J Biol Chem. 2022 Feb 08. pii: S0021-9258(22)00137-5. [Epub ahead of print] 101697
Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions (PPIs) with many co-chaperones to modulate their functions. One "hotspot" for co-chaperone binding is the EEVD motif, found at the extreme C-terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat (TPR) domain co-chaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in Class B J-domain proteins (JDPs), such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4 in parallel. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these co-chaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, while CHIP suppresses the client re-folding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse PPIs, such that competition between co-chaperones may help guide whether Hsp70-bound proteins are folded or degraded.
Keywords: J-domain protein; fluorescence polarization; molecular chaperone; peptide-protein interaction; protein folding; protein-protein interaction; tetratricopeptide repeat; ubiquitination