Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery

Ratzke, Christoph; Hellenkamp, Björn; Hugel, Thorsten

doi:10.1038/ncomms5192

Cited by 70 publications

(63 citation statements)

References 55 publications

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“…While the detailed mechanistic reasons for these observations remain to be elucidated, it is tempting to speculate that maturation of v-src may require more efficient utilization of ATP hydrolysis by Hsp90 than GR (e.g., efficient transmission of the energy of ATP hydrolysis to conformational changes that activate v-src) and thus v-src may be a more difficult client than GR to activate. Investigating this possibility in vitro is complicated because the hydrolysis of ATP by Hsp90 is strongly influenced by co-chaperones (Panaretou et al, 2002; Ratzke et al, 2014) and likely by clients, resulting in a complex relationship between in vitro and in vivo ATPase activity. The complexities of this relationship are exemplified by the in vitro ATPase activity of the nine Hsp90 variants that are capable of maturation of GR but not v-src (Fig.…”

Section: Hsp90 Mutations With Client-specific Effectsmentioning

confidence: 99%

Systematic Mutant Analyses Elucidate General and Client-Specific Aspects of Hsp90 Function

et al. 2016

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Summary To probe the mechanism of the Hsp90 chaperone that is required for the maturation of many signaling proteins in eukaryotes, we analyzed the effects of all individual amino acid changes in the ATPase domain on yeast growth rate. The sensitivity of a position to mutation was strongly influenced by proximity to the phosphates of ATP, indicating that ATPase driven conformational changes impose stringent physical constraints on Hsp90. To investigate how these constraints may vary for different clients, we performed biochemical analyses on a panel of Hsp90 mutants spanning the full range of observed fitness effects. We observed distinct effects of nine Hsp90 mutations on activation of v-src and glucocorticoid receptor (GR), indicating that different chaperone mechanisms can be utilized for these clients. These results provide a detailed guide for understanding Hsp90 mechanism and highlight the potential for inhibitors of Hsp90 that target a subset of clients.

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Section: Hsp90 Mutations With Client-specific Effectsmentioning

confidence: 99%

Systematic Mutant Analyses Elucidate General and Client-Specific Aspects of Hsp90 Function

et al. 2016

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“…Compared with two-color FRET that monitors a single distance, three-color FRET can determine three distances and therefore has great potential to obtain 3D structural information for a molecule or a molecular complex. Multicolor FRET has been demonstrated for well-known and designed molecular systems (23)(24)(25)(26)(27) and used for the molecular identification (24,28,29) and investigations of conformational changes and dynamics of proteins and nucleic acids (30)(31)(32)(33)(34)(35) and their interactions (36)(37)(38). In some cases, the experiment and analysis can be simplified by preventing the energy transfer between one dye pair (e.g., much larger separation than the Förster radius).…”

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confidence: 99%

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

Chung

Meng

Kim

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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We describe a method that combines two-and three-color singlemolecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two-and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.single-molecule spectroscopy | three-color FRET | intrinsically disordered protein | p53 oligomerization | fluorescence lifetime I t is well known that intrinsically disordered proteins (IDPs) can fold into different structures when attaching to their binding targets. This structural flexibility and binding promiscuity are required for the formation of protein-protein interaction networks in various biological processes such as signal transduction and gene transcription (1-3). On the other hand, some IDPs selfassemble and form oligomers, many of which are implicated in the development of diseases such as Alzheimer's disease (amyloid-β protein) (4, 5) and Parkinson's disease (α-synuclein) (6). An ensemble of these oligomers with different sizes and conformations is not easy to separate, and therefore, their characterization is very difficult. However, single-molecule spectroscopy can be a very powerful tool because it can probe subpopulations in a mixture without the need for separation. Single-molecule spectroscopy has been successfully used for characterizing individual molecular states such as the folded and unfolded states of proteins (7-9), intermediate states (10, 11), and transition paths (12-17) and for identifying specific molecular species and complexes (18)(19)(20)(21)(22). In this paper, we describe the development of a singlemolecule fluorescence method that probes individual oligomers in a mixture, characterizes their conformations, and determines oligomerization kinetics.We have used two-and three-color Förster resonance energy transfer (FRET) spectroscopy. Compared with two-color FRET that monitors a single distance, three-color FRET can determine three distances and therefore has great potential to obtain 3D structural information for a molecule or a molecular complex. Multicolor FRET has been d...

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“…hsp90 functions as a homodimer, with each monomer consisting of a C-terminal dimerization domain (C), a middle domain (M) that is often involved in client binding, and an N-terminal domain (N) that contains the majority of the ATP-binding site and that participates in some client-binding interactions (5,6). The hsp90 dimer functions in client maturation by undergoing large conformational rearrangements that are governed by ATP site occupancy and hydrolysis, co-chaperone proteins, and posttranslational modifications (7)(8)(9). The molecular details of hsp90-client protein interactions, and their associated structural and functional impacts, are under active investigation (6, 10 -14).…”

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confidence: 99%

Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase

Sarkar

Dai

Haque

et al. 2015

Journal of Biological Chemistry

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Background: hsp90␤ binds to heme-free sGC␤1 subunit to enable heme insertion during maturation, but the mechanism is unclear. Results: Their interaction involves the hsp90␤ M domain and the Per-Arnt-Sim (PAS) domain of apo-sGC␤1. Conclusion: hsp90␤ may modulate the apo-sGC␤1 through its PAS domain, suggesting a means to aid heme insertion. Significance: This work expands our knowledge of how hsp90 regulates PAS-containing client proteins like sGC.

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Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery

Cited by 70 publications

References 55 publications

Systematic Mutant Analyses Elucidate General and Client-Specific Aspects of Hsp90 Function

Systematic Mutant Analyses Elucidate General and Client-Specific Aspects of Hsp90 Function

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase

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