Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure

Abstract

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.Keywords: protein structure/folding; chaperonins; circular dichroism; tetratricopeptide repeatThe dynamic assembly and functional maturation of steroid receptor complexes involves the major molecular chaperones and heat shock proteins Hsp70 and Hsp90 as well as several Hsp-binding co-chaperones. Free receptor undergoes sequential association with Hsp40, Hsp70, the dual co-chaperone Hop, and ultimately Hsp90; only when Hsp90 directly binds receptor is high-affinity hormone binding established (Scherrer et al. 1990;Smith 1993). The progression of chaperone interactions observed with steroid receptors is representative of Hsp70 and Hsp90 interactions with a variety of Hsp90 client proteins, so there likely are common mechanisms underlying the manner in which Hsp90/client protein interactions are established (Wegele et al. 2004). Hop provides an important link between Hsp70 and Hsp90 since it can simultaneously bind both Abbreviations: CD, circular dichroism; HOP, Hsp70/Hsp90 organizing protein; HSP, heat shock protein; TPR, tetratricopeptide repeat.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

“…Similar results have been reported previously for the interaction of HOP with C-terminally truncated Hsp70 protein (13). Several other studies of the HOP-Hsp70 complex suggested the existence of secondary contacts strengthening the clamp-EEVD interaction between these proteins (9,35). Additionally, a model has been proposed in which the TPR domain is initially recognized by multiple Hsp70/Hsp90 sites, and this interaction is then secured by two-carboxylate clamp-EEVD motif interaction (11).…”

Hsp 70/Hsp 90 organizing protein as a nitrosylation target in cystic fibrosis therapy

“…Hop is a 60-kDa homodimer that links Hsp70 and Hsp90, central components of the regulation of CFTR trafficking, and it Co-IP's with CFTR (3). In addition, Hop is involved in cell-cycle regulation and steroid receptor maturation (24,34,35), and it is homologous to the CHIP complex, of importance to CFTR degradation (36). Hop has a conserved cysteine (C403) (23,24) located in an S-nitrosylation motif believed to be relevant to S-nitrosylation (26).…”

IFN-Induced Protein with Tetratricopeptide Repeats 2 Inhibits Migration Activity and Increases Survival of Oral Squamous Cell Carcinoma

“…Therefore, whether IFIT2 acts as an adaptor protein between heat shock proteins and CK18 to modulate the activity of CK18 needs to be addressed. Although the detailed mechanism of IFIT2-cytokeratin interactions is unknown, tetratricopeptide repeat -containing proteins have been shown to associate with heat shock protein 70 and heat shock protein 90 and act as cochaperones to regulate protein structure, stability, and degradation (28).…”