Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones

Abstract

The 70-kDa heat shock protein (Hsp70) chaperones perform a wide array of cellular functions that all derive from the ability of their N-terminal nucleotide-binding domains (NBDs) to allosterically regulate the substrate affinity of their C-terminal substrate-binding domains in a nucleotide-dependent mechanism. To explore the structural origins of Hsp70 allostery, we performed NMR analysis on the NBD of DnaK, the Escherichia coli Hsp70, in six different states (ligand-bound or apo) and in two constructs, one that retains the conserved and functionally crucial portion of the interdomain linker (residues ) and another that lacks the linker. Chemical-shift perturbation patterns identify residues at subdomain interfaces that constitute allosteric networks and enable the NBD to act as a nucleotide-modulated switch. Nucleotide binding results in changes in subdomain orientations and long-range perturbations along subdomain interfaces. In particular, our findings provide structural details for a key mechanism of Hsp70 allostery, by which information is conveyed from the nucleotide-binding site to the interdomain linker. In the presence of ATP, the linker binds to the edge of the IIA β-sheet, which structurally connects the linker and the nucleotide-binding site. Thus, a pathway of allosteric communication leads from the NBD nucleotide-binding site to the substrate-binding domain via the interdomain linker.

Fig. 1.

Nucleotide-induced conformational changes in the DnaK NBD. (A and B) Histograms showing chemical-shift differences, , for backbone atoms as a function of residue number, where Δδ_H, Δδ_N, or Δδ_CO are ¹HN, ¹⁵N, and ¹³CO chemical-shift differences between the apo and ADP-bound states of NBD³⁸⁸ (A), and between the ADP- and ATP-bound states of NBD³⁹² (B). Residues with large Δδ_tot (> 0.3 ppm) and significant chemical-shift perturbation (at least one Δδ_H, Δδ_N, or Δδ_CO value is larger than two corresponding chemical-shift errors; i.e., 0.06, 0.6, and 0.6 ppm for ¹HN and ¹⁵N, and ¹³CO atoms, respectively) are colored red and yellow, respectively; the rest are shown as cyan. The green background highlights regions that are highly affected by nucleotide binding, and the top bar shows NBD subdomains: IA (dark green), IB (light green), IIA (dark blue), IIB (light blue), crossing α-helices (red, X), the linker motif (yellow, L), and the nucleotide-binding site (black, N).

Fig. 2.

Opening of the nucleotide-binding cleft upon nucleotide dissociation. (A) Comparison of alternative conformations of subdomain IIB in X-ray structures of DnaK homologues: in green, the closed form as seen in the isolated Bos taurus Hsc70 NBD [Protein Data Bank (PDB) ID code 1KAX]; in yellow, the open form as seen in the complex of yeast Sse1 with the Bos taurus Hsc70 NBD (PDB ID code 3C7N); in red, the open form as seen in the complex of yeast Sse1 with human Hsp70 NBD (PDB ID code 3D2F). (B) Mapping of the chemical-shift differences from Fig. 1А onto the structure of the DnaK NBD (PDB ID code 1DKG:D). Residues with large chemical-shift perturbations (red) and the highly affected subdomain IIB α-helices (green) are colored as in Fig. 1А.

Fig. 3.

The cooperative effect of linker and ATP binding. (A) Relative peak intensities (Int^rel) of the C-terminal residue for Leu³⁹² for different NBD³⁹² states. The Int^rel value is the ratio of the Leu³⁹² peak height to an average peak height in an HNCO spectrum in a corresponding NBD state. (B) Blow-up of the region of the ¹⁵N TROSY spectra showing resonances corresponding to the C-terminal Leu³⁹² for different NBD³⁹² states. (C) Histograms showing combined chemical-shift differences (Δδ_tot) as a function of residue number for backbone ¹HN and ¹⁵N, and ¹³CO atoms (as in Fig. 1) between NBD³⁸⁸ and NBD³⁹² in the apo, ADP-, and ATP-bound states. Yellow and cyan colors highlight significant and insignificant chemical-shift perturbations (as defined in Fig. 1), respectively. Gray background highlights the interfaces between two lobes. The top bar is the same as for Fig. 1.

Fig. 4.

The linker binds to the hydrophobic cleft between the subdomains IA and IIA. (A) Mapping of the chemical-shift differences from Fig. 1B onto the homology model of the DnaK ATP-bound NBD structure built from the Sse1 structure (PDB ID code 2QXL:B) (Fig. S6). The regions highlighted in Fig. 1B by green background are shown in green on the structure. Residues with large chemical-shift perturbations (highlighted in red in Fig. 1B) are shown as red spheres. (B) Superposition of the ADP- (green) and ATP- (yellow) bound conformations, derived from homology models as described in the text. For the ATP-bound conformation, the C-terminal 12 residues are shown in red, and the interdomain linker () bound to the hydrophobic cleft is shown as red spheres.

Fig. 5.

An intramolecular allosteric network in the NBD. (A) Representative regions from overlaid ¹H-¹⁵N TROSY spectra of the 12 NBD states. Several examples of nonoverlapping resonances for residues with significant conformational changes are labeled to highlight their peak-walking patterns. The color code for the 12 spectra is: red (ATP.NBD³⁹²), magenta (ATPγS.NBD³⁹²), green (ADP/ADP.Pi/ADP.noMg.NBD³⁹²), light blue (apo.NBD³⁹²), orange (ATP.NBD³⁸⁸), yellow (ATPγS.NBD³⁸⁸), light green (ADP/ADP.Pi/ADP.noMg.NBD³⁸⁸), blue (apo.NBD³⁸⁸). (B) Histograms showing chemical-shift differences, , for backbone atoms as a function of residue number; where Δδ_H, Δδ_N, or Δδ_CO are the largest ¹HN, ¹⁵N, and ¹³CO chemical-shift differences between any 2 of the 12 states of NBD³⁸⁸ and NBD³⁹². Coloring and the top bar are the same as for Fig. 1.

Fig. 6.

Mechanism of intramolecular allostery in the Hsp70 NBD. (A) Mapping of allosteric hot spots onto the structure of the DnaK NBD (PDB ID code 1DKG:D): Residues with large and significant chemical-shift differences from Fig. 5B are shown in red and yellow, respectively. Cyan and gray indicate insignificant changes and residues with no data, respectively. (B) Structural model for two-way coupling pathway between the nucleotide-binding site and the interdomain linker: superposition of the IIA β-sheet and the crossing α-helices for the ATP- (yellow) and ADP- (green) bound conformations of the NBD. The ATP γ-phosphate and the linker are in red, and residues involved in nucleotide binding are shown as spheres.