Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51

Abstract

FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.

Figure 1

Biophysical characterization of C90 peptide binding to the FKBP51 TPR domain by DSF analysis. (A) DSF melting curve and first derivative curve of the FKBP51 with 0 µM (green line) and 250 µM C90 peptide (red line). The upper half of the figure shows the melting curve and the lower half shows the first derivative curve to determine the melting point. Major multiple transitions occurred at 43.8 and 57.8 °C for FKBP51 in the absence of C90 peptide. In the presence of 250 µM of C90 peptide the first melting point shifted to 48.6 °C without much change in the second melting point. A ΔTm of 4.8 °C indicates specific biding of C90 peptide to the TPR domain of FKBP51. (B) DSF melting curve and first derivative curve of the FKBP51 with 0 µM (green line) and 200 µM control peptide (DDDDDDDDDD) (red line). The upper half of the figure shows the melting curve and the lower half shows the first derivative curve to determine the melting point. The presence of peptide did not change the melting point indicating that it is not interacting with FKBP51. (C) Binding affinity of C90 peptide to TPR domain is determined by incubation with increasing (0–250 µM) concentrations of C90 peptide.

Figure 2

Crystal structure of FKBP51-Hsp90 C-terminal peptide complex. The overall structure of the complex contains FK1, FK2 and TPR domains. The C90 peptide is shown as sticks with carbons in green color.

Figure 3

Surface characteristics of the FKBP51 structure. (A) Electrostatic surface potential of FKBP51. (B) Zoomed-in view of the peptide binding pocket. The binding pocket is electropositive in nature and thus facilitates the binding of electronegative peptide. (C) +180° rotated view around x-axis. (D) Surface hydrophobicity of the FKBP51.

Figure 4

Validation of the FKBP51 model by multiple methods. (A) Ramachandran plot. (B) Qmean based structure validation which compares the structure to non-redundant set of PBDs of similar size. The FKBP51 structure indicated as a red star lies within the range of scores of similar size structures, indicating its good quality. (C) ProSa Z-score plot of the FKBP51 indicated by red arrow. (D) The local quality of the model indicated as plot of energy as a function of amino acid sequence position.

Figure 5

Electron density maps of the Hsp90 C-terminal peptide binding to the TPR domain of FKBP51. (A) The sigma weighted 2fo-fc electron density map calculated by Buster (contoured at 1.2 σ) of Hsp90 C-terminal peptide bound to the TPR domain of FKBP51. (B) The sigma weighted simulated annealing omit map (contoured at 3 σ) of Hsp90 C-terminal peptide bound to the TPR domain of FKBP51.

Figure 6

Interaction of Hsp90 C-terminal peptide with the TPR domain of FKBP51. (A) Hydrogen bond and salt bridge network of C90 peptide bound to the TPR domain of FKBP51. Yellow carbon sticks shows TPR domain and the MEEVD peptide is shown as green carbon sticks. Hydrogen bonds and salt bridges are represented as black dotted lines and are labeled. The figure is generated by PyMOL. (B) 2D ligand interaction diagram created by LigPlot+ showing the hydrogen bond network and the hydrophobic interactions of the peptide with the TPR domain of FKBP51.

Figure 7

Comparison of binding of MEEVD peptide to the FKBP51 vs FKBP52 (A) and FKBP51 vs FKBP38 (B). The C-terminal Asp amino acid is rendered as ball and stick.

Figure 8

ConSurf analysis for the TPR domains of TPR-containing proteins. The 3D structure of TPR domain of FKBP51 is rendered as surface and the bound C90 peptide is rendered as sticks. The TPR domain’s surface is color coded by its conservation grade using the color-coding bar shown in the figure, with turquoise-through-maroon indicating the variable to conserved residues. The figure indicates that the functionally important amino acid residues, taking part in the binding to the C90 peptide are highly conserved. The analysis was carried out using the solved crystal structure and the figure was generated with the help of PyMOL script output by ConSurf.

Figure 9

Qualitative analysis of the Molecular dynamics trajectory. RMSD plot of TPR domain of FKBP51 and C90 peptide (A). Radius of gyration of the complex (B). RMSF of TPR (C) and RMSF of C90 peptide (D).

Figure 10

Hydrogen bond analysis of the molecular dynamics trajectory. Total number of hydrogen bonds formed during 100 ns simulation trajectory (A). Length of HB1 and HB2 (B). Length of HB3 and HB4 (C). Length of HB5 and HB6 (D) Length of SB1 and SB2 (E) and Length of SB3 and SB4 (F).

Figure 11

Cross-RMSD matrix of the backbone. In the upper left half the coordinates belonging to same clusters (red) and different cluster (blue) represented in the lower half.

Figure 12

Median structures of the clusters obtained after MD analysis. The size of cluster is given in the parenthesis as percentage population.