Figure 1.
(A) Surface representation of the full length S. cerevisiae Hsp90 (PDB code 2CG9), showing the three different protein domains (N-terminal domain: orange, middle domain: green, C-terminal domain: blue). (B) Homology model of hHsp90 C-terminal domain (blue) overlaid with a crystal structure (PDB code 3Q6M) of the same domain (red) (C) Blow-up of the overlay highlighting the side chain orientation of residues located at the interface of helices H5 and H4′.
Figure 2.
Hot spot and cold spot prediction.
(A) Contribution to the dimer stabilization of each amino acid within the hHsp90 CTD. ΔG values are calculated by the MM-GB/SA approach [37], [83] starting from the homology model, employing a structural decomposition of the effective energy [10]. The standard error in the mean is <0.1 kcal mol−1 in all cases. Amino acids contributing to the dimerization with ΔG<−2 kcal mol−1 are considered hot spots and are indicated in the graphic by red dots. In addition two “cold spots” mentioned in the text are marked with blue dots. In the upper part of the panel, the secondary structure of the CTD is shown. The amino acids are numbered according to the full length hHsp90 α isoform (UniProt code: P07900). (B) Hot spot residues localized on H5 (red) and interacting residues on H4′ (green). (C) Helical wheel representation showing the position of hot spots (red) and cold spots (blue) on helices H5 and H4′.
Figure 3.
Thermofluor assay for investigating the stability of wild type and mutant hHsp90 CTD.
Protein stability was analyzed in 12 different pH buffers in four independent measurements. (A) Heatmap for wild type hHsp90 CTD. Melting curves of measurements at pH 7.5 with the average Tm and standard deviation are shown below for wild type hHsp90 CTD (B), hHsp90 CTD with cold spot mutants as negative control (C), as well as hot spot alanine mutants CTDY689A/I692A/L696A (D) and CTDI688A/Y689A/I692A (E). The mean value (dotted black line) was calculated from four independent measurements (yellow, red, blue, green lines) in reaction buffer with 100 mM Tris.
Table 1.
Variants of the CTD of hHsp90 investigated in this study.
Table 2.
Tm of hHsp90 CTD wild type and alanine mutants.
Figure 4.
Elution profiles of hHsp90 CTD variants using size exclusion chromatography.
Chromatograms for wild type hHsp90 CTD (A), cold spot (B), and hot spot alanine mutants (C and D). Experiments were performed in triplicates on a Superdex SD200 10/300 column in HPLC-buffer (10 mM MES/KOH, 200 mM KCl, 1 mM EDTA, 1% Glycerol) at pH 6 with 110 µL of purified hHsp90 CTD. The molecular weight of hHsp90 CTD variants was calculated based on the slope of the calibration curve obtained with standard proteins. The elution peak of the wild type corresponds to a molecular weight of 88±0.5 kDa, indicating an apparent tetrameric complex. The cold spot mutant shows the same elution profile as the wild type protein corresponding to a molecular weight of 88±0.3 kDa. Alanine mutants CTDY689A/I692A/L696A and CTDI688A/Y689A/I692A show a shift to lower molecular weights of 57–58 kDa (57±0.2 kDa for CTDY689A/I692A/L696A and 57±0.4 kDa for CTDI688A/Y689A/I692A). This indicates a smaller protein complex suggesting a weakly associated dimer or closely associated trimer configuration.
Figure 5.
Differential refractive index and molecular mass of hHsp90 variants using multi-angle light-scattering.
(A) For wild type hHsp90 CTD a species with molar mass of 45.4±0.1 kDa was determined. Molar masses of higher oligomeric species could not be specified due to insufficient separation. (B, C) Hot spot mutants of hHsp90 CTD, CTDY689A/I692A/L696A (B) and CTDI688A/Y689A/I692A (C), revealed species with molar masses of 23.5±0.2 kDa and 23.2±0.2 kDa, respectively, and additional species with molar masses of 48.7±0.5 kDa and 50.1±0.5 kDa, respectively. Furthermore, also higher oligomeric species were detectable but could not be analyzed with respect to molar masses.
Table 3.
Molecular weights and mass distributions measured by MALS.
Table 4.
Mutagenesis primers[a].
Table 5.
pH values of screening buffers [86] for Thermofluor assay.