Mechanisms of assembly and function of the Hsp70-Hsp40 chaperone machinery

Abstract

Hsp70 and Hsp40 molecular chaperones form a central machinery that remodels client proteins involved in numerous biological processes. Here, we integrated cryo-electron microscopy and nuclear magnetic resonance spectroscopy to determine the architecture of the full-length Hsp70-Hsp40 machinery. The structure of the complex in a physiologically inhibited state reveals distinct regulatory mechanisms. In the active state, the Hsp40 glycine-phenylalanine (G/F)-rich region acts as a pseudo-substrate for Hsp70, directly modulating refolding. This region also maintains Hsp40 in an autoinhibited state; upon binding to Hsp70, the inhibition is disrupted, exposing a cryptic client-binding site that enables client engagement and refolding. Transitions between these states are central to controlling refolding efficiency. Disrupting either the autoinhibited state or the G/F-Hsp70 interaction impairs function and elicits a compensatory heat shock response in cells. Our findings uncover the regulatory dynamics of a fundamental chaperone system, with broad implications for understanding protein homeostasis and the cellular response to stress.

Keywords: Hsp40; Hsp70; NMR; cryo-EM; heat shock response; molecular chaperones; protein folding.

Figure 1.. Architecture of the hetero-hexameric Hsp70-Hsp40-DafA complex

(A) Domain organization of Hsp70, Hsp40, and DafA from T. thermophilus. (B) Final model of the Hsp70-Hsp40-DafA complex shown in surface rendering (the cryo-EM map is shown in Figure S2). (C) Final model of the Hsp70-Hsp40-DafA complex shown in cartoon representation. Zoomed views of important inter-subunit contacts are shown: upper left, interaction between the Hsp70 C-tail and the Hsp40 CBD2; mid left, interaction of the DafA N-tail with the client-binding pocket in SBDβ; upper right, interaction between DafA and the J domain; lower right, dimeric interface of the two NBDs. (D) ¹H-¹³C methyl TROSY of [U-²H,¹⁵N; Met-¹³CH₃; Ile-δ1-¹³CH₃; Leu, Val-¹³CH₃/¹³CH₃;]-labelled Hsp70-Hsp40-DafA hetero-hexameric complex. Sample concentration was 0.15 mM. The zoomed views show overlaid spectra of the hetero-hexameric complex (grey) with free Hsp40 (blue) and free Hsp70 (orange). Representative residues affected in the complex are shown. (E) 2D class averages showing the NBD in a closed state (left), where the two NBDs interact with each other, and in an open state (right), where the two NBDs are far from each other. The closed state is markedly more prevalent in the particles. The cartoon depicts the conformational changes undergone by the NBDs between these two conformational states.

Figure 2.. Autoinhibition and interactions mediated by the G/F region in the Hsp70-Hsp40 complex

(A) The three distinct interacting surfaces between Hsp70 and Hsp40 in the hetero-tetramer identified by NMR are shown. The green arrows denote the interaction between Hsp70 C-tail and Hsp40 CBD2; the purple arrows denote the interaction between the Hsp70 SBDβ and Hsp40 G/F; and the orange arrows denote the interaction between the Hsp70 NBD and Hsp40 J domain. The Hsp70 and Hsp40 coordinates from the cryo-EM structure of the hetero-hexamer were used as a model. (B) NMR solution structure of the Hsp40 J-G/F fragment (residues 1–116). The Phe cluster residues and the HPD motif are labeled. The J domain and G/F region are colored cyan and purple, respectively. (C) Overlaid ¹H-¹⁵N HSQC spectra of the J domain (cyan), J-G/F (dark blue), and J-G/F^S94E (orange). Concentration of either sample was 0.2 mM. (D) NMR structure of the J-G/F fragment superimposed on the crystal structure of the complex between E. coli Hsp70 and the J domain from DnaJ (PDB ID 5NRO) indicating the steric clash between the G/F region and SBDβ. (E) ¹H-¹⁵N HSQC spectra of the J domain (left) and J-G/F (right) in the absence (cyan) and presence (pink) of Hsp70^mon bound to ATP. Concentration of either sample was 0.2 mM. (F) (Left) ¹H-¹⁵N HSQC spectra of the J-G/F^S94E in the absence (orange) and presence (green) of Hsp70. (Right) ITC profile of the J-G/F^S94E variant binding to Hsp70-ATP. (G) NMR structure of the Hsp70 SBDβ (yellow) in complex with a G/F peptide encompassing Phe (residues 103–109; purple). (H) AlphaFold structural model of J-G/F (red) in complex with Hsp70 (grey). Phe106 is positioned inside the SBDβ pocket as in our NMR structure (panel G) and the G/F region is partially unfolded. A zoomed view is shown on the right where the structure of free J-G/F (light blue) is overlaid. The comparison highlights the exposure of the Phe residues in the G/F region in the complex. (I) ¹H-¹⁵N HSQC spectra of unfolded PhoA (0.1 mM) in the absence (pink) and presence (green) of J-G/F^S94E (0.2 mM). (J) Normalized fluorescence polarization (FP) of the client peptide NR bound to Hsp70 upon addition of the specified Hsp40 variant. Lower values reflect a stronger effect in releasing the peptide. Data are presented as mean values ± standard deviation (SD) from a triplicate.

Figure 3.. Functional assays on the role of the G/F region

(A-D) Folding of denatured luciferase by the Hsp70-Hsp40-NEF tripartite chaperone machinery measured as a percentage of its enzymatic activity. Detailed graphs are shown in Figure S8. Data are shown as mean ± SD from a triplicate. Panel A shows the results of the T. thermophilus system, panel B shows the results of the E. coli system with DnaJ, panel C shows the results of the E. coli system with CbpA and panel D shows the results of the human chaperones DnaJA2, DnaJB1, and DnaJB6. (E) Growth assay in E. coli liquid culture of the indicated protein at 42 °C. The graph shows the time delay, compared to the wild type strains, required for each E. coli variant to reach OD of 0.5. (F) In vivo folding of denatured luciferase in E. coli expressing the indicated wild type and mutant proteins after heat shock at 47 °C measured as a percentage of its enzymatic activity. (G) Residual luciferase activity right after heat shock from the in vivo luciferase refolding assay shown in panel E. Detailed graphs are shown in Figure S8. (H,I) Volcano plots showing the differential gene expression analysis of ribosome profiling datasets from E. coli DnaJ double mutant strain compared to wild type at exponential (OD₆₀₀ 0.5) (panel G) and early stationary (OD₆₀₀ 2) (panel H) phases. Libraries were prepared in two biological replicates. Enhanced expression of heat shock proteins is highlighted in red.

Figure 4.. Assembly of the Hsp70-Hsp40 complex

(A) Structural model of the hetero-tetrameric complex Hsp70-Hsp40 generated as described in the text. Hsp70 is bound to ATP and thus adopts the “docked” state, which has the highest affinity for the J domain. G/F Phe106 binds inside the SBDβ pocket. (B) Structural model of the hetero-tetrameric complex Hsp70-Hsp40 complex with G/F Phe101 inside the SBDβ pocket. This binding entails the complete unfolding of the G/F region, which exposes the cluster of Phe residues. (C) Overlay of the structures of the hetero-tetrameric Hsp70-Hsp40 complex in the “docked” and “undocked” states. The color code is as in panel a and Figure 1c. The arrows denote the conformational changes undergone by the chaperones as the complex transitions between the two states. (D) Structural model of the Hsp70-Hsp40 hetero-tetramer in the “undocked” conformation. A cross-section rendering is depicted that shows the presence of a chamber lined by the client-binding sites.

Figure 5.. Client binding to the Hsp70-Hsp40 complex

(A) Hydrophobicity plot of PhoA as a function of its primary sequence. A hydrophobicity score (Roseman algorithm, window = 9) higher than zero denotes increased hydrophobicity. The sites identified by NMR to be recognized by Hsp40 and Hsp70 are highlighted in blue. The sites are labeled a to e. The mature form of PhoA (residues 23–471) was used. (B) Schematic of client folding mediated by the Hsp40-Hsp70 chaperone machinery refined by the current findings. (C) Structural model of Hsp40 (PDB ID 6PSI) bound to unfolded PhoA as determined previously. The backbone chain of PhoA is shown in a cartoon rendering. (D) Structural model of the Hsp70-Hsp40 complex in the “undocked” conformation bound to unfolded PhoA. (E) Structural model of the hetero-tetrameric Hsp70-Hsp40 complex in the “docked” conformation bound to unfolded PhoA. The cartoon model was generated from the cryo-EM structure of the hetero-hexamer by removing DafA.