Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin.

Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure-antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2-10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin.     

The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.     

The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties

DnaK is a molecular chaperone that promotes cell survival during stress by preventing protein misfolding. The chaperone activity is regulated by nucleotide binding and hydrolysis events in the N-terminal ATPase domain, which in turn mediate substrate binding and release in the C-terminal substrate binding domain. In this study we determined that ATP hydrolysis was the rate limiting step in the ATPase cycle of Agrobacterium tumefaciens DnaK (Agt DnaK); however the data suggested that Agt DnaK had a significantly lower affinity for ATP than Escherichia coli DnaK. We show for the first time that Agt DnaK was very effective at preventing thermal aggregation of malate dehydrogenase (MDH) in a concentration dependent manner. This is in contrast to E. coli DnaK which was ineffective at preventing thermal aggregation of MDH. A mutant Agt DnaK-V431F, with a blocked hydrophobic pocket in the substrate binding domain, was unable to suppress the thermosensitivty of an E. coli dnaK103 deletion strain. However the mutation did not inhibit Agt DnaK-V431F from preventing the thermal aggregation of MDH. The oligomeric state of Agt DnaK was studied using size exclusion chromatography. We demonstrated that dilution of the Agt DnaK protein, the addition of ATP and the removal of the 10kDa C-terminal alpha-helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal alpha-helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation.     

Construction and analysis of hybrid Escherichia coli-Bacillus subtilis dnaK genes

The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the B. subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E. coli. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in E. coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage lambda but abolished degradation of sigma32.     

Hsc70 contacts helix III of the J domain from polyomavirus T antigens: addressing a dilemma in the chaperone hypothesis of how they release E2F from pRb

Hsc70's expected binding site on helix II of the J domain of T antigens appears to be blocked in its structure bound to tumor suppressor pRb. We used NMR to map where mammalian Hsc70 binds the J domain of murine polyomavirus T antigens (PyJ). The ATPase domain of Hsc70 unexpectedly has its biggest effects on the NMR peak positions of the C-terminal end of helix III of PyJ. The Hsc70 ATPase domain protects the C-terminal end of helix III of PyJ from an uncharged paramagnetic probe of chelated Gd(III), clearly suggesting the interface. Effects on the conserved HPD loop and helix II of PyJ are smaller. The NMR results are supported by a novel assay of Hsc70's ATP hydrolysis showing that mutations of surface residues in PyJ helix III impair PyJ-dependent stimulation of Hsc70 activity. Evolutionary trace analysis of J domains suggests that helix III usually may join helix II in contributing specificities for cognate hsp70s. Our novel evidence implicating helix III differs from evidence that Escherichia coli DnaK primarily affects helix II and the HPD loop of DnaJ. We find the pRb-binding fragment of E2F1 to be intrinsically unfolded and a good substrate for Hsc70 in vitro. This suggests that E2F1 could be a substrate for Hsc70 recruited by T antigen to an Rb family member. Importantly, our results strengthen the chaperone hypothesis for E2F release from an Rb family member by Hsc70 recruited by large T antigen. That is, it now appears that Hsc70 can freely access helix III and the HPD motif of large T antigen bound to an Rb family member.     

Crystal structure of E. coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1) and mechanistic studies on ClpB ATPase activity

E. coli Hsp100 ClpB was recently identified as a critical part in a multi-chaperone system to play important roles in protein folding, protein transport and degradation in cell physiology. ClpB contains two nucleotide-binding domains (NBD1 and NBD2) within their primary sequences. NBD1 and NBD2 of ClpB can be classified as members of the large ATPase family known as ATPases associated with various cellular activities (AAA). To investigate how ClpB performs its ATPase activities for its chaperone activity, we have determined the crystal structure of ClpB nucleotide-binding domain 1 (NBD1) by MAD method to 1.80 A resolution. The NBD1 monomer structure contains one domain that comprises 11 alpha-helices and six beta-strands. When compared with the typical AAA structures, the crystal structure of ClpB NBD1 reveals a novel AAA topology with six-stranded beta-sheet as its core. The N-terminal portion of NBD1 structure has an extra beta-strand flanked by two extra alpha-helices that are not present in other AAA structures. Moreover, the NBD1 structure does not have a C-terminal helical domain as other AAA proteins do. No nucleotide molecule is bound with ClpB NBD1 in the crystal structure probably due to lack of the C-terminal helix domain in the structure. Isothermal titration calorimetry (ITC) studies of ClpB NBD1 and other ClpB deletion mutations showed that either ClpB NBD1 or NBD2 alone does not bind to nucleotides. However, ClpB NBD2 combined with ClpB C-terminal fragment can interact with one ADP or ATP molecule. ITC data also indicated that full-length ClpB could bind two ADP molecules or one ATP analogue ATPgammaS molecule. Further ATPase activity studies of ClpB and ClpB deletion mutants showed that only wild-type ClpB have ATPase activity. None of ClpB NBD1 domain, NBD2 domain and NBD2 with C-terminal fragment has detectable ATPase activities. On the basis of our structural and mutagenesis data, we proposed a "see-saw" model to illustrate the mechanisms by which ClpB performs its ATPase activities for chaperone functions.     

Structure-function analysis of the Escherichia coli GrpE heat shock protein.

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.     

The insect antimicrobial peptide, L-pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK

Recent reports have indicated that insect antimicrobial peptides kill bacteria by inhibiting the molecular chaperone DnaK. It was proposed that the antimicrobial peptide, all-L-pyrrhocoricin (L-PYR), binds to two sites on DnaK, the conventional substrate-binding site and the multi-helical C-terminal lid, and that inhibition of DnaK comes about from the lid mode of binding. In this report, we show using two different assays that L-PYR binds to and stimulates the ATPase activity of both wild-type and a lidless variant of DnaK. Our study shows that L-PYR interacts with DnaK much like the all-L NR (NRLLLTG) peptide, which is known to bind in the conventional substrate-binding site of DnaK. L-PYR antimicrobial activity is thus a consequence of the competitive inhibition of bacterial DnaK.     

Characterization of a lidless form of the molecular chaperone DnaK: deletion of the lid increases peptide on- and off-rate constants

The C-terminal, polypeptide binding domain of the 70-kDa molecular chaperone DnaK is composed of a unique lidlike subdomain that appears to hinder steric access to the peptide binding site. We have expressed, purified, and characterized a lidless form of DnaK to test the influence of the lid on the ATPase activity, on interdomain communication, and on the kinetics of peptide binding. The principal findings are that loss of the lid creates an activated form of DnaK which is not equivalent to ATP-bound DnaK. For example, at 25 degrees C the NR peptide (NRLLLTG) dissociates from the ADP and ATP states of DnaK with observed off-rate constants of 0.001 and 4.8 s(-1), respectively. In contrast, for DnaK that lacks most of the helical lid, residues 518-638, the NR peptide dissociates with observed off-rate constants of 0.1 and 188 s(-1). These results show that the loss of the lid does not interfere with interdomain communication, that the beta-sandwich peptide binding domain can exist in two discrete conformations, and that the lid functions to increase the lifetime of a DnaK.peptide complex. We discuss several mechanisms to explain how the lid affects the lifetime of a DnaK.peptide complex.     

Importance of the D and E helices of the molecular chaperone DnaK for ATP binding and substrate release

The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five alpha-helices (alphaA-alphaE) (residues 508-608) that is followed by a 30-residue disordered, flexible region (609-638). The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region is not known. By sequentially deleting the flexible subdomain and the individual lid helices, we deduced the importance of each structural unit to creating long-lived DnaK-peptide complexes. Here we report that (i) the alphaD helix is essential for long-lived DnaK-peptide complexes. For example, ATP triggers the dissociation of a acrylodan-labeled p5 peptide (ap5, a-CLLLSAPRR) from wtDnaK and DnaK595(A-D) with k(off) equal to 7.6 and 8.9 s(-1), respectively, whereas when the D-helix is deleted, creating DnaK578(A-C), k(off) jumps to 207 s(-1). (ii) The presence of the alphaB helix impacts the rate of the ATP-induced high-to-low affinity conformational change. For example, ATP induces this conformational change in a lidless variant, DnaK517(1/2A), with a rate constant of 442 s(-1), whereas, after adding back the B-helix (residues 518-554), ATP induces this conformational change in DnaK554(A-B) with a rate constant of 2.5 s(-1). Our interpretation is that this large decrease occurs because the B-helix of the DnaK554(A-B) is bound in the substrate-binding site. (iii) The deletion analysis also revealed that residues 596-638, which comprise the alphaE helix and the flexible subdomain, affect ATP binding. Our results are consistent with this part of the lid producing conformational heterogeneity, perhaps by binding to the ATPase domain.     

Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.     

Mapping of Apidaecin Regions Relevant for Antimicrobial Activity and Bacterial Internalization

Apidaecins are 18–20-residue long proline-rich peptides expressed in insects as part of the innate immune system. They are very active against Gram-negative bacteria, especially Enterobacteriaceae. The C-terminal sequence PRPPHPRL is highly conserved, whereas the N-terminal region is variable. By replacing all 18 residues of apidaecin 1a and apidaecin 1b individually by alanine (Ala-scan), we have shown that single mutations in the C-terminal half of the peptides drastically reduced and mostly abolished the antibacterial activity against Escherichia coli. Conversely, substitutions in the N-terminal eight residues produced no, or only minor effects. The activity loss was correlated to the ability of apidaecin 1b and its mutants to enter Gram-negative bacteria, most likely because they no longer bind to a protein transporter. This assumed binding, however, was not inhibited by truncated apidaecin peptides added at tenfold higher concentrations. Interestingly, the antibacterial activity of full length apidaecin 1b was enhanced about four times by addition of a N-terminally truncated apidaecin peptide [11–18]-apidaecin 1b, as indicated by lower MIC-values against E. coli, although the short 5(6)-carboxyfluorescein-labeled peptide did not enter the bacteria. In contrast, the activity against the Gram-positive bacterium Micrococcus luteus was not located in the C-terminal sequence of apidaecins 1a and b, but depended mostly on the presence of all four basic residues.     

Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase,protein folding,and copper binding under various salinity conditions

Previously, it was found that the dnaK1 gene of the halotolerant cyanobacterium Aphanothece halophytica encodes a polypeptide of 721 amino acids which has a long C-terminal region rich in acidic amino acid residues. To understand whether the A. halophytica DnaK1 possesses chaperone activity at high salinity and to clarify the role of the extra C-terminal amino acids, a comparative study examined three kinds of DnaK molecules for ATPase activity as well as the refolding activity of other urea-denatured proteins under various salinity conditions. DnaK1s from A. halophytica and Synechococcus sp. PCC 7942 and the C-terminal deleted A. halophytica DnaK1 were expressed in Escherichia coli and purified. The ATPase activity of A. halophytica DnaK1 was very high even at high salinity (1.0 M NaCl or KCl), whereas this activity in Synechococcus PCC 7942 DnaK1 decreased with increasing concentrations of NaCl or KCl. The salt dependence on the refolding activity of urea-denatured lactate dehydrogenase by DnaK1s was similar to that of ATPase activity of the respective DnaK1s. The deletion of the C-terminal amino acids of A. halophytica DnaK1 had no effect on the ATPase activity, but caused a significant decrease in the refolding activity of other denatured proteins. These facts indicate that the extra C-terminal region of A. halophytica DnaK1 plays an important role in the refolding of other urea-denatured proteins at high salinity. Furthermore, it was shown that DnaK1 could assist the copper binding of precursor apo-plastocyanin as well as that of mature apo-plastocyanin during the folding of these copper proteins.     

Direct comparison of a stable isolated Hsp70 substrate-binding domain in the empty and substrate-bound states

The Hsp70 family of molecular chaperones acts to prevent protein misfolding, import proteins into organelles, unravel protein aggregates, and enhance cell survival under stress conditions. These activities are all mediated by recognition of diverse hydrophobic sequences via a C-terminal substrate-binding domain. ATP-binding/hydrolysis by the N-terminal ATPase domain regulates the interconversion of the substrate-binding domain between low and high affinity conformations. The empty state of the substrate-binding domain has been difficult to study because of its propensity to bind nearly any available protein chain, even if only modestly hydrophobic. We have generated a new stable construct of the substrate-binding domain from the Escherichia coli Hsp70, DnaK, which has enabled us to compare the empty and peptide-bound conformations using NMR chemical shift analysis and hydrogen-deuterium exchange. We have determined that the empty state is, overall, quite similar to the peptide-bound state, contrary to a previous report. Peptide binding leads to a subtle alteration in the packing of the alpha-helical lid relative to the beta-subdomain. Significantly, we have shown that the chemical shifts of the substrate-binding domain and the ATPase domain do not change when they are placed together in a two-domain construct, whether or not peptide is bound, suggesting that, in the absence of nucleotide, the two domains of E. coli DnaK do not interact. We conclude that the isolated substrate-binding domain exists in a stable high affinity state in the absence of influence from a nucleotide-bound ATPase domain.     

Structure and activity of ClpB from Escherichia coli. Role of the amino-and -carboxyl-terminal domains

ClpB is a member of a protein-disaggregating multi-chaperone system in Escherichia coli. The mechanism of protein-folding reactions mediated by ClpB is currently unknown, and the functional role of different sequence regions in ClpB is under discussion. We have expressed and purified the full-length ClpB and three truncated variants with the N-terminal, C-terminal, and a double N- and C-terminal deletion. We studied the protein concentration-dependent and ATP-induced oligomerization of ClpB, casein-induced activation of ClpB ATPase, and ClpB-assisted reactivation of denatured firefly luciferase. We found that both the N- and C-terminal truncation of ClpB strongly inhibited its chaperone activity. The reasons for such inhibition were different, however, for the N- and C-terminal truncation. Deletion of the C-terminal domain inhibited the self-association of ClpB, which led to decreased affinity for ATP and to decreased ATPase and chaperone activity of the C-terminally truncated variants. In contrast, deletion of the N-terminal domain did not inhibit the self-association of ClpB and its basal ATPase activity but decreased the ability of casein to activate ClpB ATPase. These results indicate that the N-terminal region of ClpB may contain a functionally significant protein-binding site, whereas the main role of the C-terminal region is to support oligomerization of ClpB.     

Severe oxidative stress causes inactivation of DnaK and activation of the redox-regulated chaperone Hsp33

DnaK/DnaJ/GrpE constitutes the primary chaperone machinery in E. coli that functions to protect proteins against heat-induced protein aggregation. Surprisingly, upon exposure of cells to reactive oxygen species at elevated temperature, proteins are no longer protected by the DnaK system. Instead, they bind now to the redox-regulated chaperone Hsp33, which is activated by the same conditions that inactivate DnaK. The inactivation of DnaK seems to be induced by the dramatic decrease in intracellular ATP levels that occurs upon exposure of cells to reactive oxygen species. This appears to render DnaK's N-terminal ATPase domain nucleotide depleted and thermolabile. DnaK's N terminus reversibly unfolds in vivo, and DnaK loses its ability to protect proteins against stress-induced aggregation. Now, the ATP-independent chaperone holdase Hsp33 binds to a large number of cellular proteins and prevents their irreversible aggregation. Upon return to nonstress conditions, Hsp33 becomes inactivated while DnaK reactivates and resumes its task to support protein folding.     

Isolation and characterization of functional domains of UvrA.

The sequence of Escherichia coli UvrA protein suggests that it may fold into two functional domains each possessing DNA binding and ATPase activities. We have taken two approaches to physically isolate polypeptides corresponding to the two putative domains. First, a 180 base pair DNA segment encoding multiple collagenase recognition sequences was inserted into UvrA's putative interdomain hinge region. This UvrA derivative was purified and digested with collagenase, and the resulting 70-kDa N-terminal and 35-kDa C-terminal fragments were purified. Both fragments possessed nonspecific DNA binding activity, but only the N-terminal domain retained its nucleotide binding capacity as evidence by measurements of ATP hydrolysis and by ATP photo-cross-linking. Together, the two fragments failed to substitute for UvrA in reconstituting (A)BC excinuclease and, therefore, were presumed to be unable to load UvrB onto damaged DNA. Second, the DNA segments encoding the two domains were fused to the beta-galactosidase gene. The UvrA N-terminal domain-beta-galactosidase fusion protein was overproduced and purified. This fusion protein had ATPase activity, thus confirming that the amino-terminal domain does possess an intrinsic ATPase activity independent of any interaction with the carboxy terminus. Our results show that UvrA has two functional domains and that the specificity for binding to damaged DNA is provided by the proper three-dimensional orientation of one zinc finger motif relative to the other and is not an intrinsic property of an individual zinc finger domain.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL

Many proteins contain a thioredoxin (Trx)-like domain fused with one or more partner domains that diversify protein function by the modular construction of new molecules. The Escherichia coli protein YbbN is a Trx-like protein that contains a C-terminal domain with low homology to tetratricopeptide repeat motifs. YbbN has been proposed to act as a chaperone or co-chaperone that aids in heat stress response and DNA synthesis. We report the crystal structure of YbbN, which is an elongated molecule with a mobile Trx domain and four atypical tetratricopeptide repeat motifs. The Trx domain lacks a canonical CXXC active site architecture and is not a functional oxidoreductase. A variety of proteins in E. coli interact with YbbN, including multiple ribosomal protein subunits and a strong interaction with GroEL. YbbN acts as a mild inhibitor of GroESL chaperonin function and ATPase activity, suggesting that it is a negative regulator of the GroESL system. Combined with previous observations that YbbN enhances the DnaK-DnaJ-GrpE chaperone system, we propose that YbbN coordinately regulates the activities of these two prokaryotic chaperones, thereby helping to direct client protein traffic initially to DnaK. Therefore, YbbN may play a role in integrating the activities of different chaperone pathways in E. coli and related bacteria.     

Two domains of superfamily I helicases may exist as separate proteins.

DNA and RNA helicases of superfamily I are characterized by seven conserved motifs. The five N-terminal motifs are separated from the two C-terminal ones by a spacer that is highly variable in both sequence and length, suggesting the existence of two distinct domains. Using computer methods for protein sequence analysis, we show that PhoH, an ATP-binding protein that is conserved in Escherichia coli and Mycobacterium leprae, is homologous to the putative N-terminal domain of the helicases, whereas the putative E. coli protein YjhR is homologous to the C-terminal domain. These findings suggest that the N-and C-terminal domains of superfamily I helicases have distinct activities, with only the N-terminal domain having the ATPase activity. It is speculated that PhoH and YjhR have evolved from helicases through deletion of the portions of the helicase genes coding for the C- and N-terminal domain, respectively.