Basic mechanism of the autonomous ClpG disaggregase
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100460
ISSN1083-351X
AutoresPanagiotis Katikaridis, Ute Römling, Axel Mogk,
Tópico(s)Salmonella and Campylobacter epidemiology
ResumoBacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical 70-kDa heat shock protein (Hsp70)–ClpB bichaperone disaggregase, which is most widespread in bacteria. The ClpB disaggregase is a member of the ATPase associated with diverse cellular activities protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, which initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the ATPase associated with diverse cellular activities protein ClpG as an alternative disaggregase. Pseudomonas aeruginosa ClpG functions autonomously and does not cooperate with Hsp70 for substrate binding, enhanced ATPase activity, and disaggregation. With the underlying molecular basis largely unknown, the fundamental differences in ClpG- and ClpB-dependent disaggregation are reflected by the presence of sequence alterations and additional ClpG-specific domains. By analyzing the effects of mutants lacking ClpG-specific domains and harboring mutations in conserved motifs implicated in ATP hydrolysis and substrate threading, we show that the N-terminal, ClpG-specific N1 domain generally mediates protein aggregate binding as the molecular basis of autonomous disaggregation activity. Peptide substrate binding strongly stimulates ClpG ATPase activity by overriding repression by the N-terminal N1 and N2 domains. High ATPase activity requires two functional nucleotide binding domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability. Bacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical 70-kDa heat shock protein (Hsp70)–ClpB bichaperone disaggregase, which is most widespread in bacteria. The ClpB disaggregase is a member of the ATPase associated with diverse cellular activities protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, which initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the ATPase associated with diverse cellular activities protein ClpG as an alternative disaggregase. Pseudomonas aeruginosa ClpG functions autonomously and does not cooperate with Hsp70 for substrate binding, enhanced ATPase activity, and disaggregation. With the underlying molecular basis largely unknown, the fundamental differences in ClpG- and ClpB-dependent disaggregation are reflected by the presence of sequence alterations and additional ClpG-specific domains. By analyzing the effects of mutants lacking ClpG-specific domains and harboring mutations in conserved motifs implicated in ATP hydrolysis and substrate threading, we show that the N-terminal, ClpG-specific N1 domain generally mediates protein aggregate binding as the molecular basis of autonomous disaggregation activity. Peptide substrate binding strongly stimulates ClpG ATPase activity by overriding repression by the N-terminal N1 and N2 domains. High ATPase activity requires two functional nucleotide binding domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability. 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A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid.Mol Microbiol. 2021; 115: 255-271Crossref PubMed Scopus (8) Google Scholar). clpG copies are typically located on mobile genomic islands and plasmids and are coorganized with additional ORFs encoding for protein quality control factors, including chaperones (small HSPs), proteases (FtsH, DegP, HtpX), or factors involved in oxidative stress response (thioredoxin) (23Lee C. Wigren E. Lunsdorf H. Romling U. Protein homeostasis-more than resisting a hot bath.Curr. Opin. Microbiol. 2016; 30: 147-154Crossref PubMed Scopus (20) Google Scholar). The gene clusters (locus of heat resistance, transmissible locus for protein quality control) comprise up to 16 core ORFs (21Lee C. Wigren E. Trcek J. Peters V. Kim J. Hasni M.S. Nimtz M. Lindqvist Y. Park C. Curth U. Lunsdorf H. Romling U. A novel protein quality control mechanism contributes to heat shock resistance of worldwide-distributed Pseudomonas aeruginosa clone C strains.Environ. Microbiol. 2015; 17: 4511-4526Crossref PubMed Scopus (27) Google Scholar, 22Mercer R.G. Zheng J. Garcia-Hernandez R. Ruan L. Ganzle M.G. McMullen L.M. Genetic determinants of heat resistance in Escherichia coli.Front. Microbiol. 2015; 6: 932Crossref PubMed Scopus (80) Google Scholar, 32Li H. Ganzle M. Some like it hot: Heat resistance of Escherichia coli in food.Front. Microbiol. 2016; 7: 1763Crossref PubMed Scopus (49) Google Scholar). Notably, clpG can also be encoded in the core genome of bacteria, although this placement is less frequent (24Lee C. Franke K.B. Kamal S.M. Kim H. Lunsdorf H. Jager J. Nimtz M. Trcek J. Jansch L. Bukau B. Mogk A. Romling U. Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E273-E282Crossref PubMed Scopus (24) Google Scholar). P. aeruginosa clone C strains encode for two clpG copies, one located in the core chromosome (clpGGC) and a second on the transmissible locus for protein quality control island (clpGGI). As a hallmark of the ClpG subfamily of Hsp100 disaggregases, both ClpG proteins exhibit stand-alone protein disaggregation activity in vitro and can functionally replace each other in vivo, indicating similar basic functionality irrespective of gene synteny (24Lee C. Franke K.B. Kamal S.M. Kim H. Lunsdorf H. Jager J. Nimtz M. Trcek J. Jansch L. Bukau B. Mogk A. Romling U. Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E273-E282Crossref PubMed Scopus (24) Google Scholar). GlpG and ClpB can functionally replace each other in vivo for basic lethal temperature tolerance (24Lee C. Franke K.B. Kamal S.M. Kim H. Lunsdorf H. Jager J. Nimtz M. Trcek J. Jansch L. Bukau B. Mogk A. Romling U. Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E273-E282Crossref PubMed Scopus (24) Google Scholar, 33Katikaridis P. Meins L. Kamal S.M. Romling U. Mogk A. ClpG provides increased heat resistance by acting as superior disaggregase.Biomolecules. 2019; 9: 815Crossref Scopus (9) Google Scholar). However, the two disaggregases differ in fundamental mechanistic aspects as ClpB strictly requires cooperation with the Hsp70 (DnaK) chaperone system. These differences in mechanisms of disaggregation must be reflected in sequence variability of shared domains and/or the presence of extra domain(s); however, a systematic analysis of ClpG extra domain functions has not been performed. ClpG (ClpK) exhibits a similar core domain organization as compared with ClpB but possesses a distinct M domain and an N1 domain and a C-terminal extension (CTE) not present in other Hsp100 family members. Here, we dissect the roles of individual domains of P. aeruginosa ClpGGI, which represents the best characterized family member to date. By deleting individual extra domains, we confirm that solely the ClpGGI-specific N1 domain is essential for aggregate binding and subsequent disaggregation. Intriguingly, an N1 domain fusion converts ClpB into an autonomous disaggregase. ClpGGI ATPase activity is negatively regulated by both N1 and N2 domains and positively by the ATPase activity of the alternate AAA+ domain, with substrate binding to override ATPase control by N1 and N2, directly linking substrate presence to high ATPase and disaggregation activities. Mutating pore-located aromatic residues in the AAA domains reduces (AAA1) or abolishes (AAA2) disaggregation activity, confirming substrate threading as a force-generating step in disaggregation and defining the AAA2 ring as a main threading motor. Together, these findings define the basic mechanism of ClpG-mediated disaggregation and ATPase control. ClpG harbors two N domains (N1 and N2), two AAA domains (AAA1, AAA2), an AAA1-inserted M domain and a CTE (Fig. 1A, Fig. S1). Sequence conservations among ClpG homologs are very high in both AAA domains and lower in extra domains, as also observed for other Hsp100 family members (Fig. S2). The N1 domain represents a defining feature of ClpG proteins distinctively discriminating this subfamily from other Hsp100 proteins (Fig. 1A). The N2 domain of ClpG is homologous to N domains of other Hsp100 proteins such as ClpA, ClpB, and ClpC (Fig. 1A, Fig. S1). The ClpG M domain is predicted to form a coiled-coil structure composed of a single helical wing, like the M domain of ClpC but it is shorter than the ClpB M domain, which is composed of two wings (Fig. 1A, Fig. S1). ClpG also separates from ClpB by harboring a CTE including 20 to 40 residues that is typically enriched for proline and lysine residues and is predicted disordered. The role of the N1 domain has been initially characterized. N1 deletion strongly reduces ClpG disaggregation activity in vitro and in vivo and can increase basal ATPase activity (24Lee C. Franke K.B. Kamal S.M. Kim H. Lunsdorf H. Jager J. Nimtz M. Trcek J. Jansch L. Bukau B. Mogk A. Romling U. Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E273-E282Crossref PubMed Scopus (24) Google Scholar). The mechanistic basis of the crucial role of the N1 domain is not fully understood. N1 confers autonomous aggregate binding to P. aeruginosa ClpGGC as a respective ΔN1 deletion mutant does no longer bind to protein aggregates. On the other hand, ΔN1–ClpGGI still seems to interact with aggregated proteins in vitro (24Lee C. Franke K.B. Kamal S.M. Kim H. Lunsdorf H. Jager J. Nimtz M. Trcek J. Jansch L. Bukau B. Mogk A. Romling U. Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E273-E282Crossref PubMed Scopus (24) Google Scholar). Therefore, it remained unclear whether the low disaggregation activity of ΔN1–ClpG stems from deficiencies in aggregate binding or is caused by indirect effects through, for example, deregulation of ATPase control. To dissect the roles of ClpG characteristic domains, we focused our analysis on P. aeruginosa ClpGGI, which represents the best-characterized family member to date (24Lee C. Franke K.B. Kamal S.M. Kim H. Lunsdorf H. Jager J. Nimtz M. Trcek J. Jansch L. Bukau B. Mogk A. Romling U. Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E273-E282Crossref PubMed Scopus (24) Google Scholar, 33Katikaridis P. Meins L. Kamal S.M. Romling U. Mogk A. ClpG provides increased heat resistance by acting as superior disaggregase.Biomolecules. 2019; 9: 815Crossref Scopus (9) Google Scholar) and generated ClpGGI deletion mutants lacking individual domains. In addition, we deleted both N domains yielding ΔN1/2–ClpGGI. All deletion mutants were expressed in Escherichia coli ΔclpB and dnaK103 mutant cells, which lack the ClpB/DnaK (Hsp70) disaggregase and are therefore sensitive to exposure to 50 °C. ClpGGI can functionally replace ClpB and Hsp70 upon overexpression in E. coli and even provides increased heat resistance to ΔclpB cells as it exhibits higher disaggregation activity than the canonical ClpB disaggregase (24Lee C. Franke K.B. Kamal S.M. Kim H. Lunsdorf H. Jager J. Nimtz M. Trcek J. Jansch L. Bukau B. Mogk A. Romling U. Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E273-E282Crossref PubMed Scopus (24) Google Scholar, 33Katikaridis P. Meins L. Kamal S.M. Romling U. Mogk A. ClpG provides increased heat resistance by acting as superior disaggregase.Biomolecules. 2019; 9: 815Crossref Scopus (9) Google Scholar). The production levels of ClpGGI in complementation assays were similar to those of ClpB or DnaK controls produced from the same expression vector (Fig. S3A). ClpB or DnaK overexpression did not restore heat tolerance of dnaK103 or ΔclpB cells, respectively, underlining the specific function of ClpGGI as stand-alone disaggregase (Fig. S3, B and C). To test whether ClpGGI overproduction is required for complementation activity, we expressed the disaggregase in E. coli dnaK103 mutant cells in the presence of varying IPTG concentrations (0–250 μM) and determined production levels and cellular protection upon heat shock to 49 °C (Fig. S3, D and E). We observe that ClpGGI overproduction in the presence of 100- to 250-μM IPTG is necessary for providing efficient heat resistance to these cells. All ClpGGI-based deletion constructs except those lacking the N1 domain provided heat resistance to both E. coli mutant strains (Fig. 1, B and C), comparable with ClpGGI WT except for ClpGGI-ΔCTE, which provided slightly reduced heat resistance to dnaK103 cells. Expression levels of all mutant constructs were similar except for those lacking the N1 domain (Fig. S3, F and G), raising the possibility that lower levels of ΔN1–ClpGGI and ΔN1/N2–ClpGGI contribute to the lack of complementation activity in vivo. We also tested the abilities of ClpGGI deletion mutants to complement the temperature-sensitive growth phenotype of E. coli dnaK103 cells at 41 °C (Fig. S4). We again find that the N1 domain is essential for complementation activity, while N2 or M domain deletions exhibited activities similar to ClpGGI WT. A CTE deletion lowered the ability of ClpGGI to complement significantly more than in the lethal heat tolerance assay (Fig. 1C). Notably, in this assay, the highest complementation activities were observed in presence of 25- to 50-μM IPTG, while higher IPTG concentrations particularly reduced the ability of ClpGGI deletion mutants to restore growth at 41 °C. This finding suggests that high levels of these mutant proteins provoke deleterious effects in dnaK103 cells at high temperatures. To better characterize and compare the ClpGGI deletion mutants, we purified all constructs and determined their disaggregation activities toward heat-aggregated malate dehydrogenase (MDH) and firefly luciferase (Fig. 2). Deleting the N2 and M domains or the CTE still allowed for efficient reactivation of both aggregated model substrates by ClpGGI. ΔN2–ClpGGI exhibited, however, reduced activity toward aggregated luciferase and reactivated 15.5% as compared with 40.7% by ClpGGI WT (Fig. 2, D and E). Disaggregation kinetics toward luciferase aggregates were lower than aggregated MDH with lower renatured protein product, indicating that disentanglement of these aggregates is more challenging and less efficient. On the other hand, ΔN1–ClpGGI did not reactivate aggregated Luciferase and exhibited 11.6-fold reduced disaggregation activity toward MDH as compared with ClpGGI WT (Fig. 2, A–C). ΔN1/2–ClpGGI did not exhibit any disaggregation activity, suggesting that the residual MDH disaggregation activity of ΔN1–ClpGGI stems from the N2 domain. These results from in vitro disaggregation experiments largely agreed with those obtained from in vivo heat-resistance experiments. Together, these findings underline the most crucial role of the N1 domain for ClpGGI disaggregation activity in vitro and in vivo, whereas all other extra domains are not essential. We were wondering whether we could overcome ClpB dependence on DnaK and converting it into a stand-alone disaggregase by fusing N1 or N1/N2 domains of ClpGGI to the ClpB ATPase motor composed of its AAA1 and AAA2 domains and the regulatory M domain (Fig. 3A). F1–ClpB and F1/2–ClpB showed partial disaggregation activity and reactivated 20.1% and 18.2% of MDH after 120 min, respectively. Because DnaK also activates the ClpB ATPase ring by displacing repressing M domains, we additionally generated chimeras harboring the M domain mutation Y503D, which causes derepression of the AAA domains independent of DnaK (16Oguchi Y. Kummer E. Seyffer F. Berynskyy M. Anstett B. Zahn R. Wade R.C. Mogk A. Bukau B. A tightly regulated molecular toggle controls AAA+ disaggregase.Nat. Struct. Mol. Biol. 2012; 19: 1338-1346Crossref PubMed Scopus (99) Google Scholar, 34Haslberger T. Weibezahn J. Zahn R. Lee S. Tsai F.T. Bukau B. Mogk A. M domains couple the ClpB threading motor with the DnaK chaperone activity.Mol. Cell. 2007; 25: 247-260Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Purified fusion constructs were probed for reactivation of aggregated MDH. F1–ClpB–Y503D and F1/2–ClpB–Y503D exhibited MDH refolding kinetics and yields that were indistinguishable from ClpGGI or ClpB in the presence of the DnaK chaperone system (KJE: DnaK, DnaJ, GrpE). These findings imply that fusion of N1 and concurrent abolition of M domain repression converts ClpB into a highly efficient and stand-alone disaggregase. Accordingly, the disaggregation activity of F1–ClpB could be increased upon addition of DnaK507 (Fig. S4, A–C). DnaK507 lacks the C-terminal helical bundle of the DnaK substrate binding domain and activates ClpB ATPase activity by binding and displacing M domains (35Fernandez-Higuero J.A. Aguado A. Perales-Calvo J. Moro F. Muga A. Activation of the DnaK-ClpB complex is regulated by the properties of the bound substrate.Sci. Rep. 2018; 8: 5796Crossref PubMed Scopus (13) Google Scholar). In absence of its J domain protein partner, DnaJ, DnaK507 cannot bind to substrates and thus does not recruit ClpB to protein aggregates. Accordingly, DnaK507 did not allow for ClpB-mediated protein disaggregation (Fig. S5, A–C). The stimulatory effect of DnaK507 on F1–ClpB therefore only involves the displacement of repressing M domains and demands for autonomous binding of F1–ClpB to the aggregated substrate. To directly demonstrate t
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