Fig. 4. Biophysical characterization of Hsp90.

a Arg-32/Glu-33 distance from classical atomistic MD simulations, showing that the ion pair is closed in the apo-form (purple) of the Hsp90-NTD and its conformation is sensitive to nucleotide binding (ADP in grey/ATP in blue). b ¹H-¹⁵N-HSQC spectra of the Hsp90-NTD bound to ADP (grey) and ATP (blue), with close-ups of the resonances corresponding to the Arg-32 amide group (bottom right) and its sidechain Hε-Nε (bottom left), for which the ¹H traces are also included (apo shown in purple). c NOE cross-peak between Arg-32 Hε and Glu-33 Hβ in the apo-NTD, confirming the spatial proximity of the sidechains. d Left: chemical shift perturbations (CSPs) of the Hsp90-NTD bound to ADP and ATP. Negative bars represent residues that could not be assigned due to conformational dynamics. Secondary structure elements are shown on the top and altered residues next to Arg-32 are highlighted in red. Right: distribution of CSP on the NTD for ATP/ADP exchange. Amides of residues that are not visible due to conformational dynamics are represented by blue spheres, whereas unassigned residues are shown in cyan. e FRET experiments show that closing (left) in Hsp90-R32A (in red and light blue) takes place to a smaller extent in comparison with the wild type (WT, in black and orange). Right: re-opening of the closed dimer takes place faster in Hsp90-R32A (red) than in WT Hsp90 (black). The schematic figure on the top illustrates the experimental procedure. f Rates for closing and re-opening of WT Hsp90 and Hsp90-R32A obtained from mono-exponential fitting of the FRET curves shown in e. Error bars represent SD from three independent measurements (n = 3), shown as black dots. Statistical significance was assessed using a two-sided two-sample t-test and a level of significance of 0.01 (**) and 0.001 (***). The fit yields a higher closure rate for R32A relative to WT Hsp90. g ADP-release assays show that WT Hsp90 and Hsp90-R32A have similar global ATP turnover. Error bars represent SD from three independent measurements (n = 3), shown as black dots. Source data are provided as a source data file.