Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation

Abstract

The heat shock proteins Hsp70 and Hsp90 require the help of tetratricopeptide repeat (TPR) domain-containing co-chaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can only interact with a single TPR co-chaperone at a time and each member of the TPR co-chaperone family brings distinct functions into the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR co-chaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity amongst the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR co-chaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other co-chaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between Hsp70/90 and the TPR co-chaperones.

On the role, ecology, phylogeny, and structure of dual-family immunophilins

“…The binding exhibits various degrees of specificity between the two partners; Hsp90, for example, is preferred over Hsp70 to bind to the TPRs of the large FKBPs (Assimon et al 2015;Guy et al 2015). In particular, peptide-TPR co-crystal structure and mutational analysis have established that the invariant C-terminal pentapeptide, MEEVD, of Hsp90 (Gupta 1995) interacts with specific residues that are common to the TPRs of long FKBPs and CYNs (Assimon et al 2015;Cliff et al 2006;Scheufler et al 2000;Ward et al 2002;Wu et al 2004). The residues, found to be absolutely essential for MEEVD binding, are: K5, N9 in TPR1, N6 in TPR2, and K2, R6 in TPR3.…”

Predicting protein targets for drug-like compounds using transcriptomics

“…Fluorescence polarization assay: Fluorescence polarization (FP) studies were carried out as previously described (Assimon et al, 2015). Briefly, the FP tracer was composed of a peptide derived from Hsp72/HSPA1A (GSGPTIEEVD) that was coupled at the N-terminus Products were visualized using a Bio-Rad ChemiDoc XRS+ imaging system and quantified using ImageJ software.…”

Cracking the Chaperone Code: Cellular Roles for Hsp70 Phosphorylation

“…Given that T636 phosphorylation seems especially prevalent in proliferating cancer cells, this could be a prime determinant of oncoprotein stability, driving cancer cell growth (11). More recent studies have shown that T636 phosphorylation, while greatly impacting HOP and CHIP interactions had little effect on the binding of other co-chaperones such as DnaJC7, FKBP51 and FKBP52, even though they bind to Hsp70 at roughly the same location (12). The regulation of the selectivity of binding to co-chaperones remains obscure, but an interesting facet of chaperone quality control.…”