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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

Interdomain contacts between TPR2B and DP2.(a) Left: overlay of 15N-HSQC spectra for isolated TPR2B, isolated DP2 and TPR2B-DP2. Right, upper panel: chemical shift differences plotted as a function against residues. Right, bottom panel: mapping of the shifts into a model of TPR2B-DP2 generated from the isolated structures (PDB 2LLW and 3UPV) using Xplor-NIH. (b) PRE data for the interaction between TPR2B and different PROXYL labelled DP2 variants in the two-domain construct and mapping of the positions onto a model of TPR2B-DP2. The position of the spin label is indicated in green. (c) NMR/SAXS model of Sti1 TPR2A–TPR2B–DP2. The five best structures based on the fit to the experimental data of 50 calculated structures were selected and aligned to TPR2A-TPR2B (residues 262–515). The positions of the E525C spin label, chemical shift perturbations and highest PREs in TPR2B (residues coloured red) are highlighted. The TPR and DP domains are shown in blue and green, respectively. The Cα atoms of flexible residues were modelled by the program CORAL and are shown as grey spheres.
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f2: Interdomain contacts between TPR2B and DP2.(a) Left: overlay of 15N-HSQC spectra for isolated TPR2B, isolated DP2 and TPR2B-DP2. Right, upper panel: chemical shift differences plotted as a function against residues. Right, bottom panel: mapping of the shifts into a model of TPR2B-DP2 generated from the isolated structures (PDB 2LLW and 3UPV) using Xplor-NIH. (b) PRE data for the interaction between TPR2B and different PROXYL labelled DP2 variants in the two-domain construct and mapping of the positions onto a model of TPR2B-DP2. The position of the spin label is indicated in green. (c) NMR/SAXS model of Sti1 TPR2A–TPR2B–DP2. The five best structures based on the fit to the experimental data of 50 calculated structures were selected and aligned to TPR2A-TPR2B (residues 262–515). The positions of the E525C spin label, chemical shift perturbations and highest PREs in TPR2B (residues coloured red) are highlighted. The TPR and DP domains are shown in blue and green, respectively. The Cα atoms of flexible residues were modelled by the program CORAL and are shown as grey spheres.

Mentions: As the SAXS data suggested that in the Sti1 TPR2A–TPR2B–DP2 segment DP2 is in contact with TPR2B, we compared NMR spectra of 15N-labelled TPR2B-DP2 with the spectra of the isolated domains (Fig. 2a). Shifts appeared not only for residues in the region connecting the two domains (residues 519 to 525) but also for residues in the DP2 domain (Fig. 2a). These are located in the first, second and the last helix of DP2 and also in the C-terminal end of TPR2B indicating an interaction between the two domains. We also performed paramagnetic relaxation enhancement (PRE) NMR measurements on TRP2B–DP2 constructs where cysteines were introduced at various positions within the DP2 domain (E525C, K536C, Q545C, N559C and N589C) and modified with a PROXYL spin label (Fig. 2b and Supplementary Fig. 3). Relaxation enhancement was mainly observed in DP2 but also residues in TPR2B and the linker connecting the two domains were affected. The strongest effects on TPR2B were observed when the spin label was attached to E525C (in the linker between TPR2B and DP2). Moreover, when the spin label was attached to a residue in the second helix of DP2 (K536C and Q545C), PRE effects were visible in the C-terminal helix of TPR2B and the linker region. Together with the chemical shift analysis data, these observations show that interdomain contacts are formed between the C-terminal helix of TPR2B, the linker and helix 1 and 2 of DP2.


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)

Interdomain contacts between TPR2B and DP2.(a) Left: overlay of 15N-HSQC spectra for isolated TPR2B, isolated DP2 and TPR2B-DP2. Right, upper panel: chemical shift differences plotted as a function against residues. Right, bottom panel: mapping of the shifts into a model of TPR2B-DP2 generated from the isolated structures (PDB 2LLW and 3UPV) using Xplor-NIH. (b) PRE data for the interaction between TPR2B and different PROXYL labelled DP2 variants in the two-domain construct and mapping of the positions onto a model of TPR2B-DP2. The position of the spin label is indicated in green. (c) NMR/SAXS model of Sti1 TPR2A–TPR2B–DP2. The five best structures based on the fit to the experimental data of 50 calculated structures were selected and aligned to TPR2A-TPR2B (residues 262–515). The positions of the E525C spin label, chemical shift perturbations and highest PREs in TPR2B (residues coloured red) are highlighted. The TPR and DP domains are shown in blue and green, respectively. The Cα atoms of flexible residues were modelled by the program CORAL and are shown as grey spheres.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Interdomain contacts between TPR2B and DP2.(a) Left: overlay of 15N-HSQC spectra for isolated TPR2B, isolated DP2 and TPR2B-DP2. Right, upper panel: chemical shift differences plotted as a function against residues. Right, bottom panel: mapping of the shifts into a model of TPR2B-DP2 generated from the isolated structures (PDB 2LLW and 3UPV) using Xplor-NIH. (b) PRE data for the interaction between TPR2B and different PROXYL labelled DP2 variants in the two-domain construct and mapping of the positions onto a model of TPR2B-DP2. The position of the spin label is indicated in green. (c) NMR/SAXS model of Sti1 TPR2A–TPR2B–DP2. The five best structures based on the fit to the experimental data of 50 calculated structures were selected and aligned to TPR2A-TPR2B (residues 262–515). The positions of the E525C spin label, chemical shift perturbations and highest PREs in TPR2B (residues coloured red) are highlighted. The TPR and DP domains are shown in blue and green, respectively. The Cα atoms of flexible residues were modelled by the program CORAL and are shown as grey spheres.
Mentions: As the SAXS data suggested that in the Sti1 TPR2A–TPR2B–DP2 segment DP2 is in contact with TPR2B, we compared NMR spectra of 15N-labelled TPR2B-DP2 with the spectra of the isolated domains (Fig. 2a). Shifts appeared not only for residues in the region connecting the two domains (residues 519 to 525) but also for residues in the DP2 domain (Fig. 2a). These are located in the first, second and the last helix of DP2 and also in the C-terminal end of TPR2B indicating an interaction between the two domains. We also performed paramagnetic relaxation enhancement (PRE) NMR measurements on TRP2B–DP2 constructs where cysteines were introduced at various positions within the DP2 domain (E525C, K536C, Q545C, N559C and N589C) and modified with a PROXYL spin label (Fig. 2b and Supplementary Fig. 3). Relaxation enhancement was mainly observed in DP2 but also residues in TPR2B and the linker connecting the two domains were affected. The strongest effects on TPR2B were observed when the spin label was attached to E525C (in the linker between TPR2B and DP2). Moreover, when the spin label was attached to a residue in the second helix of DP2 (K536C and Q545C), PRE effects were visible in the C-terminal helix of TPR2B and the linker region. Together with the chemical shift analysis data, these observations show that interdomain contacts are formed between the C-terminal helix of TPR2B, the linker and helix 1 and 2 of DP2.

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