Allosteric Regulation of Hsp70 Chaperones by a Proline Switch Markus Vogel, Bernd Bukau, Matthias P. Mayer  Molecular Cell  Volume 21, Issue 3, Pages 359-367 (February 2006) DOI: 10.1016/j.molcel.2005.12.017 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Structural Basis for the Proposed Proline Switch (A) Ribbon representation of the bovine Hsc70 ATPase domain (left, 1HPM) and E. coli DnaK substrate binding domain in complex with a substrate peptide (right, 1DKX) arbitrarily connected with each other by a dashed line representing nine amino acids. The arrows indicate the proposed ATP-induced movements of the helical lid and the substrate-enclosing loops. (B) Zoom into the ATP binding site (gray box in [A]). In stick representation are shown the nucleotide and the residues, which were the basis for the hypothesis of this study. Putative hydrogen bonds are indicated as dashed lines. Dotted lines indicate the close proximity of Glu175OE to Pro147N and the preceding carbonyl of Val146 both at a distance of 3.3 Å. In italics are given the residue numbers of corresponding residues in E. coli DnaK. Molecular Cell 2006 21, 359-367DOI: (10.1016/j.molcel.2005.12.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Effects of Pro143, Arg151, and Glu171 Replacements on Chaperone and ATPase Activity (A) Pro143, Arg151, and Glu171 are essential for in vitro chaperone activity. Refolding of chemically denatured luciferase plotted as fraction of the native luciferase control. (B and C) Replacement of Pro143 and Arg151 affects basal and stimulated ATPase activity of DnaK. Single-turnover ATPase rates (B) and stimulation factors (C) in the absence (white bars) and presence of DnaJ (hatched bars), σ32 (crosshatched bars), or both (black bars). Error bars represent the standard error of at least three independent experiments. Molecular Cell 2006 21, 359-367DOI: (10.1016/j.molcel.2005.12.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Effects of Pro143, Arg151, and Glu171 Replacements on ATP-Stimulated Substrate Release and Intrinsic Tryptophane Fluorescence (A) Dissociation kinetics of the nucleotide-free DnaKwt-peptide complex in the absence (−nuc, open circles) and presence of ATP (+ ATP, closed circles). The inset shows a zoom into the first 10 s. (B) Peptide dissociation rates in the absence (−nuc, black bars) and presence of ATP (+ATP, gray bars). The lower part of the graph shows an enlargement in the range from 0 to 0.002 s−1. Error bars represent the standard error of at least three independent measurements. (C) Typtrophane fluorescence reveals defects in ATP-induced conformational changes in Arg151 and Pro143 replacement variants. Difference of the emission maximum of tryptophane fluorescence in the presence of ATP minus the maximum in the absence of nucleotide. Measurements in the absence of a substrate peptide, black bars; in the presence of substrate peptide, white bars. Error bars represent the standard error of at least three independent measurements. Molecular Cell 2006 21, 359-367DOI: (10.1016/j.molcel.2005.12.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Replacement of Pro143 Destabilizes the Switch that Controls the Conformation of the Substrate Binding Domain (A) Dissociation equilibrium titration of the fluorescent-labeled peptide σ32-Q132-Q144-C-AANS (0.1 μM) with 0.1–3 μM DnaKwt (open circles), DnaK-R151A (closed triangles), DnaK-P143A (closed diamonds), and DnaK-P143G (open diamonds). (Inset) Extended titration up to 9 μM. The Kd values were determined by fitting the quadratic equation to the data and yielded 78 ± 7 nM (DnaKwt), 102 ± 1 nM (DnaK-R151A), 320 ± 80 nM (DnaK-P143A), and 730 ± 60 nM (DnaK-P143G). (B) Association of σ32-Q132-Q144-C-AANS (3 μM) to DnaK-P143G (0.5 μM) in the absence of ATP and for comparison to DnaKwt in the absence and presence of ATP. Molecular Cell 2006 21, 359-367DOI: (10.1016/j.molcel.2005.12.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 The Activation Enthalpy for ATP Hydrolysis Is Significantly Lowered by the Pro143 Replacements Eyring plot of the temperature dependence of the ATPase reaction for DnaKwt, DnaK-R151A, DnaK-P143A, DnaK-P143G, and the isolated ATPase domain of DnaKwt (DnaK[2–385]). Error bars represent the standard error of two to three independent measurements. Molecular Cell 2006 21, 359-367DOI: (10.1016/j.molcel.2005.12.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Proposed Mutual Allosteric Mechanism Controlling Conformational Changes in the Substrate Binding Domain and ATP Hydrolysis (A) ATP binding is sensed by Lys70 and Glu171 through water-bridged H bonds to the γ-phosphate and the Mg2+ (ATP association encounter complex). (B) This interaction triggers the conformational change Pro143 and moves Arg151 toward the substrate binding domain leading to the opening of this domain (prehydrolysis complex). (C) Substrate and DnaJ interaction through Arg151 trigger the reversion of the conformation change in Pro143, moving Glu171 and Lys70 to an ideal position for catalysis of ATP hydrolysis (hydrolysis transition state). Dotted lines indicate H bonds; arrows indicate movement. The gray letters and boxes show the position of the immediately preceding state for comparison. Molecular Cell 2006 21, 359-367DOI: (10.1016/j.molcel.2005.12.017) Copyright © 2006 Elsevier Inc. Terms and Conditions