Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker

Abstract

Hsp70 chaperones assist in protein folding, disaggregation, and membrane translocation by binding to substrate proteins with an ATP-regulated affinity that relies on allosteric coupling between ATP-binding and substrate-binding domains. We have studied single- and two-domain versions of the E. coli Hsp70, DnaK, to explore the mechanism of interdomain communication. We show that the interdomain linker controls ATPase activity by binding to a hydrophobic cleft between subdomains IA and IIA. Furthermore, the domains of DnaK dock only when ATP binds and behave independently when ADP is bound. Major conformational changes in both domains accompany ATP-induced docking: of particular importance, some regions of the substrate-binding domain are stabilized, while those near the substrate-binding site become destabilized. Thus, the energy of ATP binding is used to form a stable interface between the nucleotide- and substrate-binding domains, which results in destabilization of regions of the latter domain and consequent weaker substrate binding.

Figure 1

The linker stimulates the ATPase activity of the isolated ATPase domain, similar to the effect of substrate on full-length DnaK. (A) Steady-state ATPase rates measured at pH 7.6. (B) pH-dependence of steady-state ATPase activities for full-length wild-type DnaK in the absence (△) and presence (▲) of p5 peptide, DnaK(1-388) (○), and DnaK(1-392) (●). Error bars represent standard deviation from ≥3 experiments.

Figure 2

Presence of the linker on the isolated ATPase domain confers enhanced stability and domain closure. (A) Thermal melts of nucleotide-free DnaK(1-388) in black and DnaK(1-392) in red monitored by CD at 222 nm. First derivative of the melting curves is shown at the bottom. (B) MABA-ADP release was monitored by fluorescence after addition of excess ADP. Full-length wild-type DnaK is shown in green, DnaK(1-388) in black, and DnaK(1-392) in red. (C) ESI-MS charge state distributions of DnaK(1-388) in black and DnaK(1-392) in red, with the most abundant charge state indicated by an arrow. The top four spectra were collected on the nucleotide-free state at the indicated pH, and the bottom spectrum on the ADP-bound state at pH 7.6.

Figure 3

The VLLL sequence of the linker docks onto the ATPase domain. (A) Stack plots of TROSYs on [¹⁵N]DnaK(1-388)·ADP (upper left) and [¹⁵N]DnaK(1-392)·ADP (lower right) . Intense peaks in DnaK(1-388) are indicated by arrows. (B) Same spectra as in (A), but shown overlaid in a contour map with 1-388 in black outlines and 1-392 in red. (C) Overlay of TROSY spectra for [¹⁵N]leucine-labeled DnaK(1-388)·ADP (black outlines) and DnaK(1-392)·ADP (red) with a 2D HNCO of [¹³C’]alanine/[¹⁵N]leucine-labeled DnaK(1-392)·ADP (blue outlines). (D) Overlay of TROSY spectra for [¹⁵N]DnaK(1-388)T199A·ATP (black) and [¹⁵N]DnaK(1-392)T199A·ATP (red). In B, C and D, the ADP-bound positions of C-terminal leucines of DnaK(1-392) are indicated. Resonances within the dashed box (panels B and D) provide an example of conformational heterogeneity in ATP-bound DnaK(1-388).

Figure 4

ATP binding to the two-domain DnaK protein induces domain docking that is largely reversed by substrate binding. Overlay of TROSY NMR spectra of the ¹⁵N-labeled ATPase domain (DnaK(1-388); red), SBD (H₆DnaK(387-552)ye; blue) and two-domain protein (DnaK(1-552)ye; black outlines) bound to ADP (A), ATP (B), ADP and p5 peptide (C), or ATP and p5 peptide (D). The T199A mutant was used for observation of ATP states. The W102 side chain amide is shown in an inset. The ADP-bound position of L390 in the two-domain protein is indicated in all panels by an arrow. In (B), SBD residues that apparently disappear upon ATP binding to the two domain protein are indicated by asterisks. In (A) and (C), the positions of selected ATPase domain resonances that shift upon linker binding are labeled with an x at their position in DnaK(1-392).

Figure 5

Hydrogen-deuterium exchange (HDX) reveals differential effects of ATP binding in the SBD. (A) Outline of HDX experiment. (B) Peak intensity ratios (I_ATP/I_ADP) for the 23 residues that could be quantified are represented on the SBD structure (PDB code 1DKZ). Residues undergoing faster exchange in ATP are indicated by red (I_ATP/I_ADP <0.5) and orange balls (I_ATP/I_ADP between 0.5 and 0.9). Residues that are more protected in ATP relative to ADP are shown as dark blue balls (I_ATP/I_ADP>1.1), and relatively unaffected residues are shown as cyan balls. Figure prepared using PyMOL (http://www.pymol.org).

Figure 6

A model for interdomain coupling in Hsp70 proteins. (A) In the ADP-bound state, the two domains are independent and connected by a flexible linker. When ATP binds, domain docking is accompanied by large conformational changes in both domains, and the linker is sequestered from solvent. In this docked state, portions of the SBD are stabilized while regions forming the substrate-binding pocket become dynamic. When both substrate (indicated in red) and ATP are bound, the domains are less intimately associated and the interdomain linker binds to the ATPase domain, stimulating ATP hydrolysis rates. (B) Determination of the linker binding site on the ATPase domain. Left panel, assigned ATPase domain resonances are shown as spheres colored according to their degree of shift upon linker binding (red, Δδ_av > 0.08 ppm; yellow, Δδ_av = 0.04–0.08 ppm; green, Δδ_av < 0.04 ppm; PDB code 1DKG). Right panel, an actin hot spot for protein interactions is indicated by red spheres (PDB code 1ATN) (Dominguez, 2004). Figure prepared using PyMOL (http://www.pymol.org).