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Coupling and Dynamics of Subunits in the Hexameric AAA+ Chaperone ClpB
Abstract
The bacterial AAA+ protein ClpB and its eukaryotic homologue Hsp104 ensure thermotolerance of their respective organisms by reactivating aggregated proteins in cooperation with the Hsp70/Hsp40 chaperone system. Like many members of the AAA+ superfamily, the ClpB protomers form ringlike homohexameric complexes. The mechanical energy necessary to disentangle protein aggregates is provided by ATP hydrolysis at the two nucleotide-binding domains of each monomer. Previous studies on ClpB and Hsp104 show a complex interplay of domains and subunits resulting in homotypic and heterotypic cooperativity. Using mutations in the Walker A and Walker B nucleotide-binding motifs in combination with mixing experiments we investigated the degree of inter-subunit coupling with respect to different aspects of the ClpB working cycle. We find that subunits are tightly coupled with regard to ATPase and chaperone activity, but no coupling can be observed for ADP binding. Comparison of the data with statistical calculations suggests that for double Walker mutants, approximately two in six subunits are sufficient to abolish chaperone and ATPase activity completely. In further experiments, we determined the dynamics of subunit reshuffling. Our results show that ClpB forms a very dynamic complex, reshuffling subunits on a timescale comparable to steady-state ATP hydrolysis. We propose that this could be a protection mechanism to prevent very stable aggregates from becoming suicide inhibitors for ClpB.
... As mutant PARL Skd3 concentration in the mixture increases, the probability of mutant PARL Skd3 incorporation into a PARL Skd3 hexamer increases (Figure 6A). This approach has revealed mechanochemical coupling mechanisms of other NTPases, including Hsp104/ClpB (DeSantis et al., 2012;Moreau et al., 2007;Shivhare et al., 2019;Werbeck et al., 2008). ...
... We also titrated PARL Skd3 with buffer over a range of concentrations to assess disaggregase activity ( Figure S5F). We selected saturating PARL Skd3 concentrations to ensure that any observed effects on disaggregase activity upon mixing WT and mutant are not caused by a mere decrease in the concentration of WT PARL Skd3 (DeSantis et al., 2012;Werbeck et al., 2008). PARL Skd3 K387A (Walker A) and PARL Skd3 Y430A (pore loop) are inactive for ATPase and disaggregase activity (Cupo and Shorter, 2020b). ...
... The binomial distribution was used to model the activity of various heterohexamer ensembles (DeSantis et al., 2012;Werbeck et al., 2008): PðkÞ = n k p k ð1 À pÞ n À k Where: P(k) is the probability of a hexamer containing k mutant subunits, n is total number of subunits (which for a hexamer, n = 6), and p is the probability that a mutant subunit is incorporated. FRET subunit mixing experiments demonstrated that mutant and WT subunits have a similar probability of being incorporated into a hexamer ( Figures S5D, 6A and 6B). ...
The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with defective protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). How Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. These advances illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.
... Cooperativity is then introduced by imposing that hexamers containing more than a certain number of mutant subunits are nonfunctional (Fig. 1C). Here we adopt this approach (35) to provide a framework to analyze the outcome of Rca subunit mixing experiments. We produced and purified a suite of rice activase variants to homogeneity (SI Appendix, Fig. S1), all of which displayed the typical concentration-dependent polydispersity when assayed by analytical size-exclusion chromatography (SI Appendix, Fig. S2). ...
... First we measured the ATPase activity of Rcaβ mixed at various proportions with the ATPaseinactive Walker B mutant D173A (43,44) of either Rcaβ or the longer Rcaα isoform (Fig. 1D) (residue numbering throughout refers to rice Rcaβ). In both cases we observed a severe nonlinear decrease in ATPase activity with increasing mutant subunit composition, indicating strong cooperativity between subunits, similar to the result seen for Walker mutants of ClpB earlier (35). Adding either mutant isoform resulted in an identical profile, suggesting that Rcaα and Rcaβ have similar affinities for each other and this property is not modulated by Rcaα's 33-amino-acid C-terminal extension (Fig. 1D). ...
... We then decided to estimate the rate of subunit exchange in actively ATP-hydrolyzing Rcaβ complexes, by measuring the time required to transition between fully functional and poisoned ensembles upon addition of the mutant subunits to Rcaβ using the method applied to ClpB previously (35). Following addition of an equimolar amount of mutant subunits to WT Rcaβ, the new steady-state ATP hydrolysis rate was achieved within 100 s, consistent with complete subunit exchange on the minute timescale (Fig. 1F). ...
During photosynthesis the AAA+ protein and essential molecular chaperone Rubisco activase (Rca) constantly remodels inhibited active sites of the CO 2 -fixing enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) to release tightly bound sugar phosphates. Higher plant Rca is a crop improvement target, but its mechanism remains poorly understood. Here we used structure-guided mutagenesis to probe the Rubisco-interacting surface of rice Rca. Mutations in Ser-23, Lys-148, and Arg-321 uncoupled adenosine triphosphatase and Rca activity, implicating them in the Rubisco interaction. Mutant doping experiments were used to evaluate a suite of known Rubisco-interacting residues for relative importance in the context of the functional hexamer. Hexamers containing some subunits that lack the Rubisco-interacting N-terminal domain displayed a ∼2-fold increase in Rca function. Overall Rubisco-interacting residues located toward the rim of the hexamer were found to be less critical to Rca function than those positioned toward the axial pore. Rca is a key regulator of the rate-limiting CO 2 -fixing reactions of photosynthesis. A detailed functional understanding will assist the ongoing endeavors to enhance crop CO 2 assimilation rate, growth, and yield.
... A mutant defective in ATP hydrolysis but not ATP binding in both AAA+modules stably binds substrate proteins 20 . The AAA1 and AAA2 modules regulate each other in a complex manner as regards ATP-binding and hydrolysis [21][22][23][24][25] , and the cooperative ATPase cycle causes structural changes to drive the threading of aggregated proteins 12,17,18 , although the threading of denatured proteins does not require ATP hydrolysis 26 . The MD protruding from the middle of AAA1 surrounds the ClpB ring and regulates both ATPase and disaggregation activities of ClpB 10,12,[27][28][29][30] . ...
... 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 . ...
... Taken together, TClpB undergoes large-scale dynamic changes in the oligomeric state, driven by the cycles of ATP binding and hydrolysis. Even events of ring disassembly into two separated parts and subunit reshuffling between oligomers were also observed (Supplementary Movies 7 and 8), as suggested from a biochemical study 22 . The dynamic nature of TClpB oligomers was also confirmed by our SV-AUC analysis (Supplementary Figure 9). ...
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.
... A single inactivated subunit would consequently have a severe effect on the rate of ATP hydrolysis, potentially stopping unfolding. To test the model, we measured the ATPase activity of VAT complexes that had been doped with different fractions (r) of catalytically-inactive protomers bearing mutations in the Walker B motifs of both NBD1 and NBD2 (E291Q, E568Q) (Werbeck et al., 2008) relative to wild type VAT ATPase activity (A). These experiments were carried out in the presence of GFP with an 11-residue ssRA tag as substrate (Barthelme et al., 2014). ...
... The slight elevation relative to the A ¼ 1 À r ð Þ 6 profile in Figure 4c and d may reflect an 'escape' mechanism that allows stalled VAT complexes to disengage and then reengage with substrate, as seen with other molecular machines (Werbeck et al., 2008). In the absence of an escape mechanism ATP hydrolysis would proceed from one protomer to the next in a cyclical manner (Figure 4b) until a defective subunit is reached, preventing further hydrolysis and, thus, subsequent substrate unfolding. ...
... Expression and purification of DN-VAT were performed following the protocol described previously for full-length VAT (Huang et al., 2016). Walker B (catalytically inactivating) mutations (Werbeck et al., 2008) in VAT were made in a pET-28b vector that encoded codon-optimized VAT with an N-terminal His 6 -tag and a tobacco etch virus (TEV) cleavage site between the His 6 -tag and the DN-VAT sequence. The mutant protein was purified in the same way as wild type VAT and DN-VAT (Huang et al., 2016). ...
AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 A ˚ stretch of unfolded protein. The structure suggests a processive handover hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.
... Thus, under these conditions, we are certain that if the number of WT subunits per hexamer exceeds five then the hexamer is inactive. We employed the approach of Reinstein and colleagues to simulate the distribution of Hsp104 and Hsp104 A503V subunits within a given population of Hsp104 hexamers as described (26,61,62,70). Thus, we employed the binomial distribution: ...
... where P is the probability that a hexamer (therefore, n=6) contains x WT subunits and p is the probability that a WT subunit is incorporated (26,61,62,70). Experiments demonstrated that WT and A503V subunits have a similar probability of being incorporated into a hexamer (26,62). ...
... 6 WT subunits can be derived (Fig. 1B) (26,61,62,70). Activity versus p plots could then be generated assuming each A503V subunit makes an equal contribution to the total activity (onesixth per subunit) (Fig. 1C) (26,61,62,70). ...
Potentiated variants of Hsp104, a protein disaggregase from yeast, can dissolve protein aggregates connected to neurodegenerative
diseases such as Parkinson's disease and amyotrophic lateral sclerosis. However, the mechanisms underlying Hsp104 potentiation
remain incompletely defined. Here, we establish that 2-3 subunits of the Hsp104 hexamer must bear an A503V potentiating mutation
to elicit enhanced disaggregase activity in the absence of Hsp70. We also define the ATPase and substrate-binding modalities
needed for potentiated Hsp104A503V activity in vitro and in vivo. Hsp104A503V disaggregase activity is strongly inhibited by the Y257A mutation that disrupts substrate binding to the nucleotide-binding
domain 1 (NBD1) pore loop, and is abolished by the Y662A mutation that disrupts substrate binding to the NBD2 pore loop. Intriguingly,
Hsp104A503V disaggregase activity responds to mixtures of ATP and ATPγS (a slowly hydrolyzable ATP analogue) differently than Hsp104.
Indeed, an altered pattern of ATP hydrolysis and altered allosteric signaling between NBD1 and NBD2 are likely critical for
potentiation. Hsp104A503V variants bearing inactivating Walker A or Walker B mutations in both NBDs are inoperative. Unexpectedly, however, Hsp104A503V retains potentiated activity upon introduction of sensor-1 mutations that reduce ATP hydrolysis at NBD1 (T317A) or NBD2 (N728A).
Hsp104T317A A503V and Hsp104A503V N728A rescue TDP-43, FUS, and α-synuclein toxicity in yeast. Thus, Hsp104A503V displays a more robust activity that is unperturbed by sensor-1 mutations that greatly reduce Hsp104 activity in vivo. Indeed, ATPase activity at NBD1 or NBD2 is sufficient for Hsp104 potentiation. Our findings will empower design of ameliorated
therapeutic disaggregases for various neurodegenerative diseases.
... Evidence for both concerted and sequential ATP hydrolysis is available. The presence of heterotypic allostery in nucleotide hydrolysis and inter-subunit coupling favors a concerted mechanism for ATP hydrolysis (Werbeck et al., 2008). Structural basis for allosteric communications in ClpB Tth is unclear, owing to the lack of high resolution hexameric structures. ...
... Hsp104/ClpB and other ATPases belonging to the AAA family typically form dynamic, nucleotide-and ionic strength-dependent, hexameric structures (Beuron et al., 1998;Bochtler et al., 2000;Ishikawa et al., 2001;Ortega et al., 2000;Sousa et al., 2000;Wang et al., 2001;Zolkiewski et al., 1999a;Haslberger et al., 2008;Werbeck et al., 2008). Hexamer formation in ClpB/Hsp104 is facilitated at high protein concentration, low ionic strength and in the presence of ATP; whereas, ADP drives the oligomeric equilibrium to lower order Schirmer et al., 1998;Zolkiewski et al., 1999;Schlee et al 2001;Schirmer et al., 2001;Hattendorf and Lindquist 2002b). ...
... An intersubunit conformational change can also explain ADP-mediated oligomer dissociation in ClpB Tth as observed in several studies. Dynamic coupling between subunits in ClpB Tth was shown to be occurring at time scales comparable to rates of ATP hydrolysis (Werbeck et al., 2008). Partial threading of substrate proteins through the central channel of ClpB also involves the dynamic nature of ClpB oligomers. ...
ClpBTth is a molecular motor, which exerts an ATP hydrolysis driven mechanical force, resulting in disaggregation of aggregated proteins. ClpBTth belonging to the AAA family of ATPases carries the signature AAA module, which comprises of a large nucleotide binding domain (NBD) and an α-helical small domain (SD). The two tandem AAA modules present per subunit of ClpBTth interact with each other and the neighboring AAA modules in the hexameric ring-like structure. The current study focuses on inter-subunit (homotypic) and intra-subunit (heterotypic) communications between the AAA modules in ClpBTth oligomer, in respect to nucleotide binding and hydrolysis. The two tandem AAA modules of ClpBTth upon isolation exhibit unique properties. The isolated AAA2 module more or less represents a building block for the full-length hexameric protein. It appears to have retained most of the key characteristics, exhibited by full-length ClpBTth, as evident by sigmoidal kinetics in ATP hydrolysis and nucleotide binding-related conformational changes. So, nucleotide binding in the isolated AAA modules was investigated using fluorescently labeled proteins to gain insights into the nucleotide-mediated oligomer dissociation. Experiments were performed using the isolated AAA modules to reduce the complexity which comes with studying full-length hexamer. Experiments provided hints for involvement of the α-helical small domain 2 in nucleotide-dependent oligomer formation. Importance of the presence of SD2 has been demonstrated; upon its deletion, isolated AAA2 domain lost nucleotide binding and hydrolysis. Studies using a mutant carrying proline mutation in a loop connecting SD2 to NBD2 in the AAA2 module revealed loss of chaperone and ATPase activity. This study pointed out at the importance of flexibility and motion in SD2 of the AAA2 module. Nucleotide binding studies hinted at a possible biphasic nature and inter-subunit communication. ADP binding in one AAA2 module appeared to have triggered a conformational change in SD2 of the neighboring AAA2 module. These results gave insights into the conformational changes involved in ADP-mediated oligomer dissociation in ClpBTth. Although oligomer dissociation has been linked to ADP binding/formation in several instances, the inter-subunit communications pattern has never been clearly discerned. This work provides a platform for further studies to investigate conformational changes that result in oligomer formation and dissociation. The isolated AAA modules of ClpBTth upon reconstitution exhibit functional higher order oligomer formation. This reconstituted complex resembles wild type ClpBTth hexamer in all aspects related to oligomerization, chaperone activity and ATP hydrolysis. So, complex formation between the isolated AAA modules was studied to understand the thermodynamics behind the communication between them. Isothermal titration calorimetry measurements were performed to study binding between the isolated AAA modules. Experiments provided hints at temperature dependency in binding between the AAA modules in ClpBTth. Allostery in ATP hydrolysis is central to the function of ClpBTth, which represents both homotypic and heterotypic communications between the AAA modules. Absence of heterotypic allostery always resulted in a loss of function in ClpBTth. Importance of heterotypic allosteric communications between the AAA modules within each subunit and their role in chaperone activity of ClpBTth was investigated in this work. This was done by alteration of the related interface by mutating amino acids involved in interface interactions. Most of the mutants resulted in subtle changes in nucleotide hydrolysis properties and heterotypic allostery. Changes in allosteric behavior did not translate into a loss in chaperone activity, as evident by no loss of function in mutants exhibiting altered allostery. Experiments in the presence of GdmCl, which acts as an uncompetitive inhibitor for ATP binding, additionally revealed interesting insights to the allostery-defective situation in ClpBTth. Furthermore, this study has shed some light on possible mechanisms involved for attaining catalytic effectiveness in ClpBTth.
... In addition to ClpB and yeast Hsp104, which both catalyse protein disaggregation, there are many AAA + enzymes that only catalyse protein remodelling reactions and do not interact with proteases [52]. It has proven difficult to show whether such enzymes fully translocate a substrate through their axial channels, only pull on exposed tails [53] or take only a few translocation steps. The difficulty lies in the fact that the substrates in these reactions both enter and leave the reaction without covalent modification. ...
... A non-processive mechanism is consistent with the observation that hexamers of ClpB exhibit fast subunit exchange [53]. It has been reported that monomers of ClpB rapidly dissociate from the hexamer. ...
... When cells are under stress, native proteins can become misfolded or aggregated. With or without the assistance of the DnaK chaperone system, multiple ClpB hexamers will recognize and bind to the exposed unstructured regions of the protein aggregates [53]. We have recently reported that multiple ClpB hexamers can bind to a polypeptide chain longer than 127 amino acids [25]. ...
Escherichia coli caseinolytic protease (Clp)B is a hexameric AAA+ [expanded superfamily of AAA (ATPase associated with various cellular activities)] enzyme that has the unique ability to catalyse protein disaggregation. Such enzymes are essential for proteome maintenance. Based on structural comparisons to homologous enzymes involved in ATP-dependent proteolysis and clever protein engineering strategies, it has been reported that ClpB translocates polypeptide through its axial channel. Using single-turnover fluorescence and anisotropy experiments we show that ClpB is a non-processive polypeptide translocase that catalyses disaggregation by taking one or two translocation steps followed by rapid dissociation. Using single-turnover FRET experiments we show that ClpB containing the IGL loop from ClpA does not translocate substrate through its axial channel and into ClpP for proteolytic degradation. Rather, ClpB containing the IGL loop dysregulates ClpP leading to non-specific proteolysis reminiscent of ADEP (acyldepsipeptide) dysregulation. Our results support a molecular mechanism where ClpB catalyses protein disaggregation by tugging and releasing exposed tails or loops.
© 2015 Authors; published by Portland Press Limited.
... The dynamics of ClpB/Hsp104 oligomers adds further complexity to ATPase control (Werbeck et al., 2008). ATP hydrolysis is decreasing ClpB/Hsp104 hexamer stability, suggesting that subunit exchange is happening during ongoing disaggregation (Aguado et al., 2015). ...
... ATP hydrolysis is decreasing ClpB/Hsp104 hexamer stability, suggesting that subunit exchange is happening during ongoing disaggregation (Aguado et al., 2015). This is supported by the immediate poisoning of ClpB-wt disaggregation activity upon addition of mutant subunits, which rapidly form mixed oligomers with wild type subunits (Haslberger et al., 2008;Werbeck et al., 2008). Oligomer dynamics might prevent jamming of the ClpB/Hsp104 oligomer by stable protein domains of aggregated substrates that cannot be further processed (Haslberger et al., 2008;Werbeck et al., 2008;Aguado et al., 2015). ...
... This is supported by the immediate poisoning of ClpB-wt disaggregation activity upon addition of mutant subunits, which rapidly form mixed oligomers with wild type subunits (Haslberger et al., 2008;Werbeck et al., 2008). Oligomer dynamics might prevent jamming of the ClpB/Hsp104 oligomer by stable protein domains of aggregated substrates that cannot be further processed (Haslberger et al., 2008;Werbeck et al., 2008;Aguado et al., 2015). Indeed, ClpB solubilizes aggregated proteins harboring tightly folded domains without unfolding them, indicating that partially threaded protein substrates are released upon oligomer dissociation (Haslberger et al., 2008). ...
Unicellular and sessile organisms are particularly exposed to environmental stress such as heat shock causing accumulation and aggregation of misfolded protein species. To counteract protein aggregation, bacteria, fungi, and plants encode a bi-chaperone system composed of ATP-dependent Hsp70 and hexameric Hsp100 (ClpB/Hsp104) chaperones, which rescue aggregated proteins and provide thermotolerance to cells. The partners act in a hierarchic manner with Hsp70 chaperones coating first the surface of protein aggregates and next recruiting Hsp100 through direct physical interaction. Hsp100 proteins bind to the ATPase domain of Hsp70 via their unique M-domain. This extra domain functions as a molecular toggle allosterically controlling ATPase and threading activities of Hsp100. Interactions between neighboring M-domains and the ATPase ring keep Hsp100 in a repressed state exhibiting low ATP turnover. Breakage of intermolecular M-domain interactions and dissociation of M-domains from the ATPase ring relieves repression and allows for Hsp70 interaction. Hsp70 binding in turn stabilizes Hsp100 in the activated state and primes Hsp100 ATPase domains for high activity upon substrate interaction. Hsp70 thereby couples Hsp100 substrate binding and motor activation. Hsp100 activation presumably relies on increased subunit cooperation leading to high ATP turnover and threading power. This Hsp70-mediated activity control of Hsp100 is crucial for cell viability as permanently activated Hsp100 variants are toxic. Hsp100 activation requires simultaneous binding of multiple Hsp70 partners, restricting high Hsp100 activity to the surface of protein aggregates and ensuring Hsp100 substrate specificity.
... Although several groups have studied how the ATPase cycle of ClpB/Hsp104 proceeds and couples with disaggregation, the details are still unclear because of its complexity and differences between species (22)(23)(24)(25)(26)(27)(28)(29). In some of these studies, experiments using heterohexamers consisting of two types of ClpB subunits were performed (24 -26, 28, 29). ...
... In previous studies, researchers showed that the subunits of ClpB functioned cooperatively in the disaggregation process; however, the details of the mechanism were unclear (24,25,29). Here, we analyzed the intersubunit cooperativity of the ClpB hexamer using a new approach in which a disulfide bond specifically formed between adjacent subunits. ...
... In addition, the ATPase activities of the ordered heterohexamers consisting of wild-type and single Walker A-mutated subunits were low, indicating that ATP binding to three AAAϩ modules in one ring was not sufficient for stimulation. This was consistent with a previous report demonstrating that the ATPase activity of wild-type TClpB was severely decreased by doping of a single Walker A-mutated subunit (24). ...
The ClpB/Hsp104 chaperone solubilizes and reactivates protein aggregates in cooperation with DnaK/Hsp70 and its cofactors. The ClpB/Hsp104 protomer has two AAA+ modules, AAA-1 and AAA-2, and forms a homohexamer. In the hexamer, these modules form a two-tiered ring in which each tier consists of homotypic AAA+ modules. By ATP binding and its hydrolysis at these AAA+ modules, ClpB/Hsp104 exerts the mechanical power required for protein disaggregation. Although ATPase cycle of this chaperone has been studied by several groups, an integrated understanding of this cycle has not been obtained due to the complexity of the mechanism and differences between species. To improve our understanding of the ATPase cycle, we prepared many ordered heterohexamers of ClpB from Thermus thermophilus, in which two subunits having different mutations were cross-linked to each other and arranged alternately, and measured their nucleotide-binding, ATP-hydrolysis, and disaggregation abilities. The results indicated that the ATPase cycle of ClpB proceeded as follows: i) the 12 AAA+ modules randomly bound ATP, ii) the binding of four or more ATP to one AAA+ ring was sensed by a conserved Arg residue and converted another AAA+ ring into the ATPase-active form, and iii) ATP hydrolysis occurred cooperatively in each ring. We also found that cooperative ATP hydrolysis in at least one ring was needed for the disaggregation activity of ClpB.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... ; Aguado et al. 2015;Werbeck, Schlee, and Reinstein 2008); consequently, the high resolution architecture of the hexameric, active complex has remained out of reach. ...
... The interface at the hexamer seam may also play critical roles in the functional plasticity of Hsp104, facilitating recognition and engagement of different aggregate structures. Finally, given known nucleotidedependent dynamics of the hexamer(Werbeck, Schlee, and Reinstein 2008), a compelling mechanism is that reorganization of the protomer offset is triggered by substrate binding, Hsp70 interaction or ATP hydrolysis to facilitate translocation.A number of different MD arrangements have been proposed based on previous lowresolution models (S. S. Lee, Choi, and Tsai 2007; S. Lee et al. 2010). Our structure reveals the clearest view to date of the MD conformation in the ATP state and identifies bridging interactions between L1 and L2 and the small and large subdomains of the adjacent protomer, potentially stabilizing the nucleotide pocket for hydrolysis. ...
Chaperone proteins are central to protein quality control and cell signaling. Three major classes of chaperones include foldases, holdases and disaggregases. Foldases aid proteins in folding to their native state, as well as prevent them from misfolding, a process that is regulated by ATP hydrolysis. Holdases are ATP-independent chaperones that respond to cellular stress, such as heat shock and oxidation, and protect proteins from aggregation. Disaggregases rescue aggregated proteins, resolubilize them and hand them off to foldases to be refolded. Together, these three classes of chaperones regulate many cellular processes and maintain proteostasis. In this study, three chaperones were characterized using electron microscopy: Hsp104 (disaggregase), Get3 (holdase), and FKBP51 (foldase). Hsp104 is a double ring AAA+ protein that is active as a hexamer and is capable of pulling apart amorphous aggregates and amyloid fibrils. AAA+ proteins have conserved ATP hydrolysis pockets and conserved translocation mechanism to thread polymers through the central channel. Previous structural work of Hsp104 and other AAA+ proteins has been limited to low resolution due to the dynamic nature of the hexamer. In this study, we have determined three high-resolution structures of Hsp104. First, an open, asymmetric spiral conformation of Hsp104:AMPPNP to 5.6Å, which revealed a large hexamer seam with a unique hetero-AAA+ interaction between the Nucleotide Binding Domain (NBD) 1 and 2. Next we determined two structures of Hsp104 bound to substrate in the presence of ATPS; the closed (4.0Å) and extended (4.1Å) states. The critical substrate binding tyrosine loops are arranged in a two-turn spiral around the substrate, showing direct interactions between the tyrosine and polypeptide chain. The closed state has only five of six protomers engaged, whereas in the extended state all six protomers are engaged with a translocation step of 6-7Å between states. From our structures, we propose a processive rotary translocation mechanism between the closed and extended states, with a non-processive step from the open to closed states upon substrate binding. These structures give insight into the translocation of the highly conserved AAA+ class of proteins. In another study, the holdase activity of Get3 was investigated. Get3 is important for ATP-dependent Tail Anchored-binding protein membrane insertion in yeast. We established that upon oxidative stress, Get3 undergoes a structural rearrangement from a reduced, ATP-dependent dimer, to an oxidized, ATP-independent tetramer with a general chaperone function. Using negative stain-EM we further characterized the Get3ox tetramer to determine that it has a ‘W’ shape, revealing a hydrophobic patch to bind substrates. Lastly, we studied the interaction between Hsp90 and its cochaperone, FKBP51, an immunophilin that is upregulated in cancer and Alzheimers disease. We determined that Hsp90 binds FKBP51 in its closed, ATP-bound state, a complex that is stabilized by another cochaperone, p23. Negative stain EM establishes the contacts between Hsp90 and FKBP51, which could be used as potential drug targets with improved specificity from Hsp90 inhibitors. We have determined the structures of three different chaperone classes, showing the very dynamic nature of molecular chaperones and elucidating functional mechanisms of these chaperones. Overall this thesis work has made advancements in all three areas studied, utilizing structure determination to correlate with function.
... When the wild-type and the mutant protein variants are mixed, they form a combination of heterohexamers ( Supplementary Fig. 3a). Compositions of such mixtures can be calculated for various protein ratios using the binominal distribution (Supplementary Fig. 3b) [35,42]. Hexamer tolerance for mutant subunits can be inferred from a comparison between the theoretical activities derived from a model and the experimental data [35,42]. ...
... Compositions of such mixtures can be calculated for various protein ratios using the binominal distribution (Supplementary Fig. 3b) [35,42]. Hexamer tolerance for mutant subunits can be inferred from a comparison between the theoretical activities derived from a model and the experimental data [35,42]. To determine the minimal number of the F508A subunits sufficient to abolish collaboration with Hsp70, we mixed Hsp104 wild type with the F508A mutant in various proportions and tested for reactivation of heat-aggregated luciferase (Fig. 3c). ...
Hsp104 is a yeast chaperone that rescues misfolded proteins from aggregates associated with proteotoxic stress and aging. Hsp104 consists of N-terminal domain, regulatory M-domain and two ATPase domains, assembled into a spiral-shaped hexamer. Protein disaggregation involves polypeptide extraction from an aggregate and its translocation through the central channel. This process relies on Hsp104 cooperation with the Hsp70 chaperone, which also plays important role in regulation of the disaggregase. Although Hsp104 protein-unfolding activity enables cells to survive stress, when uncontrolled, it becomes toxic to the cell.
In this work, we investigated the significance of the interaction between Hsp70 and the M-domain of Hsp104 for functioning of the disaggregation system. We identified phenylalanine at position 508 in Hsp104 to be the key site of interaction with Hsp70. Disruption of this site makes Hsp104 unable to bind protein aggregates and to confer tolerance in yeast cells. The use of this Hsp104 variant demonstrates that Hsp70 allows successful initiation of disaggregation only as long as it is able to interact with the disaggregase. As reported previously, this interaction causes release of the M-domain-driven repression of Hsp104. Now we reveal that, apart from this allosteric effect, the interaction between the chaperone partners itself contributes to effective initiation of disaggregation and plays important role in cell protection against Hsp104-induced toxicity. Interaction with Hsp70 shifts Hsp104 substrate specificity from non-aggregated, disordered substrates toward protein aggregates. Accordingly, Hsp70-mediated sequestering of the Hsp104 unfoldase in aggregates makes it less toxic and more productive.
... Here we found both Walker A mutants to be well assembled under our experimental conditions, which suggests that both NBDs play a role in hexamer stabilization ( Supplementary Fig. 3). However, we clearly observe that NBD2 contributes more a b c significantly to the machine activity ( Fig. 3a Fig. 13), but as expected 55,69 , no ATPase activity and therefore no disaggregation activity was observed DnaK and substrate binding regulate M domain dynamics. DnaK is the main component of the co-chaperone system in the disaggregation process, which has been shown to act both upstream and downstream of ClpB 25,30,63,70 . ...
... To obtain ClpB hexamers with only a single labeled protomer or less, we reassembled ClpB molecules by mixing labeled subunits with cysteine-less subunits. The distribution of labeled vs. unlabeled protomers in mixed ClpB molecules was calculated based on the binomial distribution as follows 69 : ...
Large protein machines are tightly regulated through allosteric communication channels. Here we demonstrate the involvement of ultrafast conformational dynamics in allosteric regulation of ClpB, a hexameric AAA+ machine that rescues aggregated proteins. Each subunit of ClpB contains a unique coiled-coil structure, the middle domain (M domain), proposed as a control element that binds the co-chaperone DnaK. Using single-molecule FRET spectroscopy, we probe the M domain during the chaperone cycle and find it to jump on the microsecond time scale between two states, whose structures are determined. The M-domain jumps are much faster than the overall activity of ClpB, making it an effectively continuous, tunable switch. Indeed, a series of allosteric interactions are found to modulate the dynamics, including binding of nucleotides, DnaK and protein substrates. This mode of dynamic control enables fast cellular adaptation and may be a general mechanism for the regulation of cellular machineries.
... The use of these and other similar variants, abolishing ATP binding (Walker A) or hydrolysis (Walker B or Sensor 1) have been used by many groups to investigate the coordination of the 12 ATP binding and hydrolysis sites within a the ClpA hexamer, as well as for the ClpB and Hsp104 hexamer. One common strategy is "mutant doping, " in which a variant is added to wild type protein in known ratios (Werbeck et al., 2008;Hoskins et al., 2009;del Castillo et al., 2010;DeSantis et al., 2012;Yamasaki et al., 2015). Many conclusions have been drawn regarding sequential, probabilistic, or concerted ATP hydrolysis mechanisms. ...
... However, this primary replicative helicase likely exhibits much higher processivity in the context of the fast moving replication fork, likely due to interactions with other proteins. With respect to ClpB and Hsp104, both enzymes have been proposed to be in "rapid subunit exchange" (Werbeck et al., 2008;DeSantis et al., 2012). Thus, loss of a subunit in a hexameric ring could also result in a dissociation event. ...
Cellular proteostasis involves not only the expression of proteins in response to environmental needs, but also the timely repair or removal of damaged or unneeded proteins. AAA+ motor proteins are critically involved in these pathways. Here, we review the structure and function of AAA+ proteins ClpA, ClpB, and Hsp104. ClpB and Hsp104 rescue damaged proteins from toxic aggregates and do not partner with any protease. ClpA functions as the regulatory component of the ATP dependent protease complex ClpAP, and also remodels inactive RepA dimers into active monomers in the absence of the protease. Because ClpA functions both with and without a proteolytic component, it is an ideal system for developing strategies that address one of the major challenges in the study of protein remodeling machines: how do we observe a reaction in which the substrate protein does not undergo covalent modification? Here, we review experimental designs developed for the examination of polypeptide translocation catalyzed by the AAA+ motors in the absence of proteolytic degradation. We propose that transient state kinetic methods are essential for the examination of elementary kinetic mechanisms of these motor proteins. Furthermore, rigorous kinetic analysis must also account for the thermodynamic properties of these complicated systems that reside in a dynamic equilibrium of oligomeric states, including the biologically active hexamer.
... This communication can be assessed by mixing experiments in which the effects of incorporation of the mutant subunits are determined. Mixing experiments are possible, as ClpB and Hsp104 form dynamic assemblies that exchange subunits on the minute time scale [23,[30][31][32] [15,17,23,30,33]. Such mixing experiments revealed unexpected differences between ClpB and Hsp104, as ClpB was highly sensitive to mutant subunit incorporation during protein disaggregation, whereas Hsp104 largely tolerated DWA subunit incorporation [23]. ...
... This communication can be assessed by mixing experiments in which the effects of incorporation of the mutant subunits are determined. Mixing experiments are possible, as ClpB and Hsp104 form dynamic assemblies that exchange subunits on the minute time scale [23,[30][31][32] [15,17,23,30,33]. Such mixing experiments revealed unexpected differences between ClpB and Hsp104, as ClpB was highly sensitive to mutant subunit incorporation during protein disaggregation, whereas Hsp104 largely tolerated DWA subunit incorporation [23]. ...
Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are members of the Hsp100 family of ring-forming hexameric AAA+ chaperones which promote solubilisation of aggregated proteins and propagation of prions. ClpB and Hsp104 cooperate with cognate Hsp70 chaperones for substrate targeting and activation of ATPase and substrate threading, achieved by transient Hsp70 binding to the repressing ClpB/Hsp104 M-domain. Fundamental differences in ATPase regulation and disaggregation mechanisms have been reported however, raising doubts regarding the working principle of this AAA+ chaperone. In particular unique functional plasticity was suggested to specifically enable Hsp104 to circumvent Hsp70 requirement for derepression in protein disaggregation and prion propagation. We show here that for both ClpB and Hsp104 cooperation with Hsp70 is crucial for efficient protein disaggregation and, in contrast to earlier claims, cannot be circumvented by activating M-domain mutations. Activation of ClpB and Hsp104 requires two signals, relief of M-domain repression and substrate binding, leading to increased ATPase subunit coupling. These data demonstrate that ClpB and Hsp104 operate by the same basic mechanism, underscore a dominant function of Hsp70 in regulating ClpB/Hsp104 activity, and explain a plethora of in vivo studies showing a crucial function of Hsp70 in proteostasis and prion propagation.
... 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). ...
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.
... To understand the functional connection between overhang-bound and unwinding-performing subunits within the multimeric eIF4A1 complex better, we aimed to probe for functional cooperativity within the eIF4A1 multimer directly. For this, we followed an approach that has been used for multimeric ATPases and helicases previously (62)(63)(64), in which, briefly, the catalytic activity of the multimeric enzyme is monitored when wildtype and an inactive variant are mixed at different fractions but at the same total protein concentration. Absence of functional cooperativity would result in a linear trend,with x + y = 1 (62), plotting activity versus fraction of the inactive variant. ...
Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5'UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5'UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.
... To understand the functional connection between overhang-bound and unwinding-303 performing subunits within the multimeric eIF4A1 complex better, we aimed to probe for functional 304 cooperativity within the eIF4A1 multimer directly. For this, we followed an approach that has been 305 used for multimeric ATPases previously (Moreau et al., 2007;Werbeck et al., 2008), in which, briefly, 306 the catalytic activity of the multimeric enzyme is monitored when wildtype and an inactive variant 307 are mixed at different fractions but at the same total protein concentration. Absence of functional 308 cooperativity would result in a linear trend ,with x + y = 1 (Moreau et al., 2007) (Pause and Sonenberg, 1992), our results show a differential level 311 of functional cooperativity for eIF4A1 unwinding between the AG-and CAA-overhang (Fig. 4A), 312 which correlates with differential unwinding activity on these substrates. ...
Oncogenic translational programmes underpin cancer development and are often driven by dysregulation of oncogenic signalling pathways that converge on the eukaryotic translation initiation (eIF) 4F complex. Altered eIF4F activity promotes translation of oncogene mRNAs that typically contain highly structured 5’UTRs rendering their translation strongly dependent on RNA unwinding by the DEAD-box helicase eIF4A1 subunit of the eIF4F complex. While eIF4A1-dependent mRNAs have been widely investigated, it is still unclear how highly structured mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation.
Here, we show that RNA sequence motifs regulate eIF4A1 unwinding activity in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5’UTR which recruit and stimulate eIF4A1 unwinding of localised RNA structure to facilitate mRNA translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA substrate resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex region that supports efficient unwinding.
Hence, we conclude a model in which mRNAs utilise AG-rich sequences to specifically recruit eIF4A1, enabling assembly of the helicase-active multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.
... However, based on the findings in this study, coordination of ATPase modules within and between the D1 and D2 rings appears to be different in ClpA (18,(48)(49)(50)(51). For example, doping just one or two ATPaseinactive subunits into a ClpB hexamer abrogates ATPase activity (52). Further, allosteric networks in Hsp104/ClpB, including the interaction of these enzymes' "middle domains" (absent in ClpA family enzymes) and folding chaperones with both rings, may make ATP hydrolysis in one module much more highly dependent on the nucleotide states of other modules in either ring (18,51,53). ...
Significance
Understanding of how ClpA and other double-ring AAA+ enzymes perform mechanical work is limited. Using site-specific cross-linking and mutagenesis, we introduced ATPase-inactive AAA+ modules at alternating positions in individual ClpA rings, or in both rings, to investigate potential active-site coordination during ClpAP degradation. ClpA variants containing alternating active/inactive ATPase modules processively unfolded, translocated, and supported ClpP degradation of protein substrates with energetic efficiencies similar to, or higher than, completely active ClpA. These results impact current models describing the mechanisms of AAA+ family enzymes. The cross-linking/mutagenesis method we employed will also be useful for answering other structure-function questions about ClpA and related double-ring enzymes.
... Similarly, single substitution mutations within Walker A (K613A), Walker B (E680A) or Arginine finger (R757A) of NBD-2 of clpB were generated and designated WA2, WB2, and Arg2. The Walker A mutation affects a lysine residue and this impairs nucleotide binding (S4 Fig) and the Walker B mutation affects a glutamate residue and this prevents the hydrolysis of ATP (S4 Fig) [43,63]. Mutants with substitutions in the arginine finger result in the interruption of ClpB oligomerization, ATP binding and hydrolysis [64]. ...
Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the ⊗clpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.
... Most commonly studied mutations are substitutions of glutamate and aspartate by glutamine or alanine. 56,110,125,131,132 Upon these mutations, ATP hydrolysis is prevented, but ATP binding is retained. ...
Nanomotors in nanotechnology are as important as engines in daily life. Many ATPases are nanoscale biomotors classified into three categories based on the motion mechanisms in transporting substrates: linear, rotating, and the recently discovered revolving motion. Most biomotors adopt a multi-subunit ring-shaped structure that hydrolyzes ATP to generate force. How these biomotors control the motion direction and regulate the sequential action of their multiple subunits are intriguing. Many ATPase are hexameric with each monomer containing a conserved arginine finger. This review focuses on recent findings on how the arginine finger controls motion direction and coordinates adjacent subunit interactions in both revolving and rotating biomotors. Mechanisms of inter-subunit interactions and sequential movements of individual subunits are evidenced by the asymmetrical appearance of one dimer and four monomers in high-resolution structural complexes. The arginine finger is situated at the interface of two subunits and extends into the ATP binding pocket of the downstream subunit. An arginine finger mutation results in deficiency in ATP binding/hydrolysis, substrate binding and transport, highlighting the importance of the arginine finger in regulating energy transduction and motor function. Additionally, the roles of channel chirality and channel size are discussed as related to controlling one-way trafficking and differentiating the revolving and rotating mechanisms. Finally, the review concludes by discussing the conformational changes and entropy conversion triggered by ATP binding/hydrolysis, offering a view different from the traditional concept of ATP-mediated mechanochemical energy coupling. The elucidation of the motion mechanism and direction control in ATPases could facilitate nanomotor fabrication in nanotechnology.
... This plasticity in loading mechanism would allow Hsp104 or ClpB to engage substrates at multiple positions, and thus not restrict the disaggregase for hunting for free amino-or carboxy-termini to initiate translocation, which could be problematic when tackling the irregular structures of protein aggregates. Moreover, Hsp104 and ClpB hexamers are dynamic and exchange subunits on the minute timescale (Haslberger et al. 2008;Werbeck et al. 2008;Aguado et al. 2015), and recent studies by atomic force microscopy identified large conformational changes and open spiral ring conformations of ClpB (Uchihashi et al. 2018). Structures of initiation complexes, however, have not yet been determined and the Hsp100-Hsp70 interaction and substrate handover mechanisms at heterogeneous surfaces of aggregates remain a significant question. ...
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.
... DnaK-mediated activation of ClpB, we have used a previously described binomial distribution model 42 . This model predicts the probability of each oligomeric species to occur upon mixing wt and mutant subunits at a defined molar ratio, allowing the comparison with the experimental data. ...
The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the β subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the β subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discrimínate different substrate proteins might allow its activation when client proteins require remodeling.
... Partial substrate threading demands a mechanism to allow the engaged polypeptide to become released from Hsp104/ClpB. This may be accomplished by either partial or complete opening of the ClpB ring, which was reported to occur on the second-tominute timescale and may allow for dissociation of partially threaded polypeptides (Haslberger et al., 2008;Werbeck et al., 2008). ...
Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins.
... An extensive molecular toolbox comprising site-directed mutants of all relevant Rca functional aspects such as ATPase activity, protein-protein interaction, and regulation needs to be constructed. The ability of Rca to rapidly exchange subunits, make this system highly amenable to powerful subunit doping experiments that have recently been extensively exploited in the study of the disaggregase Hsp104 [DeSantis et al., 2012] and a range of other AAA+ proteins [Moreau et al., 2007;Werbeck et al., 2008]. ...
The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/ oxygenase (Rubisco) is slow and non-specific. In order to maintain sufficient flux through the Calvin-Benson cycle, the enzyme is extremely abundant in photosynthetic tissue. In addition to its catalytic frailties, Rubisco forms dead-end inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. In order to remove these complexes, photoautotrophs have recruited multiple molecular chaperones of the AAA+ clade (ATPases associated with diverse cellular activities) known as the Rubisco activases. Here we outline a current perspective on the properties of Rubisco and summarize our knowledge of the green group of Rubisco activases, which is found in all higher plants. We acknowledge the rapid increase of mechanistic understanding currently developing for the molecular motors of the AAA+ class. Integrating this knowledge towards a detailed mechanistic understanding of Rubisco activase’s mechanism has the potential to contribute to the long-standing goal of enhancing photosynthetic efficiency. © 2018 by World Scientific Publishing Co. Pte. Ltd. All rights reserved.
... Interestingly in the absence of DnaK, an equal ratio of wild type and inactive subunits resulted in maximal enzymatic activity. Analogous approaches were applied to analyze the function of many ATPases, such as ClpX, ClpB, Rubisco activase Rca, viral DNA packaging ATPase gp16 of Phi29 [56,[131][132][133][134]. ...
ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.
... The disaggregase activity of ClpB-like Hsp100 proteins is generally low but can be enhanced markedly with addition of DnaK-like Hsp70s and their corresponding Hsp40s (Werbeck et al. 2008). The interaction between ClpB and DnaK is mediated by the ClpB's M-domain and is quite specific. ...
This book describes the role of heat shock proteins in the life cycle of malaria parasites. The work includes a general introduction on the structural and functional features of heat shock proteins. The main focus is on the role of heat shock protein families from Plasmodium falciparum, their role in protein folding and in the development of malaria pathology. The functions of individual families of heat shock proteins from plasmodium species and their cooperation in functional networks is described. Subcellular and extracellular organelles such as the apicoplast and the Maurer's Clefts which are associated with plasmodium species, are discussed in detail. The role of heat shock proteins in the development and function of these organelles structures are highlighted. Although conceding that heat shock proteins may not be ideal antimalarial drug targets, prospects of targeting heat shock proteins in antimalarial drug discovery either directly and/or in combination therapies are explored. © Springer Science+Business Media Dordrecht 2014. All rights are reserved.
... The disaggregase activity of ClpB-like Hsp100 proteins is generally low but can be enhanced markedly with addition of DnaK-like Hsp70s and their corresponding Hsp40s (Werbeck et al. 2008). The interaction between ClpB and DnaK is mediated by the ClpB's M-domain and is quite specific. ...
As obligate parasites, malaria parasites have developed mechanisms for survival under unfavourable conditions in host cells. The chapters in this book have extensively discussed the evidence that heat shock proteins of malaria play a key role in parasite survival in host cells. The role of the heat shock protein arsenal of the parasite is not limited to the protection of the parasite cell, as some of these proteins also promote the pathological development of malaria. This is largely due to the export of a large number of these proteins to the infected erythrocyte cytosol. Although PfEMP1 is the main virulent factor for the malaria parasite, some of the exported malarial heat shock proteins appear to augment parasite virulence (Maier et al. 2008). While this book largely delves into experimentally validated information on the role of heat shock proteins in the development and pathogenicity of malaria, some of the information is is based on hypotheses yet to be fully tested. Therefore, it is important to highlight what we know to be definite roles of malarial heat shock proteins. This will help us distill information that could provide practical insights on the options available for future research directions, including interventions against malaria that may target the role of heat shock proteins in the development of the disease. © Springer Science+Business Media Dordrecht 2014. All rights are reserved.
... The disaggregase activity of ClpB-like Hsp100 proteins is generally low but can be enhanced markedly with addition of DnaK-like Hsp70s and their corresponding Hsp40s (Werbeck et al. 2008). The interaction between ClpB and DnaK is mediated by the ClpB's M-domain and is quite specific. ...
Plasmodium falciparum has dedicated a large proportion of its genome to molecular chaperones (2∈%), with the heat shock protein 40 (Hsp40) family exhibiting evolutionary radiation into 49 members. A large number of the Hsp40s are predicted to be exported, with certain members shown experimentally to be present in the erythrocyte cytosol (PFA0660w and PFE0055c) or erythrocyte membrane (ring-infected erythrocyte surface antigen, RESA). PFA0660w and PFE0055c are associated with an exported plasmodial Hsp70 (PfHsp70-x) within novel structures called J dots, which have been proposed to be dedicated to the trafficking of key membrane proteins such as erythrocyte membrane protein 1 (PfEMP1). Yet other plasmodial Hsp40s have been shown to be essential, including the J dot Hsp40 PFA0660w, while others have been shown to be important for knob formation (PF10-0381). Here we review what is known about those plasmodial Hsp40s that have been well characterized, and may be directly or indirectly involved in the survival and pathogenesis of the malaria parasite. © Springer Science+Business Media Dordrecht 2014. All rights are reserved.
... One proposed model for Hsp104/ClpB action can be considered a reversal of the Dynamin mechanism. Hsp104/ClpB monomers could oligomerize around a protein aggregate but then subsequently deoligomerize, with each chaperone monomer pulling an associated substrate protein with it to break up the aggregate (Figure 5B) (Werbeck et al., 2008). This mechanism was effectively ruled out by an experiment in which it was shown that an Hsp104 hexamer with disulfides engineered to link the monomers together could still dissociate protein aggregates (Biter et al., 2012), demonstrating that deoligomerization was not required for Hsp104 action. ...
The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism-entropic pulling-may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context.
... We identified the NBD1-M trimer as the smallest ATP hydrolysiscompetent unit, which is formed upon ATP binding, but only if both arginines are present. The finding that trimer formation is essential is in agreement with previously performed mixing experiments showing that the random incorporation of two mutant ClpB subunits into the hexamer is sufficient to abolish activity (34). A previous study using full-length ClpB came to the conclusion that the trans-acting arginines are not involved in nucleotide binding (17). ...
The molecular chaperone ClpB/Hsp104, a member of the AAA+ superfamily (ATPases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the DnaK/Hsp70 chaperone system. ClpB/Hsp104
forms a hexameric, ring-shaped complex that functions as a tightly regulated, ATP-powered molecular disaggregation machine.
Highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of
the complex, which also harbor the catalytic sites. Several AAA+ proteins, including ClpB/Hsp104, possess a pair of such trans-acting arginines in the N-terminal nucleotide binding domain (NBD1), both of which were shown to be crucial for oligomerization
and ATPase activity. Here, we present a mechanistic study elucidating the role of this conserved arginine pair. First, we
found that the arginines couple nucleotide binding to oligomerization of NBD1, which is essential for the activity. Next,
we designed a set of covalently linked, dimeric ClpB NBD1 variants, carrying single subunits deficient in either ATP binding
or hydrolysis, to study allosteric regulation and intersubunit communication. Using this well defined environment of site-specifically
modified, cross-linked AAA+ domains, we found that the conserved arginine pair mediates the cooperativity of ATP binding and
hydrolysis in an allosteric fashion.
... These mutants are able to bind ATP yet are unable to hydrolyze it; thus, they can form so called -TRAP‖ molecules in which the high affinity state of the chaperones is indefinitely preserved and thus allows the stable formation of the chaperone-ATP-substrate complexes (Bosl et al., 2005;Lum et al., 2008). These mutations have also been useful for structural studies as they ensure the formation of a stable hexameric complex of Hsp104 or ClpB rather than the likely very dynamic and hence short lived WT hexamer Weibezahn et al., 2003;Werbeck et al., 2008). Important mutations including the Walker A and B mutants and their respective consequences are summarized in table 1. ...
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.
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.
NMR spectroscopy has found a wide range of applications in life sciences over recent decades. Providing a comprehensive amalgamation of the scattered knowledge of how to apply high-resolution NMR techniques to biomolecular systems, this book will break down the conventional stereotypes in the use of NMR for structural studies. The major focus is on novel approaches in NMR which deal with the functional interface of either protein-protein interactions or protein-lipid interactions. Bridging the gaps between structural and functional studies, the Editors believe a thorough compilation of these studies will open an entirely new dimension of understanding of crucial functional motifs. This in turn will be helpful for future applications into drug design or better understanding of systems.
The book will appeal to NMR practitioners in industry and academia who are looking for a comprehensive understanding of the possibilities of applying high-resolution NMR spectroscopic techniques in probing biomolecular interactions.
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.
The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with impaired protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). Yet how Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Unexpectedly, substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK-linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. Our findings illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.
The human ATPase p97, also known as valosin containing protein or Cdc48, is a highly abundant AAA+ engine that fuels diverse energy-consuming processes in the human cell. p97 represents a potential target for cancer therapy and its malfunction causes a degenerative disease. Here, we monitor the enzymatic activity of p97 in real time via an NMR-based approach that allows us to follow the steps that couple ATP turnover to mechanical work. Our data identify a transient reaction intermediate, the elusive ADP.Pi nucleotide state, which has been postulated for many ATPases but has so far escaped direct detection. In p97, this species is crucial for the regulation of adenosine triphosphate turnover in the first nucleotide-binding domain. We further demonstrate how the enzymatic cycle is detuned by disease-associated gain-of-function mutations. The high-resolution insight obtained into conformational transitions in both protein and nucleotide bridges the gap between static enzyme structures and the dynamics of substrate conversion. Our approach relies on the close integration of solution- and solid-state NMR methods and is generally applicable to shed light on the mechanochemical operating modes of large molecular engines.
The ability to reverse protein aggregation is vital to cells1,2. Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore3,4. This model of disaggregation is appealing, as it could explain how polypeptides entangled within aggregates can be extracted and subsequently refolded with the assistance of Hsp704,5. However, the model is also controversial, as the necessary motor activity has not been identified6–8 and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting9,10. How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore11,12. Here, by following individual ClpB–substrate complexes in real time, we unambiguously demonstrate processive translocation of looped polypeptides. We integrate optical tweezers with fluorescent-particle tracking to show that ClpB translocates both arms of the loop simultaneously and switches to single-arm translocation when encountering obstacles. ClpB is notably powerful and rapid; it exerts forces of more than 50 pN at speeds of more than 500 residues per second in bursts of up to 28 residues. Remarkably, substrates refold while exiting the pore, analogous to co-translational folding. Our findings have implications for protein-processing phenomena including ubiquitin-mediated remodelling by Cdc48 (or its mammalian orthologue p97)13 and degradation by the 26S proteasome14. A combination of optical tweezers and fluorescent-particle tracking is used to dissect the dynamics of the Hsp100 disaggregase ClpB, and show that the processive extrusion of polypeptide loops is the mechanistic basis of its activity.
Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.
Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.
The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of individual Hsps and the Hsp network; however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps (sHsps) and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.
Hsp104, a conserved AAA+ protein disaggregase, promotes survival during cellular stress. Hsp104 remodels amyloids, thereby supporting prion propagation, and disassembles toxic oligomers associated with neurodegenerative diseases. However, a definitive structural mechanism for its disaggregase activity has remained elusive. We determined the cryo-EM structure of wild-type Saccharomyces cerevisiae Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the 12 AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, thus explaining roles in ATP hydrolysis and protein disaggregation. Remarkably, substrate-binding pore loops line the channel in a spiral arrangement optimized for substrate transfer across the AAA+ domains, thereby establishing a continuous path for polypeptide translocation.
AAA+ proteases are universal barrel-like and ATP-fueled machines preventing the accumulation of aberrant proteins and regulating the proteome according to the cellular demand. They are characterized by two separate operating units, the ATPase and peptidase domains. ATP-dependent unfolding and translocation of a substrate into the proteolytic chamber is followed by ATP-independent degradation. This review addresses the structure and function of bacterial AAA+ proteases with a focus on the ATP-driven mechanisms and the coordinated movements in the complex mainly based on the knowledge of ClpXP. We conclude by discussing strategies how novel protease substrates can be trapped by mutated AAA+ protease variants. This article is protected by copyright. All rights reserved.
E. coli ClpB is a molecular chaperone with the unique ability to catalyze protein disaggregation in collaboration with the KJE system of chaperones. Like many AAA+ molecular motors, ClpB assembles into hexameric rings and this reaction is thermodynamically linked to nucleotide binding. Here we show that ClpB exists in a dynamic equilibrium of monomers, dimers, tetramers, and hexamers in the presence of both limiting and excess ATPγS. We find that ClpB monomer is only able to bind one nucleotide whereas all twelve sites in the hexameric ring are bound by nucleotide at saturating concentrations. Interestingly, dimers and tetramers exhibit stoichiometries of ~3 and 7, respectively. This is one fewer than the maximum number of binding sites, suggesting an open conformation for the intermediates based on the need for an adjacent monomer to fully form the binding pocket. We also report the protein-protein interaction constants for dimers, tetramers, and hexamers and their dependencies on nucleotide. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to co-chaperones and polypeptide substrates. Such information is essential for the interpretation of many in vitro studies. Finally, the strategies presented here are broadly applicable to a large number of AAA+ molecular motors that assemble upon nucleotide binding and interact with partner proteins.
Malaria parasites are principally intracellular pathogens of erythrocytes and hepatocytes. For the parasites to grow rapidly and to avoid host immunity they must extensively modify their host cells by exporting hundreds of proteins into them. These exported proteins specifically help permeabilise the host cell so additional nutrients can be obtained and to avoid splenic clearance, the exported proteins modify the erythrocyte surface to promote cytoadherance within the microvasculature. Since erythrocytes lack many of the protein trafficking systems present in normal cells, parasites export their own. Many exported proteins help transport, traffic and refold other exported proteins into functional complexes once they reach their final destinations in the host. Exported trafficking systems and their cargoes are best understood in the most deadly human malaria pathogen Plasmodium falciparum, because it can be cultured in the laboratory. Trafficking and refolding require protein chaperone ATPases and we will present the latest data on exported chaperones. In particular, we will discuss the role of HSP101 in the protein translocon that provides a portal for protein export into the host compartment. Plasmodium species also export a few Hsp40s that probably function co-operatively with host Hsp70s. P. falciparum is however unique in that it exports also many additional Hsp40s as well as its own Hsp70-x and the roles of these will also be discussed. © Springer Science+Business Media Dordrecht 2014. All rights are reserved.
Escherichia coli ClpB is a heat shock protein that belongs to the AAA+ protein superfamily. Studies have shown that ClpB and its homologue in yeast, Hsp104, can disrupt protein aggregates in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity. In addition, it is widely assumed that ClpB is uniformly hexameric in the presence of nucleotides. Here we report, in the absence of nucleotide, that increasing ClpB concentration leads to ClpB hexamer formation, decreasing NaCl concentration stabilizes ClpB hexamers, and the ClpB assembly reaction is best described by a monomer, dimer, tetramer equilibrium under the three salt concentration examined here. Further, we found that ClpB oligomers exhibit relatively fast dissociation on the time scale of sedimentation. We anticipate our studies on ClpB assembly to be a starting point to understand how ClpB assembly is linked to the binding and disaggregation of denatured proteins. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival during severe stress. The protein disaggregase activity of these members of the Hsp100 family is linked to the activity of chaperones from the Hsp70 and Hsp40 families. The precise mechanism by which these proteins untangle protein aggregates remains unclear. Strikingly, Hsp100 proteins are not present in metazoans. This does not mean that animal cells do not have a disaggregase activity, but that this activity is performed by the Hsp70 system and a representative of the Hsp110 family instead of a Hsp100 protein. This review describes the actual view of Hsp100-mediated aggregate reactivation, including the ATP-induced conformational changes associated with their disaggregase activity, the dynamics of the oligomeric assembly that is regulated by its ATPase cycle and the DnaK system, and the tight allosteric coupling between the ATPase domains within the hexameric ring complexes. The lack of homologues of these disaggregases in metazoans has suggested that they might be used as potential targets to develop antimicrobials. The current knowledge of the human disaggregase machinery and the role of Hsp110 are also discussed.
Copyright © 2015. Published by Elsevier Inc.
Hexameric AAA+ chaperone ClpB reactivates protein aggregates in collaboration with the DnaK system. An intriguing aspect of ClpB function is that the active hexamer is unstable and therefore questions how this chaperone uses multiple rounds of ATP hydrolysis to translocate substrates through its central channel. Here we report the use of biochemical and fluorescence tools to explore ClpB dynamics under different experimental conditions. The analysis of the chaperone activity and the kinetics of subunit exchange between protein hexamers labelled at different protein domains indicates, in contrast to the current view, that i) ATP favors assembly and ADP dissociation of the hexameric assembly; ii) subunit exchange kinetics is at least one order of magnitude slower that the ATP hydrolysis rate; iii) ClpB dynamics and activity are related proccesses; and iv) DnaK and substrate proteins regulate the ATPase activity and dynamics of ClpB. These data suggest that ClpB hexamers remain associated during several ATP hydrolysis events required to partially or completely translocate substrates through the protein central channel, and that ClpB dynamics is tuned by DnaK and substrate proteins.
Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.
Point mutations in either of the two nucleotide-binding domains
(NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function
in vivo. In vitro, NBD1 mutations
virtually eliminate ATP hydrolysis with little effect on
hexamerization; analogous NBD2 mutations reduce ATPase activity and
severely impair hexamerization. We report that high protein
concentrations overcome the assembly defects of NBD2 mutants and
increase ATP hydrolysis severalfold, changing
Vmax with little effect on
Km. In a complementary fashion, the
detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
inhibits hexamerization of wild-type (WT) Hsp104, lowering
Vmax with little effect on
Km. ATP hydrolysis exhibits a Hill
coefficient between 1.5 and 2, indicating that it is influenced by
cooperative subunit interactions. To further analyze the effects of
subunit interactions on Hsp104, we assessed the effects of mutant
Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that
hexamerizes but does not hydrolyze ATP reduces the ATPase activity of
WT Hsp104 in vitro. In vivo, this mutant
is not toxic but specifically inhibits the thermotolerance function of
WT Hsp104. Thus, interactions between subunits influence the ATPase
activity of Hsp104, play a vital role in its biological functions, and
provide a mechanism for conditionally inactivating Hsp104 function
in vivo.
The yeast non-Mendelian factor [psi+] has been suggested to be a self-modified protein analogous to mammalian prions. Here
it is reported that an intermediate amount of the chaperone protein Hsp104 was required for the propagation of the [psi+]
factor. Over-production or inactivation of Hsp104 caused the loss of [psi+]. These results suggest that chaperone proteins
play a role in prion-like phenomena, and that a certain level of chaperone expression can cure cells of prions without affecting
viability. This may lead to antiprion treatments that involve the alteration of chaperone amounts or activity.
Hsp78, a member of the family of Clp/Hsp100 proteins, exerts chaperone functions in mitochondria of S. cerevisiae which overlap with those of mitochondrial Hsp70. In the present study, the role of Hsp78 under extreme stress was analyzed. Whereas deletion of HSP78 does not affect cell growth at temperatures up to 39 decrees C and cellular thermotolerance at 50 degrees C, Hsp78 is crucial for maintenance of respiratory competence and for mitochondrial genome integrity under severe temperature stress (mitochondrial thermotolerance). Mitochondrial protein synthesis is identified as a thermosensitive process. Reactivation of mitochondrial protein synthesis after heat stress depends on the presence of Hsp78, though Hsp78 does not confer protection against heat-inactivation to this process. Hsp78 appears to act in concert with other mitochondrial chaperone proteins since a conditioning pretreatment of the cells to induce the cellular heat shock response is required to maintain mitochondrial functions under severe temperature stress. When expressed in the cytosol, Hsp78 can substitute for the homologous heat shock protein Hsp104 in mediating cellular thermotolerance, suggesting a conserved mode of action of the two proteins. Thus, proteins of the Clp/Hsp100-family located in the cytosol and within mitochondria confer compartment-specific protection against heat damage to the cell.
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.
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.
AAA ATPases(those associated with various cellular activities) play important roles in numerous cellular activities including proteolysis, protein folding, membrane trafficking, cytoskeletal regulation, organelle biogenesis, DNA replication, and intracellular motility. Recent structural and enzymatic studies are providing clues into the properties of the conserved AAA domain that defines this large protein superfamily. In many cases, AAA domains assemble into hexamic rings that are likely to change their shape during the ATPase cycle. This nucleotide-dependent conformational switch may apply tension to bound proteins and thereby allow AAA proteins to unfold polypeptides, dissociate protein-protein interactions, or generate unidirectional motion along a track. Thus, AAA proteins may represent a broad class of mechanoenzymes that have evolved unique ways of using a fundamentally similar conformational change in many different biological settings.
Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing V(max) with little effect on K(m). In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering V(max) with little effect on K(m). ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus, interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological functions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo.
Members of the AAA (for ATPases associated with various cellular activities) protein superfamily are characterized by the presence of one or two highly conserved modules of approximately 230 amino acid residues (the AAA box) that include an ATP-binding consensus. AAA proteins are found in all organisms and are essential for cell cycle functions, vesicular transport, mitochondrial functions, peroxisome assembly and proteolysis.
As a member of molecular chaperone Hsp100/Clp family, TClpB from Thermus thermophilus has two nucleotide binding domains, NBD1 and NBD2, in a single polypeptide, each containing WalkerA and WalkerB consensus
motifs. To probe their roles, mutations were introduced into the WalkerA or WalkerB motifs of each or both of the NBDs. The
results are as follows. 1) For each of the NBDs, the ability of nucleotide binding is lost by mutations in the WalkerA motif
but is retained by mutations in the WalkerB motif. 2) Each NBD has a casein-stimulatable small basic ATPase activity that
is lost when the WalkerB motif is mutated. 3) TClpB assembles into a uniform 580-kDa oligomer when ATP is present at 55 °C, and only the mutants in the WalkerA motif in
NBD1 fail to assemble, indicating that ATP binding to NBD1 but not hydrolysis is necessary and sufficient for the assembly.
4) Chaperone function ofTClpB was lost when the WalkerA motif in each of the NBDs was mutated. Mutants in the WalkerB motifs of each NBD retained some
chaperone activity.
ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system. The mechanism of protein reactivation and interaction with the DnaK system remains unclear. ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior. The role of the N-terminal domain that precedes the first nucleotide binding domain is largely unknown. We purified and characterized an N-terminal shortened ClpB variant (ClpBDeltaN; amino acids 140-854), which remained active in refolding assays with three different substrate proteins. In addition the N-terminal truncation did not significantly change the nucleotide binding affinities, the nucleotide-dependent oligomerization, and the allosteric behavior of the protein. In contrast casein binding and stimulation of the ATPase activity by kappa-casein were affected. These results suggest that the N-terminal domain is not essential for the chaperone function, does not influence the binding of nucleotides, and is not involved in the formation of intermolecular contacts. It contributes to the casein binding site of ClpB, but other substrate proteins do not necessarily interact with the N terminus. This indicates a substantial difference in the binding mode of kappa-casein that is often used as model substrate for ClpB and other possibly more suitable substrate proteins.
ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with
the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is composed of an N-terminal domain
and two AAA-domains that are separated by a “linker” region. Here we present a detailed structure-function analysis of ClpB,
dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity.
Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain,
while ATP binding to the first AAA-domains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker
A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate
in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity.
The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone
activity.
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.
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.
The molecular chaperone ClpB can rescue the heat-damaged proteins from an aggregated state in cooperation with other chaperones.
It has two nucleotide binding domains (NBD1 and NBD2) and forms a hexamer ring in a manner dependent on ATP binding to NBD1.
In the crystal structure of ClpB with both NBDs filled by nucleotides, the linker between two NBDs forms an 85-Å-long coiled-coil
that extends on the outside of the hexamer and leans to NBD1. To probe the possible motion of the coiled-coil, we tested the
accessibility of a labeling reagent, fluorescence change of a labeled dye, and cross-linking between the coiled-coil and NBD1
by using the mutants with defective NBD1 or NBD2. The results suggest that the coiled-coil is more or less parallel to the
main body of ClpB in the absence of nucleotide and that ATP binding to NBD1 brings it to the leaning position as seen in the
crystal structure. This motion results in stabilization of the hexamer form of ClpB and promotion of ATP hydrolysis at NBD2.
Bacterial heat-shock proteins, ClpB and DnaK form a bichaperone system that efficiently reactivates aggregated proteins. ClpB undergoes nucleotide-dependent self-association and forms ring-shaped oligomers. The ClpB-assisted dissociation of protein aggregates is linked to translocation of substrates through the central channel in the oligomeric ClpB. Events preceding the translocation step, such as recognition of aggregates by ClpB, have not yet been explored, and the location of the aggregate-binding site in ClpB has been under discussion. We investigated the reactivation of aggregated glucose-6-phosphate dehydrogenase (G6PDH) by ClpB and its N-terminally truncated variant ClpBDeltaN in the presence of DnaK, DnaJ, and GrpE. We found that the chaperone activity of ClpBDeltaN becomes significantly lower than that of the full-length ClpB as the size of G6PDH aggregates increases. Using a "substrate trap" variant of ClpB with mutations of Walker B motifs in both ATP-binding modules (E279Q/E678Q), we demonstrated that ClpBDeltaN binds to G6PDH aggregates with a significantly lower affinity than the full-length ClpB. Moreover, we identified two conserved acidic residues at the surface of the N-terminal domain of ClpB that support binding to G6PDH aggregates. Those N-terminal residues (Asp-103, Glu-109) contribute as much substrate-binding capability to ClpB as the conserved Tyr located at the entrance to the ClpB channel. In summary, we provided evidence for an essential role of the N-terminal domain of ClpB in recognition and binding strongly aggregated proteins.
The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the end of a 3'-extended single-stranded DNA tail of a Y-shaped substrate; binding is oriented so that the motor domain of the protein faces duplex DNA. Two candidate beta-hairpin motifs within the MCM monomer have partially redundant roles in DNA binding. Notably, however, conserved basic residues within these motifs have nonequivalent roles in the helicase activity of MCM. On the basis of these findings, we propose a model for the mechanism of the helicase activity of MCM and note parallels with SV40 T antigen.
ClpB cooperates with the DnaK chaperone system in the reactivation of protein from aggregates and is a member of the ATPases associated with a variety of cellular activities (AAA+) protein family. The underlying disaggregation reaction is dependent on ATP hydrolysis at both AAA cassettes of ClpB but the role of each AAA cassette in the reaction cycle is largely unknown. Here we analyze the activity of the separately expressed and purified nucleotide binding domains of ClpB from Thermus thermophilus. The two fragments show different biochemical properties: the first construct is inactive in ATPase activity assays and binds nucleotides weakly, the second construct has a very high ATPase activity and interacts tightly with nucleotides. Both individual fragments have lost their chaperone function and are not able to form large oligomers. When combined in solution, however, the two fragments form a stable heterodimer with oligomerization capacities equivalent to wild-type ClpB. This non-covalent complex regains activity in reactivating protein aggregates in cooperation with the DnaK chaperone system. Upon complex formation the ATPase activity of fragment 2 is reduced to a level similar to wild-type ClpB. Hence functional ClpB can be reassembled from its isolated AAA cassettes showing that covalent linkage of these domains is not a prerequisite for the chaperone activity. The observation that the intrinsically high ATPase activity of AAA2 is suppressed by AAA1 allows a hypothetical assignment of their mechanistic function. Whereas the energy gained upon ATP hydrolysis at the AAA2 is likely to drive a conformational change of the structure of ClpB, AAA1 might function as a regulator of the chaperone cycle.
Complex cellular events commonly depend on the activity of molecular "machines" that efficiently couple enzymatic and regulatory functions within a multiprotein assembly. An essential and expanding subset of these assemblies comprises proteins of the ATPases associated with diverse cellular activities (AAA+) family. The defining feature of AAA+ proteins is a structurally conserved ATP-binding module that oligomerizes into active arrays. ATP binding and hydrolysis events at the interface of neighboring subunits drive conformational changes within the AAA+ assembly that direct translocation or remodeling of target substrates. In this review, we describe the critical features of the AAA+ domain, summarize our current knowledge of how this versatile element is incorporated into larger assemblies, and discuss specific adaptations of the AAA+ fold that allow complex molecular manipulations to be carried out for a highly diverse set of macromolecular targets.
IntroductionCharacterization of ATPase Cycles of Molecular ChaperonesExperimental Protocols
Hsp78, a member of the family of Clp/Hsp100 proteins, exerts chaperone functions in mitochondria of S. cerevisiae which overlap with those of mitochondrial Hsp70. In the present study, the role of Hsp78 under extreme stress was analyzed. Whereas deletion of HSP78 does not affect cell growth at temperatures up to 39 decrees C and cellular thermotolerance at 50 degrees C, Hsp78 is crucial for maintenance of respiratory competence and for mitochondrial genome integrity under severe temperature stress (mitochondrial thermotolerance). Mitochondrial protein synthesis is identified as a thermosensitive process. Reactivation of mitochondrial protein synthesis after heat stress depends on the presence of Hsp78, though Hsp78 does not confer protection against heat-inactivation to this process. Hsp78 appears to act in concert with other mitochondrial chaperone proteins since a conditioning pretreatment of the cells to induce the cellular heat shock response is required to maintain mitochondrial functions under severe temperature stress. When expressed in the cytosol, Hsp78 can substitute for the homologous heat shock protein Hsp104 in mediating cellular thermotolerance, suggesting a conserved mode of action of the two proteins. Thus, proteins of the Clp/Hsp100-family located in the cytosol and within mitochondria confer compartment-specific protection against heat damage to the cell.
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.
Self-association of ClpB (a mixture of 95- and 80-kDa subunits) has been studied with gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. Monomeric ClpB predominates at low protein concentration (0.07 mg0mL), while an oligomeric form is highly populated at >4 mg/mL. The oligomer formation is enhanced in the presence of 2 mM ATP or adenosine 5′-O-thiotriphosphate (ATPS). In contrast, 2 mMADP inhibits full oligomerization of ClpB. The apparent size of the ATP- or ATPS-induced oligomer, as determined by gel filtration, sedimentation velocity and electron microscopy image averaging, and the molecular weight, as determined by sedimentation equilibrium, are consistent with those of a ClpB hexamer. These results indicate that the oligomerization reactions of ClpB are similar to those of other Hsp100 proteins.
A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented. This system is based on the use of the fluorescent ligand alpha,omega-di[(3' or 2')-O-(N-methylanthraniloyl)adenosine-5'] pentaphosphate (mAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A). It allows the determination of dissociation constants for any ligand in the range of 1 x 10(-9) to 5 x 10(-2) M. Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium. An analysis of the binding of bisubstrate inhibitors is proposed and applied to these data. The techniques are used to describe the properties of a mutant enzyme with Gln-28----His (Q28H) prepared by site-directed mutagenesis in comparison to those of wild-type AK from Escherichia coli. This newly introduced histidine is already present in most other adenylate kinases and was regarded to be important or even essential for the catalytic reaction of AK. Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure. However, steady-state catalytic studies and binding experiments showed that the affinities for substrates and inhibitors are elevated from 3-fold (AMP) to 5-fold (ATP) to 15-fold (AP5A) compared to those of the wild-type enzyme. Together with the results obtained by Tian et al. [Tian, G., Sanders, C. R., Kishi, F., Nakazawa, A., & Tsai, M.-D. (1988) Biochemistry 27, 5544-5552] on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E. coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity.
The concentration of proteins in solutions or cell-free extracts contaminated by tRNA's or rRNA's can be simply, rapidly, and accurately determined by measuring the absorbance at 2285 and 2345 Å, two wavelengths where the absorption due to ribonucleic acids is identical so that their interference can be eliminated. This method is sensitive and gives results which are similar to those obtained by the colorimetric method of Lowry and better than those obtained by the spectrophotometric method of Warburg and Christian or that of Groves et al.
The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3'-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.12-0.24. Their fluorescence was sensitive to the polarity of the solvent; in N,N-dimethylformamide the quantum yields were 0.83-0.93. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield in water. All anthraniloyl derivatives, as well as the N-methylanthraniloyl ones, had virtually identical fluorescent properties, regardless of their base structures. The ATP derivatives showed considerable substrate activity as a replacement of ATP with adenylate kinase, guanylate kinase, glutamine synthetase, myosin ATPase and sodium-potassium transport ATPase. The ADP derivatives were good substrates for creatine kinase and glutamine synthetase (gamma-glutamyl transfer activity). The GMP and adenosine derivatives were substrates for guanylate kinase and adenosine deaminase, respectively. All derivatives had only slightly altered Km values for these enzymes. While more fluorescent in water, the N-methylanthraniloyl derivatives were found to show relatively low substrate activities against some of these enzymes. The results indicate that these ribose-modified nucleosides and nucleotides can be versatile fluorescent substrate analogs for various enzymes.
A cDNA clone encoding a 101-kD heat shock protein (HSP101) of soybean was isolated and sequenced. Genomic DNA gel blot analysis indicated that the corresponding gene is a member of a multigene family. The mRNA for HSP101 was not detected in 2-day-old etiolated soybean seedlings grown at 28 degrees C but was induced by elevated temperatures. DNA sequence comparison has shown that the corresponding gene belongs to the Clp (caseinolytic protease) (or Hsp100) gene family, which is evolutionarily conserved and found in both prokaryotes and eukaryotes. On the basis of the spacer length between the two conserved ATP binding regions, this gene has been identified as a member of the ClpB subfamily. Unlike other Clp genes previously isolated from higher plants, the expression of this soybean Hsp101 gene is heat inducible, and it does not have an N-terminal signal peptide for targeting to chloroplasts. Transformation of the soybean Hsp101 gene into a yeast HSP104 deletion mutant complemented restoration of acquired thermotolerance, a process in which cells survive an otherwise lethal heat stress after they are given a permissive heat treatment.
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.
We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation.
ClpB belongs to the Hsp100 family and assists de-aggregation of protein aggregates by DnaK chaperone systems. It contains two Walker consensus sequences (or P-Loops) that indicate potential nucleotide binding domains (NBD). Both domains appear to be essential for chaperoning function, since mutation of the conserved lysine residue of the GX(4)GKT consensus sequences to glutamine (K204Q and K601Q) abolishes its properties to accelerate renaturation of aggregated firefly luciferase. The underlying biochemical reason for this malfunction appears not to be a dramatically reduced ATPase activity of either P-loop per se but rather changed properties of co-operativity of ATPase activity connected to oligomerization properties to form productive oligomers. This view is corroborated by data that show that structural stability (as judged by CD spectroscopy) or ATPase activity at single turnover conditions (at low ATP concentrations) are not significantly affected by these mutations. In addition nucleotide binding properties of wild-type protein and mutants (as judged by binding studies with fluorescent nucleotide analogues and competitive displacement titrations) do not differ dramatically. However, the general pattern of formation of stable, defined oligomers formed as a function of salt concentration and nucleotides and more importantly, cooperativity of ATPase activity at high ATP concentrations is dramatically changed with the two P-loop mutants described.
The yeast mitochondrial chaperone Hsp78, a homologue of yeast cytosolic Hsp104 and bacterial ClpB, is required for maintenance of mitochondrial functions under heat stress. Here, Hsp78 was purified to homogeneity and shown to form a homo-hexameric complex, with an apparent molecular mass of approximately 440 kDa, in an ATP-dependent manner. Analysis of its ATPase activity reveals that the observed positive cooperativity effect depends both on Hsp78 and ATP concentration. Site-directed mutagenesis of the two putative Hsp78 nucleotide-binding domains suggest that the first nucleotide-binding domain is responsible for ATP hydrolysis and the second one for protein oligomerization. Studies on the chaperone activity of Hsp78 show that its cooperation with the mitochondrial Hsp70 system, consisting of Ssc1p, Mdj1p and Mge1p, is needed for the efficient reactivation of substrate proteins. These studies also suggest that the oligomerization but not the Hsp78 ATPase activity is essential for its chaperone activity.
AAA proteins share a conserved active site for ATP hydrolysis and regulate many cellular processes. AAA proteins are oligomeric and often have multiple ATPase domains per monomer, which is suggestive of complex allosteric kinetics of ATP hydrolysis. Here, using wild-type Hsp104 in the hexameric state, we demonstrate that its two AAA modules (NBD1 and NBD2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis. Using mutations in the AAA sensor-1 motif of NBD1 and NBD2 that reduce the rate of ATP hydrolysis without affecting nucleotide binding, we also examine the consequences of keeping each site in the ATP-bound state. In vitro, reducing k(cat) at NBD2 significantly alters the steady-state kinetic behavior of NBD1. Thus, Hsp104 exhibits allosteric communication between the two sites in addition to homotypic cooperativity at both NBD1 and NBD2. In vivo, each sensor-1 mutation causes a loss-of-function phenotype in two assays of Hsp104 function (thermotolerance and yeast prion propagation), demonstrating the importance of ATP hydrolysis as distinct from ATP binding at each site for Hsp104 function.
AAA proteins remodel other proteins to affect a multitude of biological processes. Their power to remodel substrates must lie in their capacity to couple substrate binding to conformational changes via cycles of nucleotide binding and hydrolysis, but these relationships have not yet been deciphered for any member. We report that when one AAA protein, Hsp104, engages polypeptide at the C-terminal peptide-binding region, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2) drives a conformational change in the middle region. This, in turn, drives ATP hydrolysis in the N-terminal ATPase domain (NBD1). This interdomain communication pathway can be blocked by mutation in the middle region or bypassed by antibodies that bind there, demonstrating the crucial role this region plays in transducing signals from one end of the molecule to the other.
Proteins of thermophilic organisms are adapted to remain well structured and functional at elevated temperatures. Nevertheless like their 'cousins' that reside at medium temperatures, they require the assistance of molecular chaperones to fold properly and prevent aggregation. This review compares structural and functional properties of the DnaK/ClpB systems of Thermus thermophilus and, mainly, Escherichia coli (DnaK(Tth) and DnaK(Eco)). Many elemental properties of these systems remain conserved. However, in addition to a general increase of the thermal stability of its components, the DnaK(Tth) system shows profound differences in its regulation, and genetic as well as oligomeric organization. Whether these differences are unique or represent general strategies of adaptation to life at elevated temperatures remains to be clarified.
Active protein-disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress-induced protein aggregates in vitro and in Escherichia coli cells. K-glutamate and glycine-betaine (betaine) naturally accumulate in salt-stressed cells. In addition to providing thermo-protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone-mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K-glutamate or betaine. The combined protective, counter-negative and net activatory effects of K-glutamate and betaine, allowed protein disaggregation and refolding under heat-shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte-activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat-shock conditions.
The AAA+ ATPases are enzymes containing a P-loop NTPase domain, and function as molecular chaperones, ATPase subunits of proteases, helicases or nucleic-acid-stimulated ATPases. All available sequences and structures of AAA+ protein domains were compared with the aim of identifying the definitive sequence and structure features of these domains and inferring the principal events in their evolution. An evolutionary classification of the AAA+ class was developed using standard phylogenetic methods, analysis of shared sequence and structural signatures, and similarity-based clustering. This analysis resulted in the identification of 26 major families within the AAA+ ATPase class. We also describe the position of the AAA+ ATPases with respect to the RecA/F1, helicase superfamilies I/II, PilT, and ABC classes of P-loop NTPases. The AAA+ class appears to have undergone an early radiation into the clamp-loader, DnaA/Orc/Cdc6, classic AAA, and "pre-sensor 1 beta-hairpin" (PS1BH) clades. Within the PS1BH clade, chelatases, MoxR, YifB, McrB, Dynein-midasin, NtrC, and MCMs form a monophyletic assembly defined by a distinct insert in helix-2 of the conserved ATPase core, and additional helical segment between the core ATPase domain and the C-terminal alpha-helical bundle. At least 6 distinct AAA+ proteins, which represent the different major clades, are traceable to the last universal common ancestor (LUCA) of extant cellular life. Additionally, superfamily III helicases, which belong to the PS1BH assemblage, were probably present at this stage in virus-like "selfish" replicons. The next major radiation, at the base of the two prokaryotic kingdoms, bacteria and archaea, gave rise to several distinct chaperones, ATPase subunits of proteases, DNA helicases, and transcription factors. The third major radiation, at the outset of eukaryotic evolution, contributed to the origin of several eukaryote-specific adaptations related to nuclear and cytoskeletal functions. The new relationships and previously undetected domains reported here might provide new leads for investigating the biology of AAA+ ATPases.
The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
The oligomeric rings formed by RecA-fold proteins are mechanochemical motors that perform many important biological functions. Their RecA-fold domains convert the chemical energy of ATP into mechanical work through large nucleotide-dependent conformational changes. This review summarizes recent structural and mechanistic works on the F1-ATPase and HslU regarding to the force generation by individual RecA folds in the context of ring structures. The F1-ATPase ring for example generates the force perpendicular to the ring axis, while the HslU ring generates forces presumably parallel to it. There exists a strong correlation between the directions of forces generated and the orientation of the RecA folds, not only in these two proteins but also in T7 DNA helicase, suggesting that it should be possible to predict the direction of forces generated by other members of this family on the basis of the orientation of their RecA folds.
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.
Cell survival under severe thermal stress requires the activity of a bi-chaperone system, consisting of the ring-forming AAA+ chaperone ClpB (Hsp104) and the DnaK (Hsp70) chaperone system, which acts to solubilize and reactivate aggregated proteins. Recent studies have provided novel insight into the mechanism of protein disaggregation, demonstrating that ClpB/Hsp104 extracts unfolded polypeptides from an aggregate by threading them through its central pore. This translocation activity is necessary but not sufficient for aggregate solubilization. In addition, the middle (M) domain of ClpB and the DnaK system have essential roles, possibly by providing an unfolding force, which facilitates the extraction of misfolded proteins from aggregates.
Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.
We show that the mechanisms of DNA-dependent and -independent dTTP hydrolysis by the gene 4 protein of bacteriophage T7 differ in the pathways by which these reactions are catalyzed. In the presence of dTTP, gene 4 protein monomers assemble as a ring that binds single-stranded DNA and couples the hydrolysis of dTTP to unidirectional translocation and the unwinding of duplex DNA. When mixing wild-type monomers with monomers lacking the catalytic base for the dTTPase reaction, we observe that each wild-type subunit hydrolyzes dTTP independently in the absence of single-stranded DNA. Conversely, when either these catalytically inactive monomers or altered monomers incapable of binding single-stranded DNA are mixed with wild-type monomers, a small fraction of altered to wild-type monomers causes a sharp decline in DNA-dependent dTTP hydrolysis. We propose that sequential hydrolysis of dTTP is coupled to the transfer of single-stranded DNA from subunit to adjacent subunit.
It is now well accepted that the origin of all plastids can be traced back to a single primary endosymbiosis involving a eukaryote and a cyanobacterium. Challenging this view, a recent study provides the first evidence for a second and more recent primary endosymbiosis.
AAA+ ATPase protein machines use the power obtained from ATP hydrolysis to remodel macromolecular assemblies in the cell. Recent work in Escherichia coli on the ClpX AAA+ protein reveals fundamental mechanical principles that underlie ClpX motor function.
At the Cold Spring Harbor Meeting on 'Molecular Chaperones and the Heat Shock Response' in May 1996, Susan Lindquist presented evidence that a chaperone of yeast termed Hsp104, which her group had been investigating for several years, is able to dissolve protein aggregates (Glover, J.R., Lindquist, S., 1998. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73-82). Among many of the participants this news stimulated reactions reaching from decided skepticism to utter disbelief because protein aggregation was widely considered to be an irreversible process. Several years and publications later, it is undeniable that Susan had been right. Hsp104 is an ATP dependent molecular machine that-in cooperation with Hsp70 and Hsp40-extracts polypeptide chains from protein aggregates and facilitates their refolding, although the molecular details of this process are still poorly understood. Meanwhile, close homologues of Hsp104 have been identified in bacteria (ClpB), in mitochondria (Hsp78), and in the cytosol of plants (Hsp101), but intriguingly not in the cytosol of animal cells (Mosser, D.D., Ho, S., Glover, J.R., 2004. Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling. Biochemistry 43, 8107-8115). Observations that Hsp104 plays an essential role in the maintenance of yeast prions (see review by James Shorter in this issue) have attracted even more attention to the molecular mechanism of this ATP dependent chaperone (Chernoff, Y.O., Lindquist, S.L., Ono, B., Inge-Vechtomov, S.G., Liebman, S.W., 1995. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [PSI+]. Science 268, 880-884).
Clp ATPases are protein machines involved in protein degradation and disaggregation. The common structural feature of Clp ATPases is the formation of ring-shaped oligomers. Recent work has shown that the function of all Clp ATPases is based on an energy-dependent threading of substrates through the narrow pore at the centre of the ring. This review gives an outline of known mechanistic principles of threading machines that unfold protein substrates either before their degradation (ClpA, ClpX, HslU) or during their reactivation from aggregates (ClpB). The place of Clp ATPases within a broad AAA+ superfamily of ATPases associated with various cellular activities suggests that similar mechanisms can be used by other protein machines to induce conformational rearrangements in a wide variety of substrates.
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.