The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains

Abstract

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle.

Keywords: 70-kilodalton heat shock protein (Hsp70); DnaK; Hsp70; allosteric regulation; allostery; chaperone DnaK (DnaK); conformational simulation; molecular chaperone; molecular dynamics.

Figure 1.

A, the allosteric cycle of DnaK showing the undocked/ADP-bound state (top) and docked/ATP-bound state (bottom) (PDB codes 2KHO (7) and 4JN4 (5), respectively). B, close-up view of the docked linker in the ATP-bound state, corresponding to the region labeled in the bottom structure in A. The crossing helices are highlighted in cyan. C, the relative orientations of the NBD and SBD in ADP-bound DnaK are restricted to the gray cone, based on NMR analysis (7). In all images, the NBD is colored blue, βSBD is green, the VLLL linker sequence is orange, and the α-helical lid is red. The nucleotides, ATP in the docked state, and ADP in the undocked state, are colored pink. The substrate (superimposed from PDB entry 1DKZ (17)) is colored yellow.

Figure 2.

Representative multiple-sequence alignment for interdomain linkers of Hsp70s from a variety of organisms. The logos (19) denote how conserved each residue is within the full 640-sequence MSA (18). The division into hydrophilic and hydrophobic regions is denoted at the bottom. The residue numbers correspond to those in DnaK.

Figure 3.

Predominant secondary structures adopted by each linker residue when the linker is part of ADP-bound DnaK. A, the population is given as a fraction of the configurations sampled by MD. Extended structure is defined by ϕ dihedral angles between −200° (160°) and 25° and ψ dihedral angles between 50 and −140° (220°). Helical structure is characterized by ϕ dihedral angles between −110 and 25° and ψ angles between −125 and 85°. Lys³⁸⁷ populates two substates. One (Helical) has canonical ϕ, ψ values, and the other (Helical′) is distorted with a ϕ angle between −180 and −110°. Left-handed helix conformations are defined by ϕ dihedral angles between 25 and 160° and ψ dihedral angles between −60 and 100°. Dihedral angles not within the ranges described above are considered “other”. These other states are included in the total population count. B, ΔδCα − ΔδCβ secondary chemical shifts (23) from residue 384 to 395 in the two-domain construct, DnaK(1–552), in the ADP-bound state. Positive values indicate α-helical propensity, and negative values indicate β-structure propensity. Values greater than 2 ppm or less than −2 ppm suggest fully formed secondary structure, whereas smaller deviations often indicate partial secondary structure propensities.

Figure 4.

A, overlay of linker conformations based on minimizing root mean square deviation using all backbone atoms (top) and then (from left to right) by all backbone atoms for the hydrophilic, central hydrophobic, and C-terminal regions, separately (bottom). B, conformational landscape projected over two intralinker atom distance pairs. The conformational landscape is a 2D histogram with each bin normalized by taking the negative logarithm of the total bin population. Each basin is labeled by the state it corresponds to. C, overlays by all backbone atoms for each basin in the conformational landscape. Each overlay is labeled by the basin it occupies. D, linker–SBD conformation for each of the minima, with the SBD area contacting the linker indicated (within 5 Å, highlighted in cyan). Key residues are highlighted in blue, SBD is green, the hydrophobic linker region is orange, and the hydrophilic region is magenta.

Figure 5.

A, linker interaction with the SBD pocket in the predominant state from MD simulations. Depictions of the allosteric opening and closing of this pocket follow. B, the open pocket in the SBD crystal structure (PDB code 1DKX (16)) partially occupied by the neighboring molecule's linker; C, the open arrangement as found in ADP-bound DnaK (PDB code 2KHO (7)); D, the closed arrangement as found in ATP-bound DnaK (PDB code 4JN4 (5)); E, structure of DnaK SBD with PET-16 (fuchsia) bound to the SBD pocket (PDB code 4R5G) (24); F, SBD pocket with residues highlighted in cyan, which, from an early NMR study (25), constitute a secondary binding site for model substrate peptides. Key charged residues are highlighted in blue and labeled. The color scheme is the same as in Figs. 1 and 4.