Abstract
Stip1 (STress-Inducible Protein 1) is a 60 kD co-chaperone containing two DP (aspartate-proline) domains and three TPR (tetratricopeptide repeat) domains, and the main function of Stip1 is to assist the connection between Hsp90 (Heak shock protein 90) and Hsp70 (Heat shock protein 70). TPR domains are important for co-chaperones, such as Stip1, HIP and CHIP, to bind to Hsps. The Stip1-Hsp90-Hsp70 ternary complex is responsible for client protein folding. Interestingly, one of the DP domains in Stip1, DP2, plays a central role in client protein folding process, while the mechanism is less understood. There is no report to define the interaction between DP2 and Hsp90 or Hsp70. However, we noticed the deletion of DP2 in Stip1 decreases the ability in binding Hsp90 and Hsp70 in the pull-down assay. In addition, the last C-terminal fragment (Peptide 520) of Stip1 reduced the growth of ovarian tumor cell. In this study, we try to define the roles of DP2 and Peptide520 by NMR titration experiments. The structure information of DP2 and Peptide520 were established by NMR backbone assignment. Based on the NMR titration experiments, Stip1 TPR2A-2B showed strong interaction with Hsp90 middle and C-terminal domain, whereas DP2 remains no interaction with any domain of Hsp90 or Hsp70. We noticed the direct interaction between Hsp70 ATPase domain and Hsp90 middle and C-terminal domain. However, Peptide 520 has no effect in modulating the binding of Stip1-Hsp90 and Hsp90-Hsp70. The roles of DP2 and Peptide 520 in the Stip1-Hsp90-Hsp70 ternary complex still need to be further elucidated.