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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 and Ssa1 peptides containing the VEEVD motifITC by injecting into Tah1 (a) the pentapeptide VEEVD, (b) the heptapeptide PTVEEVD and (c) the decapeptide AEGEPTVEED, representing the extreme C-terminus of Ssa1. The results show that the Tah1 interaction with Ssa1 peptides remains weak and does not increase in affinity with increasing peptide length, in contrast with equivalent peptides based on Hsp90.
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Figure 4: ITC of Tah1 and Ssa1 peptides containing the VEEVD motifITC by injecting into Tah1 (a) the pentapeptide VEEVD, (b) the heptapeptide PTVEEVD and (c) the decapeptide AEGEPTVEED, representing the extreme C-terminus of Ssa1. The results show that the Tah1 interaction with Ssa1 peptides remains weak and does not increase in affinity with increasing peptide length, in contrast with equivalent peptides based on Hsp90.

Mentions: For Hop, the residue at position −4 of Hsp70 (I−4EEVD0 or VEEVD for SsaI) and Hsp90 (MEEVD) primarily determines the specificity for the TPR1 and TPR2a domains respectively [29]. To determine whether Tah1 ligand discrimination was similar to that of TPR2a we investigated the ability of Tah1 to bind peptides representing the C-terminal ends of Hsp90 and Ssa1. The results in Figure 3 show that the binding affinity for the C-terminal peptides of yeast Hsp90 increases with increasing peptide length (MEEVD, Kd=35.4±4.4 μM; TEMEEVD, Kd=0.95±0.04 μM; PADTEMEEVD, Kd=0.73±0.04 μM; Figures 3a–3c) and that the decapeptide most probably represents the intact binding site for Tah1 (PADTEMEEVD, Kd=0.73±0.04 μM and full-length yeast Hsp90, Kd=0.78±0.06 μM; Figures 1a and 3c respectively). Indeed, the binding of Tah1 to the human Hsp90α and Hsp90β decapeptide was also similar to that of the corresponding full-length proteins (Hsp90α decapeptide, DDTSRMEEVD, Kd=0.9±0.06 μM; Hsp90β decapeptide, EDASRMEEVD, Kd=1.0±0.19 μM; full-length Hsp90α, Kd=0.33±0.03 μM and full-length Hsp90β, Kd=0.34±0.07 μM; Figures 1c, 1d, 3d and 3e). In contrast with the Hsp90 decapeptides, peptides representing the C-terminal end of Ssa1 showed a significantly weaker affinity for Tah1 (VEEVD, Kd=48.8±5.5 μM; PTVEEVD, Kd=10.6±1.4 μM; and AEGPTVEEVD, Kd=28.9±3.1 μM; Figures 4a–4c).


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 and Ssa1 peptides containing the VEEVD motifITC by injecting into Tah1 (a) the pentapeptide VEEVD, (b) the heptapeptide PTVEEVD and (c) the decapeptide AEGEPTVEED, representing the extreme C-terminus of Ssa1. The results show that the Tah1 interaction with Ssa1 peptides remains weak and does not increase in affinity with increasing peptide length, in contrast with equivalent peptides based on Hsp90.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: ITC of Tah1 and Ssa1 peptides containing the VEEVD motifITC by injecting into Tah1 (a) the pentapeptide VEEVD, (b) the heptapeptide PTVEEVD and (c) the decapeptide AEGEPTVEED, representing the extreme C-terminus of Ssa1. The results show that the Tah1 interaction with Ssa1 peptides remains weak and does not increase in affinity with increasing peptide length, in contrast with equivalent peptides based on Hsp90.
Mentions: For Hop, the residue at position −4 of Hsp70 (I−4EEVD0 or VEEVD for SsaI) and Hsp90 (MEEVD) primarily determines the specificity for the TPR1 and TPR2a domains respectively [29]. To determine whether Tah1 ligand discrimination was similar to that of TPR2a we investigated the ability of Tah1 to bind peptides representing the C-terminal ends of Hsp90 and Ssa1. The results in Figure 3 show that the binding affinity for the C-terminal peptides of yeast Hsp90 increases with increasing peptide length (MEEVD, Kd=35.4±4.4 μM; TEMEEVD, Kd=0.95±0.04 μM; PADTEMEEVD, Kd=0.73±0.04 μM; Figures 3a–3c) and that the decapeptide most probably represents the intact binding site for Tah1 (PADTEMEEVD, Kd=0.73±0.04 μM and full-length yeast Hsp90, Kd=0.78±0.06 μM; Figures 1a and 3c respectively). Indeed, the binding of Tah1 to the human Hsp90α and Hsp90β decapeptide was also similar to that of the corresponding full-length proteins (Hsp90α decapeptide, DDTSRMEEVD, Kd=0.9±0.06 μM; Hsp90β decapeptide, EDASRMEEVD, Kd=1.0±0.19 μM; full-length Hsp90α, Kd=0.33±0.03 μM and full-length Hsp90β, Kd=0.34±0.07 μM; Figures 1c, 1d, 3d and 3e). In contrast with the Hsp90 decapeptides, peptides representing the C-terminal end of Ssa1 showed a significantly weaker affinity for Tah1 (VEEVD, Kd=48.8±5.5 μM; PTVEEVD, Kd=10.6±1.4 μM; and AEGPTVEEVD, Kd=28.9±3.1 μM; Figures 4a–4c).

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