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Escherichia coli DnaK allosterically modulates ClpB between high and low peptide affinity states Clarissa L Durie, Elizabeth C Duran, and Aaron L Lucius Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00045 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Biochemistry

Escherichia coli DnaK allosterically modulates ClpB between high and low peptide affinity states Clarissa L. Durie, Elizabeth C. Duran, Aaron L. Lucius* University of Alabama at Birmingham, Chemistry Department, Birmingham, AL *Correspondence: Aaron L. Lucius [email protected] 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 or 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. Introduction The 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 have 1 ACS Paragon Plus Environment

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been determined in which ClpB alone can resolve aggregates in vitro.2-6 The ClpB-DnaKJE cochaperone system is able to resolve aggregates that are intractable by 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 either chaperone alone. The species specificity of the co-chaperone interaction suggests the importance of a ClpBDnaK complex.11-13 ClpB-DnaK complexes have been observed by co-elution and NMR 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 made up of ClpB, DnaK, and peptide substrate. Our objective in this study is to test whether a ClpB-DnaK complex has an altered translocation mechanism compared to ClpB in the absence of DnaK. 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 T. thermophilus DnaK for ClpB has been reported as Kd = 17 µM14 and Kd = 25 µM.12 For E. coli ClpB, the Kd has been estimated as 2 ACS Paragon Plus Environment

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Biochemistry

9-30 µM.20 Interestingly, the in vivo DnaK concentration has been reported to be 27 M,1 which is strikingly similar to the Kd = 25 μM for DnaK binding ClpB.12 Despite the knowledge of the strength of this interaction constant and the cellular concentration of DnaK1, 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 M, which

remains an order of magnitude below the reported Kd12 and cellular concentration1. 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 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 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 single-turnover FRET method to test the translocation function of the ClpBDnaK 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 polypeptide substrate compared to ClpB alone. Surprisingly, we determine that DnaK causes ClpB to release 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% glycerol (v/v), 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 that remains after removal of a histidine tag, added for purification purposes. The concentration of ClpB was determined at 280 nm in H200 using the extinction coefficient ɛ = 35,562 M-1cm-1, determined following the method of Edelhoch and modified by Pace.29, 30 All protein concentrations are reported in monomer units. ClpB was covalently modified with Cy5-maleimide. The labeling reaction was carried out 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 Supplemental Methods for additional information. The Cy5ClpB had a labeling efficiency of 27%. DnaK was expressed with an N-terminal His6tag, followed by a TEV cleavage site for purification purposes. Protein concentrations are reported in monomer units using the extinction coefficient at 280 nm ε = 16,269 M-1 cm-1 determined in our lab by the method proposed by 4 ACS Paragon Plus Environment

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Biochemistry

Edolhoch and later refined by Pace et al.29, 30 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 Supplemental Methods. 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.28α-casein substrates were prepared as previously described.31 Peptides are referred to 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. Steady State Fluorescence Measurements Steady state fluorescence measurements were made 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 Cy5GClpB was collected monitoring emission at 670 nm. Sensitized emission. Emission spectra were collected for Cy5-ClpB with excitation at 494 nm, the maximum excitation wavelength for fluorescein. Next, ATPγS was added and emission spectra were collected. Finally, fluorescein labeled peptide, either RepA1-50Flu or RepAFlu1-50, was


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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 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 one hour 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 made with RepAFlu1-50 in the presence or absence of ATPγS. Additional measurements were made 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 so the minor changes in concentration due to sequential additions of components does not contribute to the anisotropy signal and no correction for dilution is needed. All reagents were equilibrated to 25 °C for approximately one hour prior to addition to the fluorescence cuvette. Fluorescence anisotropy – titration of 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 made 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 minutes prior to addition to the binding sample. Upon each addition, the cuvettes were gently inverted to mix the contents, then incubated for 5 minutes prior to collecting data. For each titration, three single point anisotropy measurements at


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Biochemistry

515 nm and one fluorescence emission spectrum from 500-600 nm were collected. The three anisotropy measurements are reported in Figure 5 as means ± s.d. where the standard deviations are measurement errors. Rapid Mixing Assays All rapid mixing experiments were performed using an Applied Photophysics SX20 stopped flow fluorimeter (Leatherhead, UK). All solutions were incubated for 1 hour at 25 °C prior to mixing. The syringes of the stopped-flow fluorimeter and the reaction cell were also maintained at 25 °C. Fluorescence of fluorescein-modified peptides was excited at 494 nm. In the FRET assay, the fluorescence emission of fluorescein was monitored using PMT R6095 with a 520 (±5) nm filter and the fluorescence emission of Cy5 was monitored using PMT 1104 with a 665 nm cut off long pass filter. In the standard assay, using unmodified ClpB and fluorescein on the peptide as previously reported by Li et al., emission of fluorescein was monitored using PMT R6095 and a 515 nm cut off 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] 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 4 shots or pushes were averaged. [DnaK] 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 M ATPS was incubated in the other syringe. The solutions were rapidly mixed. For each concentration of ATP, a minimum of 4 shots or pushes were averaged.


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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 ATP concentration dependence and DnaK concentration dependence, each averaged, normalized time course was individually fit to a sum of two exponentials (Equation (1) ) using the non-linear least squares (NLLS) fitting routine, Conlin, kindly provided by Dr. Jeremy Williams32 where f is fluorescence signal, t is 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, respectively. 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 40,000 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 6 B and 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 carried out by loading 400 μL of sample and reference solutions into each corresponding sector of Beckman 12 mm double sector Epon charcoal-filled non-meniscus matching centerpieces. Protein and reference solutions were prepared in H200 and incubated for 2 hours at 25 °C before the first sedimentation scan was collected. In samples containing DnaK, the DnaK was added after 15 minute incubation of other components.


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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 (Chad Brautigam, University of Texas Southwestern Medical Center). The corrected boundaries were then analyzed using SEDFIT version 14.4f (Peter Schuck, NIH) as previously described.35, 36 The data were analyzed between the meniscus plus 0.01 cm and 6.7 cm to minimize the contribution from gradients of glycerol and nucleotide to the sedimentation boundaries.36 Results Development of Single-Turnover FRET Strategy for Observing ClpB-catalyzed polypeptide translocation in the absence or presence of DnaK Using single-turnover stopped-flow experiments we previously showed that ClpB is a nonprocessive translocase26. We proposed a model where ClpB takes one or two steps before rapid dissociation from the polypeptide chain. We have described this as a “pull and release” mechanism. In order to study the effect of DnaK on this mechanism, we were unable to simply add DnaK into our experimental setup. This is because DnaK is also capable of binding peptide on its own.1 Thus, including DnaK in the experimental design used previously resulted in a signal not only from ClpB-peptide complex, but also from DnaK-peptide complex (data not shown). In order to remove any contributing signal from DnaK-peptide complexes, here we develop a FRET assay in which we could monitor the translocation of polypeptide by ClpB in either 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 since the peptide is predicted to enter the axial channel from the N-domain and proceed 9 ACS Paragon Plus Environment

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from nucleotide binding domain 1 to nucleotide binding domain 2. Based on our previous work on ClpB assembly,35, 36 we labeled ClpB with a Cy5-maleimide dye in 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. The peptide RepAFlu1-50 has been used in a previous study of ClpB binding avidity for peptide in the presence of various nucleotides.28 Figure 1 A shows the overlap in the Flupeptide emission spectrum (green solid line) with the Cy5ClpB excitation spectrum (blue dashed line).

Figure 1. Development of FRET assay to monitor ClpB catalyzed polypeptide translocation. (A) Emission spectrum of RepA1-50Flu (green). Excitation spectrum of Cy5ClpB (blue). (B & C) With excitation at 494 nm, emission spectra were collected for Cy5GClpB alone (blue, dashed lines), and upon addition of ATPγS (grey, dotted lines), and RepAFlu1-50 (B) or RepA1-50Flu (C) (green, solid lines).


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Biochemistry

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 then sensitized emission from Cy5 would be detected if the fluorescein on the peptide is spatially close to the Cy5 in the N-domain. Even though the excitation spectrum for Cy5 (see Fig. 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 Cy5-ClpB by exciting at 494 nm, i.e. the excitation wavelength for fluorescein and the wavelength used in FRET experiments. As seen in Figure 1 B and C (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 ATPS. Therefore, we next added 300 M ATPS to determine if the Cy5 is sensitive to this change in the assembly state. Figure 1 B and C show that the Cy5 emission does not change upon addition of ATPS. 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, which is a 50 amino acid peptide containing fluorescein at either 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 indicative of the 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 ATP-driven translocation catalyzed by ClpB.26 If ClpB translocates the polypeptide chain through its axial channel then we would predict that the


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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 in sensitized emission upon rapid mixing with ATP or a slight increase followed by a loss in signal. To test these possibilities the assembled complex (Cy5ClpB-RepAFlu1-50 complex) 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 collecting fluorescence at 665 nm and above (see Materials and Methods). This experimental design is schematized in Figure 2A. Figure 2 B and C 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 before dissociation from the polypeptide lattice. Each curve is well described by a sum of two exponentials, Equation(1)

f  A1 (1  e

 kobs ,1t

)  A2 (1  e

 kobs ,2t


)B

(1)

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Biochemistry

where f is fluorescence signal, t is 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, respectively. The fits are shown in broken black lines.

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. Broken, black traces represent the best fit lines of each time course, individually, to Equation 1 using NLLS analysis. (C) Time courses collected from the 665 nm + (Cy5 λem, max) simultaneously with those in B. Fitting is the same as for panel B. (D&E) Secondary plots for the parameters from individual fits of time courses to Equation 1 as a function of [ATP]. Parameters are (D) kobs1, and (E) kobs, 2 Parameters from the Cy5 sensitized emission channel, Panel C, are blue squares (mean ± s.d., n=3) and blue curve is fit of those data to Equation(2). Black circles are data from Li et al. (mean ± s.d., n=3) and black curve is fit of those data to Equation(2). 13 ACS Paragon Plus Environment

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Because our data in Figure 2 B and C were well described by Equation (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 invoked26. Figures 2 D and E 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 change from the fluorescein on the polypeptide substrate26. The signal is quenched when ClpB is bound and it recovers when ClpB dissociates. This happens regardless of the presence of the Cy5. Thus, it remains unclear 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. On the other hand, the sensitized emission time courses are only sensitive 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 in black circles (mean ± s. d., n = 3). The rate constant kobs,1 (mean ± s. d., n = 3) from the fit of the data in Figure 2 C, the sensitized emission channel, to Equation (1) is overlaid with the Li et al. data in Figure 2 D as blue squares. All of the data are well described by a rectangular hyperbola (Equation (2)).

kobs , x 

kobs , x ,max [ ATP] K1/2  [ ATP]

(2)

Li et al. reported kobs1,max = (0.190 ± 0.004) s-1and K1/2 = (192 ± 18) M ATP)26. From the sensitized emission of Cy5, we observe remarkably similar parameters of kobs1,max = (0.22 ± 0.01) s-1and K1/2 = (290 ± 80) M ATP.


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Biochemistry

The corresponding comparison is also performed in Figure 2 E for the rate constant kobs,2 as a function of ATP (mean ± s. d., n=3). Li et al. reported kobs2, max = (0.0516 ±0.006) s-1 and K1/2 = (226 ± 16) M ATP26. Parameters from fits of the data in Figure 2 C (sensitized emission) are shown as blue squares with the fit line shown as a blue curve. Here, we observe kobs2, max = (0.022 ±0.001) s-1 and K1/2 = (260 ± 70) M 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 M.12 Thus, to test if the ClpB-DnaK complex exhibits different translocation activity compared to ClpB alone we performed experiments as schematized in Figure 2A by adding DnaK to the syringe containing Cy5ClpB-Flu-peptide. To do this we included DnaK at a number of different [DnaK] both above and below the Kd = 25 M. 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 pre-incubation syringe, we assembled the Cy5ClpB-RepAFlu1-50 complex from which we observed sensitized emission, and rapidly mixed 15 ACS Paragon Plus Environment

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that solution with varying concentrations of DnaK as schematized in Figure 3 A. 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 would predict a DnaK concentration dependence of the time courses. Figure 3 B 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 which reports on sensitized emission of Cy5ClpB. Each time course displays a fast increase in sensitized emission (see inset in Figure 3 B), followed by a slower decrease in sensitized emission with time. Each of these time courses was well described by a sum of two exponentials as defined in Equation 1. The faster, increasing phase is represented by kobs,1 and is independent of DnaK concentration (Figure 3 C). An increase in sensitized emission could be due to the fluorescein on the peptide and the Cy5 on the N-domain of ClpB coming closer together. Perhaps DnaK binding to the ClpB hexamer causes such a change. It also can’t be ruled out that the increase in Cy5 fluorescence is due to a change in the environment of Cy5 on ClpB. For example, the binding of DnaK could cause a conformational change that causes the dye to be more solvent-exposed. The decreasing phase represented by kobs,2 and is noted to be faster with higher concentrations of DnaK (Figure 3 D). This decrease in sensitized emission is consistent with the release of peptide substrate from ClpB as observed in Figure 2D. The dependence of kobs,2 on ATP is well described by a rectangular hyperbola (Equation (2)) which is consistent with the disruption of the Cy5ClpB-RepAFlu1-50 by DnaK. The midpoint of this curve is (5 ± 2) M DnaK, slightly lower than the reported Kd for DnaK binding ClpB,12, 14, 20 suggesting that the disruption of the Cy5ClpBRepAFlu1-50 complex is due to DnaK binding ClpB. 16 ACS Paragon Plus Environment

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Biochemistry

Figure 3. Application of 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, 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 as orange, green, and blue, and the highest concentration, 104.7 µM DnaK shown in purple. The gray trace is the time course resulting from mixing the complex with ATPγS (300 µM) in the absence of DnaK. The broken black traces are fit lines to a sum of two exponentials. (C&D) Rate constants from the fits shown in B as a function of [DnaK] (mean ± s.d, 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 (D) is the fit of those data to Equation(2). 17 ACS Paragon Plus Environment

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DnaK induces ClpB to release bound peptide To 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 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 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 ATPS than 300 M ATPS; thus we used 2 mM ATPS for this experiment. As schematized in Figure 4 A, 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 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 ClpB channel. Consequently the signal should report on effects on the peptide due to DnaK binding ClpB. Figure 4 B shows the steady state time courses collected. Initially, each cuvette contained only RepA1-30Flu (final concentration 20 nM). The anisotropy values of r = 0.039 ± 0.002 (black circles, 0-57 minutes) in the absence of nucleotide and r = 0.042 ± 0.004 (red squares, 0-57 minutes) 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 2 µM). 18 ACS Paragon Plus Environment

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Biochemistry

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 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: ATPγS (2 mM or none), ClpB (2 µM), and DnaK (25 µM). Upon addition of ClpB, the anisotropy of the 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 being bound by the large ClpB hexamer. Upon the addition of ClpB in the absence of ATPγS, the anisotropy of the peptide did not change and was observed r = 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


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in free peptide and a low anisotropy value. DnaK could also bind peptide after its release from ClpB, likely resulting in a different, intermediate anisotropy value due to the size and shape differences between 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 in Figure 3. Fourth, DnaK could form a stable ternary complex with ClpB and peptide. Due to 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 anisotropy value measured. This result may be indistinguishable from the case where DnaK has no effect (first proposed possibility). In Figure 4 B, when DnaK is added to the sample that includes complex assembled in the presence of ATPγS (red squares, 180 – 360 min), the anisotropy value drops to r = 0.061 ± 0.003. This measurement is outside of error for the anisotropy value reported for free peptide as well as for the ClpB-bound peptide. Furthermore in the absence of nucleotide, the anisotropy of the peptide increases to r = 0.070 ± 0.003 upon addition of DnaK. DnaK was observed to bind this peptide in the absence of 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 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 20 ACS Paragon Plus Environment

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Biochemistry

Figure 5. Binding isotherms for the titration of RepA1-30Flu (A&B) or α-caseinFlu102 (C&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 r = 0.30, and K1/2 = 97.5 µM. (B) RepA1-30Flu was titrated with ClpB; the maximum anisotropy value is r = 0.10, and K1/2 = 2.2 µM. (C) α-caseinFlu102 was titrated with DnaK; the maximum expected anisotropy value is r = 0.15, and K1/2 = 120 µM. (D) α-caseinFlu102 was titrated with ClpB; the maximum anisotropy value is r = 0.10, and K1/2 = 2.2 µM. ClpB (panel B) from the titration of RepA1-30Flu in the presence of 1 mM ATPγS. We chose to use 1 mM ATPS because our previous quantitative study of ClpB polypeptide binding, which included incorporation of the dynamic, nucleotide-linked oligomeric assembly of ClpB, was performed with 1 mM ATPS36. ClpB resides in a dynamic equilibrium of hexamers and lower order 21 ACS Paragon Plus Environment

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oligomers35, 36, and DnaK has been observed as monomeric and dimeric in solution38-41. 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 ClpB binding to 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 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 state43. The titration curves were both fit to a rectangular hyperbola, i.e. a one-to-one binding model given by (Equation (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). x

[ x] K1/ 2  [ x ]

(3)

Notably, the titration curve for DnaK does not saturate, see Figure 5 A. The binding site on the peptide could be smaller for a DnaK monomer than for a ClpB hexamer, so it is possible 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 peptide44. Nevertheless the fit to Equation 3 yields an estimated K1/2 ≈ 97.5 µM for DnaK binding peptide in the presence of ATPγS. Since 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 5 B, we determined ClpB binds RepA130Flu with

K1/2 = 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. 22 ACS Paragon Plus Environment

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Biochemistry

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 peptide that is in complex with ClpB than it does for peptide free in solution. Nevertheless, a recently published single particle reconstruction of the S. 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 mechanism44, 45. This lead to the question of whether using ATPS to populate high peptide affinity ClpB hexamers may have the unintended effect of populating low peptide affinity DnaK. RepA130Flu titrated

with DnaK in the absence of nucleotide displayed K1/2 ≈ 76 M, which is only slightly

tighter than the binding observed in the presence of ATPS (Supporting Figure 1). This is consistent with work that has shown that ATPS prevents the release, but not the binding, of substrate by DnaK44. Both ClpB and DnaK have numerous protein clients in the cell. We repeated this titration with another commonly used model substrate, α-casein, in order to test whether the RepA fragment had unusual relative affinities for ClpB and DnaK. Figure 5 C and D show a higher initial anisotropy measurement, due to the increased length of the α-casein truncation compared to the RepA truncation (compare initial anisotropy in Figure 5 A and B to Figure 5 C and D, respectively). 23 ACS Paragon Plus Environment

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Again, we observed that the DnaK titration did not saturate and ClpB bound more tightly, K1/2 = (120 ± 40) M, than DnaK, K1/2 = (800 ± 200) M, 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. ClpB-DnaK complex exists in solution but does not bind 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 ClpBDnaK complex. We next sought to determine whether a ClpB-DnaK complex is indeed populated in these conditions. We further investigated 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 6 A shows the c(s) 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 done by monitoring interference (see Materials and Methods). Thus, the resulting c(s) distributions are sensitive to all proteins and protein complexes present in each condition. In the c(s) distribution for 5 µM ClpB in the presence of 2 mM ATPγS, (Figure 6 A, green) we observe a predominant population at approximately 11 S. This corresponds to an s20,w ~16 S, consistent with our previously reported ClpB hexamer sedimentation coefficient36. The c(s) distribution for 100 µM DnaK in the presence of 2 mM ATPγS is shown in purple. Here, we see two predominant peaks at approximately (3.2 ± 0.1) S and (4.3 ± 0.1) S, s20,w 24 ACS Paragon Plus Environment

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Biochemistry

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. ~4.5 S 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 to conclude this and that examination is outside the scope of the 25 ACS Paragon Plus Environment

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work presented here. Finally, the pink trace in Figure 6 A represents the c(s) distribution for 100 µM DnaK and 5 µM ClpB in the presence of 2 mM ATPγS. From comparison (Figure 6 A, 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 ClpB6-DnaK we labeled DnaK with fluorescein (Flu-DnaK). Here we will use fluorescein as a chromophore and monitor absorbance at 494 nm of the fluorescein on DnaK. Figure 6 B shows the c(s) distribution from sedimentation velocity experiments performed with 30 M Flu-DnaK, 5 M ClpB and 2 mM ATPS. In this experimental design the signal is only sensitive to the Flu-DnaK. Figure 6 B shows a distribution at (3.3 ± 0.2) S consistent with DnaK monomer (compare Figure 6 A, purple to Figure 6 B) as well as a population at (11.7 ± 0.1) S. As shown in Figure 6 A, DnaK does not form oligomers with sedimentation coefficients higher than 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 6 A, green and pink traces). Since the c(s) distribution in Figure 6 B only reports on 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 done in order to confirm the stoichiometry of DnaK binding to ClpB6. 26 ACS Paragon Plus Environment

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Biochemistry

We propose that DnaK binds to ClpB and reduces the ClpB affinity for polypeptide substrate. In other words, ClpB6-DnaKn exhibits a low affinity for peptide binding. 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 Cy3 labeled polypeptide substrate monitoring the absorbance of Cy3 on the peptide in the presence of DnaK, ClpB, and ATPγS. The c(s) distributions in Figure 6 C is the result of sedimentation velocity experiments collected using 18 µM RepA1-30Cy3 in the presence of 100 µM DnaK, 5 µM ClpB, and 2 mM ATPγS. In order to detect all species formed as well as only the species bound to peptide, sedimenting boundaries were collected by simultaneously monitoring interference (orange, broken 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 6 C exhibits distributions at approximately 3.2 S and 4.3 S. This is consistent with DnaK monomers and dimers observed in Figure 6 A (purple). In addition, we also observe peaks at approximately 8 S and 11.5 S. The 8 S peak, likely represents a low order ClpB oligomer, since ClpB is reported to reside in a distribution of oligomeric states35, 36. The 11.5 S peak is consistent with the ClpB6-DnaKn complex observed in Figure 6 panels A and B. Surprisingly, sedimentation profiles simultaneously collected monitoring 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. Only ClpB hexamers have been observed to bind peptide. While lower order ClpB oligomers binding peptide in the presence of nucleotide can’t be ruled out, previous experiments show that ClpB hexamers are the major, if not the only, species of ClpB 27 ACS Paragon Plus Environment

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that binds peptide.42 From comparison to the c(s) distribution for DnaK in the absence of ClpB (Figure 6 A, 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 peptide. 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 or not the ClpB-DnaK complex has different translocation activity compared to 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 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 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 ~ 9 – 30 M.12, 14, 20 And, to our knowledge, the highest concentration of DnaK used in reported experiments is ~2 M with most experiments performed in the submicromolar range. This concentration of DnaK remains an order of magnitude below the reported Kd. We would


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Biochemistry

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 M,1 which is strikingly similar to the Kd = 25 μM for DnaK binding ClpB.12 This observation suggests a regulatory role for DnaK. Based on 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 low peptide binding affinity (Figure 7 A). 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 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 7 B), 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 7 C, 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 introduction 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, 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


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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, not only is DnaK free to bind denatured client proteins, but ClpB hexamers are now in a high peptide 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 legend, bottom, for identification of symbols.


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Biochemistry

Our sedimentation velocity experiments showed the presence of a ClpB6-DnaKn complex in all conditions that included ATPγS, where ATPS is included to stabilize hexameric ClpB. Surprisingly, this ClpB6-DnaKn complex does not display an observable affinity for peptide binding. Rather, in conditions where 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, ATPS is not considered to promote a high-affinity state for DnaK since ATP bound DnaK is known to have low peptide affinity44, 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 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 Further, DnaK could also bind the peptide or aggregate after its release from ClpB. Our findings are at odds with a model recently proposed based on 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. CryoEM data were collected of this protein, saturated with ATPS, in the absence or presence of -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 peptide to ClpB. This DnaK-peptide-ClpB complex has not been observed in the Deville et al work, or any other publication to our knowledge. The present study provides an insight into why that complex has not been observed. When ClpB is bound by peptide, as in many of our experiments and the Deville et al cryo-EM 31 ACS Paragon Plus Environment

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experiments, the addition of DnaK in 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 low peptide binding affinity28. Only ATPS populated ClpB hexamers with 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 peptide for transfer from DnaK to ClpB12. 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. Acknowledgements This work was supported by the NSF (grant MCB-1412624 to ALL). We thank Nate Scull for his thoughtful commentary on the manuscript and Ethan Cagle for assistance protein labeling in a reduced oxygen environment. 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.


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Biochemistry

Supporting Information Binding isotherm of DnaK binding peptide in absence of nucleotide. Detailed methods of labeling ClpB with Cy5-maleimide, purifying and concentrating DnaK, and labeling DnaK with fluorescein-5-maleimide.


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Biochemistry

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Biochemistry

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Biochemistry


Biochemistry 1ClpB6 -peptide high 2 peptide affinity 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

excess DnaK

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1 Escherichia coli DnaK allosterically modulates ... - ACS Publications

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