Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones

  1. Salvatore Assenza
  2. Alberto Stefano Sassi
  3. Ruth Kellner
  4. Benjamin Schuler
  5. Paolo De Los Rios
  6. Alessandro Barducci  Is a corresponding author
  1. ETH Zürich, Switzerland
  2. Universidad Autónoma de Madrid, Spain
  3. École Polytechnique Fédérale de Lausanne (EPFL), Switzerland
  4. Yorktown Heights, United States
  5. University of Zurich, Switzerland
  6. Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland
  7. INSERM, CNRS, Université de Montpellier, France
7 figures and 3 additional files


Progressive expansion of substrate by multiple Hsp70 binding.

Probability density maps of substrate potential energy and radius of gyration for representative Hsp70/rhodanese complexes with one (left), three (center) and six (right) bound chaperones. The different Hsp70 chaperones have been represented with different colors to ease their discernibility.

Figure 1—source data 1

Text files containing the data used to generate the probability density maps in Figure 1.

Figure 2 with 1 supplement
Hsp70-induced swelling increases the conformational free energy of the system.

Conformational free-energy differences ΔG of the Hsp70/rhodanese complexes with respect to the unbound substrate (n = 0) plotted as a function of the corresponding radius of gyration Rg. Each point represents one of the 64 possible binding configurations with color code indicating the number of bound chaperones. The black curve was obtained using the model in Sanchez (1979) (see Appendix 2). (inset) Distribution of ΔΔG corresponding to the free-energy cost for binding an additional Hsp70 to a chaperone/substrate complex.

Figure 2—source data 1

Data from simulation results and Sanchez theory used to generate the plot in Figure 2 and the histogram in the inset of Figure 2.

Figure 2—figure supplement 1
Free energy computation of different combinations of bound chaperones from steered MD simulations.

Top: representative chaperone/rhodanese complex in the fully-stretched conformation. Bottom: Free-energy difference δG between equilibrium conformations and the fully-stretched state for rhodanese (red line) and two representative chaperone/rhodanese complexes. The double arrows indicate the final value of ΔG for the corresponding complex.

Figure 2—figure supplement 1—source data 1

Simulation data used to generate the free energy plots; the work performed by the pulling force for each trajectory is also included in the subfolders.

The relevant biochemical reactions of the Hsp70/rhodanese system can be described by a rate model.

Each chaperone binding site on rhodanese (black dots) can be either free or occupied by an Hsp70 (yellow), which in turn can be either ADP- or ATP-bound. We depict here for the sake of clarity only a representative portion of the full model, which takes into account six binding sites. The reaction cycle is governed by the rates for chaperone binding/unbinding to the substrate (konATP,konADP,koffATP,koffADP) and for hydrolysis (khs), synthesis (kss) and exchange (kex,DTeff,kex,TDeff) of nucleotides bound to the chaperones (see Materials and methods for further details). Importantly, the binding rate constants, konATP and konADP, take into account the conformational free energies, according to Equation (1).

Hsp70 binding is a non-equilibrium process that depends on ATP hydrolysis.

(Top) compound probabilities for Hsp70/substrate complexes with given number of bound chaperones n as a function of [ATP]/[ADP]. (Bottom) Mean value n as a function of [ATP]/[ADP] with (solid line) and without (dashed line) ATP hydrolysis.

Figure 4—source data 1

Data from rate model employed to generate the plots in Figure 4.

Simulation results match sm FRET experimental data.

Histograms of the radius of gyration for equilibrium (blue) and non equilibrium (red) values of [ATP]. The black dashed line indicates the average radius of unbound rhodanese. (inset) FRET transfer efficiencies as a function of the sequence separation between the fluorescent dyes. The black circles correspond to the experimental values (Kellner et al., 2014). Calculated efficiencies taking into account uncertainties are reported as blue (equilibrium conditions) and red circles (ATP excess).

Figure 5—source data 1

Molecular simulation data used to generate the plots in Figure 5.

The conversion of chemical energy into mechanical work by Hsp70s is optimal in cellular conditions thanks to the chaperone intrinsic rates.

(Top) Ratio between the conformational free energy and the free energy of ATP hydrolysis, as a function of [ATP]/[ADP]. Dark green curve results from data from molecular simulations and light green curves takes into account normally-distributed uncertainties on calculated ΔGi. (Bottom) Effective dissociation constant in the case of a single binding site normalized with respect to the corresponding value in equilibrium, as a function of [ATP]/[ADP] (solid black line). Ratio between the non-equilibrium excess of binding free energy ΔGb and the free energy of ATP hydrolysis ΔGh, as a function of [ATP]/[ADP] (dashed red line). The gray region indicates the interval corresponding to physiological conditions.

Appendix 1—figure 1
Normalized FRET efficiency histograms of the variant Δ39 (K135C/K174C).

Experiments were performed in the presence of 10 μM DnaK, 500 nM DnaJ, 1 mM ATP and either 5 mM (grey curves) or 100 mM (red curves) of KCl.

Appendix 1—figure 1—source data 1

Experimental data employed to generate the FRET histograms reported in the Appendix 1—figure 1.


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  1. Salvatore Assenza
  2. Alberto Stefano Sassi
  3. Ruth Kellner
  4. Benjamin Schuler
  5. Paolo De Los Rios
  6. Alessandro Barducci
Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones
eLife 8:e48491.