Escherichia coli DnaK Allosterically Modulates ClpB between High

May 29, 2018 - Clarissa L. Durie , Elizabeth C. Duran , and Aaron L. Lucius*. Department of Chemistry, University of Alabama at Birmingham, Birmingham...
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Article Cite This: Biochemistry 2018, 57, 3665−3675

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Escherichia coli DnaK Allosterically Modulates ClpB between Highand Low-Peptide Affinity States Clarissa L. Durie, Elizabeth C. Duran, and Aaron L. Lucius* Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294-1240, United States

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S Supporting Information *

ABSTRACT: ClpB and DnaKJE provide protection to Escherichia coli cells during extreme environmental stress. Together, this co-chaperone system can resolve protein aggregates, restoring misfolded proteins to their native form and function in solubilizing damaged proteins for removal by the cell’s proteolytic systems. DnaK is the component of the KJE system that directly interacts with ClpB. There are many hypotheses for how DnaK affects ClpB-catalyzed disaggregation, each with some experimental support. Here, we build on our recent work characterizing the molecular mechanism of ClpB-catalyzed polypeptide translocation by developing a stopped-flow FRET assay that allows us to detect ClpB’s movement on model polypeptide substrates in the absence or presence of DnaK. We find that DnaK induces ClpB to dissociate from the polypeptide substrate. We propose that DnaK acts as a peptide release factor, binding ClpB and causing the ClpB conformation to change to a low-peptide affinity state. Such a role for DnaK would allow ClpB to rebind to another portion of an aggregate and continue nonprocessive translocation to disrupt the aggregate.

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he ClpB−DnaKJE co-chaperone system in Escherichia coli protects the cell from toxic aggregates caused by environmental stressors.1 DnaK cannot dissolve aggregates without ClpB.1 ClpB is also unable to resolve aggregates in vivo in the absence of DnaK, although conditions under which ClpB alone can resolve aggregates in vitro have been determined.2−6 The ClpB−DnaKJE co-chaperone system can resolve aggregates that are intractable with either component alone.7 In fact, ClpB and DnaK together can solubilize at least 75% of thermally aggregated E. coli proteins in extracts, demonstrating their breadth of function and importance to the survival of cells.1 Three hypotheses regarding the partnership of DnaK and ClpB have been explored. These hypotheses are not mutually exclusive. One possibility is that DnaK first binds the protein substrate, modifies the substrate making it more tractable to ClpB, and then transfers the protein substrate to ClpB for disaggregation.7,8 A second possibility is that DnaK accepts the protein substrate after some remodeling, or possibly complete translocation and unfolding by ClpB.9,10 A third possibility is that DnaK and ClpB form a complex that has greatly amplified disaggregation activity compared to that of either chaperone alone. The species specificity of the co-chaperone interaction suggests the importance of a ClpB−DnaK complex.11−13 ClpB−DnaK complexes have been observed by co-elution and nuclear magnetic resonance studies.12,14,15 For this complex to have an enhanced activity with respect to the protein substrate, one would predict the presence of a ternary complex consisting of ClpB, DnaK, and the peptide substrate. Our objective in this study is to test whether a ClpB−DnaK complex has an altered translocation mechanism compared to that of ClpB in the absence of DnaK. © 2018 American Chemical Society

Researchers have identified DnaK as the component of the KJE system that directly interacts with ClpB, with DnaK binding the M domain of ClpB. Notably, the M domain is a unique feature of the disaggregases (ClpB and Hsp104)16,17 in the AAA+ motor protein superfamily and is thought to serve a regulatory role in the coordination of nucleotide hydrolysis within and between the subunits of the hexameric ring.18,19 The affinity of Thermus thermophilus DnaK for ClpB (Kd) has been reported to be 17 μM14 and 25 μM.12 For E. coli ClpB, the Kd has been estimated to be 9−30 μM.20 Interestingly, the in vivo DnaK concentration has been reported to be 27 μM,1 which is strikingly similar to the Kd of 25 μM for binding of DnaK to ClpB.12 Despite the knowledge of the strength of this interaction constant and the cellular concentration of DnaK,1 many experiments designed to test the effect of DnaK (or the DnaKJE system) on ClpB are performed using concentrations well below these dissociation constants, most in the submicromolar concentration range.6,8,10−14,20−25 In fact, the highest concentration of DnaK used in reported experiments is ∼2 μM, which remains an order of magnitude below the reported Kd12 and cellular concentration.1 Thus, conclusions about the synergy or collaboration of ClpB and DnaK are being drawn from experiments in which the reported Kd values do not predict a significant population of the ClpB−DnaK complex. Having recently published a rigorous study of ClpB-catalyzed polypeptide translocation, we are now poised to probe how DnaK affects that mechanism.26 Our recent work has shown that ClpB is a nonprocessive polypeptide translocase, taking Received: January 12, 2018 Revised: April 26, 2018 Published: May 29, 2018 3665

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Biochemistry

Steady State Fluorescence Measurements. Steady state fluorescence measurements were taken using a Fluorolog-3 spectrophotometer (HORIBA Jobin Yovin, Edison, NJ). All experiments were performed in H200 at 25 °C. FRET Pair Emission and Excitation Spectra. The emission spectrum of RepA1−50Flu was collected with excitation at 494 nm. The excitation spectrum of Cy5ClpB was collected by monitoring emission at 670 nm. Sensitized Emission. Emission spectra were collected for Cy5ClpB with excitation at 494 nm, the maximum excitation wavelength for fluorescein. Next, ATPγS was added, and emission spectra were collected. Finally, a fluorescein-labeled peptide, either RepA1−50Flu or RepAFlu1−50, was added, and emission spectra were collected. The final concentrations were 2 μM Cy5ClpB, 300 μM ATPγS, and 20 nM fluorescein-labeled peptide. These are the concentrations used to assemble the ClpB−peptide complex in previous studies of the ClpB mechanism of polypeptide translocation using a stopped-flow assay with a single fluorophore.26 All reagents were equilibrated to 25 °C for approximately 1 h prior to addition to the fluorescence cuvette. Fluorescence Anisotropy. Sequential Additions of Different Components. Steady state fluorescence anisotropy was monitored by exciting fluorescein at 494 nm and observing emission at 515 nm as previously described.28,31 Measurements were taken with RepAFlu1−50 in the presence or absence of ATPγS. Additional measurements were taken after the subsequent addition of ClpB, and finally DnaK. Final concentrations were 20 nM RepAFlu1−50, 2 mM ATPγS (or no ATPγS), 2 μM ClpB, and 25 μM DnaK. Note that anisotropy is not concentration-dependent; therefore, the minor changes in concentration due to sequential additions of components do not contribute to the anisotropy signal, and no correction for dilution is needed. All reagents were equilibrated to 25 °C for approximately 1 h prior to addition to the fluorescence cuvette. Titration of the Flu−Peptide with Proteins. Steady state fluorescence anisotropy was monitored by exciting fluorescein at 494 nm and observing emission at 515 nm as previously described.28,31 Measurements were taken with RepA1−30Flu or caseinFlu102 in the presence of 1 mM ATPγS. Titrant solutions of ClpB with 1 mM ATPγS and DnaK with 1 mM ATPγS were prepared. The fluorescently labeled peptides were then titrated with either protein solution. All samples were incubated at 25 °C for at least 45 min prior to addition to the binding sample. Upon each addition, the cuvettes were gently inverted to mix the contents and then incubated for 5 min prior to collecting data. For each titration, three single-point anisotropy measurements at 515 nm and one fluorescence emission spectrum from 500 to 600 nm were collected. The three anisotropy measurements are reported in Figure 5 as means ± the standard deviation (SD), where the standard deviations are measurement errors. Rapid Mixing Assays. All rapid mixing experiments were performed using an Applied Photophysics (Leatherhead, U.K.) SX20 stopped-flow fluorimeter. All solutions were incubated for 1 h at 25 °C prior to being mixed. The syringes of the stoppedflow fluorimeter and the reaction cell were also maintained at 25 °C. The fluorescence of fluorescein-modified peptides was excited at 494 nm. In the FRET assay, the fluorescence emission of fluorescein was monitored using a PMT R6095 instrument with a 520 ± 5 nm filter and the fluorescence emission of Cy5 was monitored using a PMT 1104 instrument

one or two kinetic steps along a polypeptide substrate before dissociating.26 This finding is in contrast to the prevailing model in the field that states that ClpB processively translocates a protein substrate, completely threading the protein substrate through the axial channel of ClpB, releasing an unfolded substrate.16,27 Here, we report the development of a singleturnover FRET method to test the translocation function of the ClpB−DnaK complex. Using DnaK concentrations that would saturate the binding to ClpB, we sought to determine if the ClpB−DnaK complex operates differently in the translocation of the polypeptide substrate compared to ClpB alone. Surprisingly, we determine that DnaK causes ClpB to release the polypeptide substrate.

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MATERIALS AND METHODS Buffers and Reagents. All buffers were prepared with distilled, deionized water from a Purelab Ultra Genetic system (Evoqua, Warrendale, PA). All chemicals were reagent grade. ATPγS was purchased from CalBiochem (La Jolla, Ca). All experiments were performed in buffer H200 at 25 °C unless otherwise noted. Buffer H200 contains 25 mM HEPES, 200 mM NaCl, 10 mM MgCl2, 2 mM 2ME, and 10% (v/v) glycerol (pH 7.5) at 25 °C. Protein and Peptides. ClpB was expressed and purified as previously reported.28 Note that this preparation of ClpB maintains a glycine at the N-terminus, which is not a part of the native sequence but remains after removal of a histidine tag, added for purification purposes. The concentration of ClpB was determined at 280 nm in H200 using an extinction coefficient ε of 35562 M−1 cm−1, determined following the method of Edelhoch modified by Pace et al.29,30 All protein concentrations are reported in monomer units. ClpB was covalently modified with Cy5-maleimide. The labeling reaction was performed in a reduced salt (100 mM NaCl) buffer to promote stable ClpB hexamers, and a reduced oxygen environment to prevent oxidation of the maleimide group. See the Supporting Information for additional information. The Cy5ClpB had a labeling efficiency of 27%. DnaK was expressed with an N-terminal His6 tag, followed by a TEV cleavage site for purification purposes. Protein concentrations are reported in monomer units using the extinction coefficient at 280 nm (ε) of 16269 M−1 cm−1 determined in our lab by the method proposed by Edolhoch and later refined by Pace et al.29,30 A GE Healthcare HiTrap Q FF column was used to concentrate DnaK for reaction conditions above the reported Kd for binding ClpB. DnaK was labeled with the fluorescent dye fluorescein 5-maleimide. Detailed protocols for purifying, concentrating, and labeling DnaK can be found in the Supporting Information. RepA peptides were prepared by CPC Scientific (Sunnyvale, CA). The peptides are made of the first 30, 40, or 50 amino acids of the N-terminal sequence of RepA, with a cysteine residue added to either the N- or C-terminus for labeling. Labeling with fluorescein 5-maleimide or with Cy3-maleimide was performed as previously described, forming a covalent bond between the peptide and the fluorophore.28 α-Casein substrates were prepared as previously described.31 Peptides are mentioned by the name of the full length protein, followed by the length of the peptide in subscript, with the fluorophore noted prior to the length to indicate N-terminal labeling and after the length to indicate C-terminal labeling. For example, a 50-amino acid peptide based on the sequence of RepA, labeled with fluorescein at the N-terminus, is called RepAFlu1−50. 3666

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with a 665 nm cutoff long pass filter. In the standard assay, using unmodified ClpB and fluorescein on the peptide as previously reported by Li et al., the emission of fluorescein was monitored using a PMT R6095 instrument and a 515 nm cutoff long pass filter (the second PMT was not used).26 All time courses displayed and fit were the average of at least six shots (or pushes, or mixing events). ATP Concentration Dependence. A solution of 2 μM ClpB, 300 μM ATPγS, and 20 nM RepA1−50Flu was incubated in one syringe of the stopped-flow fluorimeter. A solution of ATP (various concentrations) and 20 μM α-casein was incubated in the other syringe. The solutions were rapidly mixed. For each concentration of ATP, a minimum of four shots or pushes were averaged. DnaK Concentration Dependence. A solution of 2 μM ClpB, 300 μM ATPγS, and 20 nM RepA1−50Flu was incubated in one syringe of the stopped-flow fluorimeter. A solution of DnaK (various concentrations) and 300 μM ATPγS was incubated in the other syringe. The solutions were rapidly mixed. For each concentration of ATP, a minimum of four shots or pushes were averaged. NLLS Analysis of Transient State Kinetics. Averaged time courses from the 665 nm+ (sensitized emission) channel were normalized to begin at a fluorescence intensity of 1 and end at a fluorescence intensity of 0. This operation did not change the direction or shape of the time courses. For analysis of the ATP concentration dependence and DnaK concentration dependence, each averaged, normalized time course was individually fit to a sum of two exponentials (eq 1) using the nonlinear least-squares (NLLS) fitting routine, Conlin, kindly provided by J. Williams,32 where f is the fluorescence signal, t is the time, A1 and A2 are amplitude terms, B is an intercept, and kobs,1 and kobs,2 are the rate constants for each exponential phase. Sedimentation Velocity. Sedimentation velocity experiments were performed in a Beckman ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter, Brea, CA). Samples were subjected to an angular velocity of 40000 rpm, and scans were collected every 30 s at 25 °C. Samples labeled with fluorescent dyes were observed simultaneously using the absorbance and interference optical systems (Figure 6B,C). Fluorescein was monitored at 494 nm, and Cy3 was monitored at 548 nm. Samples with no fluorescent dye were monitored using the interference optical system. Experiments were performed by loading 400 μL of sample and reference solutions into each corresponding sector of Beckman 12 mm double-sector Epon charcoal-filled nonmeniscus matching centerpieces. Protein and reference solutions were prepared in H200 and incubated for 2 h at 25 °C before the first sedimentation scan was performed. In samples containing DnaK, the DnaK was added after incubation of the other components for 15 min. Analysis of Sedimentation Velocity Experiments. Interference boundaries acquired from sedimentation velocity experiments were first corrected for time stamp errors33,34 using REDATE version 0.1.7 (C. Brautigam, University of Texas Southwestern Medical Center). The corrected boundaries were then analyzed using SEDFIT version 14.4f (P. Schuck, National Institutes of Health) as previously described.35,36 The data were analyzed between the meniscus plus 0.01 and 6.7 cm to minimize the contribution from gradients of glycerol and nucleotide to the sedimentation boundaries.36

Article

RESULTS

Development of the Single-Turnover FRET Strategy for Observing ClpB-Catalyzed Polypeptide Translocation in the Absence or Presence of DnaK. Using singleturnover stopped-flow experiments, we previously showed that ClpB is a nonprocessive translocase.26 We proposed a model in which ClpB takes one or two steps before rapid dissociation from the polypeptide chain. We have described this as a “pull and release” mechanism. To study the effect of DnaK on this mechanism, we were unable to simply add DnaK to our experimental setup because DnaK is also capable of binding the peptide on its own.1 Thus, including DnaK in the experimental design used previously resulted in a signal not only from the ClpB−peptide complex but also from the DnaK−peptide complex (data not shown). To remove any contributing signal from DnaK−peptide complexes, here we develop a FRET assay in which we could monitor the translocation of the polypeptide by ClpB in the absence or presence of DnaK. ClpB contains three cysteine residues. Structural data suggest that only one cysteine would be available for labeling in the hexameric ring of ClpB and this would be in the N domain. This is ideal because the peptide is predicted to enter the axial channel from the N domain and proceed from nucleotide binding domain 1 to nucleotide binding domain 2. On the basis of our previous work on ClpB assembly,35,36 we labeled ClpB with a Cy5-maleimide dye under conditions promoting hexamer formation, thereby allowing access only to the cysteine residue in the N domain of ClpB (see Materials and Methods). Here we will use Cy5 as a FRET acceptor and pair it with fluorescein as a FRET donor. Peptide RepAFlu1−50 has been used in a previous study of ClpB binding avidity for peptide in the presence of various nucleotides.28 Figure 1A shows the overlap in the Flu-peptide emission spectrum (green solid line) with the Cy5ClpB excitation spectrum (blue dashed line). In the FRET design, we will excite fluorescein at 494 nm and observe emission from fluorescein at 515 nm and sensitized emission from Cy5 at 670 nm. We predict that when the peptide is bound to ClpB sensitized emission from Cy5 would be detected if the fluorescein on the peptide were spatially close to the Cy5 in the N domain. Even though the excitation spectrum for Cy5 (see Figure 1A) indicates very little absorbance by Cy5 at 494 nm (excitation wavelength for fluorescein), some direct excitation of Cy5 may occur. To test this, we measured an emission spectrum for Cy5ClpB by exciting at 494 nm, i.e., the excitation wavelength for fluorescein and the wavelength used in FRET experiments. As seen in panels B and C of Figure 1 (blue dashed lines), some emission is detected upon off resonance excitation of Cy5 at 494 nm. The population of hexameric ClpB will increase upon addition of ATPγS. Therefore, we next added 300 μM ATPγS to determine if the Cy5 is sensitive to this change in the assembly state. Panels B and C of Figure 1 show that the Cy5 emission does not change upon addition of ATPγS. Thus, we conclude that emission due to direct excitation will remain constant in our experimental design. To test for sensitized emission, we then added either RepAFlu1−50 or RepA1−50Flu, each of which is a 50-amino acid peptide containing fluorescein at the N- or C-terminus, respectively. Strikingly, the fluorescence emission of Cy5ClpB at 670 nm increases by 15% in the presence of RepAFlu1−50 and by 40% in the presence of RepA1−50Flu. Although it is tempting to interpret these percentages as being indicative of the 3667

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Figure 1. Development of a FRET assay for monitoring ClpBcatalyzed polypeptide translocation. (A) Emission spectrum of RepA1−50Flu (green) and excitation spectrum of Cy5ClpB (blue). (B and C) With excitation at 494 nm, emission spectra were collected for Cy5ClpB alone (blue, dashed lines) and upon addition of ATPγS (gray, dotted lines) and RepAFlu1−50 (B) or RepA1−50Flu (C) (green, solid lines).

proximity of ClpB to the N- or C-terminus, these differences are likely the consequence of the labeling efficiency differences in the peptide. Having established that sensitized emission was observed in the bound Cy5ClpB−RepAFlu1−50 complex, we next tested whether this experimental design could report on ATPdriven translocation catalyzed by ClpB.26 If ClpB translocates the polypeptide chain through its axial channel, then we would predict that the sensitized emission would increase as the peptide was pulled into the axial channel and subsequently decrease as the fluorescein moved away from the Cy5 on ClpB. On the other hand, if ClpB pulls and releases the peptide, as we have previously reported,26 we would anticipate an immediate loss of sensitized emission upon rapid mixing with ATP or a slight increase followed by a loss of signal. To test these possibilities, the assembled complex (Cy5ClpB−RepAFlu1−50) was rapidly mixed with ATP and a protein trap (α-casein), where the protein trap is included to maintain single-turnover conditions with respect to the polypeptide substrate being translocated. Fluorescein was directly excited at 494 nm. Emission from the fluorescein was monitored using an interference filter at 520 ± 5 nm, and simultaneously, sensitized emission from Cy5ClpB was monitored using a long pass filter resulting in the collection of fluorescence at ≥665 nm (see Materials and Methods). This experimental design is schematized in Figure 2A. Panels B and C of Figure 2 show the time courses for fluorescein emission and Cy5-sensitized emission, respectively, from experiments performed across a range of ATP concentrations from 30 μM ATP (red solid trace) to 5 mM ATP (blue solid trace). Consistent with the previous report using only fluorescein,26 the time courses are faster at high ATP concentrations and slower at low ATP concentrations, indicating that both signals are sensitive to ATP-driven steps

Figure 2. ATP-dependent time courses. (A) Schematic of [ATP]dependent FRET experiment. (B) Time courses collected at 520 nm (fluorescein λem,max). Data shown as solid, colored traces. Dotted black traces represent the best fit lines of each time course, individually fit, to eq 1 using NLLS analysis. (C) Time courses collected from the 665 nm + (Cy5 λem,max) simultaneously with those in panel B. Fitting is the same as for panel B. (D and E) Secondary plots for the parameters from individual fits of time courses to eq 1 as a function of [ATP]. Parameters are (D) kobs1 and (E) kobs,2. Parameters from the Cy5sensitized emission channel (C) are shown as blue squares (mean ± SD; n = 3), and the blue curve is a fit of those data to eq 1. Black circles are data from Li et al. (mean ± SD; n = 3), and the black curve is a fit of those data to eq 2.

before dissociation from the polypeptide lattice. Each curve is described well by a sum of two exponentials (eq 1) f = A1(1 − e−kobs,1t ) + A 2 (1 − e−kobs,2t ) + B

(1)

where f is the fluorescence signal, t is the time, A1 and A2 are amplitude terms, B is an intercept, and kobs,1 and kobs,2 are the rate constants for each exponential phase. The fits are shown as dotted black lines. Because our data in panels B and C of Figure 2 were described well by eq 1, we can compare the parameters directly with those published by Li et al. where only fluorescence from fluorescein was used and the same fitting strategy as applied here was invoked.26 Panels D and E of Figure 2 show the secondary plots of kobs,1 and kobs,2, respectively, as a function of [ATP]. As we have previously shown, there is a large signal 3668

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Biochemistry change from the fluorescein on the polypeptide substrate.26 The signal is quenched when ClpB is bound and recovers when ClpB dissociates. This happens regardless of the presence of Cy5. Thus, what component of the fluorescein signal is the signal normally detected in the absence of Cy5 as opposed to being due to resonance energy transfer remains unclear. On the other hand, the sensitized emission time courses are sensitive only to spatial separation of Cy5 from fluorescein. Thus, we proceed with analysis and reporting of sensitized emission from Cy5. In panels D and E, the data reported by Li et al. are shown as black circles (mean ± SD; n = 3). Rate constant kobs,1 (mean ± SD; n = 3) from the fit of the data in Figure 2C, the sensitized emission channel, to eq 1 is overlaid with the data of Li et al. in Figure 2D as blue squares. All of the data are described well by a rectangular hyperbola (eq 2). kobs, x =

kobs, x ,max[ATP] K1/2 + [ATP]

(2)

Li et al. reported a kobs1,max of 0.190 ± 0.004 s−1 and a K1/2 of 192 ± 18 μM ATP.26 From the sensitized emission of Cy5, we observe remarkably similar parameters: kobs1,max = 0.22 ± 0.01 s−1, and K1/2 = 290 ± 80 μM ATP. The corresponding comparison is also performed in Figure 2E for rate constant kobs,2 as a function of [ATP] (mean ± SD; n = 3). Li et al. reported a kobs2,max of 0.0516 ± 0.006 s−1 and a K1/2 of 226 ± 16 μM ATP.26 Parameters from fits of the data in Figure 2C (sensitized emission) are shown as blue squares with the fit line shown as a blue curve. Here, we observe a kobs2,max of 0.022 ± 0.001 s−1 and a K1/2 of 260 ± 70 μM ATP. Given the similarity in the kinetic parameters, we conclude that the FRET assay reports on the same ATP-dependent translocation and dissociation of peptide from ClpB. It is important to note that the kobs,1 and kobs,2 from sensitized emission of Cy5 are systematically lower than the corresponding rate constants we previously reported from observing only fluorescein emission. Nevertheless, with the FRET method in hand, we are now poised to introduce DnaK and observe whether or how the presence of DnaK affects the ClpB molecular mechanism. DnaK Concentration Dependence of ClpB−Peptide Dissociation in Single-Turnover FRET Experiments. It has been reported that DnaK binds to ClpB with an affinity constant of ∼25 μM.12 Thus, to test if the ClpB−DnaK complex exhibits translocation activity different from that of ClpB alone, we performed experiments as schematized in Figure 2A by adding DnaK to the syringe containing the Cy5ClpB−Flu-peptide complex. To do this, we included DnaK at a number of different DnaK concentrations both above and below the Kd of 25 μM. Surprisingly, when we included DnaK in the preincubation syringe with the Cy5ClpB−Flu-peptide complex and rapidly mixed with ATP and trap, the resulting time courses were flat (data not shown), suggesting the FRET complex was already dissociated before rapid mixing with ATP. To test if the complex is dissociated in the preincubation syringe, we assembled the Cy5ClpB−RepAFlu1−50 complex from which we observed sensitized emission and rapidly mixed that solution with varying concentrations of DnaK as schematized in Figure 3A. If the presence of DnaK causes dissociation of the Cy5ClpB−RepAFlu1−50 complex, then we should observe the loss of sensitized emission over time upon rapid mixing. Furthermore, if this signal change is due to the presence of DnaK interacting with the ClpB−peptide complex, then we

Figure 3. Application of the FRET assay to observe [DnaK]dependent complex dissociation. (A) Schematic of the FRET experiment. Cy5ClpB (2 μM), ATPγS (300 μM), and RepA1−50Flu (20 nM) were incubated in one syringe of the stopped-flow fluorimeter. DnaK (varying concentrations) with ATPγS (300 μM) was incubated in the other syringe. Upon rapid mixing, the fluorescence was excited at 494 nm (the maximum excitation wavelength of the FRET donor, fluorescein). The time course was monitored at ≥665 nm. (B) The resulting time courses show a fast increase followed by a slow decrease in sensitized emission over time. The decreasing phase is faster with higher concentrations of DnaK. Concentrations of DnaK are represented in different colored solid traces with red representing 107 nM DnaK, the intermediate concentrations colored orange, green, and blue, and the highest concentration, 104.7 μM DnaK, colored purple. The gray trace is the time course resulting from mixing the complex with ATPγS (300 μM) in the absence of DnaK. The dotted black traces are fit lines to a sum of two exponentials. (C and D) Rate constants from the fits shown in panel B as a function of [DnaK] (mean ± SD; n = 3). For the experiment without DnaK, kobs,1 = 0.00108 ± 0.0001 s−1 and kobs,2 = 0.039 ± 0.009 s−1, where errors are simulated by Monte Carlo analysis. The black curve in panel D is the fit of those data to eq 2.

would predict a DnaK concentration dependence of the time courses. Figure 3B displays the time courses collected when the Cy5ClpB−RepAFlu1−50 complex was rapidly mixed with varying concentrations of DnaK. The time courses displayed are from the channel that reports on the sensitized emission of Cy5ClpB. Each time course displays a fast increase in sensitized emission (see the inset in Figure 3B), followed by a slower decrease in sensitized emission with time. Each of these time courses was described well by a sum of two exponentials as defined in eq 1. The faster, increasing phase is represented by kobs,1 and is independent of DnaK concentration (Figure 3C). An increase in sensitized emission could be due to the fluorescein on the peptide and Cy5 on the N domain of ClpB coming closer together. Perhaps binding of DnaK to the ClpB hexamer causes such a change. The possibility that the increase in Cy5 fluorescence is due to a change in the environment of Cy5 on ClpB also cannot be ruled out. For example, the binding of DnaK could cause a conformational change that causes the dye to be more solvent-exposed. The decreasing phase is represented by kobs,2 and is noted to be faster with higher concentrations of DnaK (Figure 3D). This 3669

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Figure 4B shows the steady state time courses collected. Initially, each cuvette contained only RepA1−30Flu (final concentration of 20 nM). The anisotropy values of 0.039 ± 0.002 (black circles, 0−57 min) in the absence of nucleotide and 0.042 ± 0.004 (red squares, 0−57 min) in the presence of nucleotide are consistent with a fluorescently labeled peptide of this size tumbling freely in solution. ClpB was then added to both cuvettes (final concentration of 2 μM). Upon addition of ClpB, the anisotropy of RepA1−30Flu in the presence of ATPγS increased significantly to r = 0.80 ± 0.004 (red squares, 58−180 min), indicating slower tumbling of RepA1−30Flu, consistent with it being bound by the large ClpB hexamer. Upon addition of ClpB in the absence of ATPγS, the anisotropy of the peptide did not change and was observed to be 0.041 ± 0.003 (black circles, 58−180 min). These observations are consistent with recently published observations.28 Upon addition of DnaK to the cuvette with the ClpB− RepA1−30Flu complex, there are at least four possibilities. First, DnaK could have no effect on the complex, resulting in no change in the observed anisotropy. Second, DnaK could bind ClpB, triggering release of the peptide. This possibility is consistent with our observations reported in Figure 3. Release of the peptide could result in a free peptide and a low anisotropy value. DnaK could also bind the peptide after its release from ClpB, likely resulting in a different, intermediate anisotropy value due to the size and shape differences between the ClpB hexamer and DnaK. Third, DnaK could bind the peptide and extract it from the complex, effectively outcompeting ClpB for peptide binding. This explanation would also be consistent with our observations shown in Figure 3. Fourth, DnaK could form a stable ternary complex with ClpB and the peptide. Because of the size of the ClpB hexamer, the additional 12% increase in molecular weight may not make a detectable difference in how slowly the ternary complex tumbles or the measured anisotropy value. This result may be indistinguishable from the case in which DnaK has no effect (first proposed possibility). In Figure 4B, when DnaK is added to the sample that includes the complex assembled in the presence of ATPγS (red squares, 180−360 min), the anisotropy value drops to 0.061 ± 0.003. This measurement is outside of the error for the anisotropy value reported for the free peptide as well as for the ClpB-bound peptide. Furthermore, in the absence of a nucleotide, the anisotropy of the peptide increases to 0.070 ± 0.003 upon addition of DnaK. DnaK was observed to bind this peptide in the absence of a nucleotide (Supporting Figure 1), suggesting this intermediate anisotropy value may represent RepA1−30Flu bound by DnaK and not by ClpB. This observation is consistent with the second and third possible ClpB−DnaK interactions proposed above, that DnaK causes ClpB and RepA1−30Flu to dissociate either by binding ClpB or by binding the peptide. ClpB Binds More Tightly Than DnaK to Model Peptides. To determine if the observations indicated in Figure 4 are the result of DnaK outcompeting ClpB for peptide binding, we next sought to assess the relative peptide binding affinities of ClpB and DnaK for our model peptide. To do this, we titrated RepA1−30Flu with either DnaK or ClpB. Figure 5 shows the anisotropy as a function of increasing concentrations of DnaK (panel A) and ClpB (panel B) from the titration of RepA1−30Flu in the presence of 1 mM ATPγS. We chose to use 1 mM ATPγS because our previous quantitative study of ClpB polypeptide binding, which included incorporation of the

decrease in sensitized emission is consistent with the release of the peptide substrate from ClpB as observed in Figure 2D. The dependence of kobs,2 on DnaK is described well by a rectangular hyperbola (eq 2), which is consistent with the disruption of the Cy5ClpB−RepAFlu1−50 complex by DnaK. The midpoint of this curve is 5 ± 2 μM DnaK, slightly lower than the reported Kd for binding of DnaK to ClpB,12,14,20 suggesting that the disruption of the Cy5ClpB−RepAFlu1−50 complex is due to binding of DnaK to ClpB. DnaK Induces ClpB To Release Bound Peptide. To the best of our knowledge, it has not been suggested that DnaK might act as a peptide release factor on ClpB. To provide an alternative method to test whether the ClpB−peptide complex was in fact dissociated, we next employed a steady state anisotropy experiment. Fluorescence anisotropy is a measure of how rapidly particles tumble in solution. Smaller particles such as the free peptide tumble quickly, resulting in low anisotropy, while larger particles such as a ClpB hexamer tumble more slowly, resulting in higher anisotropy. This steady state fluorescence anisotropy technique was recently used to show that ClpB, like Hsp104, avidly binds the polypeptide substrate only in the presence of ATPγS, and not other commonly used nucleotide analogues.28 In that report, we observed that ClpB polypeptide binding was saturated more completely and quickly using 2 mM ATPγS rather than 300 μM ATPγS; thus, we used 2 mM ATPγS for this experiment. As schematized in Figure 4A,

Figure 4. Fluorescence anisotropy measurements of RepA1−30Flu in the presence (red circles) or absence (black squares) of ATPγS before and after subsequent additions of ClpB, and DnaK where indicated. (A) Schematic of the steady state fluorescence anisotropy experiment showing sequential addition of components, as listed from top to bottom. (B) Resulting anisotropy time courses of RepA1−30Flu in the presence (red circles) or absence (black squares) of ATPγS before and after subsequent additions of ClpB, and DnaK where indicated. Final concentrations of 0 or 2 mM ATPγS, 2 μM ClpB, and 25 μM DnaK.

we repeated that experiment with an additional step. After the stable ClpB−peptide complex had formed, DnaK was added. Here, we used the shorter polypeptide substrate, RepA1−30Flu. The recently published structure of yeast homologue Hsp104 with the substrate bound reveals that approximately 26 residues of the polypeptide substrate can be enclosed in the axial channel.37 Thus, the shorter polypeptide substrate limits the possibility of portions of the bound polypeptide protruding from the ClpB channel. Consequently, the signal should report on effects on the peptide due to binding of DnaK to ClpB. 3670

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Biochemistry

that multiple DnaK molecules could bind to this substrate; however, a 1:1 binding model has been established for DnaK binding a 24-amino acid peptide.44 Nevertheless, the fit to eq 3 yields an estimated K1/2 of ≈97.5 μM for DnaK binding peptide in the presence of ATPγS. Because the curve does not saturate, this number represents a lower limit. In other words, the binding affinity could be weaker but not tighter. From the fit shown in Figure 5B, we determined ClpB binds RepA1−30Flu with a K1/2 of 2.2 μM monomer in the presence of ATPγS. Thus, ClpB has a greater affinity for RepA1−30Flu than DnaK does, though DnaK is still capable of binding RepA1−30Flu. The observation that DnaK binds this peptide much more weakly than ClpB suggests that DnaK is not outcompeting ClpB for peptide binding. Likewise, it seems unlikely that a region of the peptide outside of the ClpB axial channel remains available for DnaK to bind and strip the peptide from the complex. However, it is possible that DnaK has a stronger affinity for the peptide that is in a complex with ClpB than it does for the peptide free in solution. Nevertheless, a recently published single-particle reconstruction of the Saccharomyces cerevisiae homologue of ClpB, Hsp104, in complex with a substrate revealed that approximately 26 amino acids occupy the axial channel of the hexameric ring.37 Thus, it is unlikely that RepA1−30Flu bound by ClpB has much of its sequence available for binding by DnaK. It has also been noted that the ATP-bound state of DnaK has a low peptide binding affinity, while the ADP-bound DnaK has a high peptide binding affinity. The allosteric interactions between the DnaK nucleotide binding domain and substrate binding domain are key to the DnaK mechanism.44,45 This led to the question of whether using ATPγS to populate highpeptide affinity ClpB hexamers may have the unintended effect of populating low-peptide affinity DnaK. RepA1−30Flu titrated with DnaK in the absence of a nucleotide displayed a K1/2 of ≈76 μM, which is only slightly tighter than the binding observed in the presence of ATPγS (Supporting Figure 1). This is consistent with work that has shown that ATPγS prevents the release, but not the binding, of the substrate by DnaK.44 Both ClpB and DnaK have numerous protein clients in the cell. We repeated this titration with another commonly used model substrate, α-casein, to test whether the RepA fragment had unusual relative affinities for ClpB and DnaK. Panels C and D of Figure 5 show a higher initial anisotropy measurement, caused by the increased length of the α-casein truncation compared to the RepA truncation (compare initial anisotropy in panels A and B of Figure 5 to that in panels C and D, respectively). Again, we observed that the DnaK titration did not saturate and ClpB bound more tightly (K1/2 = 120 ± 40 μM) than DnaK did (K1/2 = 800 ± 200 μM) to the peptide substrate. However, the K1/2 for DnaK represents a lower limit, and in this case, it is also possible that more than one DnaK molecule may bind the 102-amino acid peptide. The observation of DnaK binding these peptides much more weakly than ClpB suggests that DnaK is not outcompeting ClpB for peptide binding or stripping the peptide from the complex. The ClpB−DnaK Complex Exists in Solution but Does Not Bind the Peptide. Figures 3−5 provide evidence of the ClpB−peptide complex being disrupted by DnaK, and a DnaK−peptide complex being formed, at protein concentrations intended to populate a ClpB−DnaK complex. We next sought to determine whether a ClpB−DnaK complex is indeed populated under these conditions. We further investigated

Figure 5. Binding isotherms for the titration of RepA1−30Flu (A and B) or α-caseinFlu102 (C and D) with protein in the presence of ATPγS. Fluorescence anisotropy is reported. The binding curves are fit to a rectangular hyperbola. (A) RepA1−30Flu was titrated with DnaK. The maximum expected anisotropy value from the fit to a rectangular hyperbola is 0.30, and K1/2 = 97.5 μM. (B) RepA1−30Flu was titrated with ClpB. The maximum anisotropy value is 0.10, and K1/2 = 2.2 μM. (C) α-CaseinFlu102 was titrated with DnaK. The maximum expected anisotropy value is 0.15, and K1/2 = 120 μM. (D) α-CaseinFlu102 was titrated with ClpB. The maximum anisotropy value is 0.10, and K1/2 = 2.2 μM.

dynamic, nucleotide-linked oligomeric assembly of ClpB, was performed with 1 mM ATPγS.36 ClpB resides in a dynamic equilibrium of hexamers and lower-order oligomers,35,36 and DnaK has been observed to be monomeric and dimeric in solution.38−41 The concentrations of ClpB and DnaK are reported in monomer units, and here we report K1/2 as the midpoint of the binding isotherm. A quantitative measurement of the Kd of binding of ClpB to the peptide requires incorporation of the dynamic equilibrium of oligomeric states present in solution, as well as the nucleotide-linked assembly of those oligomers as was previously performed by Li et al.42 Likewise, these midpoints should not be compared directly to reported Kd values in which the ClpB hexamer concentration was assumed to be one-sixth the total ClpB monomer concentration. There are no commonly used experimental conditions in which 100% of ClpB monomers reside in the hexameric state.43 The titration curves were both fit to a rectangular hyperbola, i.e., a 1:1 binding model given by eq 3, where xbar is the fraction of ligand x bound by the macromolecule and K1/2 is the dissociation constant for the macromolecule (protein) and ligand (peptide).

x̅ =

[x] K1/2 + [x]

(3)

Notably, the titration curve for DnaK does not saturate (see Figure 5A). The binding site on the peptide could be smaller for a DnaK monomer than for a ClpB hexamer, so it is possible 3671

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Biochemistry which protein (DnaK) or protein complexes (ClpB6 or ClpB6− DnaK) have peptide binding affinity. To this end, we employed a set of sedimentation velocity experiments. We first characterized the sedimentation of protein in the presence of excess nucleotide. Figure 6A shows the c(s)

to reach this conclusion, and that examination is outside the scope of the work presented here. Finally, the pink trace in Figure 6A represents the c(s) distribution for 100 μM DnaK and 5 μM ClpB in the presence of 2 mM ATPγS. From comparison (Figure 6A, purple), we observe populations consistent with monomer and dimer DnaK as well as an additional peak at 11.3 ± 0.1 S. Using interference optics, complexes of both DnaK and ClpB would be observed. Thus, the approximately 11 S species could represent either ClpB hexamer alone, a ClpB6−DnaK complex, or a mixture of both species. To determine if the 11 S distribution represents ClpB6 or the ClpB6−DnaK complex, we labeled DnaK with fluorescein (FluDnaK). Here we will use fluorescein as a chromophore and monitor the absorbance at 494 nm of the fluorescein on DnaK. Figure 6B shows the c(s) distribution from sedimentation velocity experiments performed with 30 μM Flu-DnaK, 5 μM ClpB, and 2 mM ATPγS. In this experimental design, the signal is sensitive to only Flu-DnaK. Figure 6B shows a distribution at 3.3 ± 0.2 S consistent with the DnaK monomer (compare the purple trace in Figure 6A to Figure 6B) as well as a population at 11.7 ± 0.1 S. As shown in Figure 6A, DnaK does not form oligomers with sedimentation coefficients of >4.3 S in the absence of ClpB. In contrast, an approximately 11 S population is present for ClpB in the absence and presence of DnaK (Figure 6A, green and pink traces). Because the c(s) distribution in Figure 6B reports on only Flu-DnaK, the 11 S peak is consistent with a ClpB hexamer bound by Flu-DnaK. These data show that ClpB and DnaK form a complex, ClpB6− DnaKn, in the presence of ATPγS and in the absence of peptide. Note that the number of DnaK protomers bound to ClpB6 cannot be determined from these data, although Rosenzweig et al. suggest that a single DnaK monomer binds a ClpB hexamer.12 Accordingly, ClpB6−DnaKn represents n DnaK protomers bound to ClpB6. A more rigorous study, outside of the scope of this investigation, would need to be performed to confirm the stoichiometry of binding of DnaK to ClpB6. We propose that DnaK binds to ClpB and reduces the affinity of ClpB for the polypeptide substrate. In other words, the ClpB6−DnaKn complex exhibits a low affinity for the peptide. This predicts that no ClpB would be bound to a polypeptide substrate under conditions where ClpB is saturated by DnaK. To test this, we performed sedimentation velocity experiments with the Cy3-labeled polypeptide substrate by monitoring the absorbance of Cy3 on the peptide in the presence of DnaK, ClpB, and ATPγS. The c(s) distributions in Figure 6C are the result of sedimentation velocity experiments performed using 18 μM RepA1−30Cy3 in the presence of 100 μM DnaK, 5 μM ClpB, and 2 mM ATPγS. To detect all species formed as well as only the species bound to the peptide, sedimenting boundaries were collected by simultaneously monitoring interference (orange dashed line) and absorbance at 548 nm (orange solid line), the absorbance maximum for the fluorescent dye Cy3. In interference, the c(s) distribution in Figure 6C exhibits distributions at approximately 3.2 and 4.3 S. This is consistent with DnaK monomers and dimers observed in Figure 6A (purple). In addition, we also observe peaks at approximately 8 and 11.5 S. The 8 S peak likely represents a low-order ClpB oligomer, because ClpB is reported to reside in a distribution of oligomeric states.35,36 The 11.5 S peak is consistent with the ClpB6−DnaKn complex observed in panels A and B of Figure 6. Surprisingly, sedimentation profiles simultaneously collected by

Figure 6. c(s) distributions of various proteins formed in the presence of 2 mM ATPγS in buffer H200. (A) Distributions resulting from sedimentation profiles monitored with interference optics are shown for 5 μM ClpB alone (green), 100 μM DnaK alone (purple), and 5 μM ClpB with 100 μM DnaK (pink). To observe complexes associated with DnaK, (B) sedimentation of 30 μM Flu-DnaK was monitored using absorbance at 495 nm in the presence of 5 μM ClpB (teal). (C) Complexes associated with RepA1−30Cy3 were monitored using absorbance at 548 nm (orange solid) and interference (orange dotted) in the presence of 5 μM ClpB and 100 μM DnaK.

distributions that result from sedimentation velocity experiments performed with ClpB (green) or DnaK (purple) individually as well as in combination (pink) in the presence of 2 mM ATPγS. These experiments were performed by monitoring interference (see Materials and Methods). Thus, the resulting c(s) distributions are sensitive to all proteins and protein complexes present under each condition. In the c(s) distribution for 5 μM ClpB in the presence of 2 mM ATPγS (Figure 6A, green), we observe a predominant population at approximately 11 S. This corresponds to an s20,w of ∼16 S, consistent with our previously reported ClpB hexamer sedimentation coefficient.36 The c(s) distribution for 100 μM DnaK in the presence of 2 mM ATPγS is colored purple. Here, we see two predominant peaks at approximately 3.2 ± 0.1 and 4.3 ± 0.1 S (s20,w ∼ 4.5 and 6.2 S, respectively). DnaK has been observed as monomers as well as dimers, and we expect that these peaks represent the monomer and dimer species of DnaK.38−41 However, a more thorough examination is required 3672

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Biochemistry monitoring the maximum absorbance of the peptide (orange solid line) result in a c(s) distribution with a single predominant species at approximately 3.2 S. This shows that although multiple complexes are present under these conditions, only one species is associated with the peptide. This peptide-bound species is consistent with a DnaK monomer. Previous work has indicated that only ClpB hexamers bind the peptide. While lower-order ClpB oligomers binding the peptide in the presence of a nucleotide cannot be ruled out, previous experiments show that ClpB hexamers are the major, if not the only, species of ClpB that binds the peptide.42 From comparison to the c(s) distribution for DnaK in the absence of ClpB (Figure 6A, purple), only DnaK monomers appear to be peptide-bound, and there is no indication of a ClpB6−DnaKn− peptide complex. This observation is consistent with DnaK binding to ClpB and reducing ClpB’s affinity for the peptide.

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DISCUSSION In this study, we developed a stopped-flow FRET assay that reports on ClpB-catalyzed polypeptide translocation. The motivation for developing a FRET method is twofold. The first is to be able to test the influence of multiple different protein co-chaperones without every protein cofactor having a direct influence on the observed signal. The second is to be able to test whether the ClpB−DnaK complex has translocation activity that is different from that of ClpB in the absence of any cofactor. In the FRET design, the donor and acceptor are on the peptide and ClpB, respectively. Therefore, the signal is sensitive to the interaction between ClpB and the peptide. Thus, we expect that other protein cofactors will have a limited influence on the signal. Consequently, the signal is easier to interpret. Importantly, the sensitized emission from Cy5ClpB exhibits similar kinetic parameters as we previously reported using fluorescence from fluorescein on a polypeptide substrate.26 This indicates that the two methods are reporting on the same kinetic effects. To the best of our knowledge, the activity of the ClpB− DnaK complex has not been tested. This conclusion is based on the fact that ClpB and DnaK form a complex with a dissociation equilibrium constant (Kd) of ∼9−30 μM,12,14,20 and to the best of our knowledge, the highest concentration of DnaK used in reported experiments is ∼2 μM, with most experiments performed in the submicromolar range. This concentration of DnaK remains an order of magnitude below the reported Kd. We would predict that under those conditions, ∼10% ClpB−DnaK complex is present and the rest is free ClpB and DnaK. Although effects have been reported under these conditions, we would contend that this is not due to a ternary complex. Interestingly, the in vivo DnaK concentration has been reported to be 27 μM,1 which is strikingly similar to the Kd of 25 μM for binding of DnaK to ClpB.12 This observation suggests a regulatory role for DnaK. On the basis of the work reported here, we propose that when DnaK binds to the ClpB− peptide complex it allosterically induces ClpB to release the polypeptide substrate. Thus, we propose that the ClpB−DnaK complex has a low peptide binding affinity (Figure 7A). This would predict that under normal cellular conditions, where ClpB would be half-saturated with DnaK, the ClpB−DnaK complex would be in a low-peptide affinity state. This proposal does not rule out DnaK also binding other known binding partners, nor is DnaK the only regulatory mechanism for ClpB

Figure 7. Proposed mechanistic model for allosteric regulation of ClpB by DnaK. Panel A represents normal cell conditions in which DnaK binding to ClpB confers a low peptide binding affinity to ClpB. Panel B shows that upon heat shock, client proteins are unfolded, leading to a release of ClpB by DnaK. In panel C, DnaK is free to bind denatured client proteins and ClpB hexamers are now in a highpeptide affinity state due to the dissociation of DnaK. These ClpB hexamers can bind the loose loops or tails of denatured proteins in an aggregate and go through a limited number of ATP-dependent translocation steps to loosen the aggregate. Panel D illustrates that as unfolded client proteins are released from the aggregate, DnaK can act as a “holdase” while ClpB can bind the aggregate again in another location to continue ATP-dependent disaggregation. See the legend for the identification of symbols.

activity. We have previously shown the effects of ClpB concentration and nucleotide concentration on the population of ClpB hexamers.35,36 Under conditions of heat shock as native proteins are denatured (Figure 7B), DnaK would begin to be sequestered by a variety of misfolded proteins.1 Therefore, under heat shock conditions, the DnaK would be released from ClpB and ClpB’s activity would be unleashed (Figure 7C,D). Simultaneously, the other proposed collaborative activities of ClpB and DnaK would then dominate the mechanism. This model does not rule out the possibility, outlined in the introductory section and untested in this study, that DnaK may also bind a substrate and remodel the substrate prior to transferring it to ClpB. As previous researchers have noted, these models are not mutually exclusive. That is, DnaK could bind the protein substrate and modify the substrate making it more tractable to ClpB,7,8 or the DnaK could accept the protein substrate after some remodeling catalyzed by ClpB.9,10 Our sedimentation velocity experiments showed the presence of a ClpB6−DnaKn complex under all conditions that included ATPγS, where ATPγS is included to stabilize hexameric ClpB. Surprisingly, this ClpB6−DnaKn complex does not display an observable affinity for the peptide. Rather, under conditions under which DnaK is in sufficient excess over ClpB6, and we predict all of the ClpB should be in the ClpB6−DnaKn state, only DnaK is observed bound to the peptide. Notably, ATPγS is not considered to promote a high-affinity state for DnaK because ATP-bound DnaK is known to have a low 3673

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Biochemistry peptide affinity.44,45 In fact, DnaK is bound to the peptide even though ClpB has a tighter affinity for the peptide, which is a further indication that no free ClpB6 is present. This result is consistent with DnaK changing the conformation of ClpB to a low-peptide affinity state. In this regard, we can consider DnaK to act as a peptide release factor with respect to ClpB. In the context of disaggregation in the cell, such a release would allow ClpB to subsequently bind to another portion of the aggregate to repeat its nonprocessive translocation mechanism, tugging and pulling to disrupt the aggregate, as we have recently proposed.26 In addition, DnaK could also bind the peptide or aggregate after its release from ClpB. Our findings are at odds with a model recently proposed on the basis of cryo-electron microscopy (cryo-EM) reconstructions of a ClpB variant with both ATPase domains inactivated and with additional amino acid substitutions designed to engineer a non-native interaction with ClpP (BAP-DWB).46 Cryo-EM data were collected for this protein, saturated with ATPγS, in the absence or presence of α-casein. Although there was no DnaK included in the samples, the authors modeled a DnaK−peptide−ClpB complex and proposed a mechanism by which DnaK transfers the peptide to ClpB. This DnaK− peptide−ClpB complex has not been observed in the work of Deville et al. or any other publication to the best of our knowledge. The study presented here provides an insight into why that complex has not been observed. When ClpB is bound by the peptide, as in many of our experiments and the cryo-EM experiments of Deville et al., the addition of DnaK at concentrations high enough to mimic cellular concentrations and populate a DnaK−ClpB complex results in ClpB releasing its peptide substrate. Given the DnaK binding site on the M domain of ClpB, this peptide release function would not be surprising. Recent work showed that ClpB hexamers populated in the presence of various nucleotides and nucleotide analogues displayed a low peptide binding affinity.28 Only ATPγS populated ClpB hexamers with a high peptide binding affinity.28 When Rosenzweig et al. identified the binding site for DnaK on ClpB, they proposed that, in the context of DnaK acting on an aggregate prior to ClpB, this binding site would position the peptide for transfer from DnaK to ClpB.12 The M domain is thought to modulate the ATP hydrolysis cycle, so we suggest that DnaK binding at the M domain could also be a mechanism by which nucleotide-dependent peptide binding affinity is controlled.

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ORCID

Aaron L. Lucius: 0000-0001-8636-5411 Author Contributions

C.L.D. and A.L.L. designed stopped-flow and fluorimeter experiments. C.L.D. performed stopped-flow and fluorimeter experiments and analyzed the data. E.C.D., C.L.D., and A.L.L. designed sedimentation velocity experiments. E.C.D. performed sedimentation velocity experiments and analyzed the data. C.L.D. expressed and purified proteins. E.C.D. labeled DnaK. C.L.D. wrote the manuscript with contributions from E.C.D. and A.L.L. Funding

This work was supported by National Science Foundation Grant MCB-1412624 to A.L.L. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Nate Scull for his thoughtful commentary on the manuscript and Ethan Cagle for assistance with protein labeling in a reduced oxygen environment.

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(1) Mogk, A., Tomoyasu, T., Goloubinoff, P., Rudiger, S., Roder, D., Langen, H., and Bukau, B. (1999) Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB. EMBO J. 18, 6934−6949. (2) Doyle, S. M., Shorter, J., Zolkiewski, M., Hoskins, J. R., Lindquist, S., and Wickner, S. (2007) Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity. Nat. Struct. Mol. Biol. 14, 114−122. (3) Nagy, M., Wu, H. C., Liu, Z., Kedzierska-Mieszkowska, S., and Zolkiewski, M. (2009) Walker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB. Protein Sci. 18, 287−293. (4) Hoskins, J. R., Doyle, S. M., and Wickner, S. (2009) Coupling ATP utilization to protein remodeling by ClpB, a hexameric AAA+ protein. Proc. Natl. Acad. Sci. U. S. A. 106, 22233−22238. (5) del Castillo, U., Fernandez-Higuero, J. A., Perez-Acebron, S., Moro, F., and Muga, A. (2010) Nucleotide utilization requirements that render ClpB active as a chaperone. FEBS Lett. 584, 929−934. (6) Martin, I., Celaya, G., Alfonso, C., Moro, F., Rivas, G., and Muga, A. (2014) Crowding activates ClpB and enhances its association with DnaK for efficient protein aggregate reactivation. Biophys. J. 106, 2017−2027. (7) Zietkiewicz, S., Krzewska, J., and Liberek, K. (2004) Successive and synergistic action of the Hsp70 and Hsp100 chaperones in protein disaggregation. J. Biol. Chem. 279, 44376−44383. (8) Zietkiewicz, S., Lewandowska, A., Stocki, P., and Liberek, K. (2006) Hsp70 chaperone machine remodels protein aggregates at the initial step of Hsp70-Hsp100-dependent disaggregation. J. Biol. Chem. 281, 7022−7029. (9) Goloubinoff, P., Mogk, A., Zvi, A. P., Tomoyasu, T., and Bukau, B. (1999) Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proc. Natl. Acad. Sci. U. S. A. 96, 13732−13737. (10) Weibezahn, J., Schlieker, C., Bukau, B., and Mogk, A. (2003) Characterization of a trap mutant of the AAA+ chaperone ClpB. J. Biol. Chem. 278, 32608−32617. (11) Miot, M., Reidy, M., Doyle, S. M., Hoskins, J. R., Johnston, D. M., Genest, O., Vitery, M. C., Masison, D. C., and Wickner, S. (2011) Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation. Proc. Natl. Acad. Sci. U. S. A. 108, 6915−6920.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00045. Binding isotherm of the DnaK binding peptide in the absence of a nucleotide and detailed methods of labeling ClpB with Cy5-maleimide, purifying and concentrating DnaK, and labeling DnaK with fluorescein 5-maleimide (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 3674

DOI: 10.1021/acs.biochem.8b00045 Biochemistry 2018, 57, 3665−3675

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DOI: 10.1021/acs.biochem.8b00045 Biochemistry 2018, 57, 3665−3675