaK(ADP)) complex is a rate-limiting reaction. The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK*(ADP) conformation. We conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosphatase activity of DnaK.     

Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication

Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP. Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence. Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected. Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP. N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain. They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions. Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant. Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction. Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site.     

Nucleotide binding to the C-terminal nucleotide binding domain of ArsA. Studies with an ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine

ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker. ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain. The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA. The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum. The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite. Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis. Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment. The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further. Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment. This fragment was not protected upon incubation with ATP alone, indicating that A2 might be unoccupied. However, upon incubation with ATP and antimonite, almost complete protection from trypsin was seen. ATP and FSBA together mimicked the effect of ATP and antimonite, implying that this fully protected conformation might be the result of both sites occupied with the nucleotide. It is proposed that the A1 site in ArsA is a high affinity ATP site, whereas the allosteric ligand antimonite is required to allow ATP binding to A2, resulting in catalytic cooperativity. Thus antimonite binding may act as a switch in regulating ATP binding to A2 and hence the ATPase activity of ArsA.     

An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators

Heat Shock Protein 70 kDa (Hsp70) family molecular chaperones play critical roles in protein folding and trafficking in all eukaryotic cells. The mechanisms by which Hsp70 family chaperones are regulated, however, are only partly understood. BAG-1 binds the ATPase domains of Hsp70 and Hsc70, modulating their chaperone activity and functioning as a competitive antagonist of the co-chaperone Hip. We describe the identification of a family of BAG-1-related proteins from humans (BAG-2, BAG-3, BAG-4, BAG-5), the invertebrate Caenorhabditis elegans (BAG-1, BAG-2), and the fission yeast Schizosaccharomyces pombe (BAG-1A, BAG-1B). These proteins all contain a conserved approximately 45-amino acid region near their C termini (the BAG domain) that binds Hsc70/Hsp70, but they differ widely in their N-terminal domains. The human BAG-1, BAG-2, and BAG-3 proteins bind with high affinity (KD congruent with 1-10 nM) to the ATPase domain of Hsc70 and inhibit its chaperone activity in a Hip-repressible manner. The findings suggest opportunities for specification and diversification of Hsp70/Hsc70 chaperone functions through interactions with various BAG-family proteins.     

Characterization of D10S and K71E mutants of human cytosolic hsp70

To determine the effect of mutations at the nucleotide-binding site of recombinant Hsp70 on its interaction with protein and peptide substrates, point mutations were made at D10 and K71, two residues at the active site. The D10S mutation weakened both ATP and ADP binding, while the K71E mutation weakened only ATP binding. In binding experiments using Hsp70 with no bound nucleotide, the mutated Hsp70s interacted with clathrin and peptide just like the wild-type Hsp70. However, the D10 mutation completely abolished the effects of both ATP and ADP on peptide and clathrin binding. The K71 mutation also abolished the effect of ATP on substrate binding, but ADP, which still bound tightly, had its normal effect on substrate binding. In addition, the D10S and K71E mutants had greatly reduced ability to uncoat clathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0, and undergo polymerization induced by YDJ1 in the presence of ATP. We conclude, first, that nucleotides must bind strongly to Hsp70 to affect substrate binding and, second, that interaction of Hsp70 with DnaJ homologues may also require a strongly bound ATP.     

The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors

The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70's cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.     

Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.     

CNS1 encodes an essential p60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin 40 mutations and interacts with Hsp90

Cyclophilins are cis-trans-peptidyl-prolyl isomerases that bind to and are inhibited by the immunosuppressant cyclosporin A (CsA). The toxic effects of CsA are mediated by the 18-kDa cyclophilin A protein. A larger cyclophilin of 40 kDa, cyclophilin 40, is a component of Hsp90-steroid receptor complexes and contains two domains, an amino-terminal prolyl isomerase domain and a carboxy-terminal tetratricopeptide repeat (TPR) domain. There are two cyclophilin 40 homologs in the yeast Saccharomyces cerevisiae, encoded by the CPR6 and CPR7 genes. Yeast strains lacking the Cpr7 enzyme are viable but exhibit a slow-growth phenotype. In addition, we show here that cpr7 mutant strains are hypersensitive to the Hsp90 inhibitor geldanamycin. When overexpressed, the TPR domain of Cpr7 alone complements both cpr7 mutant phenotypes, while overexpression of the cyclophilin domain of Cpr7, full-length Cpr6, or human cyclophilin 40 does not. The open reading frame YBR155w, which has moderate identity to the yeast p60 homolog STI1, was isolated as a high-copy-number suppressor of the cpr7 slow-growth phenotype. We show that this Sti1 homolog Cns1 (cyclophilin seven suppressor) is constitutively expressed, essential, and found in protein complexes with both yeast Hsp90 and Cpr7 but not with Cpr6. Cyclosporin A inhibited Cpr7 interactions with Cns1 but not with Hsp90. In summary, our findings identify a novel component of the Hsp90 chaperone complex that shares function with cyclophilin 40 and provide evidence that there are functional differences between two conserved sets of Hsp90 binding proteins in yeast.     

BAG-1 modulates the chaperone activity of Hsp70/Hsc70

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.     

Neuronal nitric-oxide synthase is regulated by the Hsp90-based chaperone system in vivo

It is established that the multiprotein heat shock protein 90 (hsp90)-based chaperone system acts on the ligand binding domain of the glucocorticoid receptor (GR) to form a GR.hsp90 heterocomplex and to convert the receptor ligand binding domain to the steroid-binding state. Treatment of cells with the hsp90 inhibitor geldanamycin inactivates steroid binding activity and increases the rate of GR turnover. We show here that a portion of neuronal nitric-oxide synthase (nNOS) exists as a molybdate-stabilized nNOS. hsp90 heterocomplex in the cytosolic fraction of human embryonic kidney 293 cells stably transfected with rat nNOS. Treatment of human embryonic kidney 293 cells with geldanamycin both decreases nNOS catalytic activity and increases the rate of nNOS turnover. Similarly, geldanamycin treatment of nNOS-expressing Sf9 cells partially inhibits nNOS activation by exogenous heme. Like the GR, purified heme-free apo-nNOS is activated by the DE52-retained fraction of rabbit reticulocyte lysate, which also assembles nNOS. hsp90 heterocomplexes. However, in contrast to the GR, heterocomplex assembly with hsp90 is not required for increased heme binding and nNOS activation in this cell-free system. We propose that, in vivo, where access by free heme is limited, the complete hsp90-based chaperone machinery is required for sustained opening of the heme binding cleft and nNOS activation, but in the heme-containing cell-free nNOS-activating system transient opening of the heme binding cleft without hsp90 is sufficient to facilitate heme binding.     

GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1

The BAG-1 protein appears to inhibit cell death by binding to Bcl-2, the Raf-1 protein kinase, and certain growth factor receptors, but the mechanism of inhibition remains enigmatic. BAG-1 also interacts with several steroid hormone receptors which require the molecular chaperones Hsc70 and Hsp90 for activation. Here we show that BAG-1 is a regulator of the Hsc70 chaperone. BAG-1 binds to the ATPase domain of Hsc70 and, in cooperation with Hsp40, stimulates Hsc70's steady-state ATP hydrolysis activity approximately 40-fold. Similar to the action of the GrpE protein on bacterial Hsp70, BAG-1 accelerates the release of ADP from Hsc70. Thus, BAG-1 regulates the Hsc70 ATPase in a manner contrary to the Hsc70-interacting protein Hip, which stabilizes the ADP-bound state. Intriguingly, BAG-1 and Hip compete in binding to the ATPase domain of Hsc70. Our results reveal an unexpected diversity in the regulation of Hsc70 and raise the possibility that the observed anti-apoptotic function of BAG-1 may be exerted through a modulation of the chaperone activity of Hsc70 on specific protein folding and maturation pathways.     

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.