Characterization Of Hsp90/Cdc37 Interaction And Their Critical Residues Using Split Renilla Luciferase Protein-Fragment-Assisted Complementation

Hsp90 (90 kDa Heat shock protein) is a ubiquitous and essential molecular chaperone with multiple functions in eukaryotic cells under both stressed and non-stressed conditions. It plays a central role in posttranslational folding and stability of over 100 signaling proteins including steroid hormone receptors, the dioxin receptor, growth factor receptors, transcription factors, protein kinases and enzymes. Many of these Hsp90 clients are crucial in tumorigenesis, and when these proteins are disregulated, they contribute to the hallmark traits of cancer. It has also been reported that the expression of Hsp90 in cancer cells is 2-to 10-fold higher compared to their normal counterparts. Therefore, tumor cells show much more sensitivity when subjected to Hsp90 inhibition than non-transformed cells, which suggest the important function of Hsp90 in tumor progression.Hsp90 consists of three highly conserved domains:a 25-kDa N-terminal domain, a 35-kDa middle domain, and a 10-kDa C-terminal domain. A nucleotide binding pocket is located on the N-terminus, by which ATP binds Hsp90 and is subsequently hydrolyzed to induce a conformational change of Hsp90. These conformational changes help Hsp90 to interact with various cochaperones to form different complexes in the chaperoning cycles. The middle segment of Hsp90 is considered to be a major site for binding client proteins, while the C-terminus is essential for Hsp90 dimerization.Hsp90 interacts with multiple cochaperones to form a superchaperone complex, including Cdc37, Aha1, p23/Sba1, Hop, Hsp70, and Hsp40. Each component in the complex has its own function associated with different types of clients. Cdc37, originally named as p50, was reported as an accessory factor to load the subset of protein kinases to Hsp90 in the intermediate Hsp90 superchaperone complex. Silencing Cdc37 reduces expression of the Hsp90 clients ERBB2, CRAF, CDK4, CDK6 and phosphorylated AKT, which are highly relevant to cancer progression. The involvement of Hsp90/Cdc37 complex in the maturation and activity of oncogenic protein kinases makes the complex a potential therapeutic target for cancer chemotherapy. Recently, a quinone methide triterpene compound celastrol was shown to disrupt Hsp90/Cdc37 interaction and exhibited anticancer activity, supporting the potential application of disrupting the protein-protein interaction of Hsp90/Cdc37 complex in cancer therapy.Cdc37 is composed of three domains:a 15.5 kDa N-terminal domain (residues1-127), a 16 kDa middle domain (residues 147-276), and a 10.5 kDa C-terminal domain (residues283-378). Cdc37 forms a complex with N-terminus of Hsp90 through its middle and C-terminal portions. Crystal structures of yeast Hsp90 and human Cdc37 revealed that the interaction between the two proteins is through a flat hydrophobic patch and reinforced by a network of polar interactions, burying≈1056(?)2 of molecular surface.In the current study, we have utilized a bioluminescence imaging, split renilla luciferase protein-fragment-assisted complementation (SRL-PFAC), to study full length human Hsp90/human Cdc37 interaction in living cells. SRL-PFAC imaging system was developed by Gambhir et al. It is a complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in living cells. It is based on division of the full-length Renilla luciferase into two separate inactive halves that can reconstitute function upon complementation. When fused to two interacting proteins, the luciferase reporter fragments are complimented upon association of the interacting proteins, thus showing different degrees of bioluminescence due to different levels of protein interaction.SRL-PFAC imaging has been validated to monitor protein-protein interactions, including the heterodimerization between MyoD/Id, the interacting location of Y941/SH2, the interaction of each Hsp90 isoform (α/β) with p23, homodimeric formation of herpes simplex virus type 1 thymidine kinase, and dimerization of ERK2. Here, we optimized SRL-PFAC system to study full length of human Hsp90/human Cdc37 interaction, and applied this system to identify critical residues for human Hsp90/Cdc37 interactions in living cells.We created constructs to express N-terminal, C-terminal of RL, NRL-Hsp90 (full length) fusion protein, Cdc37 (full length)-CRL fusion proteins in pcDNA3.1 (+) vector. Constructed plasmids were transfected into human HEK293 cells. pcDNA3.1 (+) vector expressed FL was co-transfected with each plasmid or combination for transfection efficiency normalization. After 48 hours, the cells were collected to detect the expressed protein using western blotting. The results showed that the specific bands of recombined NRL-Hsp90 (117 kDa) and Cdc37-CRL (60 kDa) fusion proteins could be clearly detected in the transfected cells. Immunoprecipitation (IP) study was also done to show the complex formation of NRL-Hsp90 and Cdc37-CRL. We then performed the luciferase activity assay on HEK293 cells transfected with various plasmids to test the complementation of two split RL fragments by the interaction of full length of Hsp90 and Cdc37. When the cells were transfected with each fragment alone (NRL, CRL, NRL-Hsp90, Cdc37-CRL), the luciferase activity was low. When co-transfeted with different pairs of fragments:NRL and CRL, NRL-Hsp90 and CRL, NRL and Cdc37-CRL to HEK293 cells respectively, these two fragments all showed low levels of complementation compared to NRL or CRL alone. However, when the two fragments NRL-Hsp90 and Cdc37-CRL were co-transfected into HEK293 cells, the complementation of the fragments was enhanced by 170-,220-and 35-fold, compared to the transfection of NRL+CRL, NRL-Hsp90+CRL and Cdc37-CRL+NRL respectively. These data suggest that the interaction of full length Hsp90 and Cdc37 is able to complement split luciferase fragments and the SRL-PFAC method is sensitive and specific to monitor the interaction of full length human Hsp90 and Cdc37.To study in detail the interaction of Hsp90/Cdc37 and to identify the residues at the interface of Hsp90/Cdc37, we performed a 3ns MD simulation of the Hsp90/Cdc37 complex and calculated the B-factors of residues in the hydrophobic core and the polar network. Several polar and hydrophobic residues were then selected from each protein for mutation analysis, based on their B-factor and impact on interaction energy. Three residues in Hsp90 hydrophobic interaction patch were mutated:S113, A121, and F134. Two residues in Hsp90 polar interaction patch were mutated:E47 and Q133. In addition, two residues unlikely to disrupt the interaction were also mutated for negative controls in Hsp90:R46 and S50. Two Cys residues (C481 and C598), which are located in M-and C-domain of Hsp90 and distal from the interaction patch were also chosen as controls. For Cdc37, R166, R167 and Q208 in the polar interaction region, and M164, L165, A204, L205 in the hydrophobic interaction region were chosen to mutate. In addition, three Cys residues (C54, C57 and C64) in N-terminus Cdc37 were mutated individually (C54S, C57S, C64S), in pairs (C54S/C57S; C57S/C64S), and together (C54S/C57S/C64S) to test whether they would interfere the complex formation. These mutants were used for SRL-PFAC activity to confirm their importance for Hsp90/Cdc37 interaction. Our results showed that mutations in Hsp90 (Q133A, F134A, A121N) and mutations in Cdc37 (M164A, R167A, L205A and Q208A) reduced Hsp90/Cdc37 interaction by 70-95% as measured by the resorted luciferease activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A, S113A) and mutation in Cdc37 (A204E) decreased Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A and C598A) and mutation in Cdc37 (C54S, C57S, C64S) did not change Hsp90/Cdc37 interactions.To further confirm the SRL-PFAC results that mutations of critical residues in either NRL-Hsp90 or Cdc37-CRL can disrupt the complementation of NRL-Hsp90 and Cdc37-CRL, we performed immunoprecipitation assay and bioluminescence imaging in the HEK293 cells with transfection of various vectors. The results were all very well consistent with our luciferase assay data.In conclusion, our data suggest that single amino acid mutation in the interface of Hsp90/Cdc37 is sufficient to disrupt its interaction, although Hsp90/Cdc37 interactions are through large region of hydrophobic and polar interactions. These finding provides a rationale to develop inhibitors for disruption of Hsp90/Cdc37 interaction.