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Substrate refolding on the ClpB trans-side during translocation a, Translocated polypeptide length (Lt) for 2MBP. Horizontal red and orange lines, Lt = 0 and Lt = 310 aa, respectively (a 270-aa MBP core in trans, and 2 × 20 aa inside the ClpB pore). Stars, back-slip arrested at Lt = 310 aa. Circles, events illustrated in d. b, Red line, translocated length is fully released to the cis side upon back-slip (Lr = Lt). Orange line corresponds to an MBP core refolding on the trans side and then blocked from passing the ClpB pore upon back-slip (Lr = Lt – 310 aa). c, Probability of core folding (Pc). Consistently, Pc is 0 below 310 aa, when the MBP core is not fully translocated (n = 266 runs, 12 molecules, mean ± standard error of binomial distribution, see Methods). d, Cartoons of trans refolding. Blue, core MBP; red, non-core MBP. MBP cores do not refold on the cis side because tweezers constrain the polypeptide chain. In cells, aggregates may constrain substrates from folding. On the trans side, the chain is freed from these constraints and an MBP core can fold when fully translocated (light and dark green dots) and thus cannot pass through the ClpB pore when back-slip occurs (orange dot and line).
Source publication
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 subseq...
Citations
... Consistent with rapid translocation on an unfolded protein, Avellaneda et. al. reported translocation rates for ClpB(Y503D) to be ~240 and 450 aa s -1 on mechanically unfolded substrates (15). However, neither their reported rates of ATP hydrolysis nor any published rates are consistent with this hyper-fast translocation activity (16). ...
... Here we report rates of protein unfolding in the range of 1 -4 amino acids (aa) s -1 and ~60 aa are unfolded during each unfolding event. These rates of protein unfolding are approximately two orders of magnitude slower than the reported translocation rates on unfolded polypeptide chains reported by others (15,18). Thus, we propose that protein unfolding catalyzed by ClpB is rate-limiting and, upon unfolding, translocation on the newly unfolded polypeptides is much faster than unfolding. ...
... Binding Protein (MBP) using optical tweezer measurements (15). In their approach they attached the N-and C-terminus of MBP to polystyrene beads and optically trap each bead. ...
E. coli ClpB, and S. cerevisiae Hsp104, are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. Except for mitochondrial ClpB (Skd3), metazoans lack a ClpB/Hsp104 homologue. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through its axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate an unfolded loop at rates over 250 amino acids (aa) per second. Here we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s ⁻¹ with a step-size of ~60 amino acids. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.
... ClpB plays a pivotal role in protein homeostasis of bacterial cells (reviewed in (25,26)). ClpB works by using ATP hydrolysis power to thread aggregate into a channel made upon oligomerisation of six ClpB copies (27). A common substrate used to study ClpB activity is casein. ...
Since the release of AlphaFold, researchers have actively refined its predictions and attempted to integrate it into existing pipelines for determining protein structures. These efforts have introduced a number of functionalities and results at the latest Critical Assessment of protein Structure Prediction edition (CASP15), resulting in a marked improvement in the prediction of multimeric protein structures. However, AlphaFold’s capability of predicting large protein complexes is still limited and integrating experimental data in the prediction pipeline is not straightforward. In this study, we introduce AF_unmasked to overcome these limitations. Our results demonstrate that AF_unmasked can integrate experimental information to build larger or hard to predict protein assemblies with high confidence. The resulting predictions can help interpret and augment experimental data. This new approach generates near-perfect structures even when little to no evolutionary information is available and imperfect experimental structures are used as a starting point. AF_unmasked fills incomplete structures by a procedure called“structural inpainting”, which may provide insights into protein dynamics. In summary, AF_unmasked provides an easy-to-use method that efficiently integrates experiments to predict large protein complexes more confidently.
Code
github.com/clami66/AF_unmasked
... Some heat-accumulating chaperones, such as the HSP20s, do not use ATP to reduce the aggregation of heat-labile proteins [2,60,74] and to prevent the disruption of hyper-fluidized membranes [20,42]. Other conserved families of heat-induced core-chaperones, such as HSP70s, HSP100s, and HSP60s, function as ATP-fueled polypeptide unfolding enzymes [26,32,47,77] that can convert inactive, potentially toxic protein aggregates into native functional proteins, even under heat-shock temperatures [4,17,23,29,50,73]. ...
Background
Global warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a heat-responsive Arabidopsis thaliana reporter line that allows an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature.
Methods
A transgenic Arabidopsis thaliana reporter line named “Heat-Inducible Bioluminescence And Toxicity” (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and d-amino acid oxidase, whose expression is toxic in the presence of d-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of d-valine and analyzed for survival rate, bioluminescence and HSP gene expression.
Results
Whereas at 22 °C, HIBAT seedlings grew unaffected by d-valine, and all survived iterative heat treatments without d-valine, 98% died following heat treatments on d-valine. The HSP17.3B promoter was highly specific to heat as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. RNAseq analysis of heat-treated HIBAT seedlings showed a strong correlation with expression profiles of two wild type lines, confirming that HIBAT does not significantly differ from its Col-0 parent. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants, apparently defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at non-heat-shock temperatures.
Conclusion
HIBAT is a valuable candidate tool to identify Arabidopsis mutants defective in the response to high temperature stress. It opens new avenues for future research on the regulation of HSP expression and for understanding the mechanisms of plant acquired thermotolerance.
... This action effectively disrupts the interactions between the proteins, allowing them to be refolded. Single-molecule analyses have shown the pulling forces exerted by these chaperone complexes to reach values as high as 20 pN (9) and 50 pN (10). ...
The disassembly of misfolded protein aggregates is a requirement for the proper functioning of cells. It has implications in multiple neuropathologies, such as Alzheimer's and Parkinson's diseases. The active unbundling of fibrillar aggregates has recently been identified as a key, rate-limiting step in the disassembly process. Yet, the nature of the underlying molecular mechanism remains an outstanding question. Here, we develop a coarse-grained computational approach from the atomistic structural information and show that the interactions of molecules tethered to fibrils lead to entropic forces consistent with the unbundling process observed in amyloid α-synuclein disaggregation by Hsp70. We uncover two main types of entropic effects, categorized as intra- and inter-protofilament, which are differentially affected by the system parameters and conditions. Our results show that only highly efficient chaperone systems can overcome the free energy cost of the lateral association between two protofilaments. Through the analysis of cryo-electron tomography and high-speed atomic force microscopy data, we find that co-chaperone networks and ATP hydrolysis are needed to achieve the conditions for highly efficient entropic force generation. We highlight the implications of these results for the design of targeted therapies for the underlying neuropathologies.
... These enzymes can unfold heat-misfolded proteins and convert them back into native functional proteins, even under elevated temperatures that are highly unfavorable for the native state (Goloubinoff et al. 2018;De Los Rios and Goloubinoff 2016;Llamas et al. 2021;Fauvet et al. 2021;Tiwari et al. 2023;Avellaneda et al. 2020). ...
Background: Global warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature, is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a special heat-responsive Arabidopsis thaliana reporter line that allowed an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature.
Methods: A transgenic Arabidopsis thaliana reporter line named "Heat-Inducible Bioluminescence And Toxicity" (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and D-amino acid oxidase, whose expression was found to be toxic only in the presence of D-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of D-valine and analyzed for survival rate, bioluminescence and HSP gene expression.
Results: Whereas at 22C, HIBAT seedlings grew unaffected by D-valine, and all survived following iterative heat treatments without D-valine, 98% died following heat treatments on D-valine. The HSP17.3B promoter was highly specific to heat, as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. Confirming that HIBAT does not significantly differ from its Col-0 parent, RNAseq analysis of heat-treated seedlings showed a strong correlation between the two lines. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at low, non-heat-shock temperatures.
Conclusion: This study adds insights into the molecular mechanisms by which higher plants sense and adapt to rapid elevations of ambient temperatures. HIBAT was valuable tool to identify Arabidopsis mutants defective in the response to high temperature stress. Our findings open new avenues for future research on the regulation of HSP expression and understanding their role in the onset of plant acquired thermotolerance
... ClpB/Hsp101 family members contain two additional domains: the N-terminal domain and the middle domain, which forms a coiled-coil structure that is inserted into the first AAA+ module (Mogk et al., 2015). The threading activity of E. coli ClpB can initiate at the N-or C-termini or at internal sites of substrate proteins in protein aggregates, such that entire peptide loops are translocated through the pore (Avellaneda et al., 2020). Translocation is mediated by mobile loops in the central pore that contact the substrate via conserved aromatic residues (Deville et al., 2017;Rizo et al., 2019). ...
In the cytosol of plant cells, heat-induced protein aggregates are resolved by ClpB/Hsp100 family member HSP101, which is essential for thermotolerance. For chloroplast family member CLPB3 this is less clear with controversial reports on its role in conferring thermotolerance. To shed light onto this issue, we have characterized two clpb3 mutants in Chlamydomonas reinhardtii. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TIG1 and the small heat shock proteins HSP22E/F in vivo and for conferring thermotolerance under heat stress. Although CLPB3 accumulates to similarly high levels as stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the ribosomal subunit PRPL1 and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that reduction in chloroplast protein synthesis capacity and increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely to the thylakoid membrane occupied area. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.
... Biochemical studies on covalently tethered ClpX 15 and HslU 16 found that these AAA+ hexamers remained functional even with multiple inactive subunits, supporting a probabilistic mechanism. In addition, single-molecule optical tweezers assays with ClpXP 17 and ClpB 18 , and high-speed AFM experiments with Abo1 19 yielded results that were more consistent with their probabilistic activities. ...
AAA+ proteins (ATPases associated with various cellular activities) comprise a family of powerful ring-shaped ATP-dependent translocases that carry out numerous vital substrate-remodeling functions. ClpB is a AAA+ protein disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops protruding into its center and binding to substrate-proteins. It remains unknown whether these pore loops contribute only passively to substrate-protein threading or have a more active role. Recently, we have applied single-molecule FRET (smFRET) spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the microsecond timescale that are likely to be mechanistically important for disaggregation. Here, using smFRET, we study the allosteric coupling between the pore loops and the two nucleotide binding domains of ClpB (NBD1-2). By mutating the conserved Walker B motifs within the NBDs to abolish ATP hydrolysis, we demonstrate how the nucleotide state of each NBD tunes pore loop dynamics. This effect is surprisingly long-ranged; in particular, PL2 and PL3 respond differentially to a Walker B mutation in either NBD1 or NBD2, as well as to mutations in both. We characterize the conformational dynamics of pore loops and the allosteric paths connecting NBDs to pore loops by molecular dynamics simulations and find that both principal motions and allosteric paths can be altered by changing the ATPase state of ClpB. Remarkably, PL3, which is highly conserved in AAA+ machines, is found to favor an upward conformation when only NBD1 undergoes ATP hydrolysis, but a downward conformation when NBD2 is active. These results explicitly demonstrate a significant long-range allosteric effect of ATP hydrolysis sites on pore-loop dynamics. Pore loops are therefore established as active participants that undergo ATP-dependent conformational changes to translocate substrate proteins through the central pores of AAA+ machines.
Statement of Significance
Molecular machines function by coupling the energy of ATP hydrolysis to mechanical motion. How this coupling occurs and what timescales are involved remains an open question. In this study, we use a powerful single-molecule FRET technique to measure the real-time dynamics of pore loops, which are essential protein-translocating elements of the ATP-dependent disaggregation machine ClpB. Using a series of mutations of the ATP-hydrolysis motifs of ClpB, we find that, although the motions of these pore loops take place on the microsecond time scale, they are markedly affected by the much slower changes in the nucleotide state of the machine. Generally, this study shows that protein machines, such as ClpB, are wired to harness ATP binding and hydrolysis to allosterically affect distal events, such as the function-related mechanics of pore-loops.
... The research illustrated the vast capabilities of studying specific protein dynamics at the singlemolecule level. To determine the accurate mechanism for the translocation of polypeptide loops in Hsp100 disaggregases, Avellaneda et al. (2020) used optical tweezers with fluorescence [141]. By trapping and manipulating maltose-binding protein (MBP) with optical tweezers (Figure 5b), a force can be applied to prevent spontaneous refolding [141]. ...
... To determine the accurate mechanism for the translocation of polypeptide loops in Hsp100 disaggregases, Avellaneda et al. (2020) used optical tweezers with fluorescence [141]. By trapping and manipulating maltose-binding protein (MBP) with optical tweezers (Figure 5b), a force can be applied to prevent spontaneous refolding [141]. Then, the addition of the disaggregase ClpB and ATP would result in the refolding of MBP in brief bursts of contractions, as shown in Figure 5b [141]. ...
... The research illustrated the vast capabilities of studying specific protein dynamics at the single-molecule level. To determine the accurate mechanism for the translocation of polypeptide loops in Hsp100 disaggregases, used optical tweezers with fluorescence [141]. By trapping and manipulating maltose-binding protein (MBP) with optical tweezers (Figure 5b), a force can be applied to prevent spontaneous refolding [141]. ...
Understanding complex biological events at the molecular level paves the path to determine mechanistic processes across the timescale necessary for breakthrough discoveries. While various conventional biophysical methods provide some information for understanding biological systems, they often lack a complete picture of the molecular-level details of such dynamic processes. Studies at the single-molecule level have emerged to provide crucial missing links to understanding complex and dynamic pathways in biological systems, which are often superseded by bulk biophysical and biochemical studies. Latest developments in techniques combining single-molecule manipulation tools such as optical tweezers and visualization tools such as fluorescence or label-free microscopy have enabled the investigation of complex and dynamic biomolecular interactions at the single-molecule level. In this review, we present recent advances using correlated single-molecule manipulation and visualization-based approaches to obtain a more advanced understanding of the pathways for fundamental biological processes, and how this combination technique is facilitating research in the dynamic single-molecule (DSM), cell biology, and nanomaterials fields.
... 60 Single-molecule assays employed dual-trap optical tweezers in which the maltose binding protein (MBP), attached to the DNA linkers, was bound via digoxygenin−antidigoxygenin interaction on one bead and biotin−neutravidin on the other bead ( Figure 4B). 47 The enzyme ClpB was introduced into the channel and was not directly bound to any bead. The protein was mechanically unfolded, and monitoring the interbead distance revealed the conformational changes in the protein, hinting at the mechanism of action of ClpB. ...
... They translocate the polypeptides an order of magnitude faster than protein unfoldases, with former translocation velocities at ∼500 amino acids/second and the latter just around ∼30 amino acids/second. 47 One of the primary reasons for this increased speed is the larger distance ClpB advances with each step, i.e., ∼30 amino acids, compared to ∼10 amino acids for the protein unfoldases. All these studies were performed on mechanically unfolded maltose binding protein (MBP) domains. ...
... The translocation of ClpB by extrusion of loops suggests that the enzyme could target the internal segments of the aggregate and not just the free ends. 47 In addition, their results infer that the folding of polypeptide loops at the exit channel is similar to cotranslational folding during protein synthesis by ribosomes. Although single-molecule optical tweezers on disaggregases are limited to date, other techniques such as SM-FRET have elucidated the ultrafast dynamics of these enzymes various components, particularly the dynamics of the substrate binding domain, middle domain and the pore loops of ClpB. ...
Mechanoenzymes convert chemical energy from the hydrolysis of nucleotide triphosphates to mechanical energy for carrying out cellular functions ranging from DNA unwinding to protein degradation. Protein-processing mechanoenzymes either remodel the protein structures or translocate them across cellular compartments in an energy-dependent manner. Optical-tweezer-based single-molecule force spectroscopy assays have divulged information on details of chemo-mechanical coupling, directed motion, as well as mechanical forces these enzymes are capable of generating. In this review, we introduce the working principles of optical tweezers as a single-molecule force spectroscopy tool and assays developed to decipher the properties such as unfolding kinetics, translocation velocities, and step sizes by protein remodeling mechanoenzymes. We focus on molecular motors involved in protein degradation and disaggregation, i.e., ClpXP, ClpAP, and ClpB, and insights provided by single-molecule assays on kinetics and stepping dynamics during protein unfolding and translocation. Cellular activities such as protein synthesis, folding, and translocation across membranes are also energy dependent, and the recent single-molecule studies decoding the role of mechanical forces on these processes have been discussed.
... The latter two explanations both deserve consideration. Our study did not cover the other E. coli chaperone systems such as TF (3,83), small heat shock proteins (70,84), the ClpB disaggregase (85)(86)(87), or HtpG-a foldase that operates with the DnaK system (88, 89)-all of which might play important roles in refolding certain clients. Nevertheless, several additional lines of evidence support the view that the 105 chaperone-nonrefolders fold cotranslationally. ...
The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain’s intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients. Here, we develop a limited proteolysis–mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding Escherichia coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.
