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Hsp90 charged-linker truncation reverses the functional consequences of weakened hydrophobic contacts in the N domain

Abstract

Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes, as it regulates diverse signal transduction nodes that integrate numerous environmental cues to maintain cellular homeostasis. Hsp90 also is secreted from normal and transformed cells and regulates cell motility. Here, we have identified a conserved hydrophobic motif in a β-strand at the boundary between the N domain and charged linker of Hsp90, whose mutation not only abrogated Hsp90 secretion but also inhibited its function. These Hsp90 mutants lacked chaperone activity in vitro and failed to support yeast viability. Notably, truncation of the charged linker reduced solvent accessibility of this β-strand and restored chaperone activity to these mutants. These data underscore the importance of β-strand 8 for Hsp90 function and demonstrate that the functional consequences of weakened hydrophobic contacts in this region are reversed by charged-linker truncation.

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Figure 1: Identification of Ile218 and Leu220 as important for function of human HSP90α.
Figure 2: Mutation of β-strand 8 attenuates co-chaperone interaction.
Figure 3: Mutation of β-strand 8 attenuates nucleotide-dependent chaperone function.
Figure 4: Mutation of β-strand 8 inhibits ATP-induced Hsp90 conformational dynamics.
Figure 5: Truncation of the charged linker restores function to β-strand 8 Hsp90 mutants.
Figure 6: β-strand 8 mutation alters the conformational state of the charged linker.
Figure 7: Impact of β-strand 8 mutation on deuteron incorporation kinetics of Hsp82.
Figure 8: Hydrophobic interactions between β-strand 8 and the adjacent α helix.

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Acknowledgements

We are indebted to K. Beebe for comments on the manuscript. This work is supported by US National Cancer Institute intramural research funds. S.T. is partially supported by a fellowship from the Japanese Society of the Promotion of Science.

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Authors

Contributions

S.T. and M.M. performed most of the experiments and helped interpret the data, design experiments and write the manuscript; C.G., B.T.S., C.-T.L., W.X., L.H., M.H. and A.A.K. performed some of the experiments; B.P., J.B. and M.P.M. designed some of the experiments and interpreted the data collected; M.P.M. and J.B. also contributed to writing part of the manuscript; J.B.T. contributed to the design and interpretation of some of the experiments; C.P. designed and performed some of the experiments, interpreted the data collected and contributed to writing part of the manuscript; L.N. oversaw the entire project, designed experiments, interpreted the data and wrote the paper.

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Correspondence to Len Neckers.

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Tsutsumi, S., Mollapour, M., Graf, C. et al. Hsp90 charged-linker truncation reverses the functional consequences of weakened hydrophobic contacts in the N domain. Nat Struct Mol Biol 16, 1141–1147 (2009). https://doi.org/10.1038/nsmb.1682

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