Figure 1.
Nucleotide-dependent conformational states of Hsp70 s.
A: Cartoon diagram of the experimental NMR structure of E. coli ADP-Hsp70 (PDB ID: 2KHO) [70] used as input structure for the MD simulations. B: Cartoon diagram of the experimental X-ray structure of E. coli ATP-Hsp70 (PDB ID: 4B9Q) [69]. C: Schematic representation of the conformational transition as expected from experimental data and observed in MD simulations of ATP-Hsp70, starting from the closed conformation of Hsp70. The color code is the following: NBD-IA (residues 4–37, 112–182 and 363–383; red), NBD-IB (residues 38–111; green), NBD-IIA (residues 183–227 and 311–362; blue), NBD-IIB (residues 228–310; yellow), linker (residues 384–393; black), SBD-β (residues 394–502; magenta) and SBD-α (residues 503–603; cyan). These figures were prepared with PyMOL [http://www.pymol.org].
Figure 2.
Cartoon representation of the representative structures obtained by MD simulations.
A: MD runs APO1 (red) and APO2 (firebrick). B: MD runs ADP1 (blue) and ADP2 (light blue). C: MD runs ATP1 (green) and ATP2 (pale green). The NMR-derived structure used as starting point for the MD simulations (PDB ID: 2KHO) [70] is shown in transparent gray. Representative structures are extracted from a dPCA on the CGDAs γ (see Methods section). These figures were prepared with PyMOL [http://www.pymol.org].
Figure 3.
Comparison between the simulated and experimental distance distribution functions P(r) of Hsp70 s.
A: Distance distribution function P(r) of the ATP-bound (green lines) and ADP-bound (blue lines) of E. coli DnaK computed from MD simulations (solid lines) and computed from the experimental X-ray (PDB ID: 4BQ9) [69] and NMR-derived (PDB ID: 2KHO) [70] structures (dashed lines), respectively. B: Distance distribution function P(r) of the ATP-bound (green line) and ADP-bound (blue line) of bHsc70. The function P(r) of bHsc70 in the ATP and ADP states were digitized from Figure 4 of Ref. [82].
Figure 4.
Contact maps from ATP-bound and ADP-bound MD trajectories.
A: Interdomain interactions extracted from the contact map difference ATP/ADP (see text for details). Residues involved in the interdomain interactions are shown in stick representation. The color code is the same as in Fig. 1. B: Interactions between the ATP nucleotide and the NBD of DnaK from ATP-bound MD trajectories. C: Three-dimensional representation of the interactions between ATP and the NBD. Residues of the NBD in contact with ATP are shown in stick representation. The color code of the protein structure is the same as in Fig. 1. These figures were prepared with PyMOL [http://www.pymol.org].
Table 1.
List of the interdomain interactions and their corresponding average distances extracted from the contact map difference ATP/ADP (see text for details).
Figure 5.
Analysis of 1-D FEPs of CGDAs γ.
Mapping of the dissimilarity index 1-H between the FEPs of APO/ADP (top panel), APO/ATP (middle panel) and ADP/ATP (bottom panel) onto the structure of DnaK (PDB ID: 2KHO) [70]. Residues belonging to the CGDAs γ which are strongly influenced (1-H>0.7), significantly influenced (0.3≤1-H<0.7) and weakly influenced by the nucleotide (1-H≤0.3) are shown with large red, medium yellow and small cyan spheres, respectively. The color code of the protein structure is the same as in Fig. 1. These figures were prepared with PyMOL [http://www.pymol.org].
Table 2.
Statistical analysis of the dissimilarity [1-H(γi)] of the FEPs between concatenated MD runs in different nucleotide-binding states.
Figure 6.
FEPs V(γ) for γ387 in kBT unit.
V(γ) computed from APO, ADP-bound and ATP-bound MD simulations of DnaK are colored in red, blue and green respectively.
Figure 7.
A: NBD+linker. B: SBD. Each CGDA γi is represented by a sphere centered on the Cα(i) atom. The color code of the protein structure is the same as in Fig. 1. These figures were prepared with PyMOL [http://www.pymol.org].
Table 3.
List of the 27 CGDAs γ revealed by analysis of 1-D FEPs of DnaK in different nucleotide-binding states and their corresponding amino acids.
Figure 8.
Dihedral principal component analysis applied to MD simulations of ATP-bound DnaK.
FES computed for the MD run ATP1. Minima are shown with gray (green for the most probable one) diamonds and the isolines (black lines) are drawn every kBT unit. The color scale for the free-energy is in kBT units.
Figure 9.
Analysis of the FEL of ATP-bound DnaK.
A: FES generated from dPCA of MD run ATP1. Minima and saddle points are shown with gray (green for the most probable one) and red diamonds, respectively. The PME is represented by the red line. B: Free-energy VPME along the PME for the transition A1→A2→A3→A4→A5. C: Results of the clustering along the PME. Each frame is represented at the corresponding time t observed in the MD trajectory.
Figure 10.
Comparison between MD data and EPR experiments.
Histograms of the distance E430-R547 computed from the MD simulations in the APO state (panels A and C, red) and in the ATP-bound state (panels B and D, green). Panels A and B represent the distributions up to 20 Å, as in the EPR experiments and panels C and D represents the same distributions up to 40 Å. Each subpopulation in the distance distribution was fitted with a Gaussian function shown with black lines. The black arrows in panels A and B represent the experimental results from Ref. [81]. FEP V(γ504) computed from different subpopulations relative to the distance E430/R547 distribution function in the APO state (panel E) and in the ATP state (panel F). <d> represents the mean value of the Gaussian distributions shown in panel C for the APO-DnaK and D for the ATP-DnaK.
Table 4.
List of residues found to be relevant for conformational dynamics of human Hsp70 deduced from NMA of human Hsp70 [49], [92] and from dPCA of E. coli MD trajectories (present work) after sequence alignment of E. coli DnaK on human Hsp70 (Table S1).