CHAPERONING CASCADES · Chaperoning molecular cascades: Hsp90 assisted folding of cell cycle regualting kinases

Final Activity Report Summary - CHAPERONING CASCADES (Chaperoning molecular cascades: Hsp90 assisted folding of cell cycle regualting kinases)

The project was dedicated to the understanding of protein folding, which is one of the great questions in molecular biology. Progress in this field is of fundamental importance to diseases such as cancer, Alzheimer's disease and cystic fibrosis. Chaperoning Cascades addresses this topic by elucidating the mechanism of the molecular chaperone Hsp90. Chaperones organise protein folding in the cell, and Hsp90 is of particular interest since it is a key factor for the proper folding of oncogenic kinases. Due to this, Hsp90 became a hot target in cancer therapy in its own right. The disclosure of molecular mechanisms of the interaction of Hsp90 with oncogenic kinases is the major aim of Chaperoning Cascades. We use an interdisciplinary setup from experiments at atomic level by advanced NMR spectroscopy to the analysis in the living cell. CHAPERONING CASCADES focussed on five objectives to address its central aims: O1. Characterising the protein stability of oncogenic kinases O2. Determining binding conditions of kinases to its dedicated chaperone, Hsp90 O3. Analysing the structure of chaperone-bound kinases O4. Identifying the kinase binding site in Hsp90 O5. Describing the interaction between Hsp90 and kinases in the living cell. Key results of CHAPERONING KINASES O1. Characterising the protein stability of oncogenic kinases Kinases are activated by phosphorylation. We found that the stability of protein Kinase A is depending on its phosphorylation pattern, and this in turn governed Hsp90 interaction. O2. Determining binding conditions of kinases to its dedicated chaperone, hsp90 We found that kinase binding to Hsp90 differs for different kinases. Protein Kinase A bound to Hsp90 in its folded state. The kinase cSrc, which is known to be a substrate of hsp90 in vivo, did not show interaction with Hsp90 in our in vitro fluorescence experiments. This indicates that the folding path of the kinase is crucial for interaction with Hsp90. O3. Analysing the structure of chaperone-bound kinases We found that protein Kinase A interacts both in folded and in unfolded state with HSP90. We also found the natively unfolded protein tau to interact with Hsp90. We conclude that there are different modes for proteins to interact with Hsp90. O4. Identifying the kinase binding site in Hsp90 We identified the substrate binding site in Hsp90 as a surface consisting of parts of both the N-terminal and middle domains of Hsp90, which are accessible in both the Apo and the ATP-bound state. Interestingly, the ADP and ATP-bound states differ when they are bound to substrate, while they were indistinguishable without. O5. Describing the interaction between HSP90 and kinases in the living cell We used a transcription-translation system as model to study interactions of Hsp90 with kinases under controlled but close to physiological conditions. We found that Hsp90 increases the yield of kinase production even without the help of any of its co-chaperones. Technological advances While following our biological questions, we improved the methods arsenal for studying the interactions of large proteins with ligands. In particular, we established the methyl TROSY technology invented by the Kay lab for the Hsp90 system, demonstrating the NMR analysis is possible for such complex molecules. We also developed a DOSY-TROSY approach to distinguish different nucleotide-bound states in a diffusion assay.

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