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. 2017 May 26;292(21):8773-8785.
doi: 10.1074/jbc.M116.770404. Epub 2017 Apr 20.

Key features of an Hsp70 chaperone allosteric landscape revealed by ion-mobility native mass spectrometry and double electron-electron resonance

Alex L Lai  1 , Eugenia M Clerico  2 , Mandy E Blackburn  3 , Nisha A Patel  4 , Carol V Robinson  4 , Peter P Borbat  1 , Jack H Freed  1 , Lila M Gierasch  5   6
Affiliations

Key features of an Hsp70 chaperone allosteric landscape revealed by ion-mobility native mass spectrometry and double electron-electron resonance

Alex L Lai et al. J Biol Chem. .

Abstract

Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains (viz. the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the Escherichia coli Hsp70 DnaK by two complementary methods, ion-mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms.

Keywords: 70-kilodalton heat shock protein (Hsp70); Hsp70; allosteric regulation; chaperone DnaK (DnaK); conformational change; double electron-electron resonance; energy landscape; ion-mobility mass spectrometry; molecular chaperone.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Allosteric conformational rearrangement of DnaK. A, left, domain-undocked DnaK (PDB entry 2KHO (6)). Bound ADP (magenta) was introduced into the structure using PDB entry 3ATU (54), and bound peptide substrate (orange) was introduced using PDB entry 1DKZ (5). Right, domain-docked DnaK (PDB entry 4B9Q (8)). ATP is shown in cyan. In both panels, the NBD is blue, the β-subdomain of the substrate-binding domain is green, the α-helical lid is red, and the conserved hydrophobic sequence of the interdomain linker (VLLL) is yellow. *, the unstructured C-terminal tail is not shown. (Structures were prepared using PyMOL (Schrödinger, LLC, New York).) B, simplified schematic of the interconversion between the domain-docked and domain-undocked states of DnaK in an energy landscape. The addition of both ATP and substrate ligands creates an intermediate state that may or may not be a true basin on the energy landscape. The structures shown illustrate the interaction surfaces that stabilize the two end-point allosteric states of DnaK. Red surface, βSBD/αSBD interface that forms in the domain-undocked structure; blue surface, SBD/NBD interface that forms in the domain-docked structure.
Figure 2.
Figure 2.
The conformational ensembles of DnaK deduced from IMMS and DEER measurements. A, CCS distributions are shown for all ligand-bound forms of DnaK for ion +17, because this ion is populated under all ligand-bound conditions (ATDs for some ions are shown in supplemental Fig. S1). ATDs for all ions were fit to Gaussian components, as described under “Experimental procedures,” and for each component, the CCS was calculated and used to convert the x axis to CCS (Å2). The components of the same ion are indicated as docked (D), domain-docked with detached α-helical lid (D′), domain-undocked with domains close (C), undocked (U), or partially docked (P). B and C, left, estimated distance between spins in DnaK* Cys52-Cys410 and DnaK* Cys333-Cys410, respectively, based on Cβ–Cβ distance in PDB 2KHO and 4B9Q. The estimated distances do not consider the spin-label side chains. Right, baseline-subtracted and normalized data showing time evolution of the interspin coupling. Bottom panels, interspin distance distributions for spin labels on DnaK* in the indicated ligand-bound states. Solid lines, experimental data; dotted lines, fitted Gaussian components. Blue, ATP-bound; green, ADP/substrate-bound; red, ATP/substrate-bound.
Figure 3.
Figure 3.
DEER-derived distance distributions in DnaK* Cys mutants that report on dynamics of the α-helical lid. A and B, left, measured distance between spins in DnaK* Cys410-Cys517 and DnaK* Cys52-Cys517, respectively, based on Cβ–Cβ distance in PDB entries 2KHO and 4B9Q. The estimated distances do not consider spin-label conformers. Right, baseline-subtracted and normalized data showing time evolution of the interspin coupling. Bottom panels, interspin distance distributions for spin labels on DnaK* in the indicated ligand-bound states; solid lines, experimental data; dotted lines, fitted Gaussian components. Blue, ATP-bound; green, ADP/substrate-bound; red, ATP/substrate-bound. D, docked; U, undocked; DL, dynamic lid; LC, lid closed. Because of the higher uncertainty due to signal heterogeneity and dynamics, the distributions at mid-distances for the Cys52-Cys517 mutant were fit to only one Gaussian (three total components).
Figure 4.
Figure 4.
Titration of ATP-bound DnaK* Cys410-Cys517 with NR peptide. Distance distributions of DnaK* Cys410-Cys517 in the presence of ATP as NR peptide is added in increasing concentrations, at peptide/protein ratios of 0, 2, 20, and 160 (black, red, green, and blue curves, respectively). D, docked; D′, domain docked with detached α-helical lid; DL, dynamic lid; LC, lid closed.
Figure 5.
Figure 5.
Impact of mutations that modulate the stability of the interdomain interface. A, mutated residues represented as spheres in the structures of DnaK in the undocked (PDB entry 2KHO) and docked (PDB entry 4B9Q) states. B, interspin distance distributions for spin labels on ATP/substrate-bound DnaK* Cys333-Cys410 carrying mutations that stabilize (L390V and L454I) or destabilize (D480N) the interdomain interface. Labeling of the peaks is as in Fig. 3. Data are shown for DnaK* Cys333-Cys410 without the interface mutations for comparison (black lines). Distance distributions for each mutant are shown in magenta for L390V, cyan for L454I, and orange for D480N.
Figure 6.
Figure 6.
A, schematic representation of the allosterically active state. Shown is a model of the allosterically active state, where the pink spheres represent the interdomain interface formed between NBD and the linker, and between the α and β subdomains, when ATP and substrate are bound. The backbone of DnaK has been colored as in Fig. 1A. B, schematic representation of the allosteric energy landscape of DnaK under different ligand-bound conditions. The conformations of DnaK under all nucleotide and substrate conditions detected by IMMS and DEER are represented by schematics on an energy landscape. Each energy landscape contains all of the conformations present in the ensemble that were detected by DEER and IMMS. The depth of each conformational energy well is proportional to the relative abundance based on the DEER measurements.
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