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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 TPR-domain-containing proteins and the Hsp90 M705V mutationITC by injecting CHIP into (a) yeast Hsp90 (yHsp90) or (b) M705V mutant, or by injecting (c) yeast Hsp90 or (d) M705V mutant into cSti1, or by injecting (e) Tah1 into the M705V mutant. The results show that the M705V mutation of Hsp90 does not affect the binding of CHIP, but, in contrast, the binding of the cSti1 and Tah1 is significantly weaker.
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Figure 6: ITC of TPR-domain-containing proteins and the Hsp90 M705V mutationITC by injecting CHIP into (a) yeast Hsp90 (yHsp90) or (b) M705V mutant, or by injecting (c) yeast Hsp90 or (d) M705V mutant into cSti1, or by injecting (e) Tah1 into the M705V mutant. The results show that the M705V mutation of Hsp90 does not affect the binding of CHIP, but, in contrast, the binding of the cSti1 and Tah1 is significantly weaker.

Mentions: Having established with decapeptides that the methionine residue in the conserved MEEVD motif of Hsp90 confers specificity for Tah1 binding we investigated whether this was also true for the intact yeast Hsp90. We therefore made the yeast Hsp90 M705V mutation to see whether it would bind Tah1 significantly less tightly. First we tested the M705V mutation with CHIP, known to bind both Hsp90 and Hsp70, and then with the C-terminal domain of Sti1 (cSti1), which favours Hsp90 binding. As expected, the M705V mutation bound CHIP with more or less equal affinity (Kd=6.7±0.62 μM) relative to wild-type Hsp90 (Kd=4.2±0.63 μM; Figures 6a and 6b). A previous estimate for the CHIP and human Hsp90β interaction by ITC (Kd=4.9 μM) is consistent with the value obtained in the present study with yeast protein [24]. For cSti1, the M705V mutation bound less tightly (Kd=0.20±0.03 μM) than the wild-type protein (Kd=0.04±0.01 μM; Figures 6c and 6d). It is noted that previous estimates with full-length Sti1 appear to be slightly lower in affinity (Kd=0.24±0.07 μM and 0.33±0.03 μM; [35,47]) against the wild-type Hsp90, indicating that TPR1 may have a negative effect on the binding of the TPR2a domain. However, having established that the M705V mutation affected the binding of both CHIP and the cSti1 in a predictable manner, we next tested the binding of Tah1 and, as expected, we found that the M705V mutation inhibited the binding of Tah1 (Kd=13±1.7 μM) relative to wild-type protein (Kd=0.78±0.06 μM; Figures 1a and 6e).


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 TPR-domain-containing proteins and the Hsp90 M705V mutationITC by injecting CHIP into (a) yeast Hsp90 (yHsp90) or (b) M705V mutant, or by injecting (c) yeast Hsp90 or (d) M705V mutant into cSti1, or by injecting (e) Tah1 into the M705V mutant. The results show that the M705V mutation of Hsp90 does not affect the binding of CHIP, but, in contrast, the binding of the cSti1 and Tah1 is significantly weaker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2865030&req=5

Figure 6: ITC of TPR-domain-containing proteins and the Hsp90 M705V mutationITC by injecting CHIP into (a) yeast Hsp90 (yHsp90) or (b) M705V mutant, or by injecting (c) yeast Hsp90 or (d) M705V mutant into cSti1, or by injecting (e) Tah1 into the M705V mutant. The results show that the M705V mutation of Hsp90 does not affect the binding of CHIP, but, in contrast, the binding of the cSti1 and Tah1 is significantly weaker.
Mentions: Having established with decapeptides that the methionine residue in the conserved MEEVD motif of Hsp90 confers specificity for Tah1 binding we investigated whether this was also true for the intact yeast Hsp90. We therefore made the yeast Hsp90 M705V mutation to see whether it would bind Tah1 significantly less tightly. First we tested the M705V mutation with CHIP, known to bind both Hsp90 and Hsp70, and then with the C-terminal domain of Sti1 (cSti1), which favours Hsp90 binding. As expected, the M705V mutation bound CHIP with more or less equal affinity (Kd=6.7±0.62 μM) relative to wild-type Hsp90 (Kd=4.2±0.63 μM; Figures 6a and 6b). A previous estimate for the CHIP and human Hsp90β interaction by ITC (Kd=4.9 μM) is consistent with the value obtained in the present study with yeast protein [24]. For cSti1, the M705V mutation bound less tightly (Kd=0.20±0.03 μM) than the wild-type protein (Kd=0.04±0.01 μM; Figures 6c and 6d). It is noted that previous estimates with full-length Sti1 appear to be slightly lower in affinity (Kd=0.24±0.07 μM and 0.33±0.03 μM; [35,47]) against the wild-type Hsp90, indicating that TPR1 may have a negative effect on the binding of the TPR2a domain. However, having established that the M705V mutation affected the binding of both CHIP and the cSti1 in a predictable manner, we next tested the binding of Tah1 and, as expected, we found that the M705V mutation inhibited the binding of Tah1 (Kd=13±1.7 μM) relative to wild-type protein (Kd=0.78±0.06 μM; Figures 1a and 6e).

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