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Protein disaggregation by the AAA+ chaperone ClpB involves partial threading of looped polypeptide segments
Abstract
The ring-forming AAA+ chaperone ClpB cooperates with the DnaK chaperone system to reactivate aggregated proteins. With the assistance of DnaK, ClpB extracts unfolded polypeptides from aggregates via substrate threading through its central channel. Here we analyze the processing of mixed aggregates consisting of protein fusions of misfolded and native domains. ClpB-DnaK reactivated all aggregated fusion proteins with similar efficiency, without unfolding native domains, demonstrating that partial threading of the misfolded moiety is sufficient to solubilize aggregates. Reactivation by ClpB-DnaK occurred even when two stably folded domains flanked the aggregated moiety, indicating threading of internal substrate segments. In contrast with the related AAA+ chaperone ClpC, ClpB lacks a robust unfolding activity, enabling it to sense the conformational state of substrates. ClpB rings are highly unstable, which may facilitate dissociation from trapped substrates during threading.
... Threading power can be assessed by monitoring YFP fluorescence during the disaggregation process. Ec KJE/ClpB and Lm KJE/ClpB did not unfold YFP during the disaggregation process, documenting limited unfolding power, consistent with former reports (Haslberger et al., 2008;Katikaridis et al., 2019). In contrast, we observed a rapid loss of YFP fluorescence in the presence of ClpL that was even faster as compared to ClpG ( Figure 2B). ...
... nascent polypeptide chains). We performed mixing experiments using L N -ClpB* and ΔN-ClpB* as model system, since ClpB hexamers dynamically exchange subunits ensuring stochastic formation of mixed hexamers ( Figure 5A/B; Haslberger et al., 2008;Werbeck et al., 2008). We confirmed mixing of WT and mutant subunits by showing that the presence of ATPase-deficient ΔN-ClpB*-E218A/ E618A, harboring mutated Walker B motifs in both AAA domains, strongly poisoned disaggregation activity of L N -ClpB* ( Figure 5-figure supplement 1A). ...
... Purifications of Ec DnaK, DnaJ, GrpE, and firefly luciferase were performed as described previously (Haslberger et al., 2008;Oguchi et al., 2012;Seyffer et al., 2012). Pyruvate kinase of rabbit muscle and MDH of pig heart muscle were purchased from Sigma. ...
Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during, e.g., food processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.
... Several modes of disaggregation by ClpB have been proposed in the literature, such as the 'crowbar' mechanism, subunit exchange mechanism and the threading mechanism [63]. So far, no clear evidence for the first two of these was provided, whereas several biochemical and structural studies of the pore loops of NBD1 and NBD2 supported the threading mechanism [17,19,[64][65][66][67][68][69], which was also supported by the studies of other AAA+ members such as ClpA [70], NSF [60], CdC48 [61,71] and its homologue VAT [58]. Interestingly, some studies indicated that ClpB or Hsp104 perform partial threading rather than complete threading of soluble substrate, a mechanism that might allow the disaggregases to dissociate and bind to other regions of a polypeptide [66,72,73]. ...
... So far, no clear evidence for the first two of these was provided, whereas several biochemical and structural studies of the pore loops of NBD1 and NBD2 supported the threading mechanism [17,19,[64][65][66][67][68][69], which was also supported by the studies of other AAA+ members such as ClpA [70], NSF [60], CdC48 [61,71] and its homologue VAT [58]. Interestingly, some studies indicated that ClpB or Hsp104 perform partial threading rather than complete threading of soluble substrate, a mechanism that might allow the disaggregases to dissociate and bind to other regions of a polypeptide [66,72,73]. Another recent study suggested that Hsp104 can also act as a 'holdase', capturing soluble forms of amyloid substrates, a function which is different from its well-established disaggregation activity [74]. ...
... Similar events at NBD2 then engage PL3 as a pawl (step 4). Disengagement of these pawls may allow looped polypeptide segments to escape after partial threading [66,73]. It is likely that the protein harnesses the power of asynchronous pulling by neighbouring subunits to generate rapid translocation events in a Brownian-ratchet like mechanism. ...
It has been recently shown that in some proteins, tertiary‐structure dynamics occur surprisingly fast, that is on the microsecond or sub‐millisecond time scales. In this State of the Art Review, we discuss how such ultrafast domain motions relate to the function of caseinolytic peptidase B (ClpB), a AAA+ disaggregation machine. ClpB is a large hexameric protein that collaborates with cellular chaperone machinery to rescue protein chains from aggregates. We used single‐molecule FRET spectroscopy to capture the dynamics of essential structural elements within this machine. It was found that the middle domain of ClpB, known to act as its activator, toggles between two states much faster than the overall activity cycle of the protein, suggesting a novel mode of continuous, tunable switching. Motions of the N‐terminal domain were observed to restrict the conformational space of the M domain in the absence of a substrate protein, thereby preventing it from tilting and spuriously activating ClpB. Finally, microsecond dynamics of pore loops responsible for substrate pulling through ClpB's central channel, together with their response to specific perturbations, point to a Brownian‐ratchet mechanism for protein translocation. Based on our findings, we propose a two‐time‐scale model for the activity of ClpB, in which fast conformational dynamics affect slower functional steps, determined by ATP hydrolysis time. Future work on this and other proteins is likely to shed further light on the role of ultrafast dynamics on protein function.
... It is rather unlikely that N-or C-termini of aggregated proteins are accessible for recognition by bacterial disaggregases, which will therefore preferentially act on internal sequence stretches. Indeed, ClpB can thread loop structures and efficiently solubilizes protein aggregates that only offer internal segments for processing (Haslberger et al., 2008). The threading of substrate loops involves the translocation of two polypeptide arms and ClpB can switch between two-arms and single-arm translocation during the disaggregation reaction (Avellaneda et al., 2020). ...
... In the context of protein disaggregation, DnaJ targets DnaK to the surface of protein aggregates (Carrio and Villaverde, 2005;Acebron et al., 2009;Winkler et al., 2012). The coating of the aggregate surface by DnaK provides specificity for reactivating ClpB and prevents binding of Hsp100/AAA+ proteins (e.g., ClpA/ClpC) that cooperate with peptidases (e.g., ClpP) (Haslberger et al., 2008). DnaK thereby impacts triage decision and ensures that aggregated proteins will be primarily targeted to refolding pathways. ...
... The activated state of ClpB likely exists only transiently and the disaggregase turns into a partially activated state during the disaggregation reaction (Figure 3). This model is supported by biochemical findings showing that ClpB has a reduced unfolding power as compared to other Hsp100 family members (Haslberger et al., 2008). Thus, ClpB cannot unfold stably folded domains of proteins trapped in a protein aggregate. ...
Bacteria as unicellular organisms are most directly exposed to changes in environmental growth conditions like temperature increase. Severe heat stress causes massive protein misfolding and aggregation resulting in loss of essential proteins. To ensure survival and rapid growth resume during recovery periods bacteria are equipped with cellular disaggregases, which solubilize and reactivate aggregated proteins. These disaggregases are members of the Hsp100/AAA+ protein family, utilizing the energy derived from ATP hydrolysis to extract misfolded proteins from aggregates via a threading activity. Here, we describe the two best characterized bacterial Hsp100/AAA+ disaggregases, ClpB and ClpG, and compare their mechanisms and regulatory modes. The widespread ClpB disaggregase requires cooperation with an Hsp70 partner chaperone, which targets ClpB to protein aggregates. Furthermore, Hsp70 activates ClpB by shifting positions of regulatory ClpB M-domains from a repressed to a derepressed state. ClpB activity remains tightly controlled during the disaggregation process and high ClpB activity states are likely restricted to initial substrate engagement. The recently identified ClpG (ClpK) disaggregase functions autonomously and its activity is primarily controlled by substrate interaction. ClpG provides enhanced heat resistance to selected bacteria including pathogens by acting as a more powerful disaggregase. This disaggregase expansion reflects an adaption of bacteria to extreme temperatures experienced during thermal based sterilization procedures applied in food industry and medicine. Genes encoding for ClpG are transmissible by horizontal transfer, allowing for rapid spreading of extreme bacterial heat resistance and posing a threat to modern food production.
... Distinct from the proteasome and what has been shown so far with VCP, Hsp104 can have partial or complete translocation of substrate across its pore (Fig. 3B). Complete or partial translocation of substrate by Hsp104 is thought to be correlated with aggregate stability (165,167,(178)(179)(180)(181)(182). Less stable aggregates are thought to have a noncooperative mechanism of disaggregation by Hsp104 subunits, while more stable aggregates, like amyloids, have a cooperative mechanism of disaggregation by Hsp104 subunits (167,174). ...
... In the cooperative mechanism, Hsp104 subunits work together to unfold aggregates. Hsp104 can also initiate disaggregation at an internal segment of protein aggregates (Fig. 3B) (180,182). Neither VCP nor the proteasome is known to initiate substrate processing at an internal segment, thus Hsp104 may possess a unique ability that expands its disaggregation potential. It remains possible that VCP, and less likely the proteasome (due its lid subunits, Fig. 2A), may be able to initiate processing in a similar manner, but this has never been observed. ...
... It is additionally thought that maybe Hsp104 can adapt to different substrates (167). As previously noted, Hsp104 has a unique ability to initiate disaggregation at an internal segment (Fig. 3B) (180,182). Though no structure to date has shown this explicitly, the seam seen between the top subunit and its counterclockwise neighbor may allow for substrate insertion from the side of Hsp104 rather than through the top of the pore (165,196,197). As has been seen in structures of VCP, the proteasome, and Hsp104, when ATP is hydrolyzed there is increased ring flexibility to allow for large movements of a subunit to the top of the ring (14,15,33,34,165). ...
Neurodegenerative diseases are characterized by the accumulation of misfolded proteins. This protein aggregation suggests that abnormal proteostasis contributes to aging-related neurodegeneration. A better fundamental understanding of proteins that regulate proteostasis may provide insight into the pathophysiology of neurodegenerative disease and may perhaps reveal novel therapeutic opportunities. The 26S proteasome is the key effector of the ubiquitin-proteasome system responsible for degrading polyubiquitinated proteins. However, additional factors, such as valosin-containing protein (VCP/p97/Cdc48) and C9orf72, play a role in regulation and trafficking of substrates through the normal proteostasis systems of a cell. Nonhuman AAA+ ATPases, such as the disaggregase Hsp104, also provide insights into the biochemical processes that regulate protein aggregation. X-ray crystallography and cryo-electron microscopy (cryo-EM) structures not bound to substrate have provided meaningful information about the 26S proteasome, VCP, and Hsp104. However, recent cryo-EM structures bound to substrate have provided new information about the function and mechanism of these proteostasis factors. Cryo-EM and cryo-electron tomography data combined with biochemical data have also increased the understanding of C9orf72 and its role in maintaining proteostasis. These structural insights provide a foundation for understanding proteostasis mechanisms with near-atomic resolution upon which insights can be gleaned regarding the pathophysiology of neurodegenerative diseases.
... ClpB mutants that cause permanent dissociation of M-domains result in persistent cytotoxic activation of the protein [16,20]. Accordingly, ClpB activation by DnaK is only transient and ClpB exhibits reduced unfolding power as compared to other Hsp100 proteins, even when engaged in protein disaggregation [21]. ...
... Purifications of DnaK, DnaJ, GrpE, Firefly Luciferase and Luciferase-YFP were performed as described previously [16,21,32]. Pyruvate kinase of rabbit muscle, Malate Dehydrogenase of pig heart muscle, Citrate Synthase from porcine heart and α-Glucosidase from Saccharomyces cerevisiae were purchased from Sigma. ...
... Luciferase-YFP forms mixed aggregates composed of misfolded Luciferase and native YFP, which resist unfolding (Figure 7a). These aggregates are efficiently solubilized by ClpB, yet the fused YFP is not unfolded during the disaggregation reaction, indicating that ClpB has low unfolding power [21]. This low activity is caused by repressing M-domains as overruling activity control in ClpB-K476C allows for YFP unfolding [16,35]. ...
Elevation of temperature within and above the physiological limit causes the unfolding and aggregation of cellular proteins, which can ultimately lead to cell death. Bacteria are therefore equipped with Hsp100 disaggregation machines that revert the aggregation process and reactivate proteins otherwise lost by aggregation. In Gram-negative bacteria, two disaggregation systems have been described: the widespread ClpB disaggregase, which requires cooperation with an Hsp70 chaperone, and the standalone ClpG disaggregase. ClpG co-exists with ClpB in selected bacteria and provides superior heat resistance. Here, we compared the activities of both disaggregases towards diverse model substrates aggregated in vitro and in vivo at different temperatures. We show that ClpG exhibits robust activity towards all disordered aggregates, whereas ClpB acts poorly on the protein aggregates formed at very high temperatures. Extreme temperatures are expected not only to cause extended protein unfolding, but also to result in an accelerated formation of protein aggregates with potentially altered chemical and physical parameters, including increased stability. We show that ClpG exerts higher threading forces as compared to ClpB, likely enabling ClpG to process “tight” aggregates formed during severe heat stress. This defines ClpG as a more powerful disaggregase and mechanistically explains how ClpG provides increased heat resistance.
... Hsp104 (and ClpB) disaggregase activity is greatly enhanced by collaboration with the Hsp70 molecular chaperone system, which includes a J-domain chaperone (Hsp40) and a nucleotide-exchange factor (Hsp110) in addition to Hsp70 (Glover and Lindquist 1998;Cashikar et al. 2005;Haslbeck et al. 2005;Shorter and Lindquist 2008;Shorter 2011;Kaimal et al. 2017). Hsp104 couples ATP hydrolysis to the forcible extraction of polypeptides from aggregates via partial or complete translocation across its axial channel by tyrosine-bearing pore loops in NBD1 and NBD2 (Lum et al. 2004(Lum et al. , 2008Haslberger et al. 2008;Tessarz et al. 2008;Sweeny et al. 2015;Gates et al. 2017). Released polypeptides that are unfolded can then refold spontaneously or in collaboration with molecular chaperones. ...
... Although appealing, this model is complicated by observations that ClpB and DnaK compete for binding to aggregates (Weibezahn et al. 2003), that DnaK causes ClpB to dissociate from substrate (Durie et al. 2018), and that Hsp104 and ClpB can show powerful disaggregase activity in the absence of Hsp70 (DeSantis et al. 2012;Duennwald et al. 2012;Zhang et al. 2013;Jackrel et al. 2014;Krajewska et al. 2017). It is also worth noting that other members of the Hsp100 family and indeed other protein-disaggregase systems show no requirement for the Hsp70 chaperone system to drive protein disaggregation (Dougan et al. 2002;Andersson et al. 2006;Haslberger et al. 2008;Park et al. 2015;Poepsel et al. 2015;Ali et al. 2016;Baker et al. 2017;LaBreck et al. 2017;Guo et al. 2018;Lee et al. 2018;Yoshizawa et al. 2018). Collectively, these observations suggest that more nuanced models are required. ...
... All recent substrate-bound structures of AAA + translocases identify a narrow channel containing a single polypeptide chain that is coordinated by a spiral array of pore-loop interactions, thus it appears unlikely that more than one strand could be accommodated in the channel (Deville et al. 2017;Gates et al. 2017;Han et al. 2017;Puchades et al. 2017). However, previous studies identified that ClpB can process internal segments of substrates (Haslberger et al. 2008) in addition to translocating from the amino-or carboxy-termini (Doyle et al. 2007;Haslberger et al. 2008;Hoskins et al. 2009). Likewise, Hsp104 can drive disaggregation via partial translocation initiated at internal segments (Haslberger et al. 2008;Sweeny et al. 2015), and can also process substrates from the amino-or carboxy-termini (Doyle et al. 2007;Sweeny et al. 2015). ...
Hsp104 is a hexameric AAA+ ATPase and protein disaggregase found in yeast, which couples ATP hydrolysis to the dissolution of diverse polypeptides trapped in toxic preamyloid oligomers, phase-transitioned gels, disordered aggregates, amyloids, and prions. Hsp104 shows plasticity in disaggregating diverse substrates, but how its hexameric architecture operates as a molecular machine has remained unclear. Here, we highlight structural advances made via cryoelectron microscopy (cryo-EM) that enhance our mechanistic understanding of Hsp104 and other related AAA+ translocases. Hsp104 hexamers are dynamic and adopt open "lock-washer" spiral states and closed ring structures that envelope polypeptide substrate inside the axial channel. ATP hydrolysis-driven conformational changes at the spiral seam ratchet substrate deeper into the channel. Remarkably, this mode of polypeptide translocation is reminiscent of models for how hexameric helicases unwind DNA and RNA duplexes. Thus, Hsp104 likely adapts elements of a deeply rooted, ring-translocase mechanism to the specialized task of protein disaggregation.
... 1,2 Disaggregation of protein aggregates may also be achieved by translocation-mediated unfolding. 3 Furthermore, translocation can be coupled to degradation of the translocated polymer segment. This process, called digestion, is carried out by exopeptidases that process proteins 4 or exonucleases that carry out degradation of unwanted nucleic acids. ...
... These approaches are commonly used in biological organisms as well as in engineered systems. 3,9,20 Here, we study the effect of chain topology on the mechanical unfolding of a chain. Unfolding involves disruption of intrachain contacts, which can be due to direct intrachain interactions or can be mediated by a linking molecule (e.g., CTCF-mediated genome folding). ...
... Thus, unfolding by the ClpB/DnaK complex is carried out using the pulling by threading method. 3,20 Despite ClpB that needs to partner DnaK, ClpG is a stand-alone disaggregase, 31 which unfolds the proteins using the threading method ( Figure 9). According to our findings, the pulling by threading method has a higher efficiency compared to that of the threading one. ...
Biopolymer unfolding events are ubiquitous in biology and mechanical unfolding is an established approach to study the structure and function of biomolecules, yet whether and how mechanical unfolding processes depend on native state topology remain unexplored. Here, we investigate how the number of unfolding pathways via mechanical methods depends on the circuit topology of a folded chain, which categorizes the arrangement of intrachain contacts into parallel, crossing, and series. Three unfolding strategies, namely, threading through a pore, pulling from the ends, and pulling by threading, are compared. Considering that some contacts may be unbreakable within the relevant forces, we also study the dependence of the unfolding efficiency on the chain topology. Our analysis reveals that the number of pathways and the efficiency of unfolding are critically determined by topology in a manner that depends on the employed mechanical approach, a significant result for interpretation of the unfolding experiments.
... The simplest model would involve a slippage of the substrate during translocation, leading to its release from the pore ring. Such a scenario could be favored by several factors: (i) limited pulling force from a single Skd3 AAA + ring, which may be insufficient to propel the complete threading of a large substrate such as luciferase; (ii) initiation of client folding, which would impede continued threading [ClpB releases partially translocated substrates upon encountering a folded domain in the substrate (15,49)]; and (iii) interaction of parts of the substrate protein with a second Skd3 hexamer, which would oppose continued threading. Regardless of the precise mechanism, release of partially threaded substrates has been observed with ClpB and was proposed to support substrate reactivation, by avoiding unnecessary unfolding of parts of the protein and preventing additional unfolded polypeptide segments from interfering with refolding (15,49). ...
... Such a scenario could be favored by several factors: (i) limited pulling force from a single Skd3 AAA + ring, which may be insufficient to propel the complete threading of a large substrate such as luciferase; (ii) initiation of client folding, which would impede continued threading [ClpB releases partially translocated substrates upon encountering a folded domain in the substrate (15,49)]; and (iii) interaction of parts of the substrate protein with a second Skd3 hexamer, which would oppose continued threading. Regardless of the precise mechanism, release of partially threaded substrates has been observed with ClpB and was proposed to support substrate reactivation, by avoiding unnecessary unfolding of parts of the protein and preventing additional unfolded polypeptide segments from interfering with refolding (15,49). ...
Ring-forming AAA+ chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA+ chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.
... In the first model, Pex1 might start by unfolding the attached ubiquitin, as is the case for Cdc48/p97 [212]. In this model, Pex1/Pex6 must be able to process two polypeptides simultaneously upon reaching the ubiquitin attachment site, but other AAA-ATPases have been shown to thread protein loops [180,212,213]. Secondly, Pex1/Pex6 could engage a disordered loop in Pex5's N-terminal domain downstream of the ubiquitin attachment site. ...
... Pex5, Atg36, and truncated Pex15 all meet these requirements ( Figure 6). Other AAA-ATPases can engage unstructured loops and process two strands simultaneously [180,212,213], so a Pex1/Pex6 substrate might likewise have an unstructured domain flanked by folded domains. Notably, Pex1/Pex6's apparent inability to unfold GFP and methotrexate-bound DHFR suggests limited unfolding power compared to other AAA-ATPase motors [98,138]. ...
The AAA-ATPases Pex1 and Pex6 are required for the formation and maintenance of peroxisomes, membrane-bound organelles that harbor enzymes for specialized metabolism. Together, Pex1 and Pex6 form a heterohexameric AAA-ATPase capable of unfolding substrate proteins via processive threading through a central pore. Here, we review the proposed roles for Pex1/Pex6 in peroxisome biogenesis and degradation, discussing how the unfolding of potential substrates contributes to peroxisome homeostasis. We also consider how advances in cryo-EM, computational structure prediction, and mechanisms of related ATPases are improving our understanding of how Pex1/Pex6 converts ATP hydrolysis into mechanical force. Since mutations in PEX1 and PEX6 cause the majority of known cases of peroxisome biogenesis disorders such as Zellweger syndrome, insights into Pex1/Pex6 structure and function are important for understanding peroxisomes in human health and disease.
... High ATPase activities of ClpB are likely restricted to the initial phase of disaggregation and drop upon Hsp70 dissociation and redocking of M domains onto the AAA1 ring (36,45). This mechanistically explains why ClpB does not exhibit high unfolding power in contrast to other Hsp100 proteins and cannot unfold stable domains during protein disaggregation (46). Disaggregation and unfolding activities of ClpG GI are higher than those of ClpB (33), indicating persistent stimulation of ClpG GI ATPase by substrates. ...
... Purifications of DnaK, DnaJ, GrpE, and firefly luciferase were performed as described previously (11,16,46). Pyruvate kinase of rabbit muscle and MDH of pig heart muscle were purchased from Sigma. ...
Bacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical Hsp70-ClpB bichaperone disaggregase, which is most widespread in Bacteria. The ClpB disaggregase is a member of the AAA+ protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, who initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the AAA+ 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 AAA domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability.
... ClpB/Hsp104 are conserved in bacteria, fungi, plants and mitochondria and are essential for recovery of cells from heat shock and other proteotoxic stresses Mogk et al., 1999). The oligomeric ring Hsp100 proteins thread substrates through a central channel, via binding to conserved tyrosine residues on flexible loops (Weber-Ban et al., 1999;Kim et al., 2000;Lum et al., 2004;Weibezahn et al., 2004;Haslberger et al., 2008;Tessarz et al., 2008). They belong to the AAA+ (ATPases associated with various cellular activities) superfamily of ATPases, with characteristic α and β subdomains (Ogura and Wilkinson, 2001;Erzberger and Berger, 2006). ...
... We used BAP (ClpB with the ClpA tripeptide for ClpP binding), a chimera engineered to bind the ClpP protease via the replacement of a C-terminal ClpB segment with the ClpP binding region of ClpA (Weibezahn et al., 2004). This construct has been extensively used to study the ClpB disaggregation mechanism by monitoring substrate proteolysis after delivery to ClpP (Weibezahn et al., 2004;Haslberger et al., 2008;Tessarz et al., 2008;Mizuno et al., 2012;Rosenzweig et al., 2013;Figure 1A). Therefore, BAP is suitable for structural studies and has allowed us to obtain maps with visible MD densities that shed light on its regulatory mechanisms. ...
... Hsp70 modifies the surface of the aggregates by exposing disentangled regions of trapped polypeptides for Hsp100 binding (Ziȩtkiewicz et al. 2006). At the same time, Hsp70 coating of the aggregate surface restricts the access of other protein quality control machineries (Haslberger et al. 2008). Hsp100 selectively interacts with ADP-Hsp70, as association with the ATP-bound form is prevented due to steric clashes (Hayashi et al. 2017). ...
... Hsp100 recognizes exposed hydrophobic stretches of the aggregated proteins and actively displaces them from Hsp70 with pulling forces (Rosenzweig et al. 2013). Following substrate transfer, Hsp70 dissociates and restricts high disaggregase activity to initial strokes, thus some protein substrates are only partially threaded (Deville et al. 2017;Duran et al. 2017;Haslberger et al. 2008). This partial threading might enable Hsp100 to sense conformational states of aggregated substrates and stop threading when encountering tightly folded domains. ...
Chaperones of the 70 kDa heat shock protein (Hsp70) superfamily are key components of the cellular proteostasis system. Together with its co-chaperones, Hsp70 forms proteostasis subsystems that antagonize protein damage during physiological and stress conditions. This function stems from highly regulated binding and release cycles of protein substrates, which results in a flow of unfolded, partially folded and misfolded species through the Hsp70 subsystem. Specific factors control how Hsp70 makes decisions regarding folding and degradation fates of the substrate proteins. In this review, we summarize how the flow of Hsp70 substrates is controlled in the cell with special emphasis on recent advances regarding substrate release mechanisms.
... Five Hsps were selected for qPCR quantitation. These included two Hsps known to be up-regulated at the transcript level after HS in T. brucei [9,10] (Hsp70, Tb927.11.11330 and Hsp83, Tb927.10.10960), homologues to other members of the canonical Hsp70-Hsp40-Hsp110 network in yeast and mammals [31] (Hsp110, Tb927.10.12710 and Hsp40, Tb927.10.8540), and the closest T. brucei homologue to yeast Hsp104, which is also up-regulated after HS in PCF T. brucei [17,[32][33][34] (Hsp100, Tb927.2.5980). Hsps with a ≥2-fold increase were defined as up-regulated after heat shock. ...
... Five major groups are conserved across all organisms (Hsp60s, Hsp70s, Hsp90s, Hsp100s and small Hsps), and some have defined functions in eukaryotes. In yeast and mammals, the canonical Hsp70-Hsp40-Hsp110 network functions to disaggregate misfolded proteins following HS and labels them for ubiquitination and degradation [31], and Hsp104 forms a hexameric ring which threads misfolded proteins through its central pore to resolve protein aggregates that form following HS [32][33][34]. In response to 1 h HS at 41°C we observe a significant increases in the mRNA levels of Hsp70, Hsp83 and Hsp100, but not other members of the Hsp70-Hsp40-Hsp110 network. ...
... A ratchet-like pulling mechanism of substrate translocation is supported by the conformational plasticity of the ClpB hexamer [11,12]. Extracted polypeptides are released from the ClpB channel after either partial translocation or complete unfolding [13][14][15]. ...
... We used fluorescence anisotropy to investigate the peptide interactions with ClpB. Binding of a FITC-labeled peptide to hexameric ClpB [12][13][14][15][16][17] (∼570 kDa) significantly decelerates the rotational diffusion of the fluorescein dye and produces an increase in fluorescence anisotropy. Indeed, a dose-dependent increase in fluorescence anisotropy was observed during titration of B1 and B2 with the substrate-trapping ClpB variant in the presence of ATP, but not when ADP was present (Fig. 3A). ...
... The ringed oligomer formation depends on many factors, such as nucleotide binding, temperature, and protein and salt concentrations 14,15,37 . The ring easily collapses and their subunits are exchanged, even in the middle of the disaggregation reaction 22,38 . It was proposed that the ring fragility would contribute to avoiding jam of aggregated protein during threading 38 . ...
... The ring easily collapses and their subunits are exchanged, even in the middle of the disaggregation reaction 22,38 . It was proposed that the ring fragility would contribute to avoiding jam of aggregated protein during threading 38 . In addition, a recent cryo-EM study demonstrated that Hsp104 forms a two-turn spiral in which AAA1 in one protomer is linked to AAA2 in an adjacent protomer 17,39 . ...
The ATP-dependent bacterial protein disaggregation machine, ClpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of ClpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize ClpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable ClpB to disentangle protein aggregates.
... Threading power can be assessed by monitoring YFP 204 fluorescence during the disaggregation process. Ec KJE/ClpB and Lm KJE/ClpB did not unfold 205 YFP during the disaggregation process, documenting limited unfolding power, consistent with 206 former reports (Haslberger et al., 2008;Katikaridis et al., 2019). In contrast, we observed a 207 rapid loss of YFP fluorescence in presence of ClpL that was even faster as compared to ClpG 208 ( Figure 2B). ...
Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during e.g. food-processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.
... Mechanisms IIa, IIb and III require a REM with the capacity to thread more than one polypeptide chain simultaneously. Although it is becoming apparent that some AAA ATPases do have this ability [48][49][50], there are no data regarding the REM. Thus, we asked whether a Ub-PEX5 conjugate possessing a second branched structure located C-terminally to the monoubiquitination site, and thus in a region of the protein that is unfolded during extraction [29,40], can still be extracted. ...
The AAA ATPases PEX1•PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N- nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6.
... Only the expression of mcsB resulted in the (Fig 1 & Fig S1). (Goloubinoff et al., 1999;Haslberger et al., 2008;Schlothauer et al., 2003). In a control ...
In the Gram-positive model organism Bacillus subtilis, the AAA+ unfoldase ClpC associated with the ClpP protease plays an important role in cellular protein homeostasis and stress response. Here, we could demonstrate that the protein arginine kinase and adaptor protein McsB, its activator McsA, the phosphatase YwlE, and ClpC form a unique chaperone system necessary for protein aggregate removal in vivo and in vitro. Activated McsB phosphorylates and targets aggregated substrate proteins for extraction and unfolding by ClpC. Sub-stoichiometric amounts of catalytically active YwlE significantly enhanced ClpC/McsB-mediated disaggregation and facilitated the refolding of unfolded, arginine-phosphorylated substrate proteins in vitro. The refolded substrate proteins could escape the degradation by the associated ClpP protease. In this unique chaperone system, protein unfolding, coordinated with protein arginine phosphorylation and de-phosphorylation, facilitates protein refolding and homeostasis.
... By contrast, the Hsp100s like ClpB are members of the AAA + family of ATP-powered extrusion motors (Shorter and Southworth, 2019). These large homo-oligomeric, barrel-shaped proteins employ ATP hydrolysis to processively feed substrate proteins through their central pore as either linear chains or loops (Haslberger et al., 2008;Deville et al., 2017;Gates et al., 2017;Avellaneda et al., 2020). Importantly, initial substrate protein loading and activation of aggressive ATP turnover by ClpB requires, under most circumstances, direct binding between ClpB and DnaK (Acebrón et al., 2009;Oguchi et al., 2012;Winkler et al., 2012;Rosenzweig et al., 2013;Carroni et al., 2014). ...
Protein aggregation, or the uncontrolled self-assembly of partially folded proteins, is an ever-present danger for living organisms. Unimpeded, protein aggregation can result in severe cellular dysfunction and disease. A group of proteins known as molecular chaperones is responsible for dismantling protein aggregates. However, how protein aggregates are recognized and disassembled remains poorly understood. Here we employ a single particle fluorescence technique known as Burst Analysis Spectroscopy (BAS), in combination with two structurally distinct aggregate types grown from the same starting protein, to examine the mechanism of chaperone-mediated protein disaggregation. Using the core bi-chaperone disaggregase system from Escherichia coli as a model, we demonstrate that, in contrast to prevailing models, the overall size of an aggregate particle has, at most, a minor influence on the progression of aggregate disassembly. Rather, we show that changes in internal structure, which have no observable impact on aggregate particle size or molecular chaperone binding, can dramatically limit the ability of the bi-chaperone system to take aggregates apart. In addition, these structural alterations progress with surprising speed, rendering aggregates resistant to disassembly within minutes. Thus, while protein aggregate structure is generally poorly defined and is often obscured by heterogeneous and complex particle distributions, it can have a determinative impact on the ability of cellular quality control systems to process protein aggregates.
... Fold topology, the arrangement and number of the "non-breakable" bonds affect the unfolding process. Mechanical stretching can be applied with several methods: (1) by pulling on either end of the polymer; (2) by threading the polymer through a nanopore, or (3) by pulling and threading a polymer through a nanopore at the same time [22,23]. Contacts that are nested inside other contacts will only break when the covering contacts are broken. ...
In this chapter, we discuss emerging concepts and tools for engineering molecular folds. We focus on linear polymers including engineered proteins and DNA as well as polymers of non-biological origins. We outline the implications of fold topology for the kinetics of folding reactions and the stability of the synthesized fold. The relation between topology and molecular properties including mechanical response to external forces will be discussed. For the desired topology, we will examine monomer chemistry options and available synthesis protocols. The chapter will end with a perspective of future challenges and possibilities in programmed polymer folding.
... Similar events at NBD2 may then engage PL3 as a pawl (Fig. 5B, step 4). It is possible that disengagement of these pawls allows looped polypeptide segments to escape after partial threading (20,35). The protein harnesses the power of asynchronous pulling by neighboring subunits to generate rapid processive translocation events. ...
AAA+ ring–shaped machines, such as the disaggregation machines ClpB and Hsp104, mediate ATP-driven substrate translocation through their central channel by a set of pore loops. Recent structural studies have suggested a universal hand-over-hand translocation mechanism with slow and rigid subunit motions. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB during substrate threading, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond time scale and change their conformation upon interaction with substrate proteins in an ATP-dependent manner. Conformational dynamics of two of the pore loops strongly correlate with disaggregation activity, suggesting that they are the main contributors to substrate pulling. This set of findings is rationalized in terms of an ultrafast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process in ClpB and other AAA+ machines.
... 7 Prokaryotic ClpB has been shown to catalyze protein unfolding and disaggregation in the setting of cellular stress. [8][9][10] This function is dependent on adenosine triphosphate (ATP) hydrolysis and the formation of a hexameric ring through which substrate proteins are driven. Although the ATPase and disaggregase function of human CLPB has been confirmed, 11,12 and it does appear to form higher-order multimers, 12 the exact structural basis for its function may differ because vertebrate CLPB only shares homology with the prokaryotic C-terminal ATPase domain and has a unique N-terminal region. 2 CLPB localizes to mitochondria, 2,11,13,14 and it has been shown to regulate mitochondrial function. ...
Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Approximately one-third of cases do not have a known genetic cause. Exome sequencing of 104 persons with congenital neutropenia identified heterozygous missense variants of CLPB (caseinolytic peptidase B) in 5 SCN cases, with 5 more cases identified through additional sequencing efforts or clinical sequencing. CLPB encodes an adenosine triphosphatase (ATPase) implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, 3-methylglutaconic aciduria was not observed and, other than neutropenia, these clinical features were uncommon in our series. Moreover, the CLPB variants are distinct, consisting of heterozygous variants that cluster near the ATP-binding pocket. Both genetic loss of CLPB and expression of CLPB variants results in impaired granulocytic differentiation of human hematopoietic progenitors and increased apoptosis. These CLPB variants associate with wildtype CLPB and inhibit its ATPase and disaggregase activity in a dominant-negative fashion. Finally, expression of CLPB variants is associated with impaired mitochondrial function but does not render cells more sensitive to endoplasmic reticulum stress. Together, these data show that heterozygous CLPB variants are a new and relatively common cause of congenital neutropenia and should be considered in the evaluation of patients with congenital neutropenia.
... HSP70 plays a critical role in this network by mediating cooperative communications between the other core chaperones. For example, HSP70 triggers the disaggregase activity of HSP100, and jointly they disaggregate aggregated proteins and promote their subsequent refolding (72)(73)(74). In another example, HSP20 can transfer misfolded substrates to HSP70 for ATP-driven unfolding, from which they can be further transferred to HSP60 for final refolding to the native state (75). ...
Significance
Across the Tree of Life, life’s phenotypic diversity has been accompanied by a massive expansion of the protein universe. Compared with simple prokaryotes that harbor thousands of proteins, plants and animals harbor hundreds of thousands of proteins that are also longer, multidomain, and comprise a variety of folds and fold combinations, repeated segments, and beta-rich architectures that make them prone to misfolding and aggregation. Surprisingly, the relative representation of core chaperones, those dedicated to maintaining the folding quality of these increasingly complex proteomes, did not change from prokaryotic to mammalian genomes. To reconcile the expanding proteomes, core chaperones have rather increased in cellular abundance and evolved to function cooperatively as a network, combined with their supporting workforce, the cochaperones.
... Functionally, conformational asymmetry of Clp ATPases is proposed to underlie either sequential 16,[30][31][32] or probabilistic 28,33-39 substrate gripping and translocation mechanisms, whereas its role in severing mechanisms is less clear. In addition, dynamic dissociation of the hexamer is proposed to act as a mechanism for releasing substrates targeted for degradation that are trapped into configurations that require excessively long processing times 40,41 or for disengaging severing proteins from microtubules. 39 Currently, it is not well understood how these structural and functional aspects are enabled by hexamer dynamics and interprotomer interactions. ...
Disaggregation and microtubule-severing nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily assemble into ring–shaped hexamers that enable protein remodeling by coupling large–scale conformational changes with application of mechanical forces within a central pore by loops protruding within the pore. We probed these motions and intra-ring interactions that support them by performing extensive explicit solvent molecular dynamics simulations of single-ring severing proteins and the double-ring disaggregase ClpB. Simulations reveal that dynamic stability of hexamers of severing proteins and of the nucleotide binding domain 1 (NBD1) ring of ClpB, which belong to the same clade, involves a network of salt bridges that connect conserved motifs of central PL1 loops of the hexamer. Clustering analysis of ClpB highlights correlated motions of domains of neighboring protomers supporting strong inter-protomer collaboration. Severing proteins have weaker inter-protomer coupling and stronger intra-protomer stabilization through salt bridges formed between PL2 and PL3 loops. Distinct mechanisms are identified in the NBD2 ring of ClpB involving weaker inter–protomer coupling through salt bridges formed by non–canonical loops and stronger intra–protomer coupling. Pore width fluctuations associated with the PL1 constriction in the spiral states, in the presence of a substrate peptide, highlight stark differences between narrowing of channels of severing proteins and widening of the NBD1 ring of ClpB. This indicates divergent substrate processing mechanisms of remodeling and translocation by ClpB and substrate tail-end gripping and possible wedging on microtubule lattice by severing enzymes. Relaxation dynamics of the distance between the PL1 loops and the centers of mass of protomers reveals observation-time-dependent dynamics, leading to predicted relaxation times of tens of microseconds on millisecond experimental timescales. For ClpB the predicted relaxation time is in excellent agreement with the extracted time from smFRET experiments.
... In summary, our results indicate that ClpB3 targets exposed disordered segments, irrespective of the overall conformation of the substrate (molten globule-like, IDP or soluble aggregate). Even native proteins containing aggregation-prone regions are targeted by ClpB proteins, which act on the aggregated segment without altering the structure of the native moiety (Haslberger et al. 2008). ...
Key message:
The first biochemical characterization of a chloroplastic disaggregase is reported (Arabidopsis thaliana ClpB3). ClpB3 oligomerizes into active hexamers that resolubilize aggregated substrates using ATP and without the aid of partners. Disaggregases from the Hsp100/Clp family are a type of molecular chaperones involved in disassembling protein aggregates. Plant cells are uniquely endowed with ClpB proteins in the cytosol, mitochondria and chloroplasts. Chloroplastic ClpB proteins have been implicated in key processes like the unfolded protein response; however, they have not been studied in detail. In this study, we explored the biochemical properties of a chloroplastic ClpB disaggregase, in particular, ClpB3 from A. thaliana. ClpB3 was produced recombinantly in Escherichia coli and affinity-purified to near homogeneity. ClpB3 forms a hexameric complex in the presence of MgATP and displays intrinsic ATPase activity. We demonstrate that ClpB3 has ATPase activity in a wide range of pH and temperature values and is particularly resistant to heat. ClpB3 specifically targets unstructured polypeptides and mediates the reactivation of heat-denatured model substrates without the aid of the Hsp70 system. Overall, this work represents the first in-depth biochemical description of a ClpB protein from plants and strongly supports its role as the putative disaggregase chaperone in chloroplasts.
... From a metabolic point-of-view, CLPB belongs to the AAAþ (ATPases Associated with diverse cellular Activities) protein family [210,211]. The mutant CLPB is characterized by the loss of its ATPase activity. ...
The occurrence of 3-methylglutaconic aciduria (3-MGA) is a well understood phenomenon in leucine oxidation and ketogenesis disorders (primary 3-MGAs). In contrast, its genesis in non-canonical (secondary) 3-MGAs, a growing-up group of disorders encompassing more than a dozen of inherited metabolic diseases, is a mystery still remaining unresolved for three decades. To puzzle out this anthologic problem of metabolism, three clues were considered: (i) the variety of disorders suggests a common cellular target at the cross-road of metabolic and signaling pathways, (ii) the response to leucine loading test only discriminative for primary but not secondary 3-MGAs suggests these latter are disorders of extramitochondrial HMG-CoA metabolism as also attested by their failure to increase 3-hydroxyisovalerate, a mitochondrial metabolite accumulating only in primary 3-MGAs, (iii) the peroxisome is an extramitochondrial site possessing its own pool and displaying metabolism of HMG-CoA, suggesting its possible involvement in producing extramitochondrial 3-methylglutaconate (3-MG). Following these clues provides a unifying common basis to non-canonical 3-MGAs: constitutive mitochondrial dysfunction induces AMPK activation which, by inhibiting early steps in cholesterol and fatty acid syntheses, pipelines cytoplasmic acetyl-CoA to peroxisomes where a rise in HMG-CoA followed by local dehydration and hydrolysis may lead to 3-MGA yield. Additional contributors are considered, notably for 3-MGAs associated with hyperammonemia, and to a lesser extent in CLPB deficiency. Metabolic and signaling itineraries followed by the proposed scenario are essentially sketched, being provided with compelling evidence from the literature coming in their support.
... 1 3 prion state (Cox et al. 2003;Haslberger et al. 2008;Tipton et al. 2008;Winkler et al. 2012). ...
Prions are self-propagating protein isoforms that are typically amyloid. In Saccharomyces cerevisiae, amyloid prion aggregates are fragmented by a trio involving three classes of chaperone proteins: Hsp40s, also known as J-proteins, Hsp70s, and Hsp104. Hsp104, the sole Hsp100-class disaggregase in yeast, along with the Hsp70 Ssa and the J-protein Sis1, is required for the propagation of all known amyloid yeast prions. However, when Hsp104 is ectopically overexpressed, only the prion [PSI⁺] is efficiently eliminated from cell populations via a highly debated mechanism that also requires Sis1. Recently, we reported roles for two additional J-proteins, Apj1 and Ydj1, in this process. Deletion of Apj1, a J-protein involved in the degradation of sumoylated proteins, partially blocks Hsp104-mediated [PSI⁺] elimination. Apj1 and Sis1 were found to have overlapping functions, as overexpression of one compensates for loss of function of the other. In addition, overexpression of Ydj1, the most abundant J-protein in the yeast cytosol, completely blocks Hsp104-mediated curing. Yeast prions exhibit structural polymorphisms known as “variants”; most intriguingly, these J-protein effects were only observed for strong variants, suggesting variant-specific mechanisms. Here, we review these results and present new data resolving the domains of Apj1 responsible, specifically implicating the involvement of Apj1’s Q/S-rich low-complexity domain.
... This led us to propose a ''tug and release'' mechanism by which ClpB can resolve an aggregate by taking one or two translocation steps before dissociation and subsequent rebinding to repeat the cycle (23). Consistent with this conclusion, ClpB and Hsp104 have been reported to use a ''partial threading'' mechanism to disaggregate some substrates (32). Moreover, Hsp104 employs a partial translocation mechanism to dissolve Sup35 prions (3,33). ...
Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104 A503S , a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104 A503S can operate as nonprocessive translocases for soluble substrates, indicating a “partial threading” model of translocation. Remarkably, Hsp104 A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104 A503S .
... ClpB as well as Hsp104 belongs to the large AAA+ superfamily. The unifying characteristic of this family is the hydrolysis of ATP through the AAA+ domain to produce energy required to catalyze protein unfolding, disassembly and disaggregation [86][87][88]. ...
Many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Prion's disease, polyQ and Huntington's disease share abnormal folding of potentially cytotoxic protein species associated with degeneration and death of specific neuronal populations. In order to maintain cellular protein homeostasis, neurons have developed an intrinsic protein quality control system as a strategy to counteract protein aggregation and their toxicity. Heat shock proteins are an essential component for regulating protein quality control and contribute potentially in the process of protein folding, prevent protein aggregation and in disaggregation in several neurodegenerative diseases. Therefore, molecular chaperones are considered an exciting therapeutic target. In this book chapter, we will focus on the potential importance of different heat shock proteins in neurodegenerative diseases and understand their mechanisms to protect neurons form aggregates and their toxicity.
... Obviously, it still remains to be discovered to which extent natural PAs would be subjected to the same disaggregation dynamics as those from the fluorescently labelled PAs, and how this differential wake could impact the physiology of the cell and the phenotypic variability between siblings. Interestingly, evidence in both bacteria and yeast suggests that functional refolding of aggregated proteins is favored considerably over degradation (Haslberger et al. 2008;Wallace et al. 2015) and could be a strategy to preserve cellular resources and allow improved resuscitation upon stress relief (Mogk et al. 1999(Mogk et al. , 2018Weibezahn et al. 2004;Tessarz et al. 2008). Together with the observation that natural PAs seem to consist of a complex and stress-dependent mixture of proteins and associated chaperones (Wallace et al. 2015;Weids et al. 2016;Govers et al. 2018), this could imply that disaggregation, functional refolding, and subsequent dilution of this complex array of proteins could reshape the proteomes of individual cells in a deterministic lineage-dependent fashion. ...
The concept of phenotypic heterogeneity preparing a subpopulation of isogenic cells to better cope with anticipated stresses has been well established. However, less is known about how stress itself can drive subsequent cellular individualization in clonal populations. In this perspective, we focus on the impact of stress-induced cellular protein aggregates, and how their segregation and disaggregation can act as a deterministic incentive for heterogeneity in the population emerging from a stressed ancestor.
... Hsp70, Hsp40 and Hsp104 play major roles in such activity (Doyle and Wickner, 2009;Masison et al., 2009). Experimental results indicate that Hsp70 and Hsp40 interact with a Sup35 polypeptide in the fiber and hand it to Hsp104, which pulls the subunit through the central channel, dislodging it, thereby breaking the fiber in two (Kryndushkin et al., 2003;Shorter and Lindquist, 2006;Haslberger et al., 2008;Higurashi et al., 2008;Tessarz et al., 2008;Tipton et al., 2008;Winkler et al., 2012). Hsp104 also appears to trim [PSI + ] fibers, removing Sup35 monomers from the ends, further reducing their sizes (Park et al., 2014;Zhao et al., 2017;Greene et al., 2018). ...
[PSI⁺] variants are different infectious conformations of the same Sup35 protein. We show that when [PSI⁺] variants VK and VL co‐infect a dividing host, only one prevails in the end and the host genetic background is involved in winner selection. In the 5V‐H19 background, the VK variant dominates over the VL variant. The order of dominance is reversed in the 74‐D694 background, where VL can co‐exists with VK for a short period, but will eventually take over. Differential interaction of chaperone proteins with distinct prion variant conformations can influence the outcome of competition. Expanding the Glycine/Methionine‐rich domain of Sis1, an Hsp40 protein, helps the propagation of VL. Over‐expression of the Hsp70 protein Ssa2 lowers the number of prion particles (propagons) in the cell. There is more reduction for VK than VL, causing the latter to dominate in some of the 5V‐H19 and all of the 74‐D694 cells tested. Consistently, depleting Ssa1 in 74‐D694 strengthens VK. Swapping chromosomal alleles of SSA1/2 and SIS1 between 5V‐H19 and 74‐D694, including cognate promoters, is not sufficient to change the native dominance order of each background, suggesting there exist additional polymorphic factors that modulate [PSI⁺] competition.
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... En réponse au stress oxydatif, la protéine Hsp33 va prendre le relais du système DnaKJE 3 vers ClpB (i) [139]. ClpB exerce alors une force de traction sur la protéine à solubiliser, l'obligeant à traverser son pore central et à s'extraire de l'agrégat protéique dans lequel elle était piégée (ii). ...
L'îlot génomique pks code une machinerie de biosynthèse complexe synthétisant la colibactine, une génotoxine produite par certaines souches de Escherichia coli. Cette génotoxine induit des cassures double-brin de l'ADN sur les cellules eucaryotes in vitro et in vivo. La colibactine n'est pas une protéine, mais un métabolite secondaire de type polycétide/peptide non-ribosomal (PK/NRP). Des résultats préliminaires de l'équipe semblaient indiquer que certains gènes du core genome de E. coli seraient également impliqués dans la production de la colibactine. L'objectif de cette thèse était d'identifier les gènes non-essentiels de E. coli situés hors de l'îlot génomique pks impliqués dans la synthèse de colibactine, en construisant une banque de mutants par insertion de transposons. Ce criblage a permis d'identifier 29 gènes candidats, mais deux groupes de gènes ont été particulièrement étudiés dans la suite du projet : trois gènes codants des protéines chaperons, et trois gènes codant des enzymes impliquées dans le métabolisme des polyamines. Le premier projet a permis de montrer que la protéine chaperon HtpG (ou Hsp90Ec), homologue bactérien de la protéine de choc thermique eucaryote Hsp90, est requise pour la production de colibactine, mais aussi de yersiniabactine, un sidérophore (ou système bactérien de captation du fer) appartenant à la même famille chimique que la colibactine. De plus, la protéase ClpQ intervient de concert avec Hsp90Ec dans la production de colibactine et de yersiniabactine. Ces résultats confirment ainsi l'interconnexion entre la synthèse des deux facteurs de virulence de E. coli, la colibactine et la yersiniabactine. Enfin, l'analyse des effets de la mutation du gène htpG au cours d'une infection systémique chez l'animal, dans des modèles de sepsis et de méningite néonatale chez les rongeurs, démontre le rôle de la protéine de réponse au stress Hsp90Ec dans la virulence de E. coli. Le second projet a révélé que les polyamines sont impliquées dans la production de colibactine. L'étude du métabolisme des polyamines par une approche de microbiologie moléculaire a démontré que la spermidine est la polyamine nécessaire à la production de colibactine. Les résultats préliminaires de ce projet indiquent que la spermidine participerait à la régulation de l'expression de certains gènes de l'îlot génomique pks, et de fait modulerait la biosynthèse de colibactine. Des études complémentaires sont en cours pour élucider les mécanismes impliqués. Les résultats de cette thèse sont une illustration parfaite de l'intégration symbiotique d'un élément génétique mobile acquis au cours de l'évolution au sein du chromosome bactérien, grâce à plusieurs connexions bilatérales permettant la production de facteurs de virulence par E. coli.
... With the help of its auxiliary chaperone DnaJ, DnaK binds to the aggregates and subsequently recruits ClpB for interaction with substrate [8]. Interaction of oligomeric barrel shaped ClpB with the exposed ends of substrate triggers ATP hydrolysis and promotes threading of the substrate through the central channel, eventually releasing the extended polypeptide from the exit pore whereafter it is free to refold [9][10][11]. It is noteworthy that, ClpB or DnaKJE individually exhibit only weak disaggregation activity [12]. ...
Mycobacterium tuberculosis (M. tb) is known to persist in extremely hostile environments within host macrophages. The ability to withstand such proteotoxic stress comes from its highly conserved molecular chaperone machinery. ClpB, a unique member of the AAA+ family of chaperones, is responsible for resolving aggregates in M. tb and many other bacterial pathogens. M. tb produces two isoforms of ClpB, a full length and an N‐terminally truncated form (ClpB∆N), with the latter arising from an internal translation initiation site. It is not clear why this internal start site is conserved and what role the N‐terminal domain (NTD) of M. tb ClpB plays in its function. In the current study, we functionally characterized and compared the two isoforms of M. tb ClpB. We found the NTD to be dispensable for oligomerization, ATPase activity and prevention of aggregation activity of ClpB. Both ClpB and ClpB∆N were found to be capable of resolubilizing protein aggregates. However, the efficiency of ClpB∆N at resolubilizing higher order aggregates was significantly lower than that of ClpB. Further, ClpB∆N exhibited reduced affinity for substrates as compared to ClpB. We also demonstrated that the surface of the NTD of M. tb ClpB has a hydrophobic groove which contains four hydrophobic residues: L97, L101, F140, and V141. These residues act as initial contacts for the substrate and are crucial for stable interaction between ClpB and highly aggregated substrates.
... Each Hsp104 protomer contains an N-terminal domain, nucleotide-binding domain 1 (NBD1), a middle domain (MD), NBD2, and a short C-terminal domain (Fig. 1A) (Sweeny and Shorter 2016). Hsp104 assembles into asymmetric ring-like or lock-washer hexamers (Wendler et al. 2009;Yokom et al. 2016;Gates et al. 2017), which extract individual polypeptides from aggregated structures via partial or complete translocation across their central channel (Lum et al. 2004;Shorter and Lindquist 2005a;Haslberger et al. 2008;Lum, Niggemann and Glover 2008;Tessarz, Mogk and Bukau 2008;Castellano et al. 2015;Sweeny et al. 2015). Polypeptide translocation is driven via ratchet-like conformational changes of the hexamer that are coupled to ATP binding and hydrolysis (Yokom et al. 2016;Gates et al. 2017). ...
Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase separation by TDP-43, FUS and α-synuclein connected to devastating neurodegenerative disorders. Subtle missense mutations in the Hsp104 MD can enhance activity, indicating that post-translational modification of specific MD residues might also potentiate Hsp104. Indeed, several serine and threonine residues throughout Hsp104 can be phosphorylated in vivo. Here, we introduce phosphomimetic aspartate or glutamate residues at these positions and assess Hsp104 activity. Remarkably, phosphomimetic T499D/E and S535D/E mutations in the MD enable Hsp104 to counter TDP-43, FUS and α-synuclein aggregation and toxicity in yeast, whereas T499A/V/I and S535A do not. Moreover, Hsp104T499E and Hsp104S535E exhibit enhanced ATPase activity and Hsp70-independent disaggregase activity in vitro. We suggest that phosphorylation of T499 or S535 may elicit enhanced Hsp104 disaggregase activity in a reversible and regulated manner.
Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during e.g. food-processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.
Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during e.g. food-processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.
Molecular chaperones are critical to control protein quality in all living cells. Understanding chaperone function at the atomic level, and in particular its mode of interaction with client proteins, is crucial to understanding the fundamental roles chaperones play in biology. This book fills a gap in the literature by comprehensively summarizing and discussing new advanced experimental techniques for their analysis. Providing a comprehensive overview of advanced biophysical methods for the characterization of molecular mechanisms of molecular chaperones, the majority of the contributions are NMR methodology. This is the method of choice for atomic resolution studies of such systems. Additional notable biophysical approaches are considered to present all relevant current developments in exploring chaperone function and the transient and dynamic interactions with their client proteins.
The book is targeted at both current practitioners of structural biology and biophysical chemistry and scientists who are interested in entering the field. It could be useful for graduate students as supplementary reading.
Severe heat stress causes massive loss of essential proteins by aggregation, necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins, while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate-targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn²⁺-coordination site that is crucial for structural integrity and disaggregase functionality. We found that conserved hydrophobic N1 residues located on a β-strand are crucial for aggregate targeting and disaggregation activity. Analysis of mixed hexamers consisting of full-length and N1-truncated subunits revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high-disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.
Chaperones of the Hsp100/Clp family represent major components of protein homeostasis, conferring maintenance of protein activity under stress. The ClpB-type members of the family, present in bacteria, fungi, and plants, are able to resolubilize aggregated proteins. The mitochondrial member of the ClpB family in Saccharomyces cerevisiae is Hsp78. Although Hsp78 has been shown to contribute to proteostasis in elevated temperatures, the biochemical mechanisms underlying this mitochondria-specific thermotolerance are still largely unclear. To identify endogenous chaperone substrate proteins, here we generated an Hsp78-ATPase mutant with stabilized substrate binding behavior. We used two stable isotope labeling (SILAC)-based quantitative mass spectrometry approaches to analyze the role of Hsp78 during heat stress-induced mitochondrial protein aggregation and disaggregation on a proteomic level. We first identified the endogenous substrate spectrum of the Hsp78 chaperone, comprising a wide variety of proteins related to metabolic functions including energy production and protein synthesis, as well as other chaperones, indicating its crucial functions in mitochondrial stress resistance. We then compared these interaction data with aggregation and disaggregation processes in mitochondria under heat stress, which revealed specific aggregation-prone protein populations and demonstrated the direct quantitative impact of Hsp78 on stress-dependent protein solubility under different conditions. We conclude that Hsp78, together with its cofactors, represents a recovery system that protects major mitochondrial metabolic functions during heat stress as well as restores protein biogenesis capacity after the return to normal conditions.
Correct protein folding is essential for the health and function of living organisms. Yet, it is not well understood how unfolded proteins reach their native state and avoid aggregation, especially within the cellular milieu. Some proteins, especially small, single-domain and apparent two-state folders, successfully attain their native state upon dilution from denaturant. Yet, many more proteins undergo misfolding and aggregation during this process, in a concentration-dependent fashion. Once formed, native and aggregated states are often kinetically trapped relative to each other. Hence, the early stages of protein life are absolutely critical for proper kinetic channeling to the folded state and for long-term solubility and function. This review summarizes current knowledge on protein folding/aggregation mechanisms in buffered solution and within the bacterial cell, highlighting early stages. Remarkably, teamwork between nascent chain, ribosome, trigger factor and Hsp70 molecular chaperones enables all proteins to overcome aggregation propensities and reach a long-lived bioactive state.
Numerous ATPases associated with diverse cellular activities (AAA+) proteins form hexameric, ring-shaped complexes that function via ATPase-coupled translocation of substrates across the central channel. Cryo-electron microscopy of AAA+ proteins processing substrate has revealed non-symmetric, staircase-like hexameric structures that indicate a sequential clockwise/2-residue step translocation model for these motors. However, for many of the AAA+ proteins that share similar structural features, their translocation properties have not yet been experimentally determined. In the cases where translocation mechanisms have been determined, a two-residue translocation step-size has not been resolved. In this review, we explore Hsp104, ClpB, ClpA and ClpX as examples to review the experimental methods that have been used to examine, in solution, the translocation mechanisms employed by AAA+ motor proteins. We then ask whether AAA+ motors sharing similar structural features can have different translocation mechanisms. Finally, we discuss whether a single AAA+ motor can adopt multiple translocation mechanisms that are responsive to different challenges imposed by the substrate or the environment. We suggest that AAA+ motors adopt more than one translocation mechanism and are tuned to switch to the most energetically efficient mechanism when constraints are applied.
After being synthesized on the ribosome, peptide chains must fold into unique 3D structures to become functional, except for IDPs lacking folded domains. The amino acid sequence determines the folded structure. However, during the folding process, polypeptide chains often misfold either spontaneously or under cellular stress, resulting in the formation of nonfunctional proteins and aggregates. To avoid this adverse result, life has evolved molecular chaperones that promote proper folding of nascent polypeptides by facilitating folding and preventing misfolding and aggreagation. However, properly folded proteins are often subjected to several types of stress, resulting in misfolding and aggregation.
Stresses such as heat shock trigger the formation of protein aggregates and the induction of a disaggregation system composed of molecular chaperones. Recent work reveals that several cases of apparent heat-induced aggregation, long thought to be the result of toxic misfolding, instead reflect evolved, adaptive biomolecular condensation, with chaperone activity contributing to condensate regulation. Here we show that the yeast disaggregation system directly disperses heat-induced biomolecular condensates of endogenous poly(A)-binding protein (Pab1) orders of magnitude more rapidly than aggregates of the most commonly used misfolded model substrate, firefly luciferase. Beyond its efficiency, heat-induced condensate dispersal differs from heat-induced aggregate dispersal in its molecular requirements and mechanistic behavior. Our work establishes a bona fide endogenous heat-induced substrate for long-studied heat shock proteins, isolates a specific example of chaperone regulation of condensates, and underscores needed expansion of the proteotoxic interpretation of the heat shock response to encompass adaptive, chaperone-mediated regulation.
Disaggregation and microtubule-severing nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily assemble into ring-shaped hexamers that enable protein remodeling by coupling large-scale conformational changes with application of mechanical forces within a central pore by loops protruding within the pore. We probed the asymmetric pore motions and intra-ring interactions that support them by performing extensive molecular dynamics simulations of single-ring severing proteins and the double-ring disaggregase ClpB. Simulations reveal that dynamic stability of hexameric pores of severing proteins and of the nucleotide binding domain 1 (NBD1) ring of ClpB, which belong to the same clade, involves a network of salt bridges that connect conserved motifs of central pore loops. Clustering analysis of ClpB highlights correlated motions of domains of neighboring protomers supporting strong inter-protomer collaboration. Severing proteins have weaker inter-protomer coupling and stronger intra-protomer stabilization through salt bridges involving pore loops. Distinct mechanisms are identified in the NBD2 ring of ClpB involving weaker inter-protomer coupling through salt bridges formed by non-canonical loops and stronger intra-protomer coupling. Analysis of collective motions of PL1 loops indicates that the largest amplitude motions in the spiral complex of spastin and ClpB involve axial excursions of the loops, whereas for katanin they involve opening and closing of the central pore. All three motors execute primarily axial excursions in the ring complex. These results suggest distinct substrate processing mechanisms of remodeling and translocation by ClpB and spastin, compared to katanin, thus providing dynamic support for the differential action of the two severing proteins.Relaxation dynamics of the distance between the PL1 loops and the center of mass of protomers reveals observation-time-dependent dynamics, leading to predicted relaxation times of tens to hundreds of microseconds on millisecond experimental timescales. For ClpB the predicted relaxation time is in excellent agreement with the extracted time from smFRET experiments.
AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.
Stressful environments often lead to protein unfolding and the formation of cytotoxic aggregates that can compromise cell survival. The molecular chaperone heat shock protein (HSP) 101 is a protein disaggregase that co-operates with the small HSP (sHSP) and HSP70 chaperones to facilitate removal of such aggregates and is essential for surviving severe heat stress. To better define how HSP101 protects plants, we investigated the localization and targets of this chaperone in Arabidopsis (Arabidopsis thaliana). By following HSP101 tagged with GFP, we discovered that its intracellular distribution is highly dynamic and includes a robust, reversible sequestration into cytoplasmic foci that vary in number and size among cell types and are potentially enriched in aggregated proteins. Affinity isolation of HSP101 recovered multiple proteasome subunits, suggesting a functional interaction. Consistent with this, the GFP-tagged 26S proteasome regulatory particle non-ATPase (RPN) 1a transiently colocalized with HSP101 in cytoplasmic foci during recovery. In addition, analysis of aggregated (insoluble) proteins showed they are extensively ubiquitylated during heat stress, especially in plants deficient in HSP101 or class I sHSPs, implying that protein disaggregation is important for optimal proteasomal degradation. Many potential HSP101 clients, identified by mass spectrometry of insoluble proteins, overlapped with known stress granule constituents and sHSP-interacting proteins, confirming a role for HSP101 in stress granule function. Connections between HSP101, stress granules, proteasomes, and ubiquitylation imply that dynamic coordination between protein disaggregation and proteolysis is required to survive proteotoxic stress caused by protein aggregation at high temperatures.
The folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, as non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by using molecular chaperones to safeguard folding cotranslationally2. However, although most of the cellular proteome forms oligomeric assemblies3, little is known about the final step of folding: the assembly of polypeptides into complexes. In prokaryotes, a proof-of-concept study showed that the assembly of heterodimeric luciferase is an organized cotranslational process that is facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences—such as the rarity of polycistronic mRNAs and different chaperone constellations—raise the question of whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of the assembly of protein complexes in eukaryotes using ribosome profiling. We determined the in vivo interactions of the nascent subunits from twelve hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find nine complexes assemble cotranslationally; the three complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5,6,7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit, thereby counteracting its propensity for aggregation. The onset of cotranslational subunit association coincides directly with the full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains and then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for the assembly of hetero-oligomers in yeast and indicates that translation, folding and the assembly of protein complexes are integrated processes in eukaryotes.
ClpB and DnaKJE provide protection to Escherichia coli cells during extreme environmental stress. Together, this co-chaperone system can resolve protein aggregates, restoring misfolded proteins to their native form and function or solubilizing damaged proteins for removal by the cell’s proteolytic systems. DnaK is the component of the KJE system that directly interacts with ClpB. There are many hypotheses for how DnaK affects ClpB catalyzed disaggregation, each with some experimental support. Here, we build on our recent work characterizing the molecular mechanism of ClpB catalyzed polypeptide translocation by developing a stopped-flow FRET assay that allows us to detect ClpB’s movement on model polypeptide substrates in the absence or presence of DnaK. We find that DnaK induces ClpB to dissociate from the polypeptide substrate. We propose that DnaK acts as a peptide release factor, binding ClpB and causing the ClpB conformation to change to a low peptide-affinity state. Such a role for DnaK would allow ClpB to rebind to another portion of an aggregate and continue nonprocessive translocation to disrupt the aggregate.
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In eukaryotes, the N-end rule pathway is a ubiquitin-dependent, proteasome-based system that targets and processively degrades proteins bearing certain N-terminal residues. Arg-DHFR, a modified dihydrofolate reductase bearing an N-terminal arginine (destabilizing residue in the N-end rule), is short lived in ATP-supplemented reticulocyte extract. It is shown here that methotrexate, which is a folic acid analog and high affinity ligand of DHFR, inhibits the degradation but not ubiquitination of Arg-DHFR by the N-end rule pathway. The degradation of other N-end rule substrates is not affected by methotrexate. We discuss implications of these results for the mechanism of proteasome-mediated protein degradation.
The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.
Development of genetic competence in Bacillus subtilis is controlled by the competence-specific transcription factor ComK. ComK activates transcription of itself and several other genes required for competence. The activity of ComK is controlled by other genes including mecA, clpC, and comS. We have used purified ComK, MecA, ClpC, and synthetic ComS to study their interactions and have demonstrated the following mechanism for ComK regulation. ClpC, in the presence of ATP, forms a ternary complex with MecA and ComK, which prevents ComK from binding to its specific DNA target. This complex dissociates when ComS is added, liberating active ComK. ClpC and MecA function as a molecular switch, in which MecA confers molecular recognition, connecting ClpC to ComK and to ComS.
Competence is a physiological state, distinct from sporulation and vegetative growth, that enables cells to bind and internalize transforming DNA. The transcriptional regulator ComK drives the development of competence in Bacillus subtilis. ComK is directly required for its own transcription as well as for the transcription of the genes that encode DNA transport proteins. When ComK is sequestered by binding to a complex of the proteins MecA and ClpC, the positive feedback loop leading to ComK synthesis is interrupted. The small protein ComS, produced as a result of signaling by a quorum-sensing two-component regulatory pathway, triggers the release of ComK from the complex, enabling comK transcription to occur. We show here, based on in vivo and in vitro experiments, that ComK accumulation is also regulated by proteolysis and that binding to MecA targets ComK for degradation by the ClpP protease in association with ClpC. The release of ComK from binding by MecA and ClpC, which occurs when ComS is synthesized, protects ComK from proteolysis. Following this release, the rates of MecA and ComS degradation by ClpCP are increased in our in vitro system. In this novel system, MecA serves to recruit ComK to the ClpCP protease and connects ComK degradation to the quorum-sensing signal-transduction pathway, thereby regulating a key developmental process. This is the first regulated degradation system in which a specific targeting molecule serves such a function.
Functional chaperone cooperation between Hsp70 (DnaK) and Hsp104 (ClpB) was demonstrated in vitro. In a eubacterium Thermus thermophilus, DnaK and DnaJ exist as a stable trigonal ring complex (TDnaK.J complex) and the dnaK gene cluster contains a clpB gene. When substrate proteins were heated at high temperature, none of the chaperones protected them from heat inactivation, but the TDnaK.J complex could suppress the aggregation of proteins in an ATP- and TGrpE-dependent manner. Subsequent incubation of these heated preparations at moderate temperature after addition of TClpB resulted in the efficient reactivation of the proteins. Reactivation was also observed, even though the yield was low, if the substrate protein alone was heated and incubated at moderate temperature with the TDnaK.J complex, TGrpE, TClpB, and ATP. Thus, all these components were necessary for the reactivation. Further, we found that TGroEL/ES could not substitute TClpB.
The bacterial protein CIpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP. ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.
ClpB is a heat-shock protein fromEscherichia coli with an unknown function. We studied a possible molecular chaperone activity of ClpB in vitro. Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mm ATP and 0.1 mg/ml bovine serum albumin). Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native
activity) because of extensive aggregation. Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone
did not reactivate luciferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase
activity regain (up to 57% of native activity) by suppressing luciferase aggregation. This coordinated function of ClpB and
DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When
the chaperones were added to the luciferase refolding solutions after 5–25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered
the luciferase activity from preformed aggregates. Thus, we have identified a novel multi-chaperone system from E. coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast. ClpB is the only known bacterial Hsp100 protein capable of
cooperating with other heat-shock proteins in suppressing and reversing protein aggregation.
A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid binding, and of the disaggregation/refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.
We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (</=30 kDa) cytosolic proteins and include essential proteins. The DnaK system in addition acts with ClpB to form a bi-chaperone system that quantitatively solubilizes aggregates of most of these proteins. Efficient solubilization also occurred in an in vivo order-of-addition experiment in which aggregates were formed prior to induction of synthesis of the bi-chaperone system. Our data indicate that large-sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central, dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation. Keywords: chaperones/heat-shock response/Hsp70/protein denaturation/thermotolerance
The ability of organisms to acquire thermotolerance to normally lethal high temperatures is an ancient and conserved adaptive response. However, knowledge of cellular factors essential to this response is limited. Acquisition of thermotolerance is likely to be of particular importance to plants that experience daily temperature fluctuations and are unable to escape to more favorable environments. We developed a screen, based on hypocotyl elongation, for mutants of Arabidopsis thaliana that are unable to acquire thermotolerance to high-temperature stress and have defined four separate genetic loci, hot1-4, required for this process. hot1 was found to have a mutation in the heat shock protein 101 (Hsp101) gene, converting a conserved Glu residue in the second ATP-binding domain to a Lys residue, a mutation that is predicted to compromise Hsp101 ATPase activity. In addition to exhibiting a thermotolerance defect as assayed by hypocotyl elongation, 10-day-old hot1 seedlings were also unable to acquire thermotolerance, and hot1 seeds had greatly reduced basal thermotolerance. Complementation of hot1 plants by transformation with wild-type Hsp101 genomic DNA restored hot1 plants to the wild-type phenotype. The hot mutants are the first mutants defective in thermotolerance that have been isolated in a higher eukaryote, and hot1 represents the first mutation in an Hsp in any higher plant. The phenotype of hot1 also provides direct evidence that Hsp101, which is required for thermotolerance in bacteria and yeast, is also essential for thermotolerance in a complex eukaryote.
Plants are sessile organisms, and their ability to adapt to stress is crucial for survival in natural environments. Many observations suggest a relationship between stress tolerance and heat shock proteins (HSPs) in plants, but the roles of individual HSPs are poorly characterized. We report that transgenic Arabidopsis plants expressing less than usual amounts of HSP101, a result of either antisense inhibition or cosuppression, grew at normal rates but had a severely diminished capacity to acquire heat tolerance after mild conditioning pretreatments. The naturally high tolerance of germinating seeds, which express HSP101 as a result of developmental regulation, was also profoundly decreased. Conversely, plants constitutively expressing HSP101 tolerated sudden shifts to extreme temperatures better than did vector controls. We conclude that HSP101 plays a pivotal role in heat tolerance in Arabidopsis. Given the high evolutionary conservation of this protein and the fact that altering HSP101 expression had no detrimental effects on normal growth or development, one should be able to manipulate the stress tolerance of other plants by altering the expression of this protein.
Processing of integral membrane proteins in order to liberate active proteins is of exquisite cellular importance. Examples are the processing events that govern sterol regulation, Notch signaling, the unfolded protein response, and APP fragmentation linked to Alzheimer's disease. In these cases, the proteins are thought to be processed by regulated intramembrane proteolysis, involving site-specific, membrane-localized proteases. Here we show that two homologous yeast transcription factors SPT23 and MGA2 are made as dormant ER/nuclear membrane-localized precursors and become activated by a completely different mechanism that involves ubiquitin/proteasome-dependent processing. SPT23 and MGA2 are relatives of mammalian NF-kappaB and control unsaturated fatty acid levels. Intriguingly, proteasome-dependent processing of SPT23 is regulated by fatty acid pools, suggesting that the precursor itself or interacting partners are sensors of membrane composition or fluidity.
ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.
In the bacterial cytosol, ATP-dependent protein degradation is performed by several different chaperone-protease pairs, including ClpAP. The mechanism by which these machines specifically recognize substrates remains unclear. Here, we report the identification of a ClpA cofactor from Escherichia coli, ClpS, which directly influences the ClpAP machine by binding to the N-terminal domain of the chaperone ClpA. The degradation of ClpAP substrates, both SsrA-tagged proteins and ClpA itself, is specifically inhibited by ClpS. In contrast, ClpS enhanced ClpA recognition of two heat-aggregated proteins in vitro and, consequently, the ClpAP-mediated disaggregation and degradation of these substrates. We conclude that ClpS modifies ClpA substrate specificity, potentially redirecting degradation by ClpAP toward aggregated proteins.
The proteasome can actively unfold proteins by sequentially unraveling their substrates from the attachment point of the degradation signal. To investigate the steric constraints imposed on substrate proteins during their degradation by the proteasome, we constructed a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. These results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain. A degradation channel that can tolerate some steric bulk may reconcile the two opposing needs for degradation that is compartmentalized to avoid aberrant proteolysis yet able to handle a range of substrates of various sizes.
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.
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.
Saccharomyces cerevisiae Hsp104, a hexameric member of the Hsp100/Clp subfamily of AAA+ ATPases with two nucleotide binding domains (NBD1 and 2), refolds aggregated proteins in conjunction with Hsp70 molecular chaperones. Hsp104 may act as a "molecular crowbar" to pry aggregates apart and/or may extract proteins from aggregates by unfolding and threading them through the axial channel of the Hsp104 hexamer. Targeting Tyr-662, located in a Gly-Tyr-Val-Gly motif that forms part of the axial channel loop in NBD2, we created conservative (Phe and Trp) and non-conservative (Ala and Lys) amino acid substitutions. Each of these Hsp104 derivatives was comparable to the wild type protein in their ability to hydrolyze ATP, assemble into hexamers, and associate with heat-shock-induced aggregates in living cells. However, only those with conservative substitutions complemented the thermotolerance defect of a Deltahsp104 yeast strain and promoted refolding of aggregated protein in vitro. Monitoring fluorescence from Trp-662 showed that titration of fully assembled molecules with either ATP or ADP progressively quenches fluorescence, suggesting that nucleotide binding determines the position of the loop within the axial channel. A Glu to Lys substitution at residue 645 in the NBD2 axial channel strongly alters the nucleotide-induced change in fluorescence of Trp-662 and specifically impairs in protein refolding. These data establish that the structural integrity of the axial channel through NBD2 is required for Hsp104 function and support the proposal that Hsp104 and ClpB use analogous unfolding/threading mechanisms to promote disaggregation and refolding that other Hsp100s use to promote protein degradation.
Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation.
Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.
By immunostaining and transmission electron microscopy, chaperones DnaK and GroEL have been identified at the solvent-exposed
surface of bacterial inclusion bodies and entrapped within these aggregates, respectively. Functional implications of this
distinct localization are discussed in the context of Escherichia coli protein quality control.
HSP100 proteins are molecular chaperones that belong to the broader family of AAA+ proteins (ATPases associated with a variety of cellular activities) known to promote protein unfolding, disassembly of protein complexes and translocation of proteins across membranes. The ClpC form of HSP100 is an essential, highly conserved, constitutively expressed protein in cyanobacteria and plant chloroplasts, and yet little is known regarding its specific activity as a molecular chaperone. To address this point, ClpC from the cyanobacterium Synechococcus elongatus (SyClpC) was purified using an Escherichia coli-based overexpression system. Recombinant SyClpC showed basal ATPase activity, similar to that of other types of HSP100 protein in non-photosynthetic organisms but different to ClpC in Bacillus subtilis. SyClpC also displayed distinct intrinsic chaperone activity in vitro, first by preventing aggregation of unfolded polypeptides and second by resolubilizing and refolding aggregated proteins into their native structures. The refolding activity of SyClpC was enhanced 3-fold in the presence of the B. subtilis ClpC adaptor protein MecA. Overall, the distinctive ClpC protein in photosynthetic organisms indeed functions as an independent molecular chaperone, and it is so far unique among HSP100 proteins in having both "holding" and disaggregase chaperone activities without the need of other chaperones or adaptor proteins.
Exposure to temperatures over a certain limit leads to massive protein aggregation in the cell. Disaggregation of such aggregates
is largely dependent on the Hsp100 and Hsp70 chaperones. The exact role of the Hsp70 chaperone machine (composed of DnaK,
DnaJ, and GrpE) in the Hsp100-dependent process remains unknown. In this study we focused on the Hsp70 role at the initial
step of the disaggregation process. Two different aggregated model substrates, green fluorescent protein (GFP) and firefly
luciferase, were incubated with the Hsp70 machine resulting in efficient fragmentation of large aggregates into smaller ones.
Our data suggest that the observed fragmentation is achieved first by extraction of polypeptides from aggregates in Hsp70
chaperone machine-dependent manner and not by direct fragmentation of large aggregates. In the absence of Hsp100 (ClpB) these
“extracted” polypeptides were not able to fold properly and promptly reassociated into new aggregates. The extracted GFP molecules
were efficiently recognized and sequestered by a molecular trap, the mutant GroEL D87K, which binds stably to unfolded but
not to native polypeptides. The binding of extracted GFP molecules to the GroEL trap prevented their reaggregation. We propose
that the Hsp70 machine disentangles polypeptides from protein aggregates prior to Hsp100 action.
The AAA+ protein ClpC is not only involved in the removal of misfolded and aggregated proteins but also controls, through regulated proteolysis, key steps of several developmental processes in the Gram-positive bacterium Bacillus subtilis. In contrast to other AAA+ proteins, ClpC is unable to mediate these processes without an adaptor protein like MecA. Here, we demonstrate that the general activation of ClpC is based upon the ability of MecA to participate in the assembly of an active and substrate-recognizing higher oligomer consisting of ClpC and the adaptor protein, which is a prerequisite for all activities of this AAA+ protein. Using hybrid proteins of ClpA and ClpC, we identified the N-terminal and the Linker domain of the first AAA+ domain of ClpC as the essential MecA interaction sites. This new adaptor-mediated mechanism adds another layer of control to the regulation of the biological activity of AAA+ proteins.
Plants are sessile organisms, and their ability to adapt to stress is crucial for survival in natural environments. Many observations suggest a relationship between stress tolerance and heat shock proteins (HSPs) in plants, but the roles of individual HSPs are poorly characterized. We report that transgenic Arabidopsis plants expressing less than usual amounts of HSP101, a result of either antisense inhibition or cosuppression, grew at normal rates but had a severely diminished capacity to acquire heat tolerance after mild conditioning pretreatments. The naturally high tolerance of germinating seeds, which express HSP101 as a result of developmental regulation, was also profoundly decreased. Conversely, plants constitutively expressing HSP101 tolerated sudden shifts to extreme temperatures better than did vector controls. We conclude that HSP101 plays a pivotal role in heat tolerance in Arabidopsis. Given the high evolutionary conservation of this protein and the fact that altering HSP101 expression had no detrimental effects on normal growth or development, one should be able to manipulate the stress tolerance of other plants by altering the expression of this protein.
Molecular chaperones assist protein folding by facilitating their “forward” folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 Å long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.
Functional chaperone cooperation between Hsp70 (DnaK) and Hsp104 (ClpB) was demonstrated in vitro. In a eubacterium Thermus thermophilus, DnaK and DnaJ exist as a stable trigonal ring complex (TDnaK\cdot J complex) and the dnaK gene cluster contains a clpB gene. When substrate proteins were heated at high temperature, none of the chaperones protected them from heat inactivation, but the TDnaK\cdot J complex could suppress the aggregation of proteins in an ATP- and TGrpE-dependent manner. Subsequent incubation of these heated preparations at moderate temperature after addition of TClpB resulted in the efficient reactivation of the proteins. Reactivation was also observed, even though the yield was low, if the substrate protein alone was heated and incubated at moderate temperature with the TDnaK\cdot J complex, TGrpE, TClpB, and ATP. Thus, all these components were necessary for the reactivation. Further, we found that TGroEL/ES could not substitute TClpB.
Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.
ClpB is thought to be involved in proteolysis because of its sequence similarity to the ClpA subunit of the ClpA-ClpP protease. It has recently been shown that ClpP is a heat shock protein. Here we show that ClpB is the Escherichia coli heat shock protein F84.1. The F84.1 protein was overproduced in strains containing the clpB gene on a plasmid and was absent from two-dimensional gels from a clpB null mutation. Besides possessing a slower growth rate at 44 degrees C, the null mutant strain had a higher rate of death at 50 degrees C. We used reverse transcription of in vivo mRNA to show that the clpB gene was expressed from a sigma 32-specific promoter consensus sequence at both 37 and 42 degrees C. We noted that the clpB+ gene also caused the appearance of a second protein spot, F68.5, on two-dimensional gels. This spot was approximately 147 amino acids smaller than F84.1 and most probably is the result of a second translational start on the clpB mRNA. F68.5 can be observed on many published two-dimensional gels of heat-induced E. coli proteins, but the original catalog of 17 heat shock proteins did not include this spot.
A heat shock protein gene, HSP104, was isolated from Saccharomyces cerevisiae and a deletion mutation was introduced into
yeast cells. Mutant cells grew at the same rate as wild-type cells and died at the same rate when exposed directly to high
temperatures. However, when given a mild pre-heat treatment, the mutant cells did not acquire tolerance to heat, as did wild-type
cells. Transformation with the wild-type gene rescued the defect of mutant cells. The results demonstrate that a particular
heat shock protein plays a critical role in cell survival at extreme temperatures.
Intramolecular melting of troponin C, calmodulin and their proteolytic fragments has been studied microcalorimetrically at various concentrations of monovalent and divalent ions. It is shown by thermodynamic analysis of the experimentally determined excess heat capacity function that the four calcium-binding domains in these two related proteins are not integrated into a single co-operative system, as would be the case if they formed a common hydrophobic core in the molecule, but still interact with each other in a very specific way. There is a positive interaction between domains I and II, which is so strong that they actually form a single co-operative block. The interaction between domains III and IV is positive also, although much less pronounced, while the interaction between the pairs of domains (I and II) and (III and IV) is negative, as if they repel each other. The structure of the co-operative block of domains I and II at room temperature does not depend noticeably on the ionic conditions, which influence its stability to a small extent only. The same applies to domain IV of calmodulin, but in troponin C this domain is unstable in the absence of divalent ions, in solutions of low ionic strength. In both proteins, the least stable is domain III, which forms a compact ordered structure at room temperature only in the presence of Ca2+. In troponin C, calcium ions can be replaced by magnesium ions, although the compact structure of domain III formed by these two ions does not seem to be quite identical. Thus, at conditions close to physiological, with regard to temperature and ionic strength, the removal of free Ca2+ from the solution induces in both proteins a reversible transition of domain III to the non-compact disordered state. This dramatic Ca2+-induced change in the domain III conformation in troponin C and calmodulin might play a key role in the functioning of these proteins as a Ca2+-controlled switch in the molecular mechanisms of living systems.
Under destabilising conditions both heat and cold denaturation of yeast phosphoglycerate kinase (PGK) can be observed. According to previous interpretation of experimental data and theoretical calculations, the C-terminal domain should be more stable than the N-terminal domain at all temperatures. We report on thermal unfolding experiments with PGK and its isolated domains, which give rise to a revision of this view. While the C-terminal domain is indeed the more stable one on heating, it reveals lower stability in the cold. These findings are of importance, because PGK has been frequently used as a model for protein folding and mutual domain interactions.
The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.
The betagamma-crystallin superfamily consists of a class of homologous two-domain proteins with Greek-key fold. Protein S, a Ca(2+)-binding spore-coat protein from the soil bacterium Myxococcus xanthus exhibits a high degree of sequential and structural homology with gammaB-crystallin from the vertebrate eye lens. In contrast to gammaB-crystallin, which undergoes irreversible aggregation upon thermal unfolding, protein S folds reversibly and may therefore serve as a model in the investigation of the thermodynamic stability of the eye-lens crystallins. The thermal denaturation of recombinant protein S (PS) and its isolated domains was studied by differential scanning calorimetry in the absence and in the presence of Ca(2+) at varying pH. Ca(2+)-binding leads to a stabilization of PS and its domains and increases the cooperativity of their equilibrium unfolding transitions. The isolated N-terminal and C-terminal domains (NPS and CPS) obey the two-state model, independent of the pH and Ca(2+)-binding; in the case of PS, under all conditions, an equilibrium intermediate is populated. The first transition of PS may be assigned to the denaturation of the C-terminal domain and the loss of domain interactions, whereas the second one coincides with the denaturation of the isolated N-terminal domain. At pH 7.0, in the presence of Ca(2+), where PS exhibits maximal stability, the domain interactions at 20 degrees C contribute 20 kJ/mol to the overall stability of the intact protein.
The Escherichia coli ClpA and ClpP proteins form a complex, ClpAP, that catalyzes ATP-dependent degradation of proteins. Formation of stable ClpA hexamers and stable ClpAP complexes requires binding of ATP or nonhydrolyzable ATP analogues to ClpA. To understand the order of events during substrate binding, unfolding, and degradation by ClpAP, it is essential to know the oligomeric state of the enzyme during multiple catalytic cycles. Using inactive forms of ClpA or ClpP as traps for dissociated species, we measured the rates of dissociation of ClpA hexamers or ClpAP complexes. When ATP was saturating, the rate constant for dissociation of ClpA hexamers was 0.032 min(-1) (t(1/2) of 22 min) at 37 degrees C, and dissociation of ClpP from the ClpAP complexes occurred with a rate constant of 0. 092 min(-1) (t(1/2) of 7.5 min). Because the k(cat) for casein degradation is approximately 10 min(-1), these results indicate that tens of molecules of casein can be turned over by the ClpAP complex before significant dissociation occurs. Mutations in the N-terminal ATP binding site led to faster rates of ClpA and ClpAP dissociation, whereas mutations in the C-terminal ATP binding site, which cause significant decreases in ATPase activity, led to lower rates of dissociation of ClpA and ClpAP complexes. Dissociation rates for wild-type and first domain mutants of ClpA were faster at low nucleotide concentrations. The t(1/2) for dissociation of ClpAP complexes in the presence of nonhydrolyzable analogues was >/=30 min. Thus, ATP binding stabilizes the oligomeric state of ClpA, and cycles of ATP hydrolysis affect the dynamics of oligomer interaction. However, since the k(cat) for ATP hydrolysis is approximately 140 min(-1), ClpA and the ClpAP complex remain associated during hundreds of rounds of ATP hydrolysis. Our results indicate that the ClpAP complex is the functional form of the protease and as such engages in multiple rounds of interaction with substrate proteins, degradation, and release of peptide products without dissociation.
Polypeptides emerging from the ribosome must fold into stable three-dimensional structures and maintain that structure throughout
their functional lifetimes. Maintaining quality control over protein structure and function depends on molecular chaperones
and proteases, both of which can recognize hydrophobic regions exposed on unfolded polypeptides. Molecular chaperones promote
proper protein folding and prevent aggregation, and energy-dependent proteases eliminate irreversibly damaged proteins. The
kinetics of partitioning between chaperones and proteases determines whether a protein will be destroyed before it folds properly.
When both quality control options fail, damaged proteins accumulate as aggregates, a process associated with amyloid diseases.
We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol. In DeltarpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5-10% and 20-30% of total protein aggregated at 30 degrees C and 42 degrees C respectively. The aggregates contained 350-400 protein species, of which 93 were identified by mass spectrometry. The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30 degrees C, and showed strong overlap with previously identified DnaK substrates. Overproduction of the DnaK system, or low-level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to DeltarpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival. In rpoH+ cells, DnaK depletion did not lead to protein aggregation at 30 degrees C, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins. Lon was the most efficient protease in degrading misfolded proteins in DnaK-depleted cells. At 42 degrees C, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42 degrees C.
Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. ClpAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP. Here we show that these proteases seem to catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. In multidomain proteins, independently stable domains are unfolded sequentially. We show that these results can explain the limited degradation by the proteasome that occurs in the processing of the precursor of the transcription factor NF-kappaB.
Clp/Hsp100 ATPases comprise a large family of ATP-dependent chaperones, some of which are regulatory components of two-component proteases. Substrate specificity resides in the Clp protein and the current thinking is that Clp proteins recognize motifs located near one or the other end of the substrate. We tested whether or not ClpA and ClpX can recognize tags when they are located in the interior of the primary sequence of the substrate. A protein with an NH2-terminal ClpA recognition tag, plasmid P1 RepA, was fused to the COOH terminus of green fluorescent protein (GFP). GFP is not recognized by ClpA or ClpX and is not degraded by ClpAP or ClpXP. We found that ClpA binds and unfolds the fusion protein and ClpAP degrades the protein. Both the GFP and RepA portions of the fusion protein are degraded. A protein with a COOH-terminal ClpX tag, MuA, was fused to the NH2 terminus of GFP. ClpXP degrades MuA-GFP, however, the rate is 10-fold slower than that of GFP-MuA. The MuA portion but not the GFP portion of MuA-GFP is degraded. Thus, a substrate with an internal ClpA recognition motif can be unfolded by ClpA and degraded by ClpAP. Similarly, although less efficiently, ClpXP degrades a substrate with an internal ClpX recognition motif. We also found that ClpA recognizes the NH2-terminal 15 aa RepA tag, when it is fused to the COOH terminus of GFP. Moreover, ClpA recognizes the RepA tag in either the authentic or inverse orientation.
Protein unfolding is an important step in several cellular processes, most interestingly protein degradation by ATP-dependent proteases and protein translocation across some membranes. Unfolding can be catalyzed when the unfoldases change the unfolding pathway of substrate proteins by pulling at their polypeptide chains. The resistance of a protein to unraveling during these processes is not determined by the protein's stability against global unfolding, as measured by temperature or solvent denaturation in vitro. Instead, resistance to unfolding is determined by the local structure that the unfoldase encounters first as it follows the substrate's polypeptide chain from the targeting signal. As unfolding is a necessary step in protein degradation and translocation, the susceptibility to unfolding of substrate proteins contributes to the specificity of these important cellular processes.
The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and how mutations are optimally placed to improve the stability of such proteins.
ClpX binds substrates bearing specific classes of peptide signals, denatures these proteins, and translocates them through a central pore into ClpP for degradation. ClpX with the V154F po e mutation is severely defective in binding substrates bearing C-motif 1 degradation signals and is also impaired in a subsequent step of substrate engagement. In contrast, this mutant efficiently processes substrates with other classes of recognition signals both in vitro and in vivo. These results demonstrate that the ClpX pore functions in the recognition and catalytic engagement of specific substrates, and that ClpX recognizes different substrate classes in at least two distinct fashions.
The cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding of substrate proteins bearing "tag" sequences, such as the 11-residue ssrA sequence appended to proteins translationally stalled at ribosomes. Unfolding is coupled to translocation through a central channel into the associating protease, ClpP. To explore the topology and mechanism of ClpA action, we carried out chemical crosslinking and functional studies. Whereas a tag from RepA protein crosslinked proximally to the flexible N domains, the ssrA sequence in GFP-ssrA crosslinked distally in the channel to a segment of the distal ATPase domain (D2). Single substitutions placed in this D2 loop, and also in two apparently cooperating proximal (D1) loops, abolished binding of ssrA substrates and unfolded proteins lacking tags and blocked unfolding of GFP-RepA. Additionally, a substitution adjoining the D2 loop allowed binding of ssrA proteins but impaired their translocation. This loop, as in homologous nucleic-acid translocases, may bind substrates proximally and, coupled with ATP hydrolysis, translocate them distally, exerting mechanical force that mediates unfolding.
The AAA+ (ATPases associated with various cellular activities) family is a large and functionally diverse group of enzymes that are able to induce conformational changes in a wide range of substrate proteins. The family's defining feature is a structurally conserved ATPase domain that assembles into oligomeric rings and undergoes conformational changes during cycles of nucleotide binding and hydrolysis. Here, we review the structural organization of AAA+ proteins, the conformational changes they undergo, the range of different reactions they catalyse, and the diseases associated with their dysfunction.
We use single-molecule force spectroscopy to demonstrate that the mechanical stability of the enzyme dihydrofolate reductase (DHFR) is modulated by ligand binding. In the absence of bound ligands, DHFR extends at very low forces, averaging 27 pN, without any characteristic mechanical fingerprint. By contrast, in the presence of micromolar concentrations of the ligands methotrexate, nicotinamide adenine dihydrogen phosphate, or dihydrofolate, much higher forces are required (82 +/- 18 pN, 98 +/- 15 pN, and 83 +/- 16 pN, respectively) and a characteristic fingerprint is observed in the force-extension curves. The increased mechanical stability triggered by these ligands is not additive. Our results explain the large reduction in the degradation rate of DHFR, in the presence of its ligands. Our observations support the view that the rate-limiting step in protein degradation by adenosine triphosphate-dependent proteases is the mechanical unfolding of the target protein.
The proteasome degrades some proteins, such as transcription factors Cubitus interruptus (Ci) and NF-kappaB, to generate biologically active protein fragments. Here we have identified and characterized the signals in the substrate proteins that cause this processing. The minimum signal consists of a simple sequence preceding a tightly folded domain in the direction of proteasome movement. The strength of the processing signal depends primarily on the complexity of the simple sequence rather than on amino acid identity, the resistance of the folded domain to unraveling by the proteasome and the spacing between the simple sequence and folded domain. We show that two unrelated transcription factors, Ci and NF-kappaB, use this mechanism to undergo partial degradation by the proteasome in vivo. These findings suggest that the mechanism is conserved evolutionarily and that processing signals may be widespread in regulatory proteins.
The proteasome is a barrel-shaped protease that conceals its active sites within its central cavity. Proteasomes usually completely degrade substrates into small peptides, but in some cases, degradation yields biologically active protein fragments. Some transcription factors are generated from precursors by this activity, but the mechanism of proteasomal protein processing remains unclear. Here we show that proteasomal processing of the yeast NFkappaB-related transcription factors Spt23 and Mga2 is initiated by an internal cleavage, followed by bidirectional proteolysis of the polypeptides. Studies with protein fusions indicate that stable proteolytic fragments are generated if the protein contains tightly folded structures that prevent the complete degradation of the protein. Furthermore, we provide evidence that the ability of the proteasome to initiate proteolysis from an internal site and to proceed via bidirectional polypeptide degradation may be relevant for the complete degradation of proteins as well.
ATP-powered AAA+ proteases degrade specific proteins in intracellular environments occupied by thousands of different proteins. These proteases operate as powerful molecular machines that unfold stable native proteins before degradation. Understanding how these enzymes choose the "right" protein substrates at the "right" time is key to understanding their biological function. Recently, proteomic approaches have identified numerous substrates for some bacterial enzymes and the sequence motifs responsible for recognition. Advances have also been made in elucidating the mechanism and impact of adaptor proteins in regulating substrate choice. Finally, recent biochemical dissection of the ATPase cycle and its coupling to protein unfolding has revealed fundamental operating principles of this important, ubiquitous family of molecular machines.
ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB trap mutant, along with reconstructions of ClpB in the AMPPNP, ADP, and in the nucleotide-free state. We show that motif 2 of the ClpB M domain is positioned between the D1-large domains of neighboring subunits and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. We further demonstrate biochemically that ATP is essential for high-affinity substrate binding to ClpB and cannot be substituted with AMPPNP. Our structures show that in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Taken together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring before threading them through the ClpB hexamer in an ATP hydrolysis-driven step.
The ClpAP chaperone-protease complex is active as a cylindrically shaped oligomeric complex built of the proteolytic ClpP double ring as the core of the complex and two ClpA hexamers associating with the ends of the core cylinder. The ClpA chaperone belongs to the larger family of AAA+ ATPases and is responsible for preparing protein substrates for degradation by ClpP. Here, we study in real time using fluorescence and light scattering stopped-flow methods the complete assembly pathway of this bacterial chaperone-protease complex consisting of ATP-induced ClpA hexamer formation and the subsequent association of ClpA hexamers with the ClpP core cylinder. We provide evidence that ClpA assembles into hexamers via a tetrameric intermediate and that hexamerization coincides with the appearance of ATPase activity. While ATP-induced oligomerization of ClpA is a prerequisite for binding of ClpA to ClpP, the kinetics of ClpA hexamer formation are not influenced by the presence of ClpP. Models for ClpA hexamerization and ClpA-ClpP association are presented along with rate parameters obtained from numerical fitting procedures. The hexamerization kinetics show that the tetrameric intermediate transiently accumulates, forming rapidly at early time points and then decaying at a slower rate to generate the hexamer. The association of assembled ClpA hexamers with the ClpP core cylinder displays cooperativity, supporting the coexistence of interchanging ClpP conformations with different affinities for ClpA.