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Bernd Bukau

Publications and source records attributed to Bernd Bukau.

At least 37 records · Page 2Linked to original sources
PubMed2004

Substrate recognition by the AAA+ chaperone ClpB.

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

Adenosine Triphosphatases↗
PubMed2004

Trigger factor binds to ribosome-signal-recognition particle (SRP) complexes and is excluded by binding of the SRP receptor.

Trigger factor (TF) and signal recognition particle (SRP) bind to the bacterial ribosome and are both crosslinked to protein L23 at the peptide exit, where they interact with emerging nascent peptide chains. It is unclear whether TF and SRP exclude one another from their ribosomal binding site(s). Here we show that SRP and TF can bind simultaneously to ribosomes or ribosome nascent-chain complexes exposing a SRP-specific signal sequence. Based on changes of the crosslinking pattern and on results obtained by fluorescence measurements using fluorescence-labeled SRP, TF binding induces structural changes in the ribosome-SRP complex. Furthermore, we show that binding of the SRP receptor, FtsY, to ribosome-bound SRP excludes TF from the ribosome. These results suggest that TF and SRP sample nascent chains on the ribosome in a nonexclusive fashion. The decision for ribosome nascent-chain complexes exposing a signal sequence to enter SRP-dependent membrane targeting seems to be determined by the binding of SRP, which is stabilized by signal sequence recognition, and promoted by the exclusion of TF due to the binding of the SRP receptor to ribosome-bound SRP.

Bacterial Proteins↗
PubMed2004

A PDZ switch for a cellular stress response.

The accumulation of misfolded porins in the periplasm of bacteria triggers a proteolytic cascade, initiated by activation of DegS, a member of the family of HtrA proteases. Activation of DegS ultimately leads to the expression of genes encoding the periplasmic protein folding machinery. A new study now reveals that binding of exposed C-termini of unassembled porins to the PDZ domain of DegS induces structural rearrangements that activate the catalytic site of the protease domain.

Bacteria↗
PubMed2004

Influence of GrpE on DnaK-substrate interactions.

The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides. This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively. Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor. However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP. In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids. A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding. Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP. These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion.

Adenosine Triphosphate↗
PubMed2004

Molecular chaperones: structure of a protein disaggregase.

The ring-forming molecular chaperone Hsp104/ClpB is a member of the AAA+ protein family which rescues proteins from aggregated states. The newly determined crystal structure of ClpB provides new insights into the mechanism of protein disaggregation, suggesting a crowbar activity mediated by a unique coiled-coil domain.

Endopeptidase Clp↗
PubMed2004

Trigger factor peptidyl-prolyl cis/trans isomerase activity is not essential for the folding of cytosolic proteins in Escherichia coli.

The ribosome-associated Trigger Factor (TF) cooperates with the DnaK system to assist the folding of newly synthesized polypeptides in Escherichia coli. TF unifies two functions in one to promote proper protein folding in vitro. First, as a chaperone it binds to unfolded protein substrates, thereby preventing aggregation and supporting productive folding. Second, TF catalyzes the cis/trans isomerization of peptidyl-prolyl bonds, which can be a rate-limiting step in protein folding. Here, we investigated whether the peptidyl-prolyl cis/trans isomerase (PPIase) function is essential for the folding activity of TF in vitro and in vivo by separating these two TF activities through site-directed mutagenesis of the PPIase catalytic center. Of the four different TF variants carrying point mutations in the PPIase domain, only the exchange of the conserved residue Phe-198 to Ala (TF F198A) abolished the PPIase activity of TF toward both a tetrapeptide and the model protein substrate RNase T1 in vitro. In contrast, all other activities of TF F198A tested were comparable with wild type TF. TF F198A retained a similar binding specificity toward membrane-bound peptides, assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in vitro, and associated with nascent polypeptides in an in vitro transcription/translation system. Importantly, expression of the TF F198A encoding gene complemented the synthetic lethality of DeltatigDeltadnaK cells and prevented global protein misfolding at temperatures between 20 and 34 degrees C in these cells. We conclude that the PPIase activity is not required for the function of TF in folding of newly synthesized proteins.

Cytosol↗
PubMed2004

Unscrambling an egg: protein disaggregation by AAA+ proteins.

Aprotein quality control system, consisting of molecular chaperones and proteases, controls the folding status of proteins and prevents the aggregation of misfolded proteins by either refolding or degrading aggregation-prone species. During severe stress conditions this protection system can be overwhelmed by high substrate load, resulting in the formation of protein aggregates. In such emergency situations, Hsp104/ClpB becomes a key player for cell survival, as it has the extraordinary capacity to rescue proteins from an aggregated state in cooperation with an Hsp70 chaperone system. The ring-forming Hsp104/ClpB chaperone belongs to the AAA+ protein superfamily, which in general drives the assembly and disassembly of protein complexes by ATP-dependent remodelling of protein substrates. A disaggregation activity was also recently attributed to other eubacterial AAA+ proteins, while such an activity has not yet been identified in mammalian cells. In this review, we report on new insights into the mechanism of protein disaggregation by AAA+ proteins, suggesting that these chaperones act as molecular crowbars or ratchets.

Journal Article↗
PubMed2003

Dimerization of the human E3 ligase CHIP via a coiled-coil domain is essential for its activity.

The Hsp70-interacting E3-ubiquitin ligase CHIP has been implicated in the decision as to whether a target protein enters the refolding or the degradation pathway. To further characterize the activity of CHIP we purified untagged Homo sapiens and Drosophila melanogaster CHIP (hCHIP, dCHIP). In contrast to other E3-ubiquitin ligases, both hCHIP and dCHIP proteins formed homodimers at physiological concentrations. We identified a predicted coiled-coil region in a mixed charge segment of the hCHIP and dCHIP sequence and found it to be necessary and sufficient for dimer formation. A mutant of hCHIP lacking this segment (hCHIPdelta-(128-229)) was incapable of dimer formation, but the segment by itself (hCHIP-(128-229)) readily dimerized. Furthermore, we demonstrated that dimerization is a prerequisite for activity of hCHIP in the reconstituted ubiquitination assay. Control of dimerization may thus provide a mechanism for regulation of CHIP activity.

Animals↗
PubMed2003

Poly-L-lysine enhances the protein disaggregation activity of ClpB.

The Hsp100 protein ClpB is a member of the AAA+ protein family that mediates the solubilization of aggregated proteins in cooperation with the DnaK chaperone system. Unstructured polypeptides such as casein or poly-L-lysine have been shown to stimulate the ATPase activity of ClpB and thus may both act as substrates. Here we compared the effects of alpha-casein and poly-L-lysine on the ATPase and chaperone activities of ClpB. alpha-Casein stimulated ATP hydrolysis by both AAA domains of ClpB and inhibited the ClpB-dependent solubilization of aggregated proteins if present in excess. In contrast, poly-L-lysine stimulated exclusively the ATPase activity of the second AAA domain and increased the disaggregation activity of ClpB. Thus poly-L-lysine does not act as substrate, but rather represents an effector molecule, which enhances the chaperone activity of ClpB.

Adenosine Triphosphatases↗
PubMed2003

Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange.

Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, sigma 32, out-competes upon temperature up-shift the housekeeping sigma-factor, sigma 70, for binding to core RNA polymerase and initiates heat shock gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37 degrees C, indicating that sigma 32 adopts a highly flexible structure. At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock promoters. The correlated exchange is shown to constitute a reversible unfolding with a half-life of about 30 min due to a temperature-dependent decrease in stabilization energy. We propose that this gradual decrease in stabilization energy of domain sigma 2 with increasing temperatures facilitates the unfolding of sigma 32 by the AAA+ protease FtsH thereby decreasing its half-life. Taken together our data show that the sigma 2 domain of sigma 32 can act as a thermosensor, which might be important for the heat shock regulation.

Amides↗
PubMed2003

Characterization of a trap mutant of the AAA+ chaperone ClpB.

The AAA+ protein ClpB mediates the solubilization of protein aggregates in cooperation with the DnaK chaperone system (KJE). The order of action of ClpB and KJE on aggregated proteins is unknown. We describe a ClpB variant with mutational alterations in the Walker B motif of both AAA domains (E279A/E678A), which binds but does not hydrolyze ATP. This variant associates in vitro and in vivo in a stable manner with protein substrates, demonstrating direct interaction of ClpB with protein aggregates for the first time. Substrate interaction is strictly dependent on ATP binding to both AAA domains of ClpB. The unique substrate binding properties of the double Walker B variant allowed to dissect the order of ClpB and DnaK action during disaggregation reactions. ClpB-E279A/E678A outcompetes the DnaK system for binding to the model substrate TrfA and inhibits the dissociation of small protein aggregates by DnaK only, indicating that ClpB acts prior to DnaK on protein substrates.

Adenosine Triphosphatases↗
PubMed2003

Refolding of substrates bound to small Hsps relies on a disaggregation reaction mediated most efficiently by ClpB/DnaK.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones. The mechanistic basis of this refolding process is poorly defined. We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously. Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK. While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps. Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress. We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK.

Animals↗
PubMed2003

Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity.

ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structure-function analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAA-domains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.

Adenosine Triphosphatases↗
PubMed2003

MecA, an adaptor protein necessary for ClpC chaperone activity.

ClpC of Bacillus subtilis is an ATP-dependent HSP100Clp protein involved in general stress survival. A complex of ClpC with the protease ClpP and the adaptor protein MecA also controls competence development by regulated proteolysis of the transcription factor ComK. We investigated the in vitro chaperone activity of ClpC and found that the presence of MecA was crucial for the major chaperone activities of ClpC. In particular, MecA enabled ClpC to solubilize and refold aggregated proteins. Finally, in the presence of ClpP, MecA allowed the ClpC-dependent degradation of unfolded or heat-aggregated proteins. This study demonstrates that adaptor proteins like MecA through interaction with their cognate ClpC proteins can have a dual role in the protein quality-control network by rescuing, or together with ClpP, by degrading, aggregated proteins. MecA can thereby coordinate substrate targeting with ClpC activation, adding another layer to the regulation of HSP100/Clp protein activity.

Adenosine Triphosphatases↗
PubMed2003

Targeted delivery of an ssrA-tagged substrate by the adaptor protein SspB to its cognate AAA+ protein ClpX.

In the bacterial cytosol, degradation of ssrA-tagged proteins is primarily carried out by the proteolytic machine ClpXP in a process which is stimulated by a ClpX-specific adaptor protein, SspB. Here we elucidate the steps required for binding and transfer of ssrA-tagged substrates from SspB to ClpX. The N-terminal region of SspB is essential for its interaction with ssrA-tagged substrates, while a short conserved region at the C terminus of SspB interacts specifically with the N domain of ClpX. A single point mutation within the conserved C-terminal region of SspB is sufficient to abolish the SspB-mediated degradation of ssrA-tagged proteins by ClpXP. We propose that this region represents a common motif for the recognition of ClpX as the C-terminal region of SspB shares considerable homology with the other ClpX-specific adaptor protein, RssB. Through docking of SspB to the N-terminal domain of ClpX, the substrate is delivered to the substrate binding site in ClpX.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2003

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Chaperonin 60↗