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.

Structure and pro-toxic mechanism of the human Hsp90/PPIase/Tau complex

“…Human Hsp90β contains 48 isoleucine residues well spread over all domains (Supplementary Figure 2a ), including Hsp90N, the charged flexible linker (CL), which connects Hsp90N with the middle domain (Hsp90M) 37 , and Hsp90C, responsible for dimerization. FKBP51 contains two domains (termed FK1 and FK2) and three flexible TPR repeats (PDB id: 1kt0, 5njx) 64 , 65 . Previously available assignments of Hsp90 isoleucine methyl groups were confirmed and extended through site-directed mutagenesis of specific isoleucine residues in both full-length Hsp90 and isolated N–M, N, and M domains 66 .…”

“…Structural modeling was performed with the program Haddock 73 using both full-length human Hsp90β (PDB id: 5fwk) 19 and FKBP51 (PDB id: 1kt0, 5njx) 64 , 65 as template structures. For the final refinement steps, an additional atomic structure encompassing FK1 domain with an additional α-helix in the N-terminus (PDB id: 3o5e) was used.…”

“…Next, Hsp90N was added to the Hsp90MCCM/FK1–FK2 complex, and after structure inspection and refinement, used as a template for the final addition of the TPR repeats onto the full Hsp90/FK1–FK2 complex. Only a minimal rotation of the flexible TPR repeats was needed, in order to accommodate both the MEEVD-binding site 65 and Hsp90C’s flexible loop 76 – 78 in the model of the complex. The structure of the complex was validated by experimental PCSs and PREs obtained by tagging both partners of the complex (Fig.…”

In silico identification and biochemical characterization of the human dicarboxylate clamp TPR protein interaction network

“…We have previously shown that interactions between the dcTPR protein FKBP51 and peptides resembling C‐terminal sequences of Hsp90 are sequence‐specific and no interaction was observed between FKBP51 and peotide containing a stretch of 10 aspartate residues . Here in this study, we systematically searched the human protein database (https://www.ncbi.nlm.nih.gov/protein/) for the protein products containing Hsp70/Hsp90‐like sequence signatures at their C terminus.…”

“…Several structural criteria can be extracted to determine the peptide motif for interaction with dcTPR domains. First, the last five amino acids in the sequence are more conserved, a notion supported by the crystallography data where they form most of the contacts within the peptidebinding groove of the dcTPR domain [2,20]. Second, conserved C-terminal aspartate or glutamate is required for binding to the dcTPR domain [3].…”