{"doi":"10.1016/j.jbc.2022.101697","title":"Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities","abstract":"Chaperones of the heat shock protein 70 (Hsp70) family engage in protein–protein interactions with many cochaperones. One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, 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, 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. 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 cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70–CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70–DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein–protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded. Chaperones of the heat shock protein 70 (Hsp70) family engage in protein–protein interactions with many cochaperones. One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, 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, 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. 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 cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70–CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70–DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein–protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded. Members of the heat shock protein 70 (Hsp70) family of molecular chaperones play a critical role in maintaining protein homeostasis (aka proteostasis). These chaperones bind to misfolded or unfolded proteins and direct them to diverse processes such as protein folding, translocation, complex formation, and degradation (1Mayer M.P. Bukau B. Hsp70 chaperones: Cellular functions and molecular mechanism.Cell. Mol. Life Sci. 2005; 62: 670-684Google Scholar, 2Kim Y.E. Hipp M.S. Bracher A. Hayer-Hartl M. Hartl F.U. Molecular chaperone functions in protein folding and proteostasis.Annu. Rev. Biochem. 2013; 82: 323-355Google Scholar). Remarkably, this diversity of functions is enabled by a relatively simple structure: Hsp70s are composed of a nucleotide-binding domain (NBD), a substrate-binding domain (SBD), and an ⍺-helical lid domain (Fig. 1A) (3Mayer M.P. Gierasch L.M. Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones.J. Biol. Che","journal":"Journal of Biological Chemistry","year":2022,"id":246590,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":37,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9467,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":451769,"name":"Cory M. Nadel","orcid":"0000-0001-6827-3302","position":1,"is_corresponding":false},{"id":815239,"name":"Emma C. Carroll","orcid":"0000-0002-6766-4245","position":2,"is_corresponding":false},{"id":660608,"name":"Taylor Arhar","orcid":"0000-0003-0526-4876","position":3,"is_corresponding":false},{"id":275686,"name":"Jason E. Gestwicki","orcid":"0000-0002-6125-3154","position":4,"is_corresponding":false},{"id":742249,"name":"Oleta T. Johnson","orcid":"0000-0001-5170-3003","position":0,"is_corresponding":true}],"reference_count":83,"raw_metadata":null,"created_at":"2026-07-19T00:23:48.622830Z","pmid":"35148989","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}