The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the \r<br />Hsp90-p50Cdc37 complex by protein–protein interaction (PPI) inhibitors has emerged as \r<br />an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human \r<br />Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.
Olesen, S. H., Ingles, D. J., Zhu, J. Y., Martin, M. P., Betzi, S., Georg, G. I., … Schönbrunn, E. (2015). Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2. Molecules, 20(1), 1643–1660. https://doi.org/10.3390/molecules20011643