Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2
Abstract
:1. Introduction
2. Results and Discussion
2.1. In Vitro Reconstitution of the Human Hsp90β-p50Cdc37 Complex and Phosphorylation by CK2
2.2. Effect of Phosphorylation on the Interaction of Hsp90β with Nucleotides and Inhibitors
Binding Partner/Ligand | Hsp90 | Observed Kd (µM) | Reported Kd (µM) | Reference |
---|---|---|---|---|
p50Cdc37 | yHsp82-FL | n/d | 1.46 | [30] |
hΔHsp90β | 1.1 | n/a | ||
hHsp90β-N | 4.4 | n/a | ||
hHsp90β-FL | 1.2 | n/a | ||
h(p)Hsp90β-FL | 1.7 | n/a | ||
(p)p50Cdc37 | hHsp90β-FL | 5.1 | n/a | |
h(p)Hsp90β-FL | 8.6 | n/a | ||
ADP | hΔHsp90β | 11 | n/a | |
hHsp90β-FL | 8.9 | 7.2 | [31] | |
h(p)Hsp90β-FL | 13 | n/a | ||
ATP | hΔHsp90β | 43 | n/a | |
hHsp90β-FL | 4.6 | 240 | [31] | |
h(p)Hsp90β-FL | 11 | n/a | ||
AMP-PNP | hΔHsp90β | 84 | n/a | |
hHsp90β-FL | 85 | 148 | [31] | |
h(p)Hsp90β-FL | 590 | n/a | ||
NVP-AUY922 | hΔHsp90β | <0.01 | n/a | |
hHsp90β-FL | <0.01 | 0.0017 | [32] | |
h(p)Hsp90β-FL | 0.1 | n/a | ||
17-DMAG | hHsp90β-FL | n/d | 0.35 | [33] |
hΔHsp90β | 0.79 | n/a | ||
Radicicol | yHsp82-FL | n/d | 0.019 | [34] |
hΔHsp90β | <0.01 | n/a |
2.3. Evaluation of Small Molecules as PPI Inhibitors of the Hsp90β-p50Cdc37 Complex
Complex | IC50 (µM) | ||||||
---|---|---|---|---|---|---|---|
ADP | ATP | AMP-PNP | H2-GMZ | H2-GMZ | Gedunin | NVP | |
Hsp90-p50Cdc37 | 500 ± 94 | >1000 | >1000 | >1000 | >1000 | >1000 | >1000 |
(p)Hsp90-p50Cdc37 | 220 ± 24 | 450 ± 150 | >1000 | >1000 | >1000 | >1000 | >1000 |
Hsp90-(p)p50Cdc37 | 270 ± 35 | >1000 | n/d | n/d | n/d | n/d | n/d |
(p)Hsp90-(p)p50Cdc37 | 32 ± 5.6 | 230 ± 40 | >1000 | >1000 | >1000 | >1000 | >1000 |
ΔHsp90-p50Cdc37 | 400 ± 81 | >1000 | >1000 | >1000 | >1000 | >1000 | >1000 |
2.4. ADP Exerts Inhibitory Activity through Binding to the ATP Site in the N-Terminal Hsp90 Domain
2.5. Possible Structural Basis for the PPI Inhibitory Activity of ADP
3. Experimental Section
3.1. Materials
3.2. Cloning and Expression
3.3. Protein Purification
3.4. GST Pull-Down
3.5. In Vitro Phosphorylation
3.6. SDS-PAGE and Western Blot Analysis
3.7. ELISA Microtiter Plate Assay
3.8. Isothermal Titration Calorimetry (ITC)
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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- Sample Availability: Samples of the compounds are not available.
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Olesen, S.H.; Ingles, D.J.; Zhu, J.-Y.; Martin, M.P.; Betzi, S.; Georg, G.I.; Tash, J.S.; Schönbrunn, E. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2. Molecules 2015, 20, 1643-1660. https://doi.org/10.3390/molecules20011643
Olesen SH, Ingles DJ, Zhu J-Y, Martin MP, Betzi S, Georg GI, Tash JS, Schönbrunn E. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2. Molecules. 2015; 20(1):1643-1660. https://doi.org/10.3390/molecules20011643
Chicago/Turabian StyleOlesen, Sanne H., Donna J. Ingles, Jin-Yi Zhu, Mathew P. Martin, Stephane Betzi, Gunda I. Georg, Joseph S. Tash, and Ernst Schönbrunn. 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, no. 1: 1643-1660. https://doi.org/10.3390/molecules20011643