The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase.

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.

Role of J-domain Proteins in Yeast Physiology and Protein Quality Control.

The Hsp70 chaperone system is a central component of cellular protein quality control (PQC) by acting in a multitude of protein folding processes ranging from the folding of newly synthesized proteins to the disassembly and refolding of protein aggregates. This multifunctionality of Hsp70 is governed by J-domain proteins (JDPs), which act as indispensable co-chaperones that target specific substrates to Hsp70. The number of distinct JDPs present in a species always outnumbers Hsp70, documenting JDP function in functional diversification of Hsp70. In this review, we describe the physiological roles of JDPs in the Saccharomyces cerevisiae PQC system, with a focus on the abundant JDP generalists, Zuo1, Ydj1 and Sis1, which function in fundamental cellular processes. Ribosome-bound Zuo1 cooperates with the Hsp70 chaperones Ssb1/2 in folding and assembly of nascent polypeptides. Ydj1 and Sis1 cooperate with the Hsp70 members Ssa1 to Ssa4 to exert overlapping functions in protein folding and targeting of newly synthesized proteins to organelles including mitochondria and facilitating the degradation of aberrant proteins by E3 ligases. Furthermore, they act in protein disaggregation reactions, though Ydj1 and Sis1 differ in their modes of Hsp70 cooperation and substrate specificities. This results in functional specialization as seen in prion propagation and the underlying dominant role of Sis1 in targeting Hsp70 for shearing of prion amyloid fibrils.

Total Synthesis and Biological Evaluation of Modified Ilamycin Derivatives.

Ilamycins/rufomycins are marine cycloheptapeptides containing unusual amino acids. Produced by Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. The cyclic peptides target the AAA+ protein ClpC1 that, together with the peptidases ClpP1/ClpP2, forms an essential ATP-driven protease. Derivatives of the ilamycins with a simplified tryptophane unit are synthesized in a straightforward manner. The ilamycin derivative 26 with a cyclic hemiaminal structure is active in the nM-range against several mycobacterial strains and shows no significant cytotoxicity. In contrast, derivative 27, with a glutamic acid at this position, is significantly less active, with MICs in the mid µM-range. Detailed investigations of the mode of action of 26 indicate that 26 deregulates ClpC1 activity and strongly enhances ClpC1-WT ATPase activity. The consequences of 26 on ClpC1 proteolytic activities were substrate-specific, suggesting dual effects of 26 on ClpC1-WT function. The positive effect relates to ClpC1-WT ATPase activation, and the negative to competition with substrates for binding to the ClpC1 NTD.

Antibacterial peptide CyclomarinA creates toxicity by deregulating the Mycobacterium tuberculosis ClpC1-ClpP1P2 protease.

The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1-ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.

Basic mechanism of the autonomous ClpG disaggregase.

Bacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical 70-kDa heat shock protein (Hsp70)-ClpB bichaperone disaggregase, which is most widespread in bacteria. The ClpB disaggregase is a member of the ATPase associated with diverse cellular activities protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, which initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the ATPase associated with diverse cellular activities protein ClpG as an alternative disaggregase. Pseudomonas aeruginosa ClpG functions autonomously and does not cooperate with Hsp70 for substrate binding, enhanced ATPase activity, and disaggregation. With the underlying molecular basis largely unknown, the fundamental differences in ClpG- and ClpB-dependent disaggregation are reflected by the presence of sequence alterations and additional ClpG-specific domains. By analyzing the effects of mutants lacking ClpG-specific domains and harboring mutations in conserved motifs implicated in ATP hydrolysis and substrate threading, we show that the N-terminal, ClpG-specific N1 domain generally mediates protein aggregate binding as the molecular basis of autonomous disaggregation activity. Peptide substrate binding strongly stimulates ClpG ATPase activity by overriding repression by the N-terminal N1 and N2 domains. High ATPase activity requires two functional nucleotide binding domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability.

Publisher Correction: Processive extrusion of polypeptide loops by a Hsp100 disaggregase.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Processive extrusion of polypeptide loops by a Hsp100 disaggregase.

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.

Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor.

AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.

Cellular Functions and Mechanisms of Action of Small Heat Shock Proteins.

Small heat shock proteins (sHsps) constitute a diverse chaperone family that shares the α-crystallin domain, which is flanked by variable, disordered N- and C-terminal extensions. sHsps act as the first line of cellular defense against protein unfolding stress. They form dynamic, large oligomers that represent inactive storage forms. Stress conditions cause a rapid increase in cellular sHsp levels and trigger conformational rearrangements, resulting in exposure of substrate-binding sites and sHsp activation. sHsps bind to early-unfolding intermediates of misfolding proteins in an ATP-independent manner and sequester them in sHsp/substrate complexes. Sequestration protects substrates from further uncontrolled aggregation and facilitates their refolding by ATP-dependent Hsp70-Hsp100 disaggregases. Some sHsps with particularly strong sequestrase activity, such as yeast Hsp42, are critical factors for forming large, microscopically visible deposition sites of misfolded proteins in vivo. These sites are organizing centers for triaging substrates to distinct quality control pathways, preferentially Hsp70-dependent refolding and selective autophagy.

Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase.

Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including Escherichia coli ClpB, form a powerful disaggregation machine that threads aggregated polypeptides through the central pore of tandem adenosine triphosphatase (ATPase) rings. To visualize protein disaggregation, we determined cryo-electron microscopy structures of inactive and substrate-bound ClpB in the presence of adenosine 5'-O-(3-thiotriphosphate), revealing closed AAA+ rings with a pronounced seam. In the substrate-free state, a marked gradient of resolution, likely corresponding to mobility, spans across the AAA+ rings with a dynamic hotspot at the seam. On the seam side, the coiled-coil regulatory domains are locked in a horizontal, inactive orientation. On the opposite side, the regulatory domains are accessible for Hsp70 binding, substrate targeting, and activation. In the presence of the model substrate casein, the polypeptide threads through the entire pore channel and increased nucleotide occupancy correlates with higher ATPase activity. Substrate-induced domain displacements indicate a pathway of regulated substrate transfer from Hsp70 to the ClpB pore, inside which a spiral of loops contacts the substrate. The seam pore loops undergo marked displacements, along with ordering of the regulatory domains. These asymmetric movements suggest a mechanism for ATPase activation and substrate threading during disaggregation.

The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation.

Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension-the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation.

Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae.

Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum-, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum-mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes.

Mechanism of Hsp104/ClpB inhibition by prion curing Guanidinium hydrochloride.

The Saccharomyces cerevisiae AAA+ protein Hsp104 and its Escherichia coli counterpart ClpB cooperate with Hsp70 chaperones to refold aggregated proteins and fragment prion fibrils. Hsp104/ClpB activity is regulated by interaction of the M-domain with the first ATPase domain (AAA-1), controlling ATP turnover and Hsp70 cooperation. Guanidinium hydrochloride (GdnHCl) inhibits Hsp104/ClpB activity, leading to prion curing. We show that GdnHCl binding exerts dual effects on Hsp104/ClpB. First, GdnHCl strengthens M-domain/AAA-1 interaction, stabilizing Hsp104/ClpB in a repressed conformation and abrogating Hsp70 cooperation. Second, GdnHCl inhibits continuous ATP turnover by AAA-1. These findings provide the mechanistic basis for prion curing by GdnHCl.

ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.

The multicomponent type VI secretion system (T6SS) mediates the transport of effector proteins by puncturing target membranes. T6SSs are suggested to form a contractile nanomachine, functioning similar to the cell-puncturing device of tailed bacteriophages. The T6SS members VipA/VipB form tubular complexes and are predicted to function in analogy to viral tail sheath proteins by providing the energy for secretion via contraction. The ATPase ClpV disassembles VipA/VipB tubules in vitro, but the physiological relevance of tubule disintegration remained unclear. Here, we show that VipA/VipB tubules localize near-perpendicular to the inner membrane of Vibrio cholerae cells and exhibit repetitive cycles of elongation, contraction and disassembly. VipA/VipB tubules are decorated by ClpV in vivo and become static in ΔclpV cells, indicating that ClpV is required for tubule removal. VipA/VipB tubules mislocalize in ΔclpV cells and exhibit a reduced frequency of tubule elongation, indicating that ClpV also suppresses the spontaneous formation of contracted, non-productive VipA/VipB tubules. ClpV activity is restricted to the contracted state of VipA/VipB, allowing formation of functional elongated tubules at a T6SS assembly. Targeting of an unrelated ATPase to VipA/VipB is sufficient to replace ClpV function in vivo, suggesting that ClpV activity is autonomously regulated by VipA/VipB conformation.

Hsp70 proteins bind Hsp100 regulatory M domains to activate AAA+ disaggregase at aggregate surfaces.

Bacteria, fungi and plants rescue aggregated proteins using a powerful bichaperone system composed of an Hsp70 chaperone and an Hsp100 AAA+ disaggregase. In Escherichia coli, the Hsp70 chaperone DnaK binds aggregates and targets the disaggregase ClpB to the substrate. ClpB hexamers use ATP to thread substrate polypeptides through the central pore, driving disaggregation. How ClpB finds DnaK and regulates threading remains unclear. To dissect the disaggregation mechanism, we separated these steps using primarily chimeric ClpB-ClpV constructs that directly recognize alternative substrates, thereby obviating DnaK involvement. We show that ClpB has low intrinsic disaggregation activity that is normally repressed by the ClpB middle (M) domain. In the presence of aggregate, DnaK directly binds M-domain motif 2, increasing ClpB ATPase activity to unleash high ClpB threading power. Our results uncover a new function for Hsp70: the coupling of substrate targeting to AAA+ chaperone activation at aggregate surfaces.

Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation.

Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA(+) hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial. Here, we demonstrate that Hsp70 chaperones were essential for species-specific targeting of their Hsp100 partner chaperones ClpB and Hsp104, respectively, to heat-induced protein aggregates in vivo. Hsp70 inactivation in yeast also abrogated Hsp104 targeting to almost all prions tested and reduced fibril mobility, which indicates that fibril fragmentation by Hsp104 requires Hsp70. The Sup35 prion was unique in allowing Hsp70-independent association of Hsp104 via its N-terminal domain, which, however, was nonproductive. Hsp104 overproduction even outcompeted Hsp70 for Sup35 prion binding, which explains why this condition prevented Sup35 fragmentation and caused prion curing. Our findings indicate a conserved mechanism of Hsp70-Hsp100 cooperation at the surface of protein aggregates and prion fibrils.

Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion.

Ring-forming AAA(+) ATPases act in a plethora of cellular processes by remodeling macromolecules. The specificity of individual AAA(+) proteins is achieved by direct or adaptor-mediated association with substrates via distinct recognition domains. We investigated the molecular basis of substrate interaction for Vibrio cholerae ClpV, which disassembles tubular VipA/VipB complexes, an essential step of type VI protein secretion and bacterial virulence. We identified the ClpV recognition site within VipB, showed that productive ClpV-VipB interaction requires the oligomeric state of both proteins, solved the crystal structure of a ClpV N-domain-VipB peptide complex, and verified the interaction surface by mutant analysis. Our results show that the substrate is bound to a hydrophobic groove, which is formed by the addition of a single α-helix to the core N-domain. This helix is absent from homologous N-domains, explaining the unique substrate specificity of ClpV. A limited interaction surface between both proteins accounts for the dramatic increase in binding affinity upon ATP-driven ClpV hexamerization and VipA/VipB tubule assembly by coupling multiple weak interactions. This principle ensures ClpV selectivity toward the VipA/VipB macromolecular complex.

Principles of general and regulatory proteolysis by AAA+ proteases in Escherichia coli.

General and regulated proteolysis in bacteria is crucial for cellular homeostasis and relies on high substrate specificity of the executing AAA+ proteases. Here we summarize the various strategies that tightly control substrate degradation from both sides: the generation of accessible degrons and their specific recognition by AAA+ proteases and cognate adaptor proteins.

Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion.

The recently identified type VI secretion systems (T6SS) have a crucial function in the virulence of various proteobacteria, including the human pathogen Vibrio cholerae. T6SS are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of Hcp and VgrG proteins in cell culture supernatants. Here, we analysed the function of the V. cholerae T6SS member ClpV, a specialized AAA+ protein. ClpV is crucial for a functional T6SS and interacts through its N-terminal domain with the VipA/VipB complex that is composed of two conserved and essential members of T6SS. Transferring ClpV substrate specificity to a distinct AAA+ protein involved in proteolysis caused degradation of VipA but not Hcp or VgrG2, suggesting that VipA rather than Hcp/VgrG2 functions as a primary ClpV substrate. Strikingly, VipA/VipB form tubular, cogwheel-like structures that are converted by a threading activity of ClpV into small complexes. ClpV-mediated remodelling of VipA/VipB tubules represents a crucial step in T6S, illuminating an unexpected role of an ATPase component in protein secretion.

The antibiotic ADEP reprogrammes ClpP, switching it from a regulated to an uncontrolled protease.

A novel class of antibiotic acyldepsipeptides (designated ADEPs) exerts its unique antibacterial activity by targeting the peptidase caseinolytic protease P (ClpP). ClpP forms proteolytic complexes with heat shock proteins (Hsp100) that select and process substrate proteins for ClpP-mediated degradation. Here, we analyse the molecular mechanism of ADEP action and demonstrate that ADEPs abrogate ClpP interaction with cooperating Hsp100 adenosine triphosphatases (ATPases). Consequently, ADEP treated bacteria are affected in ClpP-dependent general and regulatory proteolysis. At the same time, ADEPs also activate ClpP by converting it from a tightly regulated peptidase, which can only degrade short peptides, into a proteolytic machinery that recognizes and degrades unfolded polypeptides. In vivo nascent polypeptide chains represent the putative primary target of ADEP-activated ClpP, providing a rationale for the antibacterial activity of the ADEPs. Thus, ADEPs cause a complete functional reprogramming of the Clp-protease complex.