1. An Interdomain Energetic Tug-of-War Creates the Allosterically Active State in Hsp70 Molecular Chaperones 
Anastasia Zhuravleva, Eugenia M. Clerico, Lila M. Gierasch 
Cell 
Volume 151, Issue 6, Pages (December 2012)
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3. Figure 1 Conformational Insights into the Hsp70 Allosteric Cycle
(A) The Hsp70 allosteric cycle. S, substrate.
(B and C) Ribbon representation of the structures of the two “endpoint” states of DnaK: the ADP-bound state (PDB 2kho), and the Hsp110-based homology model of the ATP-bound state (Smock et al., 2010; coordinates available upon request). Structural elements are colored as in (D). (See also Figure S1.)
(D) DnaK constructs used for NMR studies.
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4. Figure 2
NMR Fingerprints of DnaK Reveal Three Different Ligand-Bound States in Its Allosteric Cycle
(A–C) The isoleucine region of methyl-TROSY spectra of the two-domain DnaK constructs (black indicates DnaK(1-552) in A and B and full-length DnaK in C; see Figure S2 for full-length DnaK in the ADP-bound state) are overlaid with the spectra of corresponding nucleotide and substrate-bound states of the individual domains: NBD, DnaK(1-388), is in blue, and SBD, DnaK( ), is in green. (A) Nucleotide free. (B) ATP bound. (C) ATP/substrate bound. In (C), red arrows point to small but significant chemical shift differences between NBD resonances of the full-length ATP/substrate-bound DnaK and those of its isolated NBD.
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5. Figure 3
Experimental Validation of the Hsp110-Based Homology Model for the ATP-Bound, Domain-Docked State of DnaK
(A and B) Mapping of the NBD residues that were significantly affected by interaction with the β-SBD onto the structure of the Hsp110-based homology model. Residues with significant chemical shift differences (see text) between DnaK(1-552) and either DnaK(1-392) or DnaK(1-507) are shown in yellow and green, respectively. Residues showing enhanced microsecond-millisecond dynamics upon ATP binding, i.e., whose assignments were obtained for the isolated NBD but not for DnaK(1-552), are shown in red (see Extended Experimental Procedures). The unassigned residues in either construct are shown in dark gray; the rest are shown in light gray (see also Figure S3A).
(C) Ribbon representation of the Hsp110-based homology model showing effects of “soft” mutations on backbone (L454I, D481N, L390V, and E511D) and methyl (M515I) chemical shifts of ATP-bound (docked) DnaK(1-552). A site of mutation and the residues affected (see Extended Experimental Procedures) are shown as dark- and light-colored spheres, respectively (see also Figures S3B and S3C) Trp102, which becomes solvent exposed in the ADP-bound conformation of DnaK (Buchberger et al., 1995), is shown in orange sticks on right. NBD residues without backbone assignments are shown in black.
(D) Interfaces between the NBD, β-SBD and α-helical lid are shown mapped onto the two endpoint Hsp70 states, domain undocked/linker unbound (PDB 2kho) and domain docked (Hsp110-based homology model [see Experimental Procedures]): residues at the interface between the β-SBD and α-helical lid, which is stabilized by substrate binding, are in red, and residues at the interface between the NBD and either the β-SBD or the α-helical lid, which is stabilized upon cooperative ATP and linker binding, are in blue; residues participating in both interfaces are in yellow.
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6. Figure 4 The Allosterically Active DnaK Intermediate
(A) Ligand-driven changes in the DnaK conformational ensemble are shown. Blowup of a representative region of methyl-TROSY spectra of ATP-bound DnaK in the absence of substrate (gray, the domain-docked state) and with substrate (black, the domain-undocked ensemble of linker-bound and -unbound conformations), overlaid on spectra of the isolated NBD, with linker (DnaK(1-392)) (blue, the linker-bound conformation) or without linker (DnaK(1-388)) (red, the linker-unbound, domain undocked conformation). Similar results were obtained for wild-type DnaK without the T199A mutation (see Figure S4).
(B) Linker-binding site on the NBD: residues that display significant chemical shift differences (more than 0.03 and 0.3 ppm for 1HN and 15N atoms, respectively) between the NBD of ATPγS-bound DnaK(1-552) (which samples both linker-bound and linker-unbound domain-undocked conformations) and its “soft” mutant L390V are shown in yellow on the modeled structure of the allosterically active conformation. To schematically model this conformation, the SBD of Hsp110-based homology model was replaced by the SBD from the ADP-bound DnaK (PDB 2kho) (see also Figure S4).
(C) The isoleucine region of the methyl-TROSY spectrum of the two-domain allosterically defective 389DDD391 DnaK(1-605) bound to ATP and substrate (black) showing near-perfect overlap with spectra of the individual, nonlinker-bound NBD, DnaK(1-388) (red).
(D) Schematic illustration of two coupled conformational transitions in DnaK: (1) between the domain-docked conformation and the domain-undocked ensemble (corresponding to a transition between gray [ATP-bound] and black [ATP/substrate-bound] peaks on left); and (2) between linker-bound and -unbound conformations (corresponding to a transition between NBD plus linker [blue] and NBD only [red] peaks in A). Note in full-length DnaK that this transition is fast on the NMR timescale and that black peaks on the left correspond to the dynamic domain-undocked ensemble of these two conformations. Interdomain interfaces are colored as in Figure 3D.
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7. Figure 5
The Impact of Competition between the NBD-β-SBD and β-SBD-α-Helical Lid Interfaces on the Hsp70 Allosteric Landscape
(A) DnaK sequence modifications that result in perturbations in its conformational ensemble are mapped onto the modeled structure of the allosterically active conformation (modeled as for Figure 4): L390V and C-terminal truncations, which favor domain docking, are shown in magenta; K414I, which favors domain undocking, is shown in green.
(B) Destabilization of the NBD-β-SBD interface results in domain undocking even in the absence of substrate. Blowup of the amide-TROSY spectra of DnaK(1-392) (blue, representing the NBD linker-bound state) and DnaK(1-388) (red, representing the NBD linker-unbound state) overlaid on the spectra of two-domain DnaK under conditions that stabilize domain undocking, either upon substrate binding to the ATP-bound DnaK(1-605) (top panels) or upon perturbation of the NBD-β-SBD interface, viz ATP-bound DnaK(1-552)K414I (middle panels), or ATPγS-bound DnaK(1-552) (bottom panels). Resonances shown (Gly6, Tyr193, Gly229, and V340) report on long-range conformational changes in the nucleotide-binding site upon linker binding to the NBD (see Extended Experimental Procedures). The spectra of the isolated NBD constructs are with the corresponding nucleotide bound (ATP, top and middle panels) or ATPγS bound (bottom panels).
(C) Stabilization of the NBD-β-SBD interface in DnaK(1-605)L390V or (D) destabilization of the β-SBD-α-helical lid interaction in DnaK(1-552) favors the domain-docked conformation even in the presence of substrate. A representative region of the amide-TROSY spectra of the ATP-bound state of DnaK(1-552) (magenta; the domain-docked conformation) and ATP/substrate-bound DnaK(1-605) (green; the domain-undocked ensemble of linker-bound and linker-unbound conformations) overlaid with spectra of DnaK(1-605)L390V (C) and DnaK(1-552) (D), shown in black. Consistent with previous biochemical studies by Kumar et al. (2011) and Swain et al. (2007), neither the L390V mutation on the NBD-β-SBD interface nor disruption of the β-SBD-α-helical lid interface in DnaK(1-552) affects the ATP-bound conformation in the absence of substrate.
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8. Figure 6
Evolutionary Variation at the NBD-β-SBD and β-SBD-α-Helical Lid Interfaces Modulates the Hsp70 Allosteric Landscape
(A–C) Sequence conservation and diversity at the NBD-β-SBD interdomain interface in the Hsp70 family. (A) Sequence conservation between different Hsp70 family members colored from blue for fully conserved residues to red for residues with no conservations (see Experimental Procedures) shown on the structure of ADP-bound DnaK state (PDB 2kho). (B) Multiple sequence alignment (ClustalW) of DnaKs from E. coli, Bacteroides thetaiotaomicron VPI-5482 (BT), Bifidobacterium longum NCC2705 (BL), Cytophaga hutchinsonii (CH), and Streptomyces coelicolor A3(2) (SC), and four human Hsp70s (Hsc70, HspA1, endoplasmic reticulum-BiP, and mitochondrial-MtHsp70). (C) Observed amino acid substitutions identified in (B) are shown on the Hsp110-based homology model.
(D) The isoleucine region of methyl-TROSY spectra of ATP-bound full-length DnaK in the absence of substrate (blue, corresponding to the docked state) and with substrate (red, corresponding to the domain-undocked ensemble, which is comprised of a mixture of linker-bound and linker-unbound conformations). Overlaid on these are variants that partition between docked and undocked states, including ATP/substrate-bound DnaK(1-552) (green), ATP/substrate-bound DnaK(1-552)L454I (light blue), and ATP/substrate-bound DnaK(1-552)D481N (orange). ATP/substrate-bound DnaK(1-552)L484I shows very similar results to L454I (Table S1). All experiments were performed at saturating substrate concentrations (2 mM of NR peptide) (see also Figure S5).
(E) Histogram showing the degree of substrate-induced domain undocking for individual constructs, colored as in (C and D). For each DnaK variant, the degree of domain undocking was estimated as described in the Experimental Procedures.
(F and G) Tuning of DnaK functionality. (F) Equilibrium binding of a fluorescently labeled peptide substrate to DnaK(1-552) (green) and its variants L454I (cyan) and D481N (orange) in the presence of ADP (open circles) and ATP (filled circles). KD values are 5.7 ± 1.0 μM (60 ± 20 μM), 6.9 ± 1.0 μM (80 ± 20 μM), and 6.8 ± 1.0 μM (300 ± 70 μM) for the ADP-(ATP-)bound state of DnaK(1-552), DnaK(1-552)-D481N, and DnaK(1-552)-L454I, respectively (see Extended Experimental Procedures). (G) Stimulation of the ATPase activity of wild-type DnaK (red) and its variants DnaK-L454I (cyan), DnaK-D481N (orange), and DnaK(1-552) (green) by 200 μM NR peptide. Error bars show SDs from the means for three replicate experiments (see Figure S5I and Extended Experimental Procedures).
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9. Figure 7 Mechanism of Hsp70 Allostery
(A and B) Schematic illustration of Hsp70 allosteric landscapes showing how the allosterically active state serves as an intermediate between the two “endpoint” states (A) and (B) how thermodynamic coupling of Hsp70 domains determines the conformations the protein populates along its allosteric cycle (see also Figures 4D and S6): binding of ADP and substrate favors interactions between the β-SBD and α-helical lid (red, in the domain-undocked conformation); ATP-induced linker binding to the NBD favors NBD-SBD docking (blue, in the domain-docked conformation). In the presence of both ATP and substrate, an interdomain energetic “tug-of-war” results in a highly dynamic and tunable conformational ensemble. The interdomain linker and helix B provide flexible and ligand-adjustable connections among the NBD, the β-SBD, and the α-helical lid.
(C) Illustration of the roles of energetic interdomain coupling and “tunability” in the allosteric cycle. The interfaces between the NBD and the β-SBD and between the β-SBD and the α-helical lid are shown in blue and red, respectively, and in magenta are shown residues that participate in both interfaces. These interfaces define the thermodynamics and kinetics of the allosteric cycle and can be modulated by either intrinsic (sequence changes) or extrinsic (binding to nucleotide, substrates, cochaperones) factors.
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10. Figure S1
Conformations Sampled by DnaK in Its Allosteric Cycle, Related to Figure 1
(A and B) Functionality of DnaK. (A) Equilibrium binding curves of F-p5 peptide to nucleotide-free (black) and ATP-bound (red) wild-type (WT) DnaK measured by fluorescence anisotropy; the determined KD values were 0.24 (±0.1) μM and 5 (±1.0) μM in the absence and presence of ATP, respectively, in good agreement with values obtained previously (Pierpaoli et al., 1998). (B) Stimulation of the WT DnaK ATPase activity by peptide (peptide-stimulated rate in red, basal ATPase rate in gray). Error bars show standard deviations from the means for three replicate experiments.
(C) The Hsp110-based homology model predicts rigid body reorientations between the NDB, β-SBD, and α-helical lid, and relatively small changes in internal structures. Domain-undocked/linker-unbound (blue; PDB 2kho) and docked (red; the Hsp110-based homology model) conformations of DnaK are superimposed; the root-mean-square deviations (RMSDs) of Cα atoms within the domains between the two structures are 3.1 (NBD), 2.0 (β-SBD), and 1.9 (α-helical lid) Å.
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11. Figure S2
Methyl NMR Reveals Three Distinct Hsp70 Conformations, Related to Figure 2
(A) Effect of α-helical lid truncation on the DnaK conformational ensemble. The isoleucine region of methyl-TROSY spectra of full-length DnaK (black) and DnaK(1-552) (red) in the apo (left), ATP-bound (middle), and ATP/substrate-bound (right) states. Only local chemical shift perturbations near the α-helical lid truncation site (labeled with arrows and residue numbers) were observed upon truncation of the lid in the apo and ATP-bound states. In contrast, the lid truncation significantly affects the DnaK conformation in the ATP/substrate-bound state (right).
(B) The isoleucine region of methyl-TROSY spectra of full-length DnaK in the apo (black), ATP-bound (red), and ATP/substrate-bound (green) states.
(C) The isoleucine region of methyl-TROSY spectra of full-length ADP-bound DnaK constructs (black) are overlaid with spectra of the individual NBD, DnaK(1-388) (blue) and SBD, DnaK( ) (green). In the ADP-bound state of DnaK, the NBD and SBD are independent and behave as ‘two beads on a string’.
(D) ATP binding results in conformational flexibility in the DnaK ensemble: Very significant line broadening and decreased peak intensities, indicating enhanced μs-ms conformational dynamics, were observed in ATP-bound DnaK both in the presence and absence of substrate. Amide-TROSY spectra of DnaK(1-605): the ADP-bound state (left) and ATP-bound states in the absence (middle) and presence (right) of substrate. To prevent formation of soluble aggregates protein concentration was less than 100 μM; further reduction of the protein concentration had no effect on the line-widths. Even more significant line broadening was observed in DnaK(1-552), which has no propensity for aggregation.
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12. Figure S3
Characterization of the Docked, ATP-Bound DnaK Conformation, Related to Figure 3
(A) Histograms showing residue-specific chemical-shift differences, Δδ = √[(ΔδH)2 + (0.154ΔδN)2 + (0.341ΔδCO)2]; where ΔδH, ΔδN, and ΔδCO are 1HN, 15N, and 13CO chemical-shift differences between the ATP-bound state of DnaK(1-552) and either DnaK(1-392) (top panel) or DnaK(1-507) (bottom panel). Residues with significant Δδ (at least one ΔδH, ΔδN, or ΔδCO value more than two-fold larger than the corresponding chemical-shift errors, i.e., 0.08, 0.8, and 0.5 ppm for 1HN, 15N, and 13CO atoms, respectively) are colored yellow (top panel) and green (bottom panel); the residues showing enhanced μs-ms dynamics upon ATP binding, i.e., whose assignments were obtained for the isolated NBD but not for DnaK(1-552), are shown in red (see Extended Experimental Procedures); the rest are shown in gray. The top bar shows NBD subdomains: IA and IIA (gray), IB and IIB (white), crossing α helices (light gray, α), and the nucleotide-binding site (black); red arrows highlight residues from the nucleotide-binding site with very large (Δδ > 0.2 ppm) chemical shift changes.
(B–D) Analysis of ‘soft’ mutations to explore SBD-NBD interfaces. (B) The isoleucine region of methyl-TROSY spectra of DnaK(1-552) (black) and its M515I variant (red). NBD residues I115 and I160 and SBD residue I512 showing significant chemical shift perturbations upon the M151I mutation [ΔδH > 0.01 ppm or/and ΔδC > 0.1 ppm, where ΔδH, and ΔδC, are chemical shift differences for methyl 1H and 13C atoms between the ATP-bound states of DnaK(1-552) and DnaK(1-552)-M515I] are labeled. (C) The isoleucine region of methyl-TROSY spectra of the ATP-bound DnaK(1-552) (black) and a corresponding ‘soft’ mutation (red). Residues showing significant changes in methyl spectra are labeled. The high similarity between methyl and backbone spectra in the absence and presence of ‘soft’ mutations indicates that ‘soft’ mutations do not perturb protein conformation and thus result in only local effects around the mutation sites. (D) Histograms showing residue-specific backbone chemical-shift differences in the NBD of ATP-bound DnaK(1-552) upon the introduction of ‘soft’ mutations L454I, D481N, L390V, or E511D: Δδ = [√(ΔδH)2 + (0.154ΔδN)2]; where ΔδH and ΔδN are 1HN and 15N chemical-shift differences between DnaK(1-552) and its variants; to avoid peak overlap, amide chemical shifts were obtained from HNCO spectra. Residues with significant Δδ (ΔδH or/and ΔδN values greater than 0.03 and 0.3 ppm for 1HN and 15N atoms, respectively) are colored red and labeled, unperturbed residues are shown in cyan, and unassigned residues are show in black bars on the bottom.
(E and F) ATP-Induced domain-docking destabilizes helix B1 in DnaK(1-552). (E) Helix B secondary chemical shifts [i.e., differences between observed CA chemical shifts and the corresponding random-coil values (Wishart and Sykes, 1994)] for apo (black) and ATP-bound (red) DnaK(1-552). (F) Peak intensities for SBD peaks in the 3D HNCO spectrum of the docked (ATP-bound) state of DnaK(1-552). Unassigned SBD peaks and peaks corresponding to helix B of the α-helical lid were separated and sorted by their intensities. Peaks corresponding to helix B (red) were at least 5 times more intense than the majority of SBD peaks. Enhanced peak intensities (F) and the absence of helical content based on CA chemical shifts (E) indicate that helix B is mobile and significantly destabilized in the domain-docked (ATP-bound) conformation of DnaK(1-552).
(G–I) SBD docking stabilizes a new lidless β-SBD conformation, which should have low substrate affinity and fast substrate binding/release. (G) Overlay of amide-TROSY spectra for the isolated SBD [red, (DnaK( ) comprising the β-SBD and helices A and B, corresponding to the domain-undocked SBD conformation with high substrate affinity] and ATP-bound DnaK(1-552) (black). Peaks corresponding to β-SBD and helix A in the DnaK(1-552) spectrum (the docked SBD conformation with low substrate affinity) are highlighted by blue boxes. There is virtually no similarity in peak positions between the two β-SBD conformations in 3D HNCO spectra, indicating that chemical shift differences for the majority of SBD peaks in the two SBD conformations are significantly greater than ∼0.2, 1 and 0.5 ppm for 1HN, 15N, and 13CO atoms, consistent with large ( ppm) 13C-methyl chemical shift differences, (see (H) and Extended Experimental Procedures). Well dispersed proton chemical shifts indicate that both conformations are well folded. (H) The isoleucine region of methyl-TROSY spectra of the uniformly (back) and segmentally (yellow, NBD-labeled only) labeled ATP-bound full-length DnaK overlaid on the spectrum of the isolated SBD (red with labeled assignments), corresponding to the β-SBD conformation in the domain-undocked, high substrate affinity state. For the majority of β-SBD residues the 13C chemical shift differences between docked and allosterically active conformations are greater than ppm (see Extended Experimental Procedures). For example, red arrows highlight changes in peak positions for several assigned methyl groups between the two alternative SBD conformations. (I) Local effects of α-helical lid truncation on amide chemical shifts of the β-SBD. (Top) histogram showing total chemical-shift differences, Δδ = [√(ΔδH)2 + (0.154ΔδN)2] as a function of residue number; where ΔδH and ΔδN, are1HN and 15N β-SBD chemical-shift differences between the apo SBD [DnaK( ), β-SBD plus helices A and B] and apo DnaK(1-507) [NBD and β-SBD]. Significant perturbations (i.e., with Δδ > 0.2 ppm) are shown in red, with the rest in gray. (Bottom) Mapping of the residues (red) included in the analysis (top) onto the SBD structure.
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13. Figure S4
Characterization of the Domain-Undocked Conformational Ensemble, Related to Figure 4
(А) Histograms showing residue-specific chemical-shift differences in the NBD of ATPγS-bound DnaK(1-552) [in this state the protein samples the ensemble of domain-undocked conformations, i.e., linker-bound and linker unbound (see text)] upon introduction of the ‘soft’ mutation L390V: Δδ = [√(ΔδH)2 + (0.154ΔδN)2]; where ΔδH and ΔδN are 1HN and 15N chemical-shift differences between the DnaK(1-552) and its mutant. Residues with significant Δδ (ΔδH or/and ΔδN values greater than 0.03 and 0.3 ppm for 1HN and 15N atoms, respectively) are colored yellow and labeled, unperturbed residues are shown in cyan, and unassigned residues are shown in black.
(B and C) Intensities of the unbound linker resonances are reduced in ATP/substrate-bound DnaK, ATP-bound DnaK(1-552)K414I, and ATPγS-bound DnaK(1-552) relative to apo-DnaK (domain-undocked, linker-unbound), arguing that the linker is unbound in a fraction of the population and bound in the rest: (B) Blowup of the amide-TROSY spectra of (red) ATP-/substrate-bound DnaK(1-605) (left), ATP-bound DnaK(1-552)-K414I (middle), and ATPγS-bound DnaK(1-552) (right) with apo DnaK(1-552) (black) illustrating representative behavior for one of linker residues, L390. (C) Ratio of normalized HNCO peak intensities (see Extended Experimental Procedures) of interdomain linker residues V389, L390, L391, L392, and D393 between ATP/substrate-bound DnaK(1-605) (left), ATP-bound DnaK(1-552)K414I (middle), and ATPγS-bound DnaK(1-552) [comprising a mixture of the two domain-undocked conformations, linker-bound and -unbound] to their intensities in the corresponding apo DnaK constructs (sampling only the linker-unbound conformation).
(D and E) The T199A mutation does not alter the transition between the SBD-bound and unbound conformations. (D) Blowup of amide-TROSY spectra of different nucleotide-bound states of WT DnaK(1-392) [NBD+linker] and its T199A variant. Changes in peak position for the linker residue L392 reflect changes in linker environment from water-exposed in the apo state to NBD-bound in the ATP- or ATPγS-bound state. The strong similarity in the L392 resonance position for the WT protein and its T199A mutant indicates that in the presence of ATP or ATPγS the linker adopts a very similar NBD-bound conformation whether the T199A mutation is present or not. Note that the linker-bound conformation and linker-binding site on the NBD of the T199A variant of DnaK(1-392) were previously characterized by our group (Zhuravleva and Gierasch, 2011). (E) The isoleucine region of the methyl-TROSY spectrum of ATPγS-bound full-length WT DnaK (black) is overlaid on the spectrum of the individual ATPγS-bound WT DnaK(1-392) [NBD+linker, red]. The spectra of ATPγS-bound WT DnaK constructs contain resonances from the ADP-bound state (due to ADP impurities in commercially available ATPγS and/or slow ATPγS-hydrolysis by WT DnaK), and the spectra are also overlaid with ADP-bound WT DnaK(1-392) (light blue). The spectrum of the ATPγS-bound WT DnaK is almost identical to that of WT DnaK(1-392) (corresponding to the linker-bound, allosterically active NBD conformation), while a number of small but significant perturbations were observed between the ATPγS -bound WT DnaK spectrum and the spectrum of WT DnaK(1-388) [corresponding to the linker-unbound NBD conformation (red); blowup on left, same region as in Figure 4A].
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14. Figure S5
Characterization of the Equilibrium between Domain-Docked and Undocked Conformations, Related to Figure 6
(A) A stress-response assay showing that E. coli DnaK and its L454I and D481N variants efficiently rescue growth in an E. coli DnaK knockout strain under heat-shock conditions (43°C).
(B and C) The isoleucine region of methyl-TROSY spectra of ATP-bound full-length DnaK in the absence of substrate (blue, corresponding to the docked state) and with substrate (red, corresponding to the domain-undocked ensemble, which is an interconverting mixture of linker-bound and linker-unbound conformations) overlaid with spectra of ATP-bound DnaK(1-552) in the presence of (B) 2.6 or 4.6 mM of NR-peptide, and (C) 2.6 mM of NR-peptide or 1.6 mM of p5-peptide (see detailed discussion in Extended Experimental Procedures). No chemical shift perturbations were observed either at p5 concentrations higher than 1 mM or at NR concentration higher than 2.6 mM.
(D and E) Characterization of the substrate-induced transition between the domain-docked and domain-undocked conformations. (D) Blowup of methyl spectra of ATP/substrate-bound DnaK(1-552) [black, ∼100 μM protein and 2.6 mM NR peptide as a substrate] with domain-docked [in red, ATP-bound DnaK(1-552)] and domain-undocked [in blue, allosterically active, ATP-bound DnaK(1-392) [NBD+linker]] conformations at two temperatures, 15.2°C and 26°C. At 15.2°C exchange is slow and two peaks are observed for individual NBD residues with positions similar to the domain-docked and -undocked conformations, while at the higher temperature (26°C), the two peaks collapse into a single peak. (E) Traces through selected cross-peaks for the 13Cδ of Ile88, Ile462, and Ile483 from the methyl-TROSY spectra of DnaK and its variants shown and colored as in Figures 6D and 6E: ATP-bound full-length DnaK in the absence (blue, corresponds to the domain-docked conformation) and presence (red, corresponds to the domain-undocked ensemble) of substrate, ATP/substrate-bound DnaK(1-552) (green) and its variants L454I (light blue) and D481N (orange). For the majority of NBD residues, the interconversion between the domain-docked and -undocked states is fast on the NMR timescale (i.e., kex > Δδ, where kex is the exchange rate constant and Δδ is the 13Cδ chemical shift difference between the two conformations), while for the SBD residues the interconversion is in the slow-to-intermediate regime (kex ≤ Δδ). (Further details are in the Extended Experimental Procedures.) (Bottom) traces through cross-peaks for the 13Cδ of residue Ile88 for the methyl-TROSY spectra for ATP/substrate-bound DnaK(1-507) (black), DnaK(1-552) (green), and full-length DnaK (red).
(F–I) Fluorescence and ATPase assays (for assay details see Extended Experimental Procedures) show good agreement with NMR results in terms of the degree to which NR and p5 peptide substrates shift the equilibrium between the domain-docked and –undocked conformations in different ATP-bound DnaK constructs. (F) Populations of the domain-undocked conformation(s) as a function of NR (left) and p5 (right) peptide concentrations obtained from time-resolved tryptophan fluorescence analysis. (G) Blowup of I88 regions of methyl spectra shown on Figures 6D and S6C (right). (H) Difference between the emission maximum of tryptophan fluorescence in the presence of ADP [corresponding the SBD-unbound conformation(s)] and ATP either without NR peptide (corresponding to the SBD-bound conformation, white bars) or with NR peptide (gray bars) for the WT full-length DnaK, WT DnaK(1-552) and WT DnaK(1-552) variants L454I and D481N. Error bars show standard deviations from the mean for three replicate experiments. (I) Stimulation of the ATPase activity of the WT DnaK (red) and its variants DnaK-L454I (cyan), DnaK-D481N (orange), and DnaK(1-552) (green) at different NR peptide concentrations, demonstrating that stimulation of the ATPase activity significantly varies for these DnaK constructs even under substrate-saturated conditions. Error bars show standard deviations from the mean for three replicate experiments.
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15. Figure S6
Conformational Selection Model for the Substrate-Induced Transitions between Domain-Docked and -Undocked Conformations, Related to Figure 7
(A) Schematic illustration of the DnaK allosteric cycle comprising three structurally and functionally different conformations: the domain-docked conformation and the two domain-undocked conformations, linker-bound (allosterically active) and linker-unbound, the distributions of which depend on the ligand-bound state (an extended version of Figure 4C).
(B and C) Theoretical dependences of the population of the domain-undocked conformation as a function of (B) substrate concentration and (C) the population of the domain-undocked conformation in the absence of substrate (see Extended Experimental Procedures for model details). Parameters similar to the experimental ones were used for simulations, KDU = 10 μM, KDD = 100 or 500 μM (gray), or alternatively, no binding to the domain-docked conformation was considered (black). The total concentration of DnaK was 100 μM, the total substrate concentration was 2 mM (for C), and the ratio between the undocked and docked conformation in the absence of substrate, K, equals 0.02 (for B). For (C) the red arrow illustrates that even a small (e.g., 1%) increase in population of the domain-undocked conformation (corresponding to changes in the SBD-NBD affinity expected to result from the interdomain energetic ‘tug-of-war’ between the NBD-SBD interface and interactions between the β-SBD and α-helical lid) in the absence of substrate may result in a very significant increase (tens of %) in the degree of domain undocking upon substrate binding (shown by blue arrows).
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