Limits...
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

Hsp70-binding sites of Sti1.(a) SAXS data showing a comparison of the experimental radial density distributions of Hsp70 at increasing stoichiometric ratios of wild-type Sti1 as indicated. (b) Affinities of Sti1 variants towards yHsp70 determined by surface plasmon resonance spectroscopy with an Hsp70-coupled chip, normalized to wild-type Sti1 levels. Error bars indicate fitting error. (c–e) Formation of ternary Hsp90–Sti1–Hsp70 complexes using (c) wild-type Sti1, (d) Sti1 N39A and (e) Sti1 TPR2A-TPR2B. The 0.5 μM fluorescein-labelled yHsp70 and 3 μM yHsp90 were incubated with 0.5 (black), 1 (orange), 2 (blue) and 4 μM (cyan) Sti1 variant in 10 mM potassium phosphate at pH 7.5. Analytical ultracentrifugation was performed at 20 °C and 42 000 r.p.m. Sedimentation profiles were converted into dc/dt plots using standard procedures46 and fitted with bi-Gaussian functions. For clarity, only fits are shown, s.e. values were below 1%. As orientation, the lines indicate observed sedimentation coefficients (Hsp70* alone 4.2 S, left, binary complexes of Hsp70* with Sti1 5.6 S and ternary complexes of Hsp70* with Sti1 and Hsp90 8–10 S) from previous studies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4403447&req=5

f3: Hsp70-binding sites of Sti1.(a) SAXS data showing a comparison of the experimental radial density distributions of Hsp70 at increasing stoichiometric ratios of wild-type Sti1 as indicated. (b) Affinities of Sti1 variants towards yHsp70 determined by surface plasmon resonance spectroscopy with an Hsp70-coupled chip, normalized to wild-type Sti1 levels. Error bars indicate fitting error. (c–e) Formation of ternary Hsp90–Sti1–Hsp70 complexes using (c) wild-type Sti1, (d) Sti1 N39A and (e) Sti1 TPR2A-TPR2B. The 0.5 μM fluorescein-labelled yHsp70 and 3 μM yHsp90 were incubated with 0.5 (black), 1 (orange), 2 (blue) and 4 μM (cyan) Sti1 variant in 10 mM potassium phosphate at pH 7.5. Analytical ultracentrifugation was performed at 20 °C and 42 000 r.p.m. Sedimentation profiles were converted into dc/dt plots using standard procedures46 and fitted with bi-Gaussian functions. For clarity, only fits are shown, s.e. values were below 1%. As orientation, the lines indicate observed sedimentation coefficients (Hsp70* alone 4.2 S, left, binary complexes of Hsp70* with Sti1 5.6 S and ternary complexes of Hsp70* with Sti1 and Hsp90 8–10 S) from previous studies.

Mentions: As demonstrated previously, Hsp70 can bind alternatively to TPR1 and TPR2B22. This is consistent with our SAXS analysis where Hsp70 is shown to bind TPR1 and TPR2B individually (Supplementary Fig. 2b,f). Our SAXS experiments also show that the molecular weight of the main complex between full-length Sti1 and Hsp70 does not change comparing a 1:1 to a 1:2 stoichiometry (Fig. 3a). As we used concentrations above both individual binding affinities, we conclude that full-length Sti1, although containing two Hsp70-binding sites, binds to Hsp70 in a 1:1 stoichiometry (Fig. 3a). This is in accordance with previous findings on the stoichiometry of the interaction of Hop and Hsp70 (refs 35, 36).


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)

Hsp70-binding sites of Sti1.(a) SAXS data showing a comparison of the experimental radial density distributions of Hsp70 at increasing stoichiometric ratios of wild-type Sti1 as indicated. (b) Affinities of Sti1 variants towards yHsp70 determined by surface plasmon resonance spectroscopy with an Hsp70-coupled chip, normalized to wild-type Sti1 levels. Error bars indicate fitting error. (c–e) Formation of ternary Hsp90–Sti1–Hsp70 complexes using (c) wild-type Sti1, (d) Sti1 N39A and (e) Sti1 TPR2A-TPR2B. The 0.5 μM fluorescein-labelled yHsp70 and 3 μM yHsp90 were incubated with 0.5 (black), 1 (orange), 2 (blue) and 4 μM (cyan) Sti1 variant in 10 mM potassium phosphate at pH 7.5. Analytical ultracentrifugation was performed at 20 °C and 42 000 r.p.m. Sedimentation profiles were converted into dc/dt plots using standard procedures46 and fitted with bi-Gaussian functions. For clarity, only fits are shown, s.e. values were below 1%. As orientation, the lines indicate observed sedimentation coefficients (Hsp70* alone 4.2 S, left, binary complexes of Hsp70* with Sti1 5.6 S and ternary complexes of Hsp70* with Sti1 and Hsp90 8–10 S) from previous studies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Hsp70-binding sites of Sti1.(a) SAXS data showing a comparison of the experimental radial density distributions of Hsp70 at increasing stoichiometric ratios of wild-type Sti1 as indicated. (b) Affinities of Sti1 variants towards yHsp70 determined by surface plasmon resonance spectroscopy with an Hsp70-coupled chip, normalized to wild-type Sti1 levels. Error bars indicate fitting error. (c–e) Formation of ternary Hsp90–Sti1–Hsp70 complexes using (c) wild-type Sti1, (d) Sti1 N39A and (e) Sti1 TPR2A-TPR2B. The 0.5 μM fluorescein-labelled yHsp70 and 3 μM yHsp90 were incubated with 0.5 (black), 1 (orange), 2 (blue) and 4 μM (cyan) Sti1 variant in 10 mM potassium phosphate at pH 7.5. Analytical ultracentrifugation was performed at 20 °C and 42 000 r.p.m. Sedimentation profiles were converted into dc/dt plots using standard procedures46 and fitted with bi-Gaussian functions. For clarity, only fits are shown, s.e. values were below 1%. As orientation, the lines indicate observed sedimentation coefficients (Hsp70* alone 4.2 S, left, binary complexes of Hsp70* with Sti1 5.6 S and ternary complexes of Hsp70* with Sti1 and Hsp90 8–10 S) from previous studies.
Mentions: As demonstrated previously, Hsp70 can bind alternatively to TPR1 and TPR2B22. This is consistent with our SAXS analysis where Hsp70 is shown to bind TPR1 and TPR2B individually (Supplementary Fig. 2b,f). Our SAXS experiments also show that the molecular weight of the main complex between full-length Sti1 and Hsp70 does not change comparing a 1:1 to a 1:2 stoichiometry (Fig. 3a). As we used concentrations above both individual binding affinities, we conclude that full-length Sti1, although containing two Hsp70-binding sites, binds to Hsp70 in a 1:1 stoichiometry (Fig. 3a). This is in accordance with previous findings on the stoichiometry of the interaction of Hop and Hsp70 (refs 35, 36).

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