Processive extrusion of polypeptide loops by a Hsp100 disaggregase

Abstract

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 identified6,7,8 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.

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Fig. 1: ClpB is a processive translocase.
Fig. 2: Optical tweezers with fluorescence reveals ClpB translocation of both loop arms.
Fig. 3: Translocation steps by ClpB.
Fig. 4: Substrate refolding on the ClpB trans-side during translocation.

Data availability

The data that support the findings of this study are available from the corresponding authors on reasonable request.

Code availability

All data were analysed using a custom Python package that is available online and can be downloaded upon request to the corresponding author.

Change history

  • 08 February 2020

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

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Acknowledgements

We thank E. Koers for help with the substrate constructs and M. E. Aubin-Tam, K. Ganzinger, S. Werner and F. Wruck for comments and critical reading of the manuscript. This study was supported by the Netherlands Organization for Scientific Research (NWO) and by grants of the Deutsche Forschungsgemeinschaft (BB617/17-2, MO970/4-2 and MO 970/4-3) to B.B. and A.M., and the AmPro program of the Helmholtz Society to B.B.

Author information

M.J.A., B.B., A.M. and S.J.T. conceived the research. K.B.F. and A.M. purified all chaperone variants and performed the biochemical assays. M.J.A. and S.J.T. designed the single-molecule experiments. M.J.A. and V.S. generated the substrate constructs. M.J.A. carried out the single-molecule experiments, developed the Python software and performed the data analysis. M.J.A. and S.J.T. wrote the manuscript with the input and discussion of all authors.

Correspondence to Sander J. Tans.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Toshio Ando, Eilika Weber-Ban, Gijs Wuite and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Mechanical unfolding of substrates and extended length description.

a, c, e, Force-extension curves showing the characteristic unfolding pattern: MBP (a), the 2MBP (c) and the 4MBP construct (e), with an initial gradual and discrete unfolding of C-terminal α-helices (Extended Data Fig. 8a) followed by a sharp unfolding of the cores. Grey lines show WLC fits to the data. Red indicates pulling and blue indicates relaxing of the protein chain. b, d, f, The corresponding extended length Le of MBP (b), the 2MBP (d) and the 4MBP construct (f). Le reflects the contour length along the polypeptide backbone, but only of the unfolded part of the protein that is compliant (that is, unfolded and at the cis-side of ClpB). Le is determined from the measured force and extension (distance between beads), and using the WLC model of a non-interacting chain. Grey lines, contour length values obtained from the WLC fits. At low forces, the WLC curves of different contour lengths converge, yielding noisy data.

Extended Data Fig. 2 Translocation by ClpB variants.

a, ClpB monomer structure indicating all tested variants. These variants (except K467C) were generated in the constitutively active Y503D background. Variants E279A and E678A are Walker B mutants in the nucleotide-binding domains NBD1 and NBD2, respectively. These mutations abolish ATP hydrolysis at NBD1 or NBD2. Variants Y251A and Y653A are pore-loop mutants in NBD1 and NBD2, respectively. These mutations affect substrate interaction in the ClpB pore at either NBD1 or NBD2. The K476C variant undocks the middle domain (MD), mimicking the effect of Hsp70 (DnaK) activation. MD undocking in the Y503D variant is more pronounced, and therefore activation is more robust. An additional construct (ClpB(ΔN)) lacked the N-terminal domain (NTD), hindering initial substrate binding. Finally, the variant E731C harbours a cysteine at the bottom of NBD2 for fluorophore labelling. b, Fraction of time showing activity (fA) for different mutants (in Y503D background, except K476C and wild type (WT)). c, Average translocation speed for all ClpB variants tested. KJE is the DnaK system (DnaK, DnaJ and GrpE). The median is displayed as a horizontal line within the box, and the mean as a white square. Whiskers indicate the lowest datum still within 1.5 interquartile range (IQR) of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile. Sample sizes: n = 1,139 (Y503D), n = 24 (K476C), n = 7 (wild type) runs. d, Translocation example for ClpB(K476C). Scale bars correspond to 200 aa and 10 s. e, Translocation example for wild-type ClpB with the DnaK system (DnaK, DnaJ and GrpE). Scale bars correspond to 200 aa and 5 s. f, g, Absolute ATPase rate (f) and ATPase substrate-stimulation (g) for the three ClpB variants and different substrate conditions (mean ± s.d.). ATPase activity is higher and more strongly stimulated for Y503D, followed by K476C and wild type. The lower activities observed in the presence of denatured MBP–DM with respect to casein may reflect lower affinity and lower concentrations due to aggregation. The ATPase activity assay was repeated three times for all conditions in f and g, except for K476C, WT + MBP2 and Y503D + casein, for which it was repeated two times.

Extended Data Fig. 3 Translocation runs for different constructs and molecules.

Traces of protein extended length contractions in the presence of ClpB(Y503D) and ATP. a, MBP (Lc = 360 aa). b, 2MBP (Lc = 720 aa). c, 4MBP (Lc = 1440 aa). d, Speed distribution of translocation runs for the three different constructs (number of runs: n = 213 (MBP), n = 287 (2MBP), n = 306(4MBP)). All show a similar range of speeds, as expected, with one main peak (at v ≈ 240 aa s−1) and a second peak or shoulder at twice the magnitude (2v). A slight change in the ratio is observed between the two peak heights, with 2v becoming more pronounced in the longer constructs. This difference could reflect that distances between the initial ClpB binding site and the arresting DNA handles is then larger, and hence double-arm translocation more likely (see also Extended Data Fig. 4). e, Translocation speed distributions from three different substrate molecules (number of runs: n1 = 218 (purple), n2 = 102 (orange) and n3 = 114 (green)), which show no significant variability between individual substrates. f, Translocation speed distributions for three different translocation bursts, which show continuous run–slip–run activity, and are thus surmised to reflect the action of individual ClpB hexamers (number runs: n1 = 25 (purple), n2 = 26 (orange) and n3 = 49 (green)). Distributions are for ClpB(Y503D) and ATP, at approximately 8 pN. The data indicate no significant variability in the translocation activity between ClpB hexamers. The burst duration varied between 5 and 80 s, whereas the time between bursts varied between 5 and 150 s, for the 2MBP construct. g, Example translocation run of MBP showing the definitions of run length and run duration. Run duration is calculated as the time from the start of a run until the next back-slipping event, including the pause after translocation and before the next back-slip. Run length is calculated as the length difference between the start of a run and the next back-slipping event. h, i, Run length (h) and run duration (i) (see g) distribution for constructs of different lengths. Notably, the run duration distributions are similar for the constructs of different length, which suggests that the moment ClpB loses grip on one of the arms and causes the back-slip is determined by events that are intrinsic to the ClpB hexamer, and do not depend on the substrate nor the encounter with blockades (such as the DNA tether). This would make functional sense in the physiological context, as ClpB can then continue to push in an attempt to disrupt aggregated structures. By contrast, the switch between double- and single-arm translocation is directly triggered by such blockades, though without losing grip on either of the two arms.

Extended Data Fig. 4 Speed characterization of translocation runs.

a, Translocated length (Lt) during threading of 2MBP by ClpB(Y503D). Raw data (light grey, 500 Hz) is filtered using a Savitzky–Golay filter (black line). b, Local translocation speed calculated as the time derivative of the translocated length after Savitzky–Golay filtering. Negative slopes below −50 nm s−1 (horizontal line) are considered back-slipping events (orange areas, also in a) and help in determining isolated translocation runs. c, Identification of different speeds within a single translocation run. Linear fits are used to calculate the speed of the run (green and magenta lines), most times revealing two main velocities, one double that of the other. d, Time derivative of the filtered translocated length for a single run, with solid black lines indicating the threshold speeds to distinguish no translocation from single- and double-translocation velocities and green and magenta indicating the fitted velocity values (also shown in c).

Extended Data Fig. 5 Integrated tweezers and fluorescence particle tracking method.

ad, Synchronization of fluorescence and tweezers signals. a, Confocal scanning kymograph of two trapped beads. b, Intensity profile of a scanning line (blue in a), with a Gaussian fit of the edge of the moving bead (bottom) in blue. c, Offset between the fluorescence detection of bead movement as shown in b (blue dots), and high-resolution tweezers signal of trap and bead movement (black line) signals. d, Root mean square deviation (r.m.s.d.) between the signals for different time shifts τ. The minimum is marked with a triangle and represents the best estimation of the offset between the signals. e, f, Force clamp and computation of the two length components. e, Scheme of the lengths involved. DL and DR, distances between beads and ClpB; xL and xR, distances between protein termini and ClpB. Note that these distances are not contour lengths. f, Bead and ClpB position changes for left-arm (left) and right-arm (right) translocation. g, Kymograph underlying data in Fig. 2i. h, Corresponding tracked position of ClpB. Horizontal lines indicate extreme ClpB positions. Top line, ClpB is positioned at the left-hand terminus (see e and f). Bottom line, ClpB at the right-hand terminus; no polypeptide is translocated (the complete polypeptide is thus on the cis side of ClpB). Deviations from the top line consistently occur at back-slip moments detected by the tweezers (j; see the two shorter back-slips), which shows that the left arm (red) back-slips. Some back-slips detected by the tweezers do not show a corresponding ClpB movement, which is expected because right-arm back-slips should not change the ClpB position. i, Corresponding lengths of left arm (red) and right arm (blue) against time, as determined from fluorescence tracking (g, h) and tweezers (j) data. j, Corresponding tweezers data showing the distance between termini (contour length of cis segments). k, Distribution of the different translocation and back-slipping events observed in the fluorescence experiments (number of events n = 127, 5 molecules).

Extended Data Fig. 6 Initial ClpB binding site estimation.

a, Fraction of runs showing double speed when considering all runs (n = 1,704) and first runs only (n = 30). Data are mean ± standard error of a binomial distribution (see Methods). b, Example of first translocation run. ClpB binds at a certain location on the polypeptide, starts translocating both strands yielding the double speed (green) until it encounters the closest terminus, when it switches to single strand translocation (red). At the switch, the length translocated thus equals the distance between the initial binding site and the closest terminus (LB), but times two because ClpB also translocated the other arm. Afterwards, the second terminus is also reached, and translocation stalls and a back-slip occurs, although this is not relevant here. c, Kernel density estimation (KDE) distribution of the inferred binding locations based on first runs, as described in b (n = 30). The distribution is symmetric because N and C termini cannot be distinguished. d, Peptide library data indicating regions of MBP that are bound by ClpB(NTD). e, Spot intensities were quantified using a custom script in Python. For direct comparison with c, the spot intensity distribution was also mirrored.

Extended Data Fig. 7 Single translocation steps by ClpB.

ad, Analysis of step periodicity, related to Fig. 3. a, b, Autocorrelation of the pairwise length distribution for single-speed (a) and double-speed (b) runs from Fig. 3 (black dots). The red line is a fit, yielding period values of 14 and 28 aa, respectively. c, d, Power spectrum analysis of the pairwise length distribution for a (c) and for b (d), showing a peak that fitted to a Gaussian distribution (red) yields 0.071 and 0.037 aa−1, respectively. This translates to 14 and 27 aa steps, in excellent agreement with the values obtained from the autocorrelation. e, The average step size is 14.6 ± 0.9 aa for single-speed translocation and 29 ± 3 aa for double-speed translocation (mean ± s.e.m., ns = 8 and nd = 4, 4 molecules), and statistically different (P = 10−7; two-sided t-test). f, Example run in the presence of ATP–ATPγS mixture (1 mM each). Longer pauses are observed during translocation because ATPγS is hydrolysed much more slowly than ATP, and therefore can result in stalling. The prolonged stalling seen here is in line with a sequential ATP-hydrolysis along ClpB subunits. gi, Notably, in these conditions, step-sizes smaller than 14 aa are now observed. These findings provide further support for the 14-aa steps being produced by the rapid consecutive action of multiple or all 6 ClpB subunits, whose individual 2-aa sub-steps would remain unresolved. After starting, a hydrolysis sequence moving along the ClpB hexamer would then arrest prematurely when encountering a ATPγS-bound subunit, and hence yield a smaller step size.

Extended Data Fig. 8 Trans-refolding does not occur in single MBP and a mutant 2MBP construct.

a, Structure of MBP (PDB ID: 2MV0), showing the C-terminal helices (red; around 90 residues) not required for core folding28 (blue). b, Cartoon representation of the extended MBP chain showing the C terminal domain in red. After translocation arrest at the termini, segments at the N- and C termini (approximately 20 aa each) remain stuck inside the ClpB pore, and are thus not available for folding. Whereas the C-terminal segment (red) is not crucial for core formation, the N-terminal segment (blue) is. Thus, trans-refolding of single-MBP is not expected and indeed not observed. c, Cartoon representation of the extended 2MBP. The second MBP core (blue, 2) can fold in trans, since it now can translocate fully. d, Translocation run-and-slip activity for a tandem repeat of double mutant MBP (2MBP(DM)), which is compromised in refolding. Grey line indicates 720 aa, red line corresponds to 0 aa and the orange line corresponds to 310 aa, the length of one MBP core plus the two approximately 20-aa segments inside the pore. Back-slipping arrests at the orange line, as seen for 2MBP (Fig. 4), are no longer observed. e, Corresponding length distribution. Upon slipping, the released length (Lr) is now typically equal to the previously translocated length (blue data follows red line, n = 203 runs, 6 molecules).

Extended Data Fig. 9 Disruption of folded and aggregated structures by ClpB.

a, Extension length (Le) of the 4MBP construct plotted against time in the presence of ClpB(Y503D) and ATP. b, Cartoons of event sequence suggested in a. (1) One MBP core is unfolded by increasing the force, immediately followed by relaxation to 5 pN to avoid unfolding other MBP cores. Some C-terminal helices also unfolded in this process. (2) After a waiting period, ClpB binds the unfolded part and translocates it completely. (3) ClpB reaches the neighbouring folded MBP domain (and the DNA tether), and hence no longer changes Le. (4) After a short pause, Le increases in a discrete step of 270 aa, indicating the unfolding of one MBP core, which has precisely that length. (5) ClpB(Y503D) translocates briefly immediately afterwards, further indicating the bound ClpB, and (6) back-slipping occurs. Note that the length of the unfolded chain has increased by 270 aa, the length of one MBP core, as expected (star). (7) Translocation continues. c, d, To create a misfolded or aggregated state, the 4MBP construct was unfolded and rapidly relaxed (green trace). This sometimes produced non-native structures characterized by being compact and highly resistant to force (red trace). The tether was then relaxed to low force. e, Subsequent measurement of extension length against time. f, Cartoons of event sequence suggested in e: (1) the length remains unchanged, for example, owing to waiting for ClpB binding. (2) The length increases abruptly by about 600 aa, which is more than one MBP core (270 aa), suggesting that ClpB disrupted a non-native (aggregated) structure that contained more than one MBP repeat. (3) ClpB translocation is observed immediately afterwards. This is consistent with the model, because one-step disruption of structures by ClpB (pushing) action can yield unfolded polypeptide segments directly on the cis side of ClpB that are then available for translocation. Note that polypeptide may also be liberated on the other side of the misfolded structure, which is not immediately available for translocation. Subsequently, further translocation and slipping behaviour is observed. Note that the structure becomes almost fully disrupted, as it nears the maximum length of 1,440 aa.

Extended Data Fig. 10 Loop extrusion as a disaggregation principle.

Insertion and translocation of loops promotes efficient disaggregation, because aggregates may display few accessible polypeptide termini at the surface. Translocation by Hsp100s of polypeptides entangled in aggregates generates pulling forces that promote their dissociation, cooperative disruption of larger structures, and extraction. The ability of Hsp100s to switch between translocation modes is relevant to prevent pore jamming when encountering structures that resist immediate disruption. To dissolve such resistive structures and larger aggregates, many translocation actions are probably required, involving multiple Hsp100 hexamers and other chaperones such as Hsp70, acting at different moments in time and at different locations within the aggregate. The random non-processive action of Hsp70s probably inherently requires multiple Hsp70s working together, in a manner that does not generate large pulling forces, while exploiting rapid binding and unbinding. In contrast, the processive nature of ClpB translocation enables fast, deterministic, and forced dissociation, which further limits re-aggregation and degradation when in combination with rapid refolding.

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Avellaneda, M.J., Franke, K.B., Sunderlikova, V. et al. Processive extrusion of polypeptide loops by a Hsp100 disaggregase. Nature 578, 317–320 (2020). https://doi.org/10.1038/s41586-020-1964-y

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