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Chaperone ligand-discrimination by the TPR-domain protein Tah1.

Millson SH, Vaughan CK, Zhai C, Ali MM, Panaretou B, Piper PW, Pearl LH, Prodromou C - Biochem. J. (2008)

Bottom Line: Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein.Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70.In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.

ABSTRACT
Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

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ITC of Tah1 with Hsp90 and Ssa1ITC by injecting Tah1 into (a) yeast Hsp90 (yHsp90), (b) the C-terminal domain of yeast Hsp90 (C-yHsp90), (c) Hsp90α, (d) Hsp90β and (e) Ssa1. The results show that the Tah1 interaction with full-length yeast Hsp90, Hsp90α, Hsp90β and C-yHsp90 is significantly tighter than against the yeast Hsp70, Ssa1.
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Figure 1: ITC of Tah1 with Hsp90 and Ssa1ITC by injecting Tah1 into (a) yeast Hsp90 (yHsp90), (b) the C-terminal domain of yeast Hsp90 (C-yHsp90), (c) Hsp90α, (d) Hsp90β and (e) Ssa1. The results show that the Tah1 interaction with full-length yeast Hsp90, Hsp90α, Hsp90β and C-yHsp90 is significantly tighter than against the yeast Hsp70, Ssa1.

Mentions: Tah1 has been identified as a TPR-domain protein that interacts with Hsp90. Our sequence alignments suggest that Tah1 is most similar to the TPR2b-domain of Hop/Sti1 (Supplementary Figure S1 at http://www.BiochemJ.org/bj/413/bj4130261add.htm), which influences the binding of Hop to both Hsp70 and Hsp90 [39]. In order to investigate the ability of Tah1 to bind both Ssa1 and Hsp90 we used ITC to determine the extent of any interaction. Figure 1(a) shows that Tah1 binds to the full-length yeast Hsp90 (Kd=0.78±0.06 μM) with a stoichiometry of 1:1, whereas Figure 1(b) shows that the binding site is solely located in the C-terminal domain of Hsp90 (Kd=0.32±0.03 μM). Table 1 summarizes all ITC data in the present study. Tah1 was also shown to be able to interact with the human Hsp90α (Kd=0.33±0.03 μM) and Hsp90β (Kd=0.34±0.07 μM) paralogues with a similar affinity (Figures 1c and 1d). However, its interaction with the yeast Ssa1 was significantly weaker (Kd=16±2.5 μM; Figure 1e), where the strength of the interaction is reflected in the shape of the binding curve for which a steeper gradient in the observed curve indicates tighter binding (compare Figures 1a and 1e), and suggests that Tah1 is an Hsp90-specific co-chaperone.


Chaperone ligand-discrimination by the TPR-domain protein Tah1.

Millson SH, Vaughan CK, Zhai C, Ali MM, Panaretou B, Piper PW, Pearl LH, Prodromou C - Biochem. J. (2008)

ITC of Tah1 with Hsp90 and Ssa1ITC by injecting Tah1 into (a) yeast Hsp90 (yHsp90), (b) the C-terminal domain of yeast Hsp90 (C-yHsp90), (c) Hsp90α, (d) Hsp90β and (e) Ssa1. The results show that the Tah1 interaction with full-length yeast Hsp90, Hsp90α, Hsp90β and C-yHsp90 is significantly tighter than against the yeast Hsp70, Ssa1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2865030&req=5

Figure 1: ITC of Tah1 with Hsp90 and Ssa1ITC by injecting Tah1 into (a) yeast Hsp90 (yHsp90), (b) the C-terminal domain of yeast Hsp90 (C-yHsp90), (c) Hsp90α, (d) Hsp90β and (e) Ssa1. The results show that the Tah1 interaction with full-length yeast Hsp90, Hsp90α, Hsp90β and C-yHsp90 is significantly tighter than against the yeast Hsp70, Ssa1.
Mentions: Tah1 has been identified as a TPR-domain protein that interacts with Hsp90. Our sequence alignments suggest that Tah1 is most similar to the TPR2b-domain of Hop/Sti1 (Supplementary Figure S1 at http://www.BiochemJ.org/bj/413/bj4130261add.htm), which influences the binding of Hop to both Hsp70 and Hsp90 [39]. In order to investigate the ability of Tah1 to bind both Ssa1 and Hsp90 we used ITC to determine the extent of any interaction. Figure 1(a) shows that Tah1 binds to the full-length yeast Hsp90 (Kd=0.78±0.06 μM) with a stoichiometry of 1:1, whereas Figure 1(b) shows that the binding site is solely located in the C-terminal domain of Hsp90 (Kd=0.32±0.03 μM). Table 1 summarizes all ITC data in the present study. Tah1 was also shown to be able to interact with the human Hsp90α (Kd=0.33±0.03 μM) and Hsp90β (Kd=0.34±0.07 μM) paralogues with a similar affinity (Figures 1c and 1d). However, its interaction with the yeast Ssa1 was significantly weaker (Kd=16±2.5 μM; Figure 1e), where the strength of the interaction is reflected in the shape of the binding curve for which a steeper gradient in the observed curve indicates tighter binding (compare Figures 1a and 1e), and suggests that Tah1 is an Hsp90-specific co-chaperone.

Bottom Line: Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein.Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70.In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.

ABSTRACT
Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

Show MeSH