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Hsp90 regulates the dynamics of its cochaperone Sti1 and the transfer of Hsp70 between modules.

Röhl A, Wengler D, Madl T, Lagleder S, Tippel F, Herrmann M, Hendrix J, Richter K, Hack G, Schmid AB, Kessler H, Lamb DC, Buchner J - Nat Commun (2015)

Bottom Line: In the presence of Hsp90, Hsp70 shifts its preference.The linker connecting the two modules is crucial for the interaction with Hsp70 and for client activation in vivo.Our results suggest that the interaction of Hsp70 with Sti1 is tightly regulated by Hsp90 to assure transfer of Hsp70 between the modules, as a prerequisite for the efficient client handover.

View Article: PubMed Central - PubMed

Affiliation: Center for integrated protein science (CIPSM) at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.

ABSTRACT
The cochaperone Sti1/Hop physically links Hsp70 and Hsp90. The protein exhibits one binding site for Hsp90 (TPR2A) and two binding sites for Hsp70 (TPR1 and TPR2B). How these sites are used remained enigmatic. Here we show that Sti1 is a dynamic, elongated protein that consists of a flexible N-terminal module, a long linker and a rigid C-terminal module. Binding of Hsp90 and Hsp70 regulates the Sti1 conformation with Hsp90 binding determining with which site Hsp70 interacts. Without Hsp90, Sti1 is more compact and TPR2B is the high-affinity interaction site for Hsp70. In the presence of Hsp90, Hsp70 shifts its preference. The linker connecting the two modules is crucial for the interaction with Hsp70 and for client activation in vivo. Our results suggest that the interaction of Hsp70 with Sti1 is tightly regulated by Hsp90 to assure transfer of Hsp70 between the modules, as a prerequisite for the efficient client handover.

No MeSH data available.


Related in: MedlinePlus

SpFRET measurements of fluorescently labelled Sti1.(a) The secondary structure of the different domains of Sti1. The residues mutated to a cysteine are shown in red (grey: not resolved in the structure). (b) Scheme of the different cysteine double mutants. The star marks the position of the fluorescent dye. (c) SpFRET efficiency plots of the mutants in panel (b). The 20 pM Sti1 was measured alone or mixed together with 10 μM Hsp90 or/and 25 μM Hsp70. The area under the curves was normalized. (d) FRET efficiency for the G193C-G309C mutant versus fluorescence lifetime of the donor in the presence of the acceptor. Multiple populations can be observed. The solid red line describes the expected relationship between donor lifetime and FRET efficiency when the populations are static, whereas the dashed green line indicates the theoretical curve when molecules undergo dynamic transitions between populations within a burst. In the absence of Hsps, the bursts fall symmetrically about the static line. In the presence of Hsps, a small deviation from the static line is observed for part of the population, suggesting there may be dynamic transitions between populations during a burst. (e) Representative data from spFRET TIRF experiments. Time traces of the donor and acceptor intensity (upper graph), total intensity corrected for the differences in sensitivity between the donor and acceptor channels (IT =γID+IA where γ is the detection correction factor, middle graph) and FRET efficiency (lower graph) of the G193C–G309C Sti1 mutant in the absence and presence of Hsp70 and Hsp90. Left: a representative spFRET trace for the G193C–G309C Sti1 mutant in the absence of Hsps. Most of the traces of Sti1 in the absence of Hsps show a static FRET efficiency until either the donor or acceptor molecule photobleaches. In this case, the acceptor fluorophore photobleached first. Right: representative spFRET traces for the G193C–G309C Sti1 mutant in the presence of 25 μM Hsp70 and 10 μM Hsp90. A dynamic FRET signal is detectable in a significant fraction of the measured molecules. The fluctuations in the donor and acceptor fluorescence intensities indicate changes in the FRET efficiency due to the movement of Sti1 between different conformations. The donor fluorophore photobleached before the acceptor molecule and the total intensity dropped to background levels (see also Supplementary Fig. 6).
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f6: SpFRET measurements of fluorescently labelled Sti1.(a) The secondary structure of the different domains of Sti1. The residues mutated to a cysteine are shown in red (grey: not resolved in the structure). (b) Scheme of the different cysteine double mutants. The star marks the position of the fluorescent dye. (c) SpFRET efficiency plots of the mutants in panel (b). The 20 pM Sti1 was measured alone or mixed together with 10 μM Hsp90 or/and 25 μM Hsp70. The area under the curves was normalized. (d) FRET efficiency for the G193C-G309C mutant versus fluorescence lifetime of the donor in the presence of the acceptor. Multiple populations can be observed. The solid red line describes the expected relationship between donor lifetime and FRET efficiency when the populations are static, whereas the dashed green line indicates the theoretical curve when molecules undergo dynamic transitions between populations within a burst. In the absence of Hsps, the bursts fall symmetrically about the static line. In the presence of Hsps, a small deviation from the static line is observed for part of the population, suggesting there may be dynamic transitions between populations during a burst. (e) Representative data from spFRET TIRF experiments. Time traces of the donor and acceptor intensity (upper graph), total intensity corrected for the differences in sensitivity between the donor and acceptor channels (IT =γID+IA where γ is the detection correction factor, middle graph) and FRET efficiency (lower graph) of the G193C–G309C Sti1 mutant in the absence and presence of Hsp70 and Hsp90. Left: a representative spFRET trace for the G193C–G309C Sti1 mutant in the absence of Hsps. Most of the traces of Sti1 in the absence of Hsps show a static FRET efficiency until either the donor or acceptor molecule photobleaches. In this case, the acceptor fluorophore photobleached first. Right: representative spFRET traces for the G193C–G309C Sti1 mutant in the presence of 25 μM Hsp70 and 10 μM Hsp90. A dynamic FRET signal is detectable in a significant fraction of the measured molecules. The fluctuations in the donor and acceptor fluorescence intensities indicate changes in the FRET efficiency due to the movement of Sti1 between different conformations. The donor fluorophore photobleached before the acceptor molecule and the total intensity dropped to background levels (see also Supplementary Fig. 6).

Mentions: The above results suggest that crosstalk between the two modules is mediated by the linker and that the linker is important for the function of Sti1. To investigate the flexibility of the linker and conformational changes induced by Hsp binding, we performed spFRET experiments on constructs where the FRET pair spanned the linker region (G193C–S258C, S2C–G309C, G131C–G309C and G193C–G309C) (Fig. 6a). When labelling the ends of the linker region (G193C–S258C), no significant changes in the peak of the spFRET distribution were observed on the binding of Hsp70 and/or Hsp90 (Fig. 6b,c). Interestingly, there is an increase in the width of the spFRET distribution on binding of Hsp70, or Hsp70 and Hsp90, which indicates that chaperone binding increases the flexibility of Sti1. For the other mutants, a significant conformational change in Sti1 to lower FRET values was observed in the presence of Hsp90, indicating an increase in the distance between the two dyes (Fig. 6c and Supplementary Fig. 1). A detailed analysis of the G193C–G309C construct revealed the presence of at least three different conformations (Fig. 6c,d). In one conformation, the distance between the labels on the DP1 and TPR2A domains is extended and a low-FRET signal is detected (low-FRET conformation with a FRET efficiency of 15%). A second high-FRET conformation (E=95%) is also observable where the two domains are close to each other. A third intermediate-FRET conformation can be seen at E∼50%, particularly in the presence of Hsp70 or Hsp90 or both Hsps. Together this suggests that different conformations of Sti1 exist and that Hsp90 and Hsp70 modulate the equilibrium between these conformations.


Hsp90 regulates the dynamics of its cochaperone Sti1 and the transfer of Hsp70 between modules.

Röhl A, Wengler D, Madl T, Lagleder S, Tippel F, Herrmann M, Hendrix J, Richter K, Hack G, Schmid AB, Kessler H, Lamb DC, Buchner J - Nat Commun (2015)

SpFRET measurements of fluorescently labelled Sti1.(a) The secondary structure of the different domains of Sti1. The residues mutated to a cysteine are shown in red (grey: not resolved in the structure). (b) Scheme of the different cysteine double mutants. The star marks the position of the fluorescent dye. (c) SpFRET efficiency plots of the mutants in panel (b). The 20 pM Sti1 was measured alone or mixed together with 10 μM Hsp90 or/and 25 μM Hsp70. The area under the curves was normalized. (d) FRET efficiency for the G193C-G309C mutant versus fluorescence lifetime of the donor in the presence of the acceptor. Multiple populations can be observed. The solid red line describes the expected relationship between donor lifetime and FRET efficiency when the populations are static, whereas the dashed green line indicates the theoretical curve when molecules undergo dynamic transitions between populations within a burst. In the absence of Hsps, the bursts fall symmetrically about the static line. In the presence of Hsps, a small deviation from the static line is observed for part of the population, suggesting there may be dynamic transitions between populations during a burst. (e) Representative data from spFRET TIRF experiments. Time traces of the donor and acceptor intensity (upper graph), total intensity corrected for the differences in sensitivity between the donor and acceptor channels (IT =γID+IA where γ is the detection correction factor, middle graph) and FRET efficiency (lower graph) of the G193C–G309C Sti1 mutant in the absence and presence of Hsp70 and Hsp90. Left: a representative spFRET trace for the G193C–G309C Sti1 mutant in the absence of Hsps. Most of the traces of Sti1 in the absence of Hsps show a static FRET efficiency until either the donor or acceptor molecule photobleaches. In this case, the acceptor fluorophore photobleached first. Right: representative spFRET traces for the G193C–G309C Sti1 mutant in the presence of 25 μM Hsp70 and 10 μM Hsp90. A dynamic FRET signal is detectable in a significant fraction of the measured molecules. The fluctuations in the donor and acceptor fluorescence intensities indicate changes in the FRET efficiency due to the movement of Sti1 between different conformations. The donor fluorophore photobleached before the acceptor molecule and the total intensity dropped to background levels (see also Supplementary Fig. 6).
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403447&req=5

f6: SpFRET measurements of fluorescently labelled Sti1.(a) The secondary structure of the different domains of Sti1. The residues mutated to a cysteine are shown in red (grey: not resolved in the structure). (b) Scheme of the different cysteine double mutants. The star marks the position of the fluorescent dye. (c) SpFRET efficiency plots of the mutants in panel (b). The 20 pM Sti1 was measured alone or mixed together with 10 μM Hsp90 or/and 25 μM Hsp70. The area under the curves was normalized. (d) FRET efficiency for the G193C-G309C mutant versus fluorescence lifetime of the donor in the presence of the acceptor. Multiple populations can be observed. The solid red line describes the expected relationship between donor lifetime and FRET efficiency when the populations are static, whereas the dashed green line indicates the theoretical curve when molecules undergo dynamic transitions between populations within a burst. In the absence of Hsps, the bursts fall symmetrically about the static line. In the presence of Hsps, a small deviation from the static line is observed for part of the population, suggesting there may be dynamic transitions between populations during a burst. (e) Representative data from spFRET TIRF experiments. Time traces of the donor and acceptor intensity (upper graph), total intensity corrected for the differences in sensitivity between the donor and acceptor channels (IT =γID+IA where γ is the detection correction factor, middle graph) and FRET efficiency (lower graph) of the G193C–G309C Sti1 mutant in the absence and presence of Hsp70 and Hsp90. Left: a representative spFRET trace for the G193C–G309C Sti1 mutant in the absence of Hsps. Most of the traces of Sti1 in the absence of Hsps show a static FRET efficiency until either the donor or acceptor molecule photobleaches. In this case, the acceptor fluorophore photobleached first. Right: representative spFRET traces for the G193C–G309C Sti1 mutant in the presence of 25 μM Hsp70 and 10 μM Hsp90. A dynamic FRET signal is detectable in a significant fraction of the measured molecules. The fluctuations in the donor and acceptor fluorescence intensities indicate changes in the FRET efficiency due to the movement of Sti1 between different conformations. The donor fluorophore photobleached before the acceptor molecule and the total intensity dropped to background levels (see also Supplementary Fig. 6).
Mentions: The above results suggest that crosstalk between the two modules is mediated by the linker and that the linker is important for the function of Sti1. To investigate the flexibility of the linker and conformational changes induced by Hsp binding, we performed spFRET experiments on constructs where the FRET pair spanned the linker region (G193C–S258C, S2C–G309C, G131C–G309C and G193C–G309C) (Fig. 6a). When labelling the ends of the linker region (G193C–S258C), no significant changes in the peak of the spFRET distribution were observed on the binding of Hsp70 and/or Hsp90 (Fig. 6b,c). Interestingly, there is an increase in the width of the spFRET distribution on binding of Hsp70, or Hsp70 and Hsp90, which indicates that chaperone binding increases the flexibility of Sti1. For the other mutants, a significant conformational change in Sti1 to lower FRET values was observed in the presence of Hsp90, indicating an increase in the distance between the two dyes (Fig. 6c and Supplementary Fig. 1). A detailed analysis of the G193C–G309C construct revealed the presence of at least three different conformations (Fig. 6c,d). In one conformation, the distance between the labels on the DP1 and TPR2A domains is extended and a low-FRET signal is detected (low-FRET conformation with a FRET efficiency of 15%). A second high-FRET conformation (E=95%) is also observable where the two domains are close to each other. A third intermediate-FRET conformation can be seen at E∼50%, particularly in the presence of Hsp70 or Hsp90 or both Hsps. Together this suggests that different conformations of Sti1 exist and that Hsp90 and Hsp70 modulate the equilibrium between these conformations.

Bottom Line: In the presence of Hsp90, Hsp70 shifts its preference.The linker connecting the two modules is crucial for the interaction with Hsp70 and for client activation in vivo.Our results suggest that the interaction of Hsp70 with Sti1 is tightly regulated by Hsp90 to assure transfer of Hsp70 between the modules, as a prerequisite for the efficient client handover.

View Article: PubMed Central - PubMed

Affiliation: Center for integrated protein science (CIPSM) at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.

ABSTRACT
The cochaperone Sti1/Hop physically links Hsp70 and Hsp90. The protein exhibits one binding site for Hsp90 (TPR2A) and two binding sites for Hsp70 (TPR1 and TPR2B). How these sites are used remained enigmatic. Here we show that Sti1 is a dynamic, elongated protein that consists of a flexible N-terminal module, a long linker and a rigid C-terminal module. Binding of Hsp90 and Hsp70 regulates the Sti1 conformation with Hsp90 binding determining with which site Hsp70 interacts. Without Hsp90, Sti1 is more compact and TPR2B is the high-affinity interaction site for Hsp70. In the presence of Hsp90, Hsp70 shifts its preference. The linker connecting the two modules is crucial for the interaction with Hsp70 and for client activation in vivo. Our results suggest that the interaction of Hsp70 with Sti1 is tightly regulated by Hsp90 to assure transfer of Hsp70 between the modules, as a prerequisite for the efficient client handover.

No MeSH data available.


Related in: MedlinePlus