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. 2012 Aug 17;287(34):28470-9.
doi: 10.1074/jbc.M112.383091. Epub 2012 Jun 28.

DnaK chaperone-dependent disaggregation by caseinolytic peptidase B (ClpB) mutants reveals functional overlap in the N-terminal domain and nucleotide-binding domain-1 pore tyrosine

Shannon M Doyle  1 , Joel R Hoskins, Sue Wickner
Affiliations

DnaK chaperone-dependent disaggregation by caseinolytic peptidase B (ClpB) mutants reveals functional overlap in the N-terminal domain and nucleotide-binding domain-1 pore tyrosine

Shannon M Doyle et al. J Biol Chem. .

Abstract

Protein disaggregation in Escherichia coli is carried out by ClpB, an AAA(+) (ATPases associated with various cellular activities) molecular chaperone, together with the DnaK chaperone system. Conformational changes in ClpB driven by ATP binding and hydrolysis promote substrate binding, unfolding, and translocation. Conserved pore tyrosines in both nucleotide-binding domain-1 (NBD-1) and -2 (NBD-2), which reside in flexible loops extending into the central pore of the ClpB hexamer, bind substrates. When the NBD-1 pore loop tyrosine is substituted with alanine (Y251A), ClpB can collaborate with the DnaK system in disaggregation, although activity is reduced. The N-domain has also been implicated in substrate binding, and like the NBD-1 pore loop tyrosine, it is not essential for disaggregation activity. To further probe the function and interplay of the ClpB N-domain and the NBD-1 pore loop, we made a double mutant with an N-domain deletion and a Y251A substitution. This ClpB double mutant is inactive in substrate disaggregation with the DnaK system, although each single mutant alone can function with DnaK. Our data suggest that this loss in activity is primarily due to a decrease in substrate engagement by ClpB prior to substrate unfolding and translocation and indicate an overlapping function for the N-domain and NBD-1 pore tyrosine. Furthermore, the functional overlap seen in the presence of the DnaK system is not observed in the absence of DnaK. For innate ClpB unfolding activity, the NBD-1 pore tyrosine is required, and the presence of the N-domain is insufficient to overcome the defect of the ClpB Y251A mutant.

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Figures

FIGURE 1.
FIGURE 1.
Central pore tyrosines are conserved among Hsp100 proteins. A, top view of a model of the E. coli ClpB hexamer with bound ATP (8, 46). The image was prepared using PyMOL. The position of ATP in black is shown as a CPK model, and pore loops (red) are added by hand. B, schematic illustrating the location of the 12 pore loop tyrosines in two rings of six each in a hexamer of ClpB wild type (ClpB(WT)). The 12 sites are superimposed on a model of a ClpB hexamer that was prepared as in A. C, sequence alignments of pore loop amino acids in NBD-1 and NBD-2 of several Hsp100 proteins. The pore loop tyrosine in NBD-1 and NBD-2 is indicated in red. D and E, schematics illustrating the single pore tyrosine substitutions in pore ring-1, Y251A (D), and in pore ring-2, Y653A (E). Tyrosines substituted with alanines are in green (D) and purple (E) and wild type tyrosines are in black.
FIGURE 2.
FIGURE 2.
DnaK-dependent chaperone activity and ATPase activity of ClpB mutant proteins. A, reactivation of heat-aggregated GFP-38 by ClpB(WT), ClpB(Y1), ClpB(ΔN), and ClpB(ΔN-Y1) with the DnaK system was measured over time as described under “Experimental Procedures.” A representative experiment of three replicates is shown. ClpB(WT) reactivates ∼75% of heat-aggregated GFP-38 at an initial rate of 1.7 ± 0.1% min−1. B, ATPase activity of ClpB mutants. Basal ATPase activity of ClpB(WT), ClpB(Y1), ClpB(Y2), ClpB(ΔN), and ClpB(ΔN-Y1) was measured as described under “Experimental Procedures.” Data are means ± S.E. (n = 3).
FIGURE 3.
FIGURE 3.
Substrate binding by ClpB(WT) and ClpB mutants. A, FITC-casein binding by ClpB mutants was measured by fluorescence anisotropy as described under “Experimental Procedures.” Double Walker B mutant (Trap) variants of ClpB(WT), ClpB(Y1), ClpB(ΔN), and ClpB(ΔN-Y1) were used. B, GFP-15 binding by double Walker B mutant (Trap) variants of ClpB(WT), ClpB(Y1), ClpB(Y2), ClpB(ΔN), and ClpB(ΔN-Y1). Binding was measured by ultrafiltration as described under “Experimental Procedures.” Data for A and B are means ± S.E. (n = 3).
FIGURE 4.
FIGURE 4.
Involvement of ClpB pore tyrosines and the N-domain in substrate disaggregation with the DnaK system. A, B, and D, rate of GFP-38 reactivation by mixtures of ClpB(WT) and ClpB(Y1) (A), ClpB(WT) and ClpB(ΔN) (B), or ClpB(WT) and ClpB(Y2) (D) plotted as a function of percent ClpB mutant. ClpB(WT) reactivated ∼75% of heat-aggregated GFP-38. C, MDH disaggregation activity by 1:1 mixtures of ClpB(WT) and buffer, ClpB(WT) and ClpB(Y1), or ClpB(WT) and ClpB(Y2) in collaboration with the DnaK system and measured following a 60-min incubation. A–D, data are means ± S.E. (n = 3). Some error bars are covered by the plot symbols. A line corresponding with a linear decrease in activity was added to aid the eye (gray dashed line) in A, B, and D.
FIGURE 5.
FIGURE 5.
Alanine substitution at position Tyr-251 or Tyr-653 causes a loss of ClpB unfolding activity. A and E, unfolding of 15-GFP by ClpB(WT), ClpB(Y1), or ClpB(Y2) in the presence of ATP and ATPγS. A representative experiment of three replicates is shown. ClpB(WT) unfolds 15-GFP at an initial rate of 0.7 ± 0.1% min−1, whereas ClpB(Y1) or ClpB(Y2) have initial unfolding rates below the level of detection. B and C, rate of 15-GFP (B) or GFP-15 (C) unfolding by mixtures of ClpB(WT) and ClpB(Y1) plotted as a function of percent ClpB(Y1) in the mixture. D, unfolding of GFP-15 by ClpB(WT), ClpB(ΔN), or ClpB(ΔN-Y1) in the presence of ATP and ATPγS. A representative experiment of three replicates is shown. ClpB(WT) unfolds GFP-15 at an initial rate of 6.0 ± 0.3% min−1. ClpB(ΔN) unfolds GFP-15 similar to ClpB(WT), whereas ClpB(ΔN-Y1) has an initial unfolding rate below the level of detection. F and G, rate of 15-GFP (F) or GFP-15 (G) unfolding by mixtures of ClpB(WT) and ClpB(Y2) plotted as a function of percent ClpB(Y2) in the mixture. B, C, F, and G data are means ± S.E. (n = 3). Some error bars are covered by the plot symbols. A line corresponding with a linear decrease in activity was added to aid the eye (gray dashed line). AU, arbitrary units.
FIGURE 6.
FIGURE 6.
NBD-1 and NBD-2 pore tyrosines within a ClpB protomer function cooperatively in protein unfolding. A, rate of GFP-15 unfolding in the presence of ATP and ATPγS by mixtures of ClpB(Y1) and ClpB(Y2) at either 1 μm (open circles) or 10 μm (filled circles) final ClpB concentration plotted as a function of percent ClpB(Y1) in the mixture. B, rate of GFP-15 unfolding by mixtures of ClpB(WT) and ClpB(Y1,Y2) at 1 μm final ClpB concentration plotted as a function of percent ClpB(Y1,Y2) in the mixture. A and B, data are means ± S.E. (n = 3). Some error bars are covered by the plot symbols. A line corresponding with a linear decrease in activity was added in B to aid the eye (gray dashed line).
FIGURE 7.
FIGURE 7.
Participation of ClpB pore tyrosines in substrate interaction and translocation. A, rate of GFP-15 unfolding in the presence of ATP as the sole nucleotide by mixtures of ClpB(WT) and ClpB(Trap) (gray dashed line), ClpB(Y1) and ClpB(Trap) (green circles), or ClpB(WT) and ClpB(Y1-Trap) (blue circles) plotted as a function of percent ClpB(Trap) or ClpB(Y1-Trap) in the mixture. B, rate of GFP-15 unfolding by mixtures of ClpB(Y2) and ClpB(Trap) (purple circles) or ClpB(WT) and ClpB(Y2-Trap) (black circles) plotted as a function of percent ClpB(Trap) or ClpB(Y2-Trap) in the mixture. A and B, data are means ± S.E. (n = 3). Some error bars are covered by the plot symbols.
FIGURE 8.
FIGURE 8.
Model of protein disaggregation by ClpB and the DnaK chaperone system. ClpB is depicted using a cutaway view. Pore loops in NBD-1 and NBD-2 are represented by projections into the central pore. Briefly, the DnaK system binds aggregates. Through interactions between DnaK and ClpB, the aggregate is delivered to ClpB. ClpB engages the aggregate through exposed regions on the surface of the aggregate via the ClpB N-domain and pore loop-1. Following engagement, the polypeptide is translocated through the channel of ClpB in a reaction requiring ATP hydrolysis. Finally the unfolded polypeptide is released, and it refolds spontaneously or with the help of other chaperones. See “Discussion” for details of the model.
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