E. coli ClpB is a Robust and Processive Protein Unfoldase

Reviewer #1 (Public Review):

In this study, the authors used a stopped-flow method to investigate the kinetics of substrate translocation through the channel in hexameric ClpB, an ATP-dependent bacterial protein disaggregase. They engineered a series of polypeptides with the N-terminal RepA ClpB-targeting sequence followed by a variable number of folded titin domains. The authors detected translocation of the substrate polypeptides by observing the enhancement of fluorescence from a probe located at the substrate's C-terminus. The total time of the substrates' translocation correlated with their lengths, which allowed the authors to determine the number of residues translocated by ClpB per unit time.

Strengths:

This study confirms a previously proposed model of processive translocation of polypeptides through the channel in ClpB. The novelty of this work is in the clever design of a series of kinetic experiments with an engineered substrate that includes stably folded domains. This approach produced a quantitative description of the reaction rates and kinetic step sizes. Another valuable aspect is that the method can be used for other translocases from the AAA+ family to characterize their mechanism of substrate processing.

Weaknesses:

The main limitation of the study is in using a single non-physiological substrate of ClpB, which does not replicate the physical properties of the aggregated cellular proteins and includes a non-physiological ClpB-targeting sequence. Another limitation is in the use of ATPgammaS to stimulate the substrate processing. It is not clear how relevant the results are to the ClpB function in living cells with ATP as the source of energy, a multitude of various aggregated substrates without targeting sequences that need ClpB's assistance, and in the presence of the co-chaperones.

The authors do not attempt to correlate the kinetic step sizes detected during substrate translocation and unfolding with the substrate's structure, which should be possible, given how extensively the stability and unfolding of the titin I27 domain were studied before. Also, since the substrate contains up to three I27 domains separated with unstructured linkers, it is not clear why all the translocation steps are assumed to occur with the same rate constant.

Some conclusions presented in the manuscript are speculative:

The notion that the emission from Alexa Fluor 555 is enhanced when ClpB approaches the substrate's C-terminus needs to be supported experimentally. Also, evidence that ATPgammaS without ATP can provide sufficient energy for substrate translocation and unfolding is missing in the paper.

Reviewer #2 (Public Review):

Summary:

The current work by Banwait et al. reports a fluorescence-based single turnover method based on protein-induced fluorescence enhancement (PIFE) to show that ClpB is a processive motor. The paper is a crucial finding as there has been ambiguity on whether ClpB is a processive or non-processive motor. Optical tweezers-based single-molecule studies have shown that ClpB is a processive motor, whereas previous studies from the same group hypothesized it to be a non-processive motor. As co-chaperones are needed for the motor activity of the ClpB, to isolate the activity of ClpB, they have used a 1:1 ratio ATP and ATPgS, where the enzyme is active even in the absence of its co-chaperones, as previously observed. A sequential mixing stop-flow protocol was developed, and the unfolding and translocation of RepA-TitinX, X = 1,2,3 repeats was monitored by measuring the fluorescence intensity with the time of Alexa F555 which was labelled at the C-terminal Cysteine. The observations were a lag time, followed by a gradual increase in fluorescence due to PIFE, and then a decrease in fluorescence plausibly due to the dissociation from the substrate allowing it to refold. The authors observed that the peak time depends on the substrate length, indicating the processive nature of ClpB. In addition, the lag and peak times depend on the pre-incubation time with ATPgS, indicating that the enzyme translocates on the substrates even with just ATPgS without the addition of ATP, which is plausible due to the slow hydrolysis of ATPgS. From the plot of substrate length vs peak time, the authors calculated the rate of unfolding and translocation to be ~0.1 aas-1 in the presence of ~1 mM ATPgS and increases to 1 aas-1 in the presence of 1:1 ATP and ATPgS. The authors have further performed experiments at 3:1 ATP and ATPgS concentrations and observed ~5 times increase in the translocation rates as expected due to faster hydrolysis of ATP by ClpB and reconfirming that processivity is majorly ATP driven. Further, the authors model their results to multiple sequential unfolding steps, determining the rate of unfolding and the number of amino acids unfolded during each step. Overall, the study uses a novel method to reconfirm the processive nature of ClpB.

Strengths:

(1) Previous studies on understanding the processivity of ClpB have primarily focused on unfolded or disordered proteins; this study paves new insights into our understanding of the processing of folded proteins by ClpB. They have cleverly used RepA as a recognition sequence to understand the unfolding of titin-I27 folded domains.

(2) The method developed can be applied to many disaggregating enzymes and has broader significance.

(3) The data from various experiments are consistent with each other, indicating the reproducibility of the data. For example, the rate of translocation in the presence of ATPgS, ~0.1 aas-1 from the single mixing experiment and double mixing experiment are very similar.

(4) The study convincingly shows that ClpB is a processive motor, which has long been debated, describing its activity in the presence of only ATPgS and a mixture of ATP and ATPgS.

(5) The discussion part has been written in a way that describes many previous experiments from various groups supporting the processive nature of the enzyme and supports their current study.

Weaknesses:

(1) The authors model that the enzyme unfolds the protein sequentially around 60 aa each time through multiple steps and translocates rapidly. This contradicts our knowledge of protein unfolding, which is generally cooperative, particularly for titinI27, which is reported to unfold cooperatively or utmost through one intermediate during enzymatic unfolding by ClpX and ClpA.

(2) It is also important to note that the unfolding of titinI27 from the N-terminus (as done in this study) has been reported to be very fast and cannot be the rate-limiting step as reported earlier(Olivares et al, PNAS, 2017). This contradicts the current model where unfolding is the rate-limiting step, and the translocation is assumed to be many orders faster than unfolding.

(3) The model assumes the same time constant for all the unfolding steps irrespective of the secondary structural interactions.

(4) Unlike other single-molecule optical tweezer-based assays, the study cannot distinguish the unfolding and translocation events and assumes that unfolding is the rate-limiting step.

Reviewer #3 (Public Review):

Summary:

The authors have devised an elegant stopped-flow fluorescence approach to probe the mechanism of action of the Hsp100 protein unfoldase ClpB on an unfolded substrate (RepA) coupled to 1-3 repeats of a folded titin domain. They provide useful new insight into the kinetics of ClpB action. The results support their conclusions for the model setup used.

Strengths:

The stopped-flow fluorescence method with a variable delay after mixing the reactants is informative, as is the use of variable numbers of folded domains to probe the unfolding steps.

Weaknesses:

The setup does not reflect the physiological setting for ClpB action. A mixture of ATP and ATPgammaS is used to activate ClpB without the need for its co-chaperones, Hsp70. Hsp40 and an Hsp70 nucleotide exchange factor. This nucleotide strategy was discovered by Doyle et al (2007) but the mechanism of action is not fully understood. Other authors have used different approaches. As mentioned by the authors, Weibezahn et al used a construct coupled to the ClpA protease to demonstrate translocation. Avellaneda et al used a mutant (Y503D) in the coiled-coil regulatory domain to bypass the Hsp70 system. These differences complicate comparisons of rates and step sizes with previous work. It is unclear which results, if any, reflect the in vivo action of ClpB on the disassembly of aggregates.