Split Renilla Luciferase Protein Fragment-assisted Complementation (SRL-PFAC) to Characterize Hsp90-Cdc37 Complex and Identify Critical Residues in Protein/Protein Interactions

Abstract

Hsp90 requires cochaperone Cdc37 to load its clients to the Hsp90 superchaperone complex. The purpose of this study was to utilize split Renilla luciferase protein fragmentassisted complementation (SRL-PFAC) bioluminescence to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/ Cdc37 interaction in living cells. SRL-PFAC showed that fulllength human Hsp90/Cdc37 interaction restored dramatically high luciferase activity through Hsp90-Cdc37-assisted complementation of the N and C termini of luciferase (compared with the set of controls). Immunoprecipitation confirmed that the expressed fusion proteins (NRL-Hsp90 and Cdc37-CRL) preserved their ability to interact with each other and also with native Hsp90 or Cdc37. Molecular dynamic simulation revealed several critical residues in the two interaction patches (hydrophobic and polar) at the interface of Hsp90/Cdc37. Mutagenesis confirmed the critical residues for Hsp90-Cdc37 complex formation. SRL-PFAC bioluminescence evaluated the contributions of these critical residues in Hsp90/Cdc37 interaction. The results showed that mutations in Hsp90 (Q133A, F134A, and A121N) and mutations in Cdc37 (M164A, R167A, L205A, and Q208A) reduced the Hsp90/Cdc37 interaction by 70 -95% as measured by the resorted luciferase activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A and S113A) and a mutation in Cdc37 (A204E) decreased the Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A, and C598A) and mutations in Cdc37 (C54S, C57S, and C64S) did not change Hsp90/Cdc37 interactions. The 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 regions of hydrophobic and polar interactions. These findings provides a rationale to develop inhibitors for disruption of the Hsp90/Cdc37 interaction.The 90-kDa heat shock protein (Hsp90) 4 is a ubiquitous and essential molecular chaperone with multiple functions in eukaryotic cells under both stressed and nonstressed conditions (1, 2). It plays a central role in post-translational folding and stability of over 100 signaling proteins, including steroid hormone receptors, the dioxin receptor, growth factor receptors, transcription factors, protein kinases, and enzymes (3). Many of these Hsp90 clients are crucial in tumorigenesis, and when these proteins are dysregulated, they contribute to the hallmark traits of cancer. It has also been reported that the expression of Hsp90 in cancer cells is 2-10-fold higher compared with their normal counterparts (4). Therefore, tumor cells show much more sensitivity when subjected to Hsp90 inhibition than nontransformed cells (5), which suggests the important function of Hsp90 in tumor progression.Hsp90 consists of the following three highly conserved domains: a 25-kDa N-terminal domain, a 35-kDa middle domain, and a 10-kDa C-terminal domain (6). A nucleotide bindin...

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Natural plant flavonoid apigenin directly disrupts Hsp90/Cdc37 complex and inhibits pancreatic cancer cell growth and migration

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